FORMULATION AND STABILITY OF ASCORBIC ACID IN LIQUID AND SEMISOLID PREPARATIONS

Thesis

presented by

MUHAMMAD ALI SHERAZ BPharm M Phil (BMU) RPh

for the degree of

Doctor of Philosophy

in

Baqai Medical University

Department of Pharmaceutics Faculty of Pharmaceutical Sciences Baqai Medical University Karachi Pakistan October 2009

CERTIFICATE

This is to certify that the work presented in this thesis entitled ldquoFormulation and

Stability of Ascorbic Acid in Liquid and Semisolid Preparationsrdquo is original and has

been conducted by Mr Muhammad Ali Sheraz under my supervision as fulfilment of

the requirement of PhD degree from the Faculty of Pharmaceutical Sciences Baqai

Medical University Karachi

Professor Dr Iqbal Ahmad

Faculty of Pharmaceutical Sciences

Baqai Medical University Karachi

iv

ABSTRACT

The present investigation is based on a study of the photodegradation of ascorbic

acid (vitamin C) in organic solvents and in oil-in-water cream preparations containing a

combination of emulsifying agents and humectants It also involves the study of the effect

of other vitamins (riboflavin nicotinamide and alpha-tocopherol) and certain compounds

acting as stabilizing agents (citric acid tartaric acid and boric acid) on the rate of

photodegradation of ascorbic acid in cream preparations The photodegradation of

ascorbic acid in organic solvents and cream preparations (pH 40ndash70) leads to the

formation of dehydroascorbic acid which is also biologically active The kinetics of

photodegradation of ascorbic acid alone and in combination with other vitamins in

creams has been studied using a UV spectrophotometric method and the official

iodimetric method respectively These methods were validated in the presence and

absence of other vitamins stabilizing agents under the experimental conditions

employed The recoveries of ascorbic acid in creams are in the range of 90ndash96 and the

reproducibility of the analytical methods is within plusmn 5

The apparent first-order rate constants (kobs) for the photodegradation of ascorbic

acid in aqueous organic solvents (029ndash040 times 10ndash3

minndash1

) and in creams (044ndash142 times

10ndash3

minndash1

) have been determined A linear relationship has been observed between kobs

and solvent dielectric constant reciprocal of solvent viscosity indicating the dependence

of the rate of photodegradation on solvent characteristics

In the creams the photodegradation of ascorbic acid appears to be affected by the

concentration of other vitamins pH of the medium carbon chain length of the

emulsifying agents (myristic acid palmitic acid and stearic acid) viscosity of the

v

humectant (ethylene glycol propylene glycol and glycerin) and redox potentials of

ascorbic acid The study indicates that the relative polar character of the emulsifying

agent and the ionized state and redox potential of ascorbic acid at a particular pH are

important factors in the photodegradation of ascorbic acid in creams

The second-order rate constants (kprime) (320 times 10ndash2

ndash 189 Mndash1

minndash1

) for the

photochemical interaction of ascorbic acid and the individual vitamins (riboflavin

nicotinamide alpha-tocopherol) along with the values of k0 obtained from the intercepts

of the plots of kobs versus vitamin concentration are also reported The values of k0

indicate that riboflavin and nicotinamide act as photosensitizing agents and alpha-

tocopherol acts as a stabilizing agent in the photodegradation of ascorbic acid in the

creams The kobs verses pH profiles for the photodegradation of ascorbic acid in creams

represents sigmoid type curves indicating the oxidation of the ionized form (AHndash) of

ascorbic acid (pKa1 41) with pH The AHndash species appears to be more susceptible to

photooxidation than the non-ionized form of ascorbic acid The effect of stabilizing

agents on the photodegradation of ascorbic acid has been found to be in the order of citric

acid gt tartaric acid gt boric acid The low activity of boric acid may be to some extent due

to its interaction with the emulsifying agents and humectants The polarity of the

emulsifying acids also plays a part in the rate of degradation of ascorbic acid Reaction

schemes for the photodegradation of ascorbic acid and its photochemical interaction with

riboflavin nicotinamide and alpha-tocopherol have been presented

vi

ACKNOWLEDGMENTS

I am highly grateful to All Mighty Allah who guided me in all difficulties and

provided me strength to overcome the problems during this work

Words are confined and inefficacious to express my immense gratitude to my

respectable supervisor Prof Dr Iqbal Ahmad Department of Pharmaceutical

Chemistry for his guidance encouragement keen interest and above all giving his

valuable time suggestions and attention His personality has been a source of constant

inspiration through out my research work

I would like to extend my sincere thanks to Prof Lt Gen (R) Dr Syed Azhar

Ahmed Vice Chancellor Baqai Medical University for his personal interest and

constant encouragement through out the study

It is my great desire to express my gratitude to Prof Dr Syed Fazal Hussain

CEO Baqai Institute of Pharmaceutical Sciences for his cooperation and attention and

providing all the facilities of the Institute at my disposal during the research work

I am also thankful to Mrs Shaukat Khalid Dean Faculty of Pharmaceutical

Sciences for her support during the study

I feel honored to express my sincere thanks and indebtedness to Prof Dr

Khursheed Ali Khan Department of Pharmaceutics Prof Dr Aminuddin Department

of Pharmaceutical Chemistry and Dr Faiyaz H M Vaid Chairman Department of

Pharmaceutical Chemistry Faculty of Pharmacy University of Karachi who helped me

selflessly with their invaluable suggestions through out the research work

vii

I feel immense pleasure to pay my sincere and special thanks to Ms Sofia

Ahmed Assistant Professor and In charge Department of Pharmaceutics who lent all

sort of cooperation and spared no effort in helping me during this work

Special thanks are due to Mr Saif-ur-Rehman Khatak Deputy Drug Controller

for his cooperation and help during this study

I acknowledge with sincere thanks the contribution of Tabros Pharmaceutical

Industry Karachi for providing me the opportunity to use their facilities for certain

measurements without which the completion of this work would not have been possible

I highly appreciate the technical services rendered by Mr Anees Mr Wajahat

and Mr Sajjad in pursuance of this study

I am very grateful to Mrs Prof Dr Iqbal Ahmad for her kindness and generous

hospitality during my innumerable visits to their residence

Last but not the least I would like to express my immense indebtedness to My

Gracious Parents Beloved Brothers and Sisters for their moral support kindness and

encouragement throughout my career

I am also thankful to all my students for their affectionate feelings

M A S

viii

To

My Beloved Parents amp

Late Prof Dr S Sabir Ali for their interest and endless support

ix

CONTENTS

Chapter Page

ABSTRACT iv

ACKNOWLEDGEMENTS vi

I INTRODUCTION 1

11 HISTORICAL BACKGROUND 2

12 PHYSICOCHEMICAL CHARACTERISTICS OF

ASCORBIC ACID

2

13 CHEMISTRY OF ASCORBIC ACID 3

131 Nomenclature and Structure 3

132 Chemical Stability 3

14 BIOCHEMICAL FUNCTIONS 7

15 ANTIOXIDANT ACTIVITY 8

16 PHOTOSTABILITY OF DRUGS 9

17 KINETIC TREATMENTS OF PHOTOCHEMICAL

REACTIONS

12

18 LITERATURE ON ASCORBIC ACID 15

II PHOTODEGRADATION REACTIONS AND ASSAY OF

ASCORBIC ACID

17

21 PHOTODEGRADATION REACTIONS 18

211 Photodegradation of Ascorbic Acid 18

212 Effect of Various Substances on Photodegradation of Ascorbic

Acid

20

213 Photosensitized Oxidation of Ascorbic Acid 22

214 Ascorbic Acid Interaction with Other Water-Soluble Vitamins 25

22 ASSAY OF ASCORBIC ACID 26

221 Spectrophotometric Methods 26

222 Fluorimetric Methods 28

x

223 Mass spectrometric Methods 28

224 Chromatographic Methods 28

225 Enzymatic Methods 29

226 Commercial Kits for Clinical Analysis 30

227 Analysis in Creams 30

III FORMULATION AND STABILITY OF CREAM

PREPARATIONS

31

31 FORMULATION OF CREAM PREPARATIONS 32

311 Choice of Emulsion Type 32

312 Choice of Oil Phase 33

313 Emulsion Consistency 33

314 Choice of Emulsifying Agent 34

315 Formulation by the HLB Method 34

316 Concept of Relative Polarity Index 35

32 FORMULATION OF ASCORBIC ACID CREAMS 37

33 STABILITY OF CREAMS 39

331 Physical Stability 39

332 Chemical Stability 39

333 Microbial Stability 40

334 Stability of Ascorbic Acid in Liquid Formulations 41

335 Stability of Ascorbic Acid in Emulsions and Creams 41

336 Stability Testing of Emulsions 45

3361 Macroscopic examination 46

3362 Globule size analysis 46

3363 Change in viscosity 46

3364 Accelerated stability tests 46

337 FDA Guidelines for Semisolid Preparations 46

xi

OBJECT OF PRESENT INVESTIGATION 48

IV MATERIALS AND METHODS 51

41 MATERIALS 52

42 METHODS 55

421 Cream Formulations 55

422 Preparation of Creams 56

423 Thin-Layer Chromatography 57

424 pH Measurements 57

425 Ultraviolet and Visible Spectrometry 58

426 Photolysis of Ascorbic Acid 59

4261 Creams 59

4262 Aqueous and organic solvents 59

4263 Storage of creams in dark 59

427 Measurement of Light Intensity 59

428 Procedure 60

4281 Calculation 62

429 Viscosity Measurements 63

4210 Assay method 65

42101 UV spectrophotometric method for the assay of creams

containing ascorbic acid alone

65

42102 Iodimetric method for the assay of ascorbic acid in creams

containing riboflavin nicotinamide and alpha-tocopherol 65

42103 Spectrophotometric method for the assay of ascorbic acid in

aqueous and organic solvents

67

V PHOTODEGRADATION OF ASCORBIC ACID IN

ORGANIC SOLVENTS AND CREAM FORMULATIONS

68

51 INTRODUCTION 69

52 PHOTOPRODUCTS OF ASCORBIC ACID 71

xii

53 SPECTRAL CHARACTERISTICS OF PHOTOLYSED

SOLUTIONS

71

54 ASSAY OF ASCORBIC ACID IN CREAMS AND

SOLUTIONS

73

55 EFFECT OF SOLVENT 74

56 EFFECT OF CONCENTRATION 80

57 EFFECT OF CARBON CHAIN LENGTH OF

EMULSIFYING AGENT

88

58 EFFECT OF VISCOSITY 94

59 EFFECT OF pH 94

510 EFFECT OF REDOX POTENTIAL 96

511 PRIMARY PHOTOCHEMICAL REACTIONS IN THE

OXIDATION OF ASCORBIC ACID

97

512 DEGRADATION OF ASCORBIC ACID IN THE DARK 98

VI PHOTOCHEMICAL INTERACTION OF ASCORBIC

ACID WITH RIBOFLAVIN NICOTINAMIDE AND

ALPHA-TOCOPHEROL IN CREAM FORMULATIONS

109

61 INTRODUCTION 110

62 ABSORPTION CHARACTERISTICS OF PHOTOLYSED

CREAMS

114

63 PHOTOPRODUCTS OF ASCORBIC ACID AND OTHER

VITAMINS

114

64 ASSAY METHOD 116

65 KINETICS OF PHOTODEGRADATION OF ASCORBIC

ACID

117

66 INTERACTION OF RIBOFLAVIN WITH ASCORBIC ACID 128

67 INTERACTION OF NICOTINAMIDE WITH ASCORBIC

ACID

129

68 INTERACTION OF ΑLPHA-TOCOPHEROL WITH

ASCORBIC ACID

130

69 EFFECT OF CARBON CHAIN LENGTH OF

EMULSIFYING AGENT

130

xiii

610 EFFECT OF VISCOSITY OF CREAMS 132

611 DEGRADATION OF ASCORBIC ACID IN THE PRESENCE

OF OTHER VITAMINS IN THE DARK

135

VII STABILIZATION OF ASCORBIC ACID WITH CITRIC

ACID TARTARIC ACID AND BORIC ACID IN CREAM

FORMULATIONS

141

71 INTRODUCTION 142

72 CREAM FORMULATIONS 142

73 PRODUCTS OF ASCORBIC ACID

PHOTODEGRADATION

145

74 SPECTRAL CHANGES IN PHOTODEGRADED CREAMS 145

75 ASSAY OF ASCORBIC ACID IN CREAMS 145

76 KINETICS OF PHOTODEGRADATION 146

77 EFFECT OF STABILIZING AGENTS 146

78 DEGRADATION OF ASCORBIC ACID IN PRESENCE OF

STABILIZING AGENTS IN THE DARK

162

79 EFFECT OF ADDITIVES ON TRANSMISSION OF

ASCORBIC ACID

163

CONCLUSIONS AND SUGGESTIONS 179

CONCLUSIONS 180

SUGGESTIONS 184

REFERENCES 185

AUTHORrsquoS PUBLICATIONS 226

CHAPTER I

INTRODUCTION

2

11 HISTORICAL BACKGROUND

The disease scurvy which now is known as a condition due to a deficiency of

ascorbic acid in the diet has considerable historical significance (Schick 1943

Carpenter 1986 Bardolph and Taylor 1997 Thomas 1997 Bors 2005) Zilva (1932)

isolated the antiscorbutic activity factor from a crude fraction of lemon and showed that

the activity was destroyed by oxidation and protected by reducing agents Waugh and

King (1932) isolated crystalline vitamin C from lemon juice and showed it to be the

antiscorbutic factor Szent-Gyorgyi (1928) had isolated the same factor from pepper in

connection with his biological oxidation-reduction studies Hirst and Zilva (1933)

identified the antiscorbutic factor as ascorbic acid Early work on the chemical

identification and elucidation of the structure of ascorbic acid has been well documented

(Carpenter 1986) The first synthesis of L-ascorbic acid was achieved almost

simultaneously by Ault et al (1933) and Reichstein et al (1933)

Plants and most animals synthesize their own vitamin C but humans lack this

ability due to the deficiency in an enzyme L-gulono-gamma-lactone oxidase that

catalyzes the terminal step in ascorbic acid biosynthesis (Nishikimi et al 1994)

Therefore humans obtain this vitamin from diet and or vitamin supplements to not only

avoid the development of scurvy but also for overall well being (Stone 1969 Lewin

1976 Davies et al 1991) The minimal daily requirement for ascorbic acid in healthy

adults is 40ndash60 mg (Truswell 2003 Mason 2007 Eitenmiller et al 2008 Elia 2009)

12 PHYSICOCHEMICAL CHARACTERISTICS OF ASCORBIC ACID

The important physicochemical characteristics of ascorbic acid (Table 1) involved

in its identification and degradation are described by many authors (Connors et al 1986

3

OrsquoNeil 2001 Moffat et al 2004 Sinko 2006 Johnston et al 2007) The most

important chemical property of ascorbic acid is the reversible oxidation to semidehydro-

L-ascorbic acid and further oxidation to dehydro-L-ascorbic acid This property is the

basis for its physiological activity In addition the proton on oxygenndash3 is acidic (pKa1 =

417) which contributes to the acidic nature of ascorbic acid (1)

13 CHEMISTRY OF ASCORBIC ACID

131 Nomenclature and Structure

The IUPAC-IUB Commission on Biochemical Nomenclature changed the name

vitamin C (2-oxo-L-theo-hexono-4-lactone-23-enediol) to ascorbic acid or L-ascorbic

acid in 1965 (Johnston et al 2007) The chemical structure of ascorbic acid (1) is

HO OH

O

OHHO

H

(1)

O

The molecule has a near planar five-membered ring with two chiral centers

which contain four stereoisomers

132 Chemical Stability

Ascorbic acid is sensitive to air and light and is kept in a well-closed container

protected from light (British Pharmacopoeia 2009) The degradation reactions of

ascorbic acid in aqueous solution depend on a number of factors such as pH temperature

presence of oxygen or metal It is not very stable in aqueous media at room

temperature and undergoes oxidative degradation to dehydroascorbic acid and

4

Table 1 Physicochemical characteristics of ascorbic acid

Empirical formula C6H8O6

Molar mass 17613

Crystalline form Monoclinic mix of platelets and needles

Melting point 190 to 192 degC

[α]25

+205deg to +215deg

pH

5 mg ml

50 mg ml

~3

~2

pKa 417 1157 (20deg)

Redox potential

(dehydroascorbic acid ascorbate)

(H+ ascorbate

ndash)

ndash174 mV

+282 mV

Solubility g ml

Water

Ethanol absolute

Ether chloroform benzene

033

002

Insoluble

UV spectrum

Absorption maximum [A(1 1 cm)]

pH 20

pH 70

245 nm [695]

265 nm [940]

Infrared spectrum

Principal peaks (Nujol mull)

1026 (CminusOH str) 1111(CminusOminusC str) 1312

(minusCminusOminus str) 1653 (C=O str) 990 (C=C str)

cmndash1

Mass spectrum

Principal ions at mz

29 41 39 42 69 116 167 168

D

5

23-diketogulonic acid The stability of ascorbic acid and dehydroascorbic acid can be

improved by lowering the pH below 2 (Wechtersbach and Cigic 2007) Above pH 7

alkali-catalyzed degradation by cleavage at Cndash1 or Cndash2 results in a number of

compounds mainly monondash dindash and tricarboxylic acids (Connors et al 1986 Bors and

Buettner 1997 Halliwell and Whiteman 1997) The oxidative degradation of ascorbic

acid and dehydroascorbic acid in parenteral nutrition mixtures is catalyzed by trace

elements particularly copper (Allwood 1984ab Allwood et al 1992 Allwood and

Kearney 1998 Kearney et al 1998 Gibbons et al 2001) Stabilized ascorbic acid

preparations in hydroalcoholic vehicle (Kaplan et al 1989) and aquaculture feeds

(OrsquoKeefe 2001) have been reported The various oxidation products of ascorbic acid are

shown in Fig 1

It is interesting to note that in addition to redox and acid-base properties ascorbic

acid can exist as a free radical (Bielski et al 1981 Bielski 1982 Halliwell 1996 Bors

and Buettner 1997) The ascorbate radical anion is an important intermediate in the

reactions involving oxidants and ascorbic acidrsquos antioxidant activity Rate constants for

the generation of ascorbate radicals are in the range of 104ndash10

8 s

ndash1 When ascorbate

radicals are generated by oxyanions the rate constants are of the order of 104ndash10

7 s

ndash1

when generated by halide radicals 106ndash10

8 s

ndash1 and when generated by tocopherols and

flavonoids radicals 106ndash10

8 s

ndash1 (Bielski 1982 Halliwell and Whiteman 1997) The

ascorbate radicals decay usually by disproportionation However a change in ionic

strength or pH can influence the rate of dismutation of ascorbic acid Certain oxyanions

such as phosphates accelerate dismutation (Bielski et al 1981) The acceleration is

attributed to the activity of various protonated forms of phosphate to donate a proton

6

Fig 1 Oxidation products of ascorbic acid

O

OHOH

H

OO

OHOH

H

OO

OHOH

H

O

Ascorbyl radical anion

(interm ediate)

Ascorbic acid

(1)

-e- -2H

+

+e- +2H

+

-e-

+e-

Dehydroascorbic acid

(2)

23-diketo-L-gulonic acid

O xalic acid

+

L-Threonic acid

L-Xylose

+

C O 2

CO 2

L-Xylonic acid

+

L-Lyxonic acid CO 2

HO OH O O-

O O

7

efficiently to the ascorbate radical particularly the dimer form of ascorbate

The unusual stability of the ascorbate radical in biological systems dictates that

accessory enzymatic systems be made available to reduce the potential transient

accumulation of ascorbate radical The excess ascorbate radicals may initiate a chain of

free-radicals reactions In plants NADHmonodehydroascorbate reductase maintains

ascorbic acid in its reduced form NADHmonodehydroascorbate reductase plays a major

role in stress related responses in plants Glutathione dehydroascorbate reductase serves

this purpose in animal tissues Such enzymes keep ascorbic acid operating at maximum

efficiency so that other enzyme systems may take advantage of the univalent redox

cycling capacity of ascorbate (Asard et al 2004 Johnston et al 2007)

The anaerobic degradation of ascorbic acid has been studied by Finholt et al

(1963) Under these conditions the molecule is dehydrated and hydrolyzed in aqueous

solution to give furfural and carbon dioxide The rate of degradation is maximum at pH

41 corresponding to the pKa of ascorbic acid This has been suggested due to the

formation of a saltndashacid complex in solution The reaction is dependent on buffer

concentration but has relatively small effect of ionic strength

14 BIOCHEMICAL FUNCTIONS

Ascorbic acid plays an essential role in the activities of several enzymes It is vital

for the growth and maintenance of healthy bones teeth gums ligaments and blood

vessels It is important for the manufacture of certain neurotransmitters and adrenal

hormones Ascorbic acid is required for the utilization of folic acid and the absorption of

iron It is also necessary for normal immune responses to infection and for wound healing

(Henry 1997)

8

Ascorbic acid deprivation and scurvy include a range of signs and symptoms that

involves defects in specific enzymatic processes (Johnston et al 2007) The

administration of ascorbic acid improves most of the signs of chemically induced

glutathione (L-γ-glutamyl-L-cysteine-glycine GSH) deficiency (Meister 1994) The

effect is very pronounced in newborn rats which do not efficiently synthesize ascorbic

acid in contrast to adult rats and guinea pigs When L-buthionine-(SR)-sulphoxime is

administered in addition to the loss in GSH there is a marked increase in

dehydroascorbic acid This has led to the hypothesis that GSH is very important to

dehydroascorbic acid reduction and as a sequence to ascorbic acid recycling (Meister

1995)

Ascorbic acid also possesses pro-oxidant properties and may cause apoptosis

lymphoid and myeloid cells It has been shown that dehydroascorbic acid also stimulates

the antioxidant defenses in some cells by preferentially importing dehydroascorbate over

ascorbate (Braun et al 1997 Banhegyi et al 1998 Puskas et al 2000 2002)

15 ANTIOXIDANT ACTIVITY

Ascorbic acid is known to readily scavenge reactive oxygen and nitrogen species

such as superoxide and hydroperoxyl radicals aqueous peroxyl radicals singlet oxygen

ozone peroxynitrite nitrogen dioxide nitroxide radicals and hypochlorous acid Excess

of such products has been associated with lipids (Niki and Noguchi 1997 Carr et al

2000 Urso and Clarkson 2003) DNA (Fraga et al 1991 1996 Lindahl 1993) and

protein oxidation (Stadtman 1991 Berlett and Stadtman 1997 Dean et al 1997

Ortwerth and Monnier 1997 Padayatty et al 2003)

9

The electron donor character of ascorbate may be responsible for many of its

known biological functions Inspite of the availability of ascorbic acid to influence the

production of hydroxyl and alkoxyl radicals it remains uncertain whether this is the

principal effect or mechanism that occurs in vivo There is a good evidence that ascorbic

acid protects lipids in biological fluids as an antioxidant (Johnston et al 2007) A

detailed account of the function of ascorbate as an antioxidant and its reactions with

reactive nitrogen species and singlet oxygen has been reported by Packer et al (2002)

and Buettner and Schafer (2004)

Ascorbic acid (Eordm ndash0115 V pH 52 Sinko 2006) has been used as an antioxidant

for the stabilization of drugs with a higher oxidation potential These drugs include

morphine (Yeh and Lach 1961) vitamin A (Wright 1986) rifampin (Maggi et al

1966) cholecalciferol (Nerlo et al 1968 Sawicka 1991) promethazine (Underberg

1978) and sulphacetamide and sulphanilamide (Ahmad and Ahmad 1983)

16 PHOTOSTABILITY OF DRUGS

Many drug substances are sensitive to light (British Pharmacopoeia 2009) and

may degrade in pharmaceutical formulations to inactive or toxic compounds This could

make a product therapeutically inactive while in use by the patients The

photodegradation (photolysis) of drug substances may occur not only during storage but

also during the use of the product It may involve several mechanisms including

oxidation reduction hydrolysis decarboxylation isomerization rearrangement and other

reactions Normal sunlight or room light may cause substantial degradation of drug

molecules The study of degradation of drug substances under the action of UVvisible

light is relevant to the process of drug development for several reasons such as

10

Exposure to light can influence the stability of a drug formulation resulting in the

loss of potency

Inappropriate exposure to light of the raw material or the final product can lead to

the formation of toxic photoproducts that are dangerous to health

Information about the stability of drug substances and formulations is needed to

predict the shelf-life of the final product (Tonnesen and Moore 1993)

The development of light-activated drugs involves activation of the compound

through photochemical reactions (Tonnesen 1991)

Adverse effects due to the formation of minor degradation products during

storage and administration have been reported (de Vries et al 1984) The drugs

substances may also cause light-induced side effects after administration to the patient by

interaction with endogenous substances The study of the photochemical properties of

drug substances and formulated products is an integral part of formulation development

to ensure the safety and efficacy of the product

The photodegradation of drug substances occurs as a result of the absorption of

radiation energy by a molecule (A) to produce an excited state species (A) (11) The

absorbed energy can be lost either by a radiative process involving fluorescence or

phosphorescence (12) or by a physical or chemical radiationless process The physical

process results in the loss of energy as heat (13) or by collisional quenching (14) The

chemical decay leads to the formation of a new species (15) The whole process is

represented as

11

A A (11)

A A + hυprime (12)

A A + heat (13)

A + A 2A (14)

A product (s) (15)

According to the Stark-Einstein law the absorption of one quantum of radiation

results in the formation of one excited molecule which may take part in several

photochemical processes [Eqs (11)ndash(15)] The quantum yield φ for any one of these

processes is defined by

Number of molecules undergoing the photochemical process φ =

Number of quanta absorbed

Considering a pure photochemical reaction the quantum yield has a value of 0ndash1

however if A is a radical that can take part in a free-radical chain reaction so that the

absorption of energy simply initiates the reaction then each quantum of energy may

result in the decomposition of molecules and φ may appear to be greater than 1 (Connors

et al 1986)

Detailed information on the photostability and photodegradation of drug

substances including vitamins alone or in solid or liquid formulations is available in the

reviews published by DeRitter (1982) Albini and Fasani (1998) Sequeira and Vozone

(2000) Tonnesen (2002 2004) Yoshioka and Stella (2002) Min and Boff (2002) Reed

et al (2003) Fasani and Albini (2005) and Sinko (2006) The photostability of cosmetic

materials has been reviewed by Sugden (1985) Important aspects dealing with the

photostability testing of drug substances have been dealt by Anderson et al (1991)

k1

k2

k3

k4

hυ

12

Tonnesen and Moore (1993) Tonnesen and Karlsen (1997) Riehl et al (1995) ICH

(1997) Singh and Bakshi (2000) Valvani (2000) Thatcher et al (2001ab) Fasani and

Albini (2005) Klick et al (2005) Singh (2006) and Ahmad and Vaid (2006)

17 KINETIC TREATMENT OF PHOTOCHEMICAL REACTIONS

The kinetic treatment of photochemical reactions with reference to the

photostability of drug substances has been considered by Moore (2004) and is presented

in this section

The photostability testing of a drug substance at the preformulation stage involves

a study of the drugrsquos rate of degradation in solution on exposure to light for a period of

time The value of the degradation rate constant depends very much on the design of the

experimental conditions (eg concentration solvent pH irradiation source oxygen

content) The factors that determine the rate of a photochemical reaction are simply the

rate at which the radiation is absorbed by the test sample (ie the number N of photons

absorbed per second) and the efficiency of the photochemical process (ie the quantum

yield of the reaction φ) For a monochromatic photon source the number of photons

absorbed depends upon the intensity of the photon source and the absorbance at that

wavelength of the absorbing species The rate of a photochemical reaction is defined as

Rate = number of molecules transformed per second = N φ (16)

In the first instance the rate can be determined for a homogeneous liquid sample

in which the only photon absorption is due to the drug molecule undergoing

transformation with the restriction that the concentration is low so that the drug does not

absorb all of the available radiation in the wavelength range corresponding to its

13

absorption spectrum The value of N can be derived at a particular wavelength λ and is

given by

Nλ = Iλ ndash It = Iλ (1 ndash 10ndashA

) (17)

where Iλ and It are the incident and transmitted radiation intensities respectively and A is

the absorbance of the sample at the wavelength of irradiation This expression can be

expanded as a power series

Nλ = 2303 Iλ (A + A22 + A

36 + hellip) (18)

When the absorbance is low (Alt 002) the second- and higher-order terms are negligible

and the expression simplifies to the first term in Eq 18 Given the Beerrsquos law relation

between absorbance and concentration N can be seen to be directly proportional to

concentration

Nλ = 2303 Iλ A = 2303 Iλ ελ b C (19)

where ελ is the molar absorptivity at wavelength λ C the molar concentration of the

absorbing species and b the optical path length of the reaction vessel Now Iλ and ελ vary

with wavelength so the expression must be integrated over the relevant wavelength range

where each has a non-zero value

N = 2303 b C int (Iλ ελ) dλ integrated from λ1 to λ2 (110)

Thus

Rate = 2303 b C φ int (Iλ ελ) dλ (111)

Now the overlap integral (int Iλ ελ dλ) is a constant for a particular combination of photon

source and absorbing substance b is determined by the reaction vessel chosen and φ is a

characteristic of the reaction Thus by grouping the constant terms into an overall

constant k1 the expression is simplified to a first-order kinetic equation

14

Rate = ndashd [Drug] dt = k1C (112)

The integrated form of Eq 112 can be expressed in exponential form (Eq 113) or

logarithmic form (Eq 114)

[Drug]t = [Drug]0 endashk1t

(113)

ln [Drug]t = ln [Drug]0 ndash k1t (114)

Verification of first-order kinetics is obtained when a plot of the logarithm of the

concentration of drug remaining is linear with slope equal to (ndashk1)

Eq 112 predicts that a photodegradation reaction studied at low concentrations in

solution will follow first-order kinetics however the rate constant derived from a study

performed in one laboratory will not be the same as that found in another The reason for

this is the inherent difficulty in reproducing exactly the experimental arrangement of

photon source and sample irradiation geometry Therefore the relative values of the rate

constants are useful in a given experimental arrangement for making comparisons of

degradation of the absorbing substance in different formulations eg those containing

ingredients designed to inhibit the photoreaction The use of rate constants is helpful for

comparative purposes when studying a number of different reaction mixtures under the

same irradiation conditions such as the effect of pH on the degradation of a drug

However the reaction order and numerical values of the rate constants are relative to the

specific conditions used

15

18 LITERATURE ON ASCORBIC ACID

A large number of reviews have been published on various aspects of ascorbic

acid A list of important reviews is given below

Chemistry biochemical functions and related aspects

Rosenberg (1945) Burns (1961) King and Burns (1975) Sim (1972) Hanck

(1982) Zaeslein (1982) Seib and Tolbert (1982) Carpenter (1986) Levine

(1986) Davies et al (1991) Halliwell and Whiteman (1997) Ortega and Delgado

(1998) Asard et al (2004) Hickey and Roberts (2004) Johnston et al (2007)

Eitenmiller (2008)

Chemical and pharmaceutical stability

Macek (1960) Garrett (1967) Carstensen (1972) Dale and Booth (1976) Hashmi

(1973) Litner (1973) DeRitter (1982) Allwood (1984ab) Allwood and Kearney

(1998) Connors et al (1986) Smith et al (1988) Racz (1989) Roth et al 1991

Ball (2006) Eitenmiller et al (2008) Sweetman (2009)

Methods of assay and chromatography

Mader (1961) Gyorgy and Pearson (1967) Bolliger and Konig (1969) Hashmi

(1973) Al-Meshal and Hassan (1982) Pelletier (1985) Lambert and deLeenheer

(1992) Halver and Felton (2001) Moffat et al (2004) Ball (2006) Eitenmiller et

al (2008)

Pharmacology and related aspects

Levine (1986) Dollery (1999) Sauberlich (1994ab) McDowell (2000)

Kaushansky and Kipps (2006) Sweetman (2009)

16

Antioxidant activity

Basu et al (1999) Shacter (2000) Thiele et al (2000) Cadenas and Packer

(2002) Packer et al (2002) Padayathy et al (2003) Parker and Parker (2003)

Burke (2006) Johnston et al (2007)

Cosmetic Preparations

Barel et al (2001) Salvador and Chisvert (2007) Rosen (2005) Bissett (2006)

Chaudhri and Jain (2009)

CHAPTER II

PHOTODEGRADATION

REACTIONS AND ASSAY

OF ASCORBIC ACID

18

21 PHOTODEGRADATION REACTIONS

211 Photodegradation of Ascorbic Acid

Aqueous ascorbic acid (1) solutions are degraded by UV light to give

dehydroascorbic acid (2) (Arcus and Zilva 1940) Ascorbic acid degradation at a

concentration of 52 and 50 mg on UV irradiation for 2 hours gave a loss of 43 and 8

respectively Dehydroascorbic acid solutions are more stable to UV light than the

ascorbic acid (Kitagawa 1968) In many natural products the vitamin is oxidized on

exposure to air and light (OrsquoNeil 2001) When solutions of multivitamin preparations are

exposed to light H2O2 as well as organic peroxides are generated and specific

byproducts that differ from dehydroascorbic acid and 23-diketogulonic acid (3) are

produced (Lavoie et al 2004)

In aqueous neutral or alkaline solution ascorbic acid (1) undergoes chemical or

photochemical oxidation to dehydroascorbic acid (2) which upon saponification of the

lactone ring under the influence of the base water produces 23-diketo-L-gulonic acid (an

α szlig- diketogulonic acid) (3) This acid undergoes further oxidation to oxalic acid (4) and

L-threonic acid (5) (Racz 1989) (Fig 2a) At room temperature oxalic acid (4) is also

formed along with threonolactone (6) by photochemical degradation of ascorbic acid (1)

in the presence of singlet oxygen (1O2) (Silva and Quina 2006) (Fig 2a) The low-

temperature photooxygenation of ascorbic acid (1) gives a mixture of unstable

hydroperoxide ketones (7) and (8) which on standing interconvert and cyclize to

hydroperoxyhemiketal (9) The hydroperoxyhemiketal breaks down on warming to

produce the oxalate esters of threonic acid (10) (Fig 2b) (Kwon and Foote 1988)

19

COOH

COOH

O

OHHO

O

HOH2C

HO2

O

O

HO

OO

O O2H

OHHO

O

HOH2C

OH

O

O

OH

O2H

OO

HO O2CCO2H

(1)hv

room temperature

(4)(6)

(1)hv

85 oC

(7)

(a)

(8)

+

cyclization

(9)

ring cleavage

(b)

(10)

(2)

OH O

OHHO

OH O O

(3)

OH OH

OH

OH O

O

OH

1O2 [O]

+

(5)

COOH

COOH

(4)

+

OH

Fig 2 Photooxidation of ascorbic acid at room and low temperature

20

An important consideration in the stability of ascorbic acid in total parenteral

nutrition (TPN) solutions is the generation of hydrogen peroxide in the presence of light

(Laborie et al 1998 1999 2000 2002 Chessex et al 2002) This may result from the

oxidation of ascorbate anion by molecular oxygen (Homann and Gaffron 1964 Taqui

Khan and Martell 1967 Mushran and Agarwal 1977 Hughes 1985 De La Rochette et

al 2000) leading to further degradation of ascorbic acid (Deutsch 1998a 1998b

1998c) The kinetics and mechanism of oxidation reactions of ascorbic acid have been

studied by several workers (Taqui Khan and Martell 1967 Ogata and Kosugi 1969

Blaugh and Hajratwala 1972 Fessenden and Verma 1978 Abe et al 1986 Kwon et al

1989 Fornaro and Coicher 1998 Njus et al 2001)

The photostability of various ascorbic acid tablets on exposure to UV light has

been studied and the influence of antioxidants and moisture on the potency loss of

ascorbic acid has been evaluated The physical characteristics of ascorbic acid tablets are

also affected on UV irradiation (Ahmad et al 1973 Jamil et al 1980ab Jamil and

Ahmad 1984)

212 Effect of Various Substances on Photodegradation of Ascorbic Acid

The oxidation-reduction reactions of ascorbic acid in the presence of riboflavin at

pH 8ndash9 under the influence of light have been studied Under these conditions ascorbic

acid is a more active H donor to riboflavin than phenolphthalein (Sibi et al 1953)

Riboflavin has been found to catalyze the photodegradation of ascorbic acid solutions

during exposure to light and air The losses of ascorbic acid are markedly increased by

the presence of Cu2+

and Fe3+

ions under light exposed and unexposed conditions (Sattar

et al 1977) A spectral study of the UV photolysis of ascorbic acid solutions in the

21

presence of riboflavin has shown that the degradation of ascorbic acid is enhanced to the

extent of about 15 (Vaid et al 2005) The influence of DL- methionine on the

photostability of ascorbic acid solutions has also been studied DL- methionine (10 mg

) enhances the photostability of ascorbic acid (40 mg ) in acetate and phosphate

buffers but not in citrate buffer at pH 45 The photoprotective action of DL-methionine

on ascorbic acid appears to be influenced by its concentration pH of the medium and the

buffer species (Asker et al 1985)

The degradation of ascorbic acid solutions on irradiation with simulated sunlight

in the presence of the food dye quinolone yellow (E 104) is enhanced However this

effect is reversed by the addition of mannitol indicating that this dye facilitates the

photogeneration of hydroxyl radicals which may cause degradation of the vitamin The

incorporation of triplet quenchers enhances the stability of substrate solutions suggesting

that the dye acts as a triplet sensitizer to facilitate the reaction (Sidhu and Sugden 1992)

The photostability of ascorbic acid solutions is enhanced by sweetening agents (mannitol

sorbitol sucrose dextrose and Canderal) at 5 wv concentration However the addition

of stoichiometric amounts of hydrogen peroxide as a source of hydroxyl radicals and 2

2rsquo-azobis (2-amidopropane) as a source of hydroperoxyl radicals results in diminished

stability of ascorbic acid solutions The diminished activity may be due to the action of

hydroperoxyl radicals in the presence of hydroxyl radical scavengers (Ho et al 1994)

Metal-complexing agents (eg disodium ethylenediaminetetraacetic acid N-

hydroxylethyl ethylenediaminetetraacetic acid 8-hydroxyquinoline) have a stabilizing

effect on the photolysis of ascorbic acid injectable solutions (Kassem et al 1969ab

22

1972) This may be due to the interaction of these agents with metal ions and other

impurities

213 Photosensitized Oxidation of Ascorbic Acid

In the presence of visible light a photosensitizer such as riboflavin can exhibit

photosensitizing properties through a mixed Type IndashType II mechanism (Yoshimura and

Ohno 1988 Foote 1991 Silva et al 1994 Silva and Quina 2006) as presented below

Type I mechanism (low oxygen concentration)

RF + hυ rarr 1RF rarr

3RF (21)

3RF + SH rarr RF

middot ndash + SH

middot + rarr RFH

middot + S

middot (22)

RFmiddot ndash

+ O2 rarr RF + O2middot ndash

(23)

2RFHmiddot rarr RF + RFH2 (24)

RFH2 + O2 rarr RF + H2O2 (25)

H2O2 + O2middot ndashrarr

ndashOH +

middotOH + O2 (26)

Smiddot and or SH

middot +

+ H2O2 O2middot ndash

rarr Soxid (27)

Type II mechanism (high oxygen concentration)

RF + hυ rarr 1RF rarr

3RF (28)

3RF + O2 rarr RF +

1O2 (29)

SH + 1O2 rarr Soxid (210)

In these reactions RF 1RF and

3RF represent RF in the ground state and in the excited

singlet and triplet states respectively RFmiddot ndash

RFHmiddot and RFH2 are the radical anion the

radical and the reduced form of RF SH is the reduced substrate and SHmiddot

+ S

middot and Soxid

23

represent the intermediate radical cation the radical and the oxidized form of the

substrate respectively

An early study of the riboflavin-sensitized photooxidation of ascorbic acid has

been carried out by flash photolysis (Heelis et al 1981) ESR spectrometry has been

used to investigate the photosensitized formation of ascorbate radicals by riboflavin (Kim

et al 1993) The photochemical behavior of a system consisting of ascorbate ion (AHndash)

and riboflavin has been studied by Mancini et al (2000) and De La Rochette et al (2000

2003) The photosensitized processes were examined as a function of oxygen pressure

and the efficiency of RF induced degradation of AHndash

at various oxygen concentrations

was compared on the basis of the respective initial photosensitization quantum yields

(Table 2)

In this reaction a Type I photosensitization mechanism (Karlsen 1996) implies a

direct electron transfer between AHndash and the RF triplet-excited state followed by the

oxidation of semioxidized ascorbyl radical (AHmiddot) by molecular oxygen or some other

reactive species On the contrary in a Type II photosensitization mechanism singlet

oxygen is produced directly by energy transfer from the RF triplet-excited state to

molecular oxygen and the singlet oxygen then oxidizes the AHndash Thus by irradiating

under increasing oxygen pressure it is possible to control the relative prevalence and

efficiency of Type I or Type II mechanisms The absence of a linear relationship between

the quantum yields of ascorbate degradation and oxygen concentration indicates that the

photosensitization mechanism involved in not exclusively Type II

24

Table 2 Initial quantum yield (φ) for ascorbate (AHndash) degradation during

photosensitization by RF (35 microM) in solutions irradiated at 365 nm and

37ordmC

O2 103 times φ (AH

ndash)a

0

5

20

14 plusmn 06

1670 plusmn 220

1940 plusmn 200

a Data are the mean plusmn SD of three independent experiments

25

In the presence of RF and O2 the quantum yields for degradation of ascorbate ion

have been found to be greater than one suggesting the participation of chain reactions

initiated by the ascorbyl radical as given by the following reactions

3RF + AH

ndash rarr RFmiddot

ndash + AHmiddot (211)

AHmiddot + O2 rarr A + HO2middot (212)

HO2middot + AHndash rarr H2O2 + AHmiddot (213)

The generation of the ascorbyl radical by the reaction between the RF excited-

triplet state and the ascorbate ion (Eq 211) is the only step that requires the absorption of

photons (to form the excited-triplet state of RF) The subsequent reactions (Eqs 212 and

213) are independent of light and lead to further degradation of the ascorbate ion In the

presence of transition metal ions such as Fe3+

in trace amounts in the buffer solution

containing RF and ascorbate ions further oxidation of ascorbate ion could also occur As

a result the reduced form of the metal ion (ie Fe2+

) can be generated by the metal

catalyzed oxidation of ascorbate ion This has been confirmed by the significant decrease

in the AHndash photooxidation quantum yield in the presence of the metal chelator EDTA

which inactivates the trace amounts of iron present in the buffer solution The quantum

yields for the photosensitized oxidation of ascorbate ion are decreased twofold at 20 O2

and fourfold at 5 O2 concentration in the presence of EDTA (Silva and Quina 2006)

Amino acids have been found to affect the photosensitized oxidation of ascorbic acid

(Jung et al 1995)

214 Ascorbic Acid Interaction with Other Water-Soluble Vitamins

The stability of ascorbic acid is reported to be enhanced in syrups containing B-

complex vitamins (Connors et al 1986) This may be due to the increased viscosity of

the syrups inhibiting the oxidation of ascorbic acid The rate of photolysis in solution

26

containing cyanocobalamin and ascorbic acid is reported to decrease with an increase in

pH (Ansari et al 2004) where as use of certain halide salts has been reported to be

beneficial in stabilizing pharmaceutical products and dietary supplements when vitamin

B12 and vitamin C are combined in solution (Ichikawa et al 2005) When a solution of

multivitamins is exposed to light it is reported that organic peroxidases are generated and

the concentration of ascorbic acid decreases (Lavoie et al 2004)

22 ASSAY OF ASCORBIC ACID

Recent accounts of the development and application of analytical methods to the

determination of ascorbic acid in pharmaceuticals biological samples and food materials

are reported in the literature (Rumsey and Levine 2000 Halver and Felton 2001 Moffat

et al 2004 Ball 2006 Sheraz et al 2007 Eitenmiller et al 2008 Salkic and Kubicek

2008) Most of these methods are based on the application of spectrophotometric

fluorimetric and chromatographic techniques to suit the requirements of a particular assay

and are summarized below

221 Spectrophotometric Methods

Spectrophotometric methods are the most widely used methods for the assay of

ascorbic acid in aqueous solution Ascorbic acid exhibits strong absorption in the

ultraviolet region (absorption maxima 243 nm at pH 2 and 265 nm at pH 4ndash10 OrsquoNeil

2001 Moffat et al 2004 British Pharmacopoeia 2009) This is the basis of

spectrophotometric methods for the determination of the vitamins in pure solutions and in

sample preparations where no interference is observed from UV absorbing impurities

The value of A (1 1 cm) at the analytical wavelength of 245 nm (pH 20) is high (695)

which makes the method very sensitive for the determination of mg quantities of the

27

vitamin Treatment of the material to be analyzed with ascorbic acid oxidase is often used

as a blank to correct for the presence of interfering substances in biological samples (Liu

et al 1982) A spectrophotometric method for the determination of ascorbic acid in

pharmaceuticals by background correction (245 nm) has been reported (Verma et al

1991) The direct determination of ascorbic acid in mixtures involves the use of 22prime-

dipyridyl as a colorimetric reagent The method is based on the reduction of Fe (III) by

ascorbic acid to Fe (II) which reacts with 2 2prime-dipyridyl to form a colored complex

(absorption maximum 510 nm) that can be used for quantitative determination (Margolis

and Schmidt 1996) A spectrophotometric method has been developed for the

determination of ascorbic acid and its oxidation product dehydroascorbic acid in

biological samples (Moeslinger et al 1995) A sensitive method has been reported for

the determination of ascorbic acid in pharmaceutical formulations and fruit juices by

interaction with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (Br-PADAP) (Ferreira

et al 1997) A novel UV method has been developed for the analysis of ascorbic acid in

methanol at 245 nm in various formulations (Zeng et al 2005)

Ascorbic acid in aqueous solutions has been assayed at 244 nm (pH ~2) (Ogata

and Kosugi 1969) 245 nm (pH 35) (Blaugh and Hajratwala 1972) 264 nm (pH 7)

(Salkic et al 2007) 265 nm (pH 7) (Hashmi 1973) 275 nm (pH 41 and 70) (Heelis et

al 1981) 265 nm (pH 7) (Al-Meshal and Hassan 1982) 245 nm (pH ~2) (Verma et al

1991) and 265 nm (pH ~7) (Erb et al 2004) Dehydroascorbic acid and 23-

diketogulonic acid do not significantly absorb in this region (Pelletier 1985 Davies et

al 1991 Rumsey and Levine 2000) and therefore do not interfere with the assay of

ascorbic acid in degraded solutions

28

222 Fluorimetric Methods

Fluorimetry is a highly sensitive technique for the determination of fluorescent

compounds or fluorescent derivatives of non-fluorescent compounds The technique has

been used for the detection of microg quantities of ascorbic acid Methods based on

fluorimetric (Kampfenkel et al 1995) and chemiluminescence detection (Zhang and

Chen 2000) provide highly sensitive methods for the determination of ascorbic acid in

plant and other materials

223 Mass Spectrometric Methods

Conventional and isotope mass spectrometric techniques have also been used for

the analysis of ascorbic acid Isotope ratio mass spectrometry is particularly useful and

sensitive when 13

C ascorbic acid is used as a standard in the analysis of complex matrices

(Gensler et al 1995)

224 Chromatographic Methods

High-performance liquid chromatographic (HPLC) methods have extensively

been employed for the determination of ascorbic acid in biological samples These

methods include ion exchange reversed phase and ion-pairing HPLC protocols

Spectrophotometric fluorimetric and electrochemical detection has been used in the

HPLC analysis of ascorbic acid The electrochemical detection is used for the

simultaneous determination of ascorbic acid dehydroascorbic acid and their isomers and

derivatives A number of HPLC methods have been developed for the detection and

determination of ascorbic acid and its oxidation products and derivatives in biological

samples and plant materials (Tsao and Young 1985 Tangney 1988 Dabrowski and

Huiterleitner 1989 Thomson and Trenerry 1995 Kimoto et al 1997 Kall and

29

Anderson 1999 Rumelin et al 1999 Lykkesfeldt 2000 Zhang et al 2000 Pastore et

al 2001 Frenich et al 2005) The limit of detection of ascorbic acid in plasma or urine

with UV detection lies in the range of 100-120 microg (Liau et al 1993 Manoharan and

Schwille 1994) Fluorescence detection of ascorbic acid and dehydroascorbic acid in

plasma and its comparison with coulometric detection has been reported (Tessier et al

1996) A liquid chromatography-diode-array detection (LCndashDAD) method has been

reported for the determination of 10 water-soluble and 10 fat-soluble vitamins including

ascorbic acid in pharmaceutical preparations with a coefficient of variation lt 65

(Konings 2006)

Liquid chromatography methods based on precolumn and o-phenylenediamine

(OPD) derivatization have been used for the determination of total vitamin C and total

isovitamin C in foods and dehydro forms of the vitamin Isoascorbic acid has been used

as an internal standard in the analysis (Speek et al 1985 Vanderslice et al 1990

Dodsun et al 1992 Vanderslice and Higgs 1988 1993 Hagg et al 1994 1995) The

limits of detection of ascorbic acid by HPLC using different detectors are in the range of

16ndash400 microgl (Capellmann and Bolt 1992 Iwase and Ono 1994 Karatepe 2004)

225 Enzymatic Methods

Enzymatic methods using ascorbate oxidase are specific and have the advantage

of selectively measuring the biological activity of ascorbic acid in serum or plasma (Liu

et al 1982) Ascorbate oxidase and OPD derivatization has been used to develop a rapid

automated method for the routine assay of ascorbic acid in serum and plasma The

method has a sample throughput of 100h (Ihara et al 2000)

30

226 Commercial Kits for Clinical Analysis

Commercial kits (eg Immunodiagnostic Germany Biovision USA) are also

used for the determination of ascorbic acid in biological samples (serum or plasma) in

clinical laboratories

227 Analysis in Creams

The general methods for the analysis of active ingredients and excipients in

cosmetic products including creams are described by Salvador and Chisvert (2007)

Ascorbic acid and derivatives in creams have been determined by liquid chromatography

(Irache et al 1993 Varvaresou et al 2006) gas chromatography-mass spectrometry

(Leveque et al 2005) and electrochemical methods (Beissenhirtz et al 2003 Guitton et

al 2007)

CHAPTER III

FORMULATION AND

STABILITY OF CREAM

PREPARATIONS

32

31 FORMULATION OF CREAM PREPARATIONS

Traditionally emulsions have been defined as dispersions of macroscopic droplets

of one liquid in another liquid with a droplet diameter approximately in the range of 05-

100 microm (Becher 1965) According to the definition of International Union of Pure and

Applied Chemistry (IUPAC) (1971) ldquoIn an emulsion liquid droplets and or liquid

crystals are dispersed in a liquidrdquo

Creams are semisolid emulsions intended for external applications They are often

composed of two phases Oil-in-water (ow) emulsions are most useful as water-washable

bases whereas water-in-oil (wo) emulsions are emollient and cleansing agents The

active ingredient is often dissolved in one or both phases thus creating a three-phase

system Patients often prefer a wo cream to an ointment because the cream spreads more

readily is less greasy and the evaporating water soothes the inflamed tissue OW creams

(vanishing creams) rub into the skin the continuous phase evaporates and increases the

concentration of a water-soluble drug in the adhering film The concentration gradient for

drug across the stratum corneum therefore increases promoting percutaneous absorption

(Barry 2002 Betageri and Prabhu 2002)

The various factors involved in the formulation of emulsions and topical products

have been discussed by Block (1996) Barry (2002) and Jain et al (2006) and are briefly

presented in the following sections

311 Choice of Emulsion Type

Oil-in-water emulsions are used for the topical application of water-soluble drugs

mainly for local effect They do not have the greasy texture associated with oily bases

and are therefore pleasant to use and easily washed from skin surfaces Moisturizing

33

creams designed to prevent moisture loss from the skin and thus inhibit drying of the

stratum corneum are more efficient if formulated as ow emulsions which produce a

coherent water-repellent film

312 Choice of Oil Phase

Many emulsions for external use contain oils that are present as carriers for the

active ingredient It must be realized that the type of oil used may also have an effect both

on the viscosity of the product and on the transport of the drug into the skin (Barry

2002) One of the most widely used oils for this type of preparation is liquid paraffin

This is one of a series of hydrocarbons which also includes hard paraffin soft paraffin

and light liquid paraffin They can be used individually or in combination with each other

to control emulsion consistency This will ensure that the product can be spread easily but

will be sufficiently viscous to form a coherent film over the skin The film-forming

capabilities of the emulsion can be further modified by the inclusion of various waxes

such as bees wax carnauba wax or higher fatty alcohols

313 Emulsion Consistency

A consideration of the texture or feel of a product intended for external use is

important A wo preparation will have a greasy texture and often exhibits a higher

apparent viscosity than ow emulsions This fact imparts a feeling of richness to many

cosmetic formulations Oil-in-water emulsions will however feel less greasy or sticky on

application to the skin will be absorbed more readily because of their lower oil content

and can be more easily washed from skin surface Ideally emulsions should exhibit the

rheological properties of plasticity pseudoplasticity and thixotropy Emulsions of high

apparent viscosity for external use (cream) are of a semisolid consistency There are

34

several methods by which the rheological properties of an emulsion can be controlled

(Billany 2002)

314 Choice of Emulsifying Agent

The choice of emulgent to be used would depend on factors such as its

emulsifying ability route of administration and toxicity Most of the non-ionic emulgents

are less irritant and less toxic than their anionic and cationic counter parts Some

emulgents such as the ionic alkali soaps often have a high pH and are thus unsuitable for

application to broken skin Even in normal intact skin with a pH of 55 the application of

such alkaline materials can cause irritation Some emulsifiers in particular wool fat can

cause sensitizing reactions in susceptible people The details of various types of

emulsifying agents are available in the literature (Betageri and Prabhu 2002 Billany

2002 Swarbrick et al 2006)

315 Formulation by the HLB Method

The physically stable emulsions are best achieved by the presence of a condensed

layer of emulgent at the oil water interface and the complex interfacial films formed by a

blend of an oil-soluble emulsifying agent with a water-soluble one produces the most

satisfactory emulsions

It is possible to calculate the relative quantities of the emulgents necessary to

produce the most physically stable emulsions for a particular formulation with water

combination This approach is called the hydrophilic-lipophilic balance (HLB) method

Each surfactant is allocated an HLB number representing the relative properties of the

lipophilic and hydrophilic parts of the molecule High numbers (up to a theoretical

number of 20) therefore indicates a surfactant exhibiting mainly hydrophilic or polar

35

properties whereas low numbers represent lipophilic or non-polar characteristics Each

type of oil requires an emulgent of a particular HLB number in order to ensure a stable

product For an ow emulsion the more polar the oil phase the more polar must be the

emulgent system (Billany 2002 Im-Emsap et al 2002 Swarbrick et al 2006)

316 Concept of Relative Polarity Index

In the ingredient selection in cosmetic formulations a new concept of relative

polarity index (RPI) has been presented (Wiechers 2005) The physicochemical

characteristics of the ingredients determine their skin delivery to a greater extent than the

formulation type The cosmetic formulation cannot change the chemistry of the active

molecule that needs to penetrate to a specific site within the skin However the

formulation type can be selected based on the polarity of the active ingredient and the

desired site of action for the active ingredient For optimum skin delivery the solubility of

the active ingredient needs to be as high as possible (to create a large concentration

gradient) and as small as possible (to create a large partition coefficient) To achieve this

it is necessary to determine the following parameters

The total amount dissolved in the formulation that is available for skin penetration

the higher this amount the more will penetrate until a solution concentration is

reached in the skin therefore a high absolute solubility in the formulation is required

The polarity of the formulation relative to that of the stratum corneum if an active

ingredient dissolves better in the stratum corneum than in the formulation then the

partition of the active ingredient will favour the stratum corneum therefore a low

(relative to that in the stratum corneum) solubility in the formulation is required

(Wiechers 2005)

36

These requirements can be met by considering the concept of RPI (Wiechers

2003 2005) In this systematic approach it is essential to consider the stratum corneum

as another solvent with its own polarity The stratum corneum appears to behave very

similarly to and in a more polar fashion than butanol with respect to its solubilizing

ability for active ingredients (Scheuplein and Blank 1973) The polarity of stratum

corneum as expressed by its octanol water partition coefficient is 63

The relative polarity index may be used to compare the polarity of an active

ingredient with both that of the skin and that of the oil phase of a cosmetic formulation

predominantly consisting of emollients It may be visualized as a vertical line with a high

polarity at the top and a high lipophilicity at the bottom The polarity is expressed as the

log10 of the octanol water coefficient For example the relative polarity index values of

glycerin and isostearyl isostearate are -176 and 2698 respectively (Wiechers 2005) In

order to use the concept of the relative polarity index three numbers (on log10 scale) are

required

The polarity of the stratum corneum is set at 08 However in reality this value will

change with the hydration state of the stratum corneum that is determined in part by

the external relative humidity (Bonwstra et al 2003)

The polarity of the active molecule

The polarity of the formulation

For multiphase or multipolarity systems like emulsions the active ingredient is dissolved

in the phase For example in an ow emulsion where a lipophilic active ingredient is

dissolved in the oil phase it is the polarity of the homogenous mixture of the lipophilic

active ingredient and internal oil For the same lipophilic active in a wo emulsion it is

37

the polarity of the homogenous mixture of the lipophilic active ingredients and external

oil For water-soluble active ingredients it is the polarity of the homogenous mixture of

the hydrophilic active ingredient and the aqueous phase regardless whether it is internal

(wo emulsions) or external (ow emulsions)

Once the active ingredient and the formulation type have been chosen it is

necessary to create the delivery system that will effectively deliver the molecule The

concept of relative polarity index allows the formulator to select the polarity of the phase

in which the active ingredient is incorporated on the basis of its own properties and those

of the stratum corneum In order to achieve maximum delivery the polarity of the active

ingredient and the stratum corneum need to be considered In order to improve the skin

delivery of active ingredients the first step involves selecting a primary emollient with a

polarity close to that of the active ingredient in which it will have a high solubility The

second step is to reduce the solubility of the active ingredient in the primary emollient via

the addition of a secondary emollient with a different polarity and therefore lower

solubility for the active ingredient This approach has shown a 3-4 fold increase in skin

penetration with out increasing the amount of active ingredients in the formulation

(Wiechers 2005)

32 FORMULATION OF ASCORBIC ACID CREAMS

Ascorbic acid is a water-soluble material and is included frequently in skin care

formulations to restore skin health It is very unstable and is easily oxidized in aqueous

solution This vitamin is known to be a reducing agent in biological systems and causes

the reduction of both oxygen- and nitrogen- based free radicals (Higdon and Frei 2002)

It can also act as a co-antioxidant with the tocopheroxyl radical to regenerate alpha-

38

tocopherol (Packer et al 1979 Buettner 1993 Peyrat-Maillard et al 2001) In this

reaction the two vitamins act synergistically Alpha-tocopherol first functions as the

primary antioxidant that reacts with an organic free radical Thereafter ascorbic acid

reacts with the free radical tocopheroxyl to general alpha-tocopherol In physiological

conditions the ascorbyl radical formed by regenerating tocopherol is then converted back

to ascorbate by the redox cycle (Davies et al 1991) The interaction of ascorbic acid

with a redox partner such as alpha-tocopherol has been found useful to slow its oxidation

and prolong its action

The instability of ascorbic acid makes this antioxidant active ingredient a

formulation challenge to deliver to the skin and retain its effective form In addition to its

use in combination with alpha-tocopherol in cream formulations the stability of ascorbic

acid may be improved by its use in the form of a fatty acid ester such as ascorbyl

palmitate Ascorbyl palmitate has been used in thixogel formulations and is typically

incorporated into the mineral oil phase Preliminary experiments have shown that it could

be slowly released from the starch-oil emulsion matrix and act as an antioxidant (Wille

2005)

Various physical and chemical factors involved in the formulation of cream

preparations have been discussed in the above sections For polar and air light sensitive

compounds such as ascorbic acid it is important to consider factors such as the choice of

formulation ingredients polar character of formulation HLB value pH viscosity etc to

achieve stability

39

33 STABILITY OF CREAMS

331 Physical Stability

The most important consideration with respect to pharmaceutical and cosmetic

emulsions (creams) is the stability of the finished product The stability of a

pharmaceutical emulsion is characterized by the absence of coalescence of the internal

phase absence of creaming and maintenance of elegance with respect to appearance

odor color and other physical properties An emulsion is a dynamic system however

any flocculation and resultant creaming represent potential steps towards complete

coalescence of the internal phase In pharmaceutical emulsions creaming results as a lack

of uniformity of drug distribution and poses a problem to the pharmaceutical

compounder Another important factor in the stabilization of emulsions is phase inversion

which involves the change of emulsion type from ow to wo or vice versa and is

considered as a case of instability The four major phenomena associated with the

physical instability of emulsions are flocculation creaming coalescence and breaking

These have been discussed by Garti and Aserin (1996) Im-Emsap et al (2002) and Sinko

(2006)

332 Chemical Stability

The instability of a drug may lead to the loss of its concentration through a

chemical reaction under normal or stress conditions This results in a reduction of the

potency and is a well-recognized cause of poor product quality The degradation of the

drug may make the product esthetically unacceptable if significant changes in color or

odor have occurred The degradation product may also be a toxic substance The various

pathways of chemical degradation of a drug depend on the structural characteristics of the

40

drug and may involve hydrolysis dehydration isomerization and racemization

decarboxylation and elimination oxidation photodegradation drug-excipients and drug-

drug interactions Factors determining the chemical stability of drug substances include

intrinsic factors such as molecular structure of the drug itself and environmental factors

such as temperature light pH buffer species ionic strength oxygen moisture additives

and excipients The application of well-established kinetic principles may throw light on

the role of each variable in altering the kinetics of degradation and to provide valuable

insight into the mechanism of degradation (Baertschi and Alsante 2005 Yoshioka and

Stella 2002 Lachman et al 1986) The chemical stability of individual components

within an emulsion system may be very different from their stability after incorporation

into other formulation types For example many unsaturated oils are prone to oxidation

and their degree of exposure to oxygen may be influenced by factors that affect the extent

of molecular dispersion (eg droplet size) This could be particularly troublesome in

emulsions because emulsification may introduce air into the product and because of the

high interfacial contact area between the phases (Barry 2002) The use of antioxidants

retards oxidation of unsaturated oils minimizes changes in color and texture and prevents

rancidity in the formulation Moreover they can retard the degradation of certain active

ingredients such as vitamin C (Vimaladevi 2005) The stability problems of dispersed

systems and the factors leading to these stability problems have been discussed by

Weiner (1996) and Lu and Flynn (2009)

333 Microbial Stability

Topical bases often contain aqueous and oily phases together with carbohydrates

and proteins and are susceptible to bacterial and fungal attack Microbial growth spoils

41

the formulation and is a potential toxic hazard Therefore topical formulations need

appropriate preservatives to prevent microbial growth and to maintain their quality and

shelf-life (Barry 2002 Arger et al 1996) Cream formulations may contain fats and oils

with high percentage of unsaturated linkages that are susceptible to oxidation degradation

and development of rancidity The addition of antioxidants retards oxidation of fats and

oils minimizes changes in color and texture and prevents rancidity in the formulation

Moreover they can retard the degradation of certain active ingredients such as vitamin C

These aspects in relation to dermatological formulations have been discussed by Barry

(1983 2002) and Vimaladevi 2005)

334 Stability of Ascorbic Acid in Liquid Formulations

Ascorbic acid is very unstable in aqueous solution Different workers have studied

the stability of ascorbic acid in liquid formulations (Connors et al 1986 Austria et al

1997) Its shelf-life can be prolonged by appropriate choice of vehicle and control of

other variables such as pH stabilizers temperature light and oxygen (Table 3)

Similarly the stability of various concentrations of ascorbic acid in solution form may

vary depending upon the type of solvent used (Table 4) (Connors et al 1986 Satoh et

al 2000 Lee et al 2004 Zeng et al 2005)

335 Stability of Ascorbic Acid in Emulsions and Creams

Ascorbic acid exerts several functions on skin such as collagen synthesis

depigmentation and antioxidant activity Ultraviolet radiation generates reactive oxygen

species (ROS) which produce some harmful effects on the skin including photocarcinoma

and photoaging In order to combat these problems ascorbic acid as an antioxidant has

42

Table 3 Effect of vehicles on the stability of ascorbic acid ( ascorbic acid remaining in

solutions after storage at room temperature) (Connors et al 1986)

Storage Time (days) No Vehicle

30 60 90 120 180 240 360

1 Corn Syrup 965 925 920 920 900 860 760

2 Sorbitol 990 990 990 970 960 925 890

3 4 Carboxymethyl

Cellulose

840 680 565 380 ndash ndash ndash

4 Glycerin 100 100 990 990 970 935 920

5 Propylene glycol 995 990 980 945 920 900 900

6 Syrup USP 100 100 980 980 930 900 880

7 Syrup 212 gL 880 810 775 745 645 590 440

8 25 Tragacanth 785 620 510 320 ndash ndash ndash

9 Saturated solution of

Dextrose

990 935 875 800 640 580 510

10 Distilled Water 900 815 745 675 405 185 ndash

11 50 Propylene glycol +

50 Glycerin

980 ndash 960 ndash 933 ndash ndash

12 25 Distilled Water +

75 Sorbo (70 solution

of Sorbitol)

955 954 ndash 942 930 ndash ndash

13 50 Glycerin + 50

Sorbo

982 984 978 ndash ndash 914 ndash

43

Table 4 Stability of various concentrations of ascorbic acid in water propylene glycol

and USP syrup at room temperature ( of ascorbic acid remaining in solution)

(Connors et al 1986)

Storage Time (days) Concentration

(mg ml)

Solvent

30 60 90 120 180 240 360

10 Water 930 840 820 670 515 410 ndash

50 Water 940 920 880 795 605 590 300

100 Water 970 930 910 835 705 680 590

10 Propylene glycol 100 985 980 975 960 920 860

50 Propylene glycol 100 970 980 980 980 965 935

100 Propylene glycol 100 100 100 100 990 100 925

10 Syrup 100 100 980 990 970 960 840

50 Syrup 100 100 100 100 990 100 960

100 Syrup 100 100 100 100 100 100 995

44

been used in various dosage forms and in different concentrations (Darr et al 1996

Gallarate et al 1999 Zhang et al 1999 Pinnell et al 2001 Lee et al 2004 Raschke

et al 2004 Elmore 2005 Farahmand et al 2006 Maia et al 2006) Ascorbic acid has

good photoprotective ability against UVA-mediated phototoxicity (Darr et al 1996) A

variety of formulations containing ascorbic acid or its derivatives have been studied in

order to evaluate their stability and delivery through the skin (Gallarate et al 1999

Zhang et al 1999 Ozer et al 2000 Pinnell et al 2001 Lee et al 2004 Raschke et al

2004 Farahmand et al 2006) Formulations containing derivatives of ascorbic acid are

found to be more stable than ascorbic acid but they do not produce the same effect as that

of the parent compound probably due to the lack of redox properties (Heber et al 2006)

Effective delivery of ascorbic acid through topical preparations is a major factor that

should be critically evaluated as it may be dependent upon the nature or type of the

formulation (Gallarate et al 1999 Pinnell et al 2001) The pH of the formulation

should be on the acidic side (~ pH 35) for effective penetration of the vitamin in the skin

(Pinnell et al 2001) and for its stabilization in the formulation (Gallarate et al 1999)

Some other antioxidants such as alpha-tocopherol ferulic acid and sodium metabisulphite

have also been used in combination with ascorbic acid for the purpose of its stabilization

in topical formulations and to produce some synergistic effects (Darr et al 1996 Lin et

al 2005 Maia et al 2006 Tournas et al 2006) Effect of some rheological properties

such as viscosity and dielectric constant on the stability of ascorbic acid in emulsions has

also been investigated (Connors et al 1986) Viscosity of the medium is an important

factor that should be considered for the purpose of ascorbic acid stability as higher

viscosity formulations have shown some degree of protection (Ozer et al 2000

45

Szymula 2005) Along with other factors formulation type also plays an important role in

the stability of ascorbic acid It is reported that ascorbic acid is more stable in emulsified

system as compared to aqueous solutions (Gallarate et al 1999 Lee et al 2004) In

multiemulsions ascorbic acid is reported to be more stable as compared to simple

emulsions (Gallarate et al 1999 Ozer et al 2000 Lee et al 2004 Farahmand et al

2006)

Ascorbic acid and its derivatives have been used in a variety of cosmetic

formulations as an antioxidant pH adjuster anti-aging and photoprotectant (Elmore

2005) The control of instability of ascorbic acid poses a significant challenge in the

development of cosmetic formulations It is also reported that certain metal ions or

enzyme systems effectively convert ascorbic acidrsquos antioxidant action to pro-oxidant

activity (Elmore 2005) Therefore utilization of an effective antioxidant system is

required to maintain the stability of vitamin C in various preparations (Zhang et al 1999

Pinnell et al 2001 Maia et al 2006) The chemical stability of ascorbic acid has been

studied in emulsions and creams by several workers (Darr et al 1996 Gallarate et al

1999 Lee et al 2004 Raschke et al 2004 Elmore 2005 Farahmand et al 2006)

however there is a lack of information on the photostability of ascorbic acid in cream

formulations

336 Stability Testing of Emulsions

The stability testing of emulsions (creams) may be carried out by performing the

following tests (Billany 2002)

46

3361 Macroscopic examination

The assessment of the physical stability of an emulsion is made by an

examination of the degree of creaming or coalescence occurring over a period of time

This involves the calculation of the ratio of the volume of the creamed or separated part

of the emulsion and the total volume A comparison of these values can be made for

different products

3362 Globule size analysis

An increase in mean globule size with time (coupled with a decrease in globule

numbers) indicates that coalescence is the cause of this behavior This can be used to

compare the rates of coalescence for a variety of emulsion formulations For this purpose

microscopic examination or electronic particle counting devices (coulter counter) or

laser diffraction sizing are widely used

3363 Change in viscosity

Many factors may influence the viscosity of emulsions A change in apparent

viscosity may result from any variation in globule size or number or in the orientation or

migration of emulsifier over a period of time

3264 Accelerated stability tests

In order to compare the relative stabilities of a range of similar products it is

necessary to speed up the processes of creaming and coalescence by storage at elevated

temperatures and then carrying out the tests described in the above sections

337 FDA guidelines for semisolid preparations

According to FDA draft guidelines to the industry (Shah 1997) semisolid

preparations (eg creams) should be evaluated for appearance clarity color

47

homogencity odour pH consistency viscosity particle size distribution (when feasible)

assay degradation products preservative and antioxidant content (if present) microbial

limits sterility and weight loss when appropriate Additionally samples from

production lot or approved products are retained for stability testing in case of product

failure in the field Retained samples can be tested along with returned samples to

ascertain if the problem was manufacturing or storage related Appropriate stability data

should be provided for products supplied in closed-end tubes to support the maximum

anticipated use period during patient use and after the seal is punctured allowing product

contact with the cap cap lever Creams in large containers including tubes should be

assayed by sampling at the surface top middle and bottom of the container In addition

tubes should be sampled near the crimp The objective of stability testing is to determine

whether the product has adequate shelf-life under market and use conditions

48

OBJECT OF PRESENT INVESTIGATION

Ascorbic acid (vitamin C) is extensively used as a single ingredient or in

combination with vitamin B complex and other vitamins in the form of drops injectables

lotions and syrups It is an ingredient of anti-aging cosmetic products alone or along with

alpha-tocopherol (vitamin E) Ascorbic acid exerts several functions on the skin as

collagen synthesis depigmentation and antioxidant activity It protects the signs of

degenerative skin conditions caused by oxy-radical damage In solutions and creams

ascorbic acid is susceptible to air and light and undergoes oxidative degradation to

dehydroascorbic acid and inactive products The degradation is influenced by

temperature viscosity and polarity of the medium and is catalysed by metal ions

particularly Cu+2

Fe+2

and Zn+2

One of the major problems faced in cream preparations is the instability of

ascorbic acid as it may be exposed to light during formulation manufacturing and

storage and the possibility of photochemical degradation can not be neglected The

behaviour of ascorbic acid in light is of particular interest since no systematic kinetic

studies have been conducted on its photodegradation in these preparations under various

conditions The study of the formulation variables such as emulsifier humectants and pH

may throw light on the photostabilization of ascorbic acid in creams

The main object of this investigation is to study the behaviour of ascorbic acid in

cream preparations on exposure to UV light in the pharmaceutically useful pH range An

important aspect of the work is to study the interaction of ascorbic acid with other

vitamins such as riboflavin nicotinamide and alpha-tocopherol and the effect of certain

stabilizers such as citric acid tartaric acid and boric acid on its photodegradation In

49

addition it is intended to study the photolysis of ascorbic acid in organic solvents to

evaluate the effect of solvent characteristics (eg dielectric constant and viscosity) on the

stability of the vitamin The study of all these aspects may provide useful information to

improve the photostability and efficacy of ascorbic acid in cream preparations

An outline of the proposed plan of work is presented as follows

1 To prepare a number of oil-in-water cream formulations based on different

emulsifying agents and humectants containing ascorbic acid alone and in

combination with other vitamins and stabilizing agents

2 To perform photodegradation studies on ascorbic acid in creams using a UV

irradiation source with emission corresponding to the absorption maximum of

ascorbic acid

3 To identify the photoproducts of ascorbic acid in creams using chromatographic

and spectrophotometric methods

4 To apply appropriate and validated analytical methods for the assay of ascorbic

acid alone and in combination with other vitamins and stabilizing agents

5 To study the effect of solvent characteristics such as dielectric constant and

viscosity on the photolysis of ascorbic acid in aqueous and organic solvents

6 To evaluate the kinetics of photodegradation of ascorbic acid and its interactions

with other vitamins (riboflavin nicotinamide and alpha-tocopherol) in creams

7 To evaluate the effect of carbon chain length of the emulsifying agent and the

viscosity of the humectant on the photodegradation of ascorbic acid

50

8 To develop relationships between the rate of photodegradation of ascorbic acid

and the concentration pH carbon chain length of emulsifier viscosity of the

creams

9 To determine the effect of compounds such as citric acid tartaric acid and boric

acid used as stabilizing agents on the rate of photodegradation and stabilization

of ascorbic acid in creams

10 To present reaction schemes for the photodegradation of ascorbic acid and its

interactions with other vitamins

CHAPTER IV

MATERIALS

AND

METHODS

52

41 MATERIALS

Vitamins and Related Compounds

L-Ascorbic Acid vitamin C (5R)-5-[(1S)-12-dihydroxyethyl]-34-dihydroxyfuran-2(5H)-

one Merck

C6H8O6 Mr 1761

Dehydroascorbic Acid L-threo-23-hexodiulosonic acid γ-lactone Sigma

C6H6O6 Mr 1741

23-Diketogulonic Acid

C6H8O7 Mr 192

It was prepared according to the method of Homann and Gaffron (1964) by the

hydrolysis of dehydroascorbic acid

Riboflavin vitamin B2 (310-dihydro-78-dimethyl-10-[(2S3S4R)-2345-

tetrahydroxypentyl] benzopteridine-24-dione) Merck

C17H20N4O6 Mr 3764

Nicotinamide vitamin B3 (pyridine-3-carboxamide) Merck

C6H6N2O Mr 1221

Alpha-Tocopherol vitamin E ((2R)-2578-tetramethyl-2-[(4R8R)-4812-

trimethyltridecyl]-34-dihydro-2H-1-benzopyran-6-ol) Merck

C29H50O2 Mr 4307

Formylmethylflavin (78-dimethyl-10-formylmethylisoalloxazine)

C14H12N4O3 Mr 2843

53

Formylmethylflavin was synthesized according to the method of Fall and Petering

(1956) by the periodic acid oxidation of riboflavin It was recrystallized from absolute

methanol dried in vacuo and stored in the dark in a refrigerator

Lumichrome (78-dimethylalloxazine) Sigma

C12H10N4O2 Mr 2423

It was stored in the dark in a desiccator

Stabilizers

Boric Acid orthoboric acid Merck

H3BO3 Mr 618

Citric Acid 2-hydroxypropane-123-tricarboxylic acid Merck

C6H8O7H2O Mr 2101

L-Tartaric acid [(2R3R)-23-dihydroxybutanedioic acid] Merck

C4H6O6 Mr 1501

Emulsifying Agents

Stearic Acid (95) octadecanoic acid Merck

C18H36O2 Mr 2845

Palmitic Acid hexadecanoic acid Merck

C16H32O2 Mr 2564

Myristic Acid tetradecanoic acid Merck

C14H28O2 Mr 2284

Cetyl alcohol hexadecan-1-ol Merck

C16H34O Mr 2424

54

Humectants

Glycerin glycerol (propane-123-triol) Merck

C3H8O3 Mr 921

Propylene glycol (RS)-propane-12-diol Merck

C3H8O2 Mr 7610

Ethylene glycol ethane-12-diol Merck

C2H6O2 Mr 6207

Potassium Ferrioxalate Actinometry

Potassium Ferrioxalate

K3Fe(C2O4)3 3H2O Mr 4912

Potassium Ferrioxalate was prepared according to the method of Hatchard and

Parker (1956) Three volumes of 15 M potassium oxalate was mixed with one volume of

15 M ferric chloride with vigorous stirring The yellow green precipitate of potassium

ferrioxalate was recrystallized twice from water dried at 45 ordmC and stored in the dark in a

desiccator

Reagents

All the reagents and solvents used were of analytical grade obtained from BDH

Merck

Water

Freshly boiled distilled water was used throughout the work

55

42 METHODS

421 Cream Formulations

On the basis of the various cream formulations reported in the literature (Block

1996 Flynn 2002 Betageri and Prabhu 2002 Vimaladevi 2005 EIRI Board Lu and

Flynn 2009) the following basic formula was used for the preparation of oil-in-water

creams containing ascorbic acid

Oil phase Percentage (ww)

Emulsifier

Myristic palmitic stearic acid

Cetyl alcohol

120

30

Aqueous phase

Humectant

Ethylene glycol propylene glycol glycerin

50

Active ingredient

Ascorbic acid

20 (0114 M)

Neutralizer

Potassium hydroxide

10

Continuous phase

Distilled water

QS

Additional ingredientsa

Vitamins

Riboflavin (Vitamin B2)

Nicotinamide (Vitamin B3)

Alpha-Tocopherol (Vitamin E)

0002ndash001 (053ndash266times10ndash4

M)

028ndash140 (0023ndash0115 M)

017ndash086 (0395ndash200times10ndash2

M)

Stabilizers

Citric acid

Tartaric acid

Boric acid

010ndash040 (0476ndash190times10ndash2

M)

010ndash040 (067ndash266times10ndash2

M)

010ndash040 (0016ndash0065 M)

a The vitamin stabilizer concentrations used were found to be effective in promotion

inhibition of photodegradation of ascorbic acid in cream formulations

56

422 Preparation of Creams

The emulsifiers were melted at 70ndash80 ordmC in a glass jar immersed in a water bath

Ascorbic acid was separately dissolved in a small portion of distilled water Potassium

hydroxide and humectant were dissolved in the remaining portion of water and mixed

with the oily phase with constant stirring until the formation of a thick white mass It was

cooled to ~40 ordmC and the ascorbic acid solution was added The thick mass was mixed

using a mechanical mixer with a glass stirrer at 1000 rpm for 5 minutes The pH of the

cream was adjusted to the desired value and the contents again mixed for 10 minutes at

500 rpm All the creams were prepared under uniform conditions to maintain their

individual physical characteristics and stored at room temperature in airtight glass

containers protected from light

In the case of other vitamins nicotinamide was dissolved along with ascorbic acid

in water and added to the cream Riboflavin or alpha-tocopherol were directly added to

the cream and mixed thoroughly according to the procedure described above

In the case of stabilizing agents (citric tartaric and boric acids) the individual

compounds were dissolved in the ascorbic acid solution and added to the cream followed

by the procedure described above

The details of the various cream formulations used in this study are given in

chapters 5ndash7

57

423 Thin-Layer Chromatography (TLC)

The following TLC systems were used for the separation and identification of

ascorbic acid and photodegradation products

Adsorbent a) Silica gel GF 254 (250-microm) precoated plates

(Merck)

Solvent systems S1 acetic acid-acetone-methanol-benzene

(552070 vv) (Ganshirt and Malzacher 1960)

S2 ethanol-10 acetic acid-water (9010 vv)

(Bolliger and Konig 1969)

S3 acetonitrile-butylnitrile-water (66332 vv)

(Saari et al 1967)

Temperature 25ndash27 ordmC

Location of spots Ascorbic acid UV light 254 nm (Uvitec lamp

UK)

Dehydroascorbic acid Spray with a 3 aqueous

phenylhydrazine hydrochloride solution

424 pH Measurements

The measurements of pH of aqueous solutions and cream formulations were

carried out using an Elmetron LCD display pH meter (modelndashCP501 sensitivity plusmn 001

pH units) (Poland) with a combination electrode The electrode was calibrated

automatically in the desired pH range (25 ordmC) using the following buffer solutions

58

Phthalate pH 4008

Phosphate pH 6865

Disodium tetraborate pH 9180

The electrode was immersed directly into the cream (British Pharmacopoeia

2009) kept for few seconds to equilibrate and the pH adjusted in the range of 40ndash70

with phosphoric acid sodium hydroxide solution

425 Ultraviolet and Visible Spectrometry

The absorbance measurements and spectral determinations were performed on

Shimadzu UVndashVisible recording spectrophotometer (model UVndash1601) using matched

silica cells of 10 mm path length The cells were employed always in the same orientation

using appropriate control solutions in the reference beam The baseline was automatically

corrected by the built-in baseline memory at the initializing period Auto-zero adjustment

was made by a one-touch operation The instrument checked the wavelength calibration

(6561 nm) using the deuterium lamp at the initializing period The absorbance scale was

periodically checked using the following calibration standard (British Pharmacopoeia

2009)

0057ndash0063 gl of potassium dichromate in 0005 M sulphuric acid

The specific absorbance [A(1 1 cm)] of the solution should match the

following values with the stated limit of tolerance

Wavelength

(nm)

Specific absorbance

A (1 1 cm)

Maximum

tolerance

235 1245 1229ndash1262

257 1445 1428ndash1462

313 486 470ndash503

350 1073 1056ndash109

430 159 157ndash161

59

426 Photolysis of Ascorbic Acid

4261 Creams

A 2 g quantity of the cream was uniformly spread on several rectangular glass

plates (5 times 15 cm) covered with a 1 cm tape on each side to give a 1 mm thick layer The

plates were irradiated in a dark chamber using a Philips 30 watt TUV tube (100

emission at 254 nm the wavelength absorbed by ascorbic acid at pH 4ndash7) fixed

horizontally at a distance of 30 cm from the centre of the plates Each plate was removed

at appropriate interval and the cream was subjected to spectrophotometric assay and

chromatographic examination

4262 Aqueous and organic solvents

A 10ndash3

M solution of ascorbic acid (50 ml) prepared in water (pH 70 005 M

phosphate buffer) or in an organic solvent in a 100 ml beaker (Pyrex) was placed in a

water bath maintained at 20 plusmn 1 ordmC The solution was irradiated with the Philips 30 watt

TUV tube in a dark chamber as stated above Samples were withdrawn at appropriate

intervals for assay and chromatography

4263 Storage of creams in dark

In order to determine the stability of various cream formulations in the dark

samples were stored at room temperature in a cupboard protected from light for a period

of three months The samples were analyzed periodically for the content of ascorbic acid

and the presence of any degradation product

427 Measurement of Light Intensity

The potassium ferrioxalate actinometry was used for the measurement of light

intensity of the radiation source employed in this work This actinometer has been

60

developed by Parker (1953) and Hatchard and Parker (1956) and is considered as the

most useful actinometer over a wide range of wavelengths (254ndash577 nm) It has been

used by Holmstrom and Oster (1961) Byrom and Turnbull (1967) McBride and Moore

(1967) Ahmad (1968) Ahmad (1978) Ahmad et al (2004a 2004b 2005 2006a

2006b 2008 2009ab) Fasihullah (1988) Vaid (1998) Ansari (2002) and Ahmad (2009)

for the measurement of light intensity

The irradiation of potassium ferrioxalate solutions in sulphuric acid results in the

reduction of ferric ion to ferrous ion according to the following reaction

2Fe [(C2O4)3]3ndash

rarr 2 Fe (C2O4) + 3 (C2O4)2ndash

+ 2CO2 (31)

The amount of Fe2+

ions formed in the reaction may be determined by

complexation with 110-phenanthroline to give a red colored complex The absorbance of

the complex is measured at 510 nm

428 Procedure

An oxygen free 0006 M solution of potassium ferrioxalate (2947 gl) in 01 N

H2SO4 was placed in the reaction vessel and irradiated with the lamp used for the

photolysis of riboflavin The irradiation was carried out under nitrogen (90ndash120

bubblesminute) which also caused stirring of the solution The temperature of the

reaction vessel was maintained at 25 plusmn 1 ordmC during the reaction

An aliquot of the photolysed solution (1ndash2 ml) was pipetted out at suitable

intervals (up to 30 minutes) into a 10 ml volumetric flask to which was then added 09

ml of N H2SO4 + 1 ml (01) 110-phenanthroline + 05 ml buffer (60 ml N CH3COONa

+ 36 ml N H2SO4 made up to 100 ml with distilled water) The flask was made up

to the mark with distilled water (final pH 35) thoroughly shaken to mix the contents and

61

Fig 3 Spectral power distribution of TUV 30 W tube (Philips)

62

allowed to stand for one hour in the dark to develop the colorndashcomplex The absorbance

of the phenanthrolinendashferrous complex was measured in a 1 cm cell at 510 nm using the

appropriate solution as blank The amount of Fe2+

ions formed was determined from the

calibration graph The calibration graph was constructed in a similar manner using

several dilutions of 1 times 10ndash6

mole ml Fe2+

in 01 N H2SO4 (freshly prepared by dilution

from standardized 01 M FeSO4 in 01 N H2SO4) (Fig 8) The experimental value of the

molar absorptivity of Fe2+

complex as determined from the slope of the calibration graph

is equal to 111 times 104 M

ndash1 cm

ndash1 and is in agreement with the value reported by Parker

(1953) Using the values of the known quantum yield for ferrioxalate actinometer at

different wavelengths (Hatchard and Parker 1956) the number of Fe2+

ions formed

during photolysis the time of exposure and the fraction of the light absorbed by the

length of the actinometer solution employed the light intensity incident just inside the

front window of the photolysis cell can be calculated In the present case total absorption

of the light has been assumed

4281 Calculation

The number of Fe2+

ions formed during photolysis (nFe

2+) is given by the

equation

6023 times 1020

V1 V3 A Σ

n Fe

2+ =

V2 1 ε (32)

where V1 is the volume of the actinometer solution irradiated (ml)

V2 is the volume of the aliquot taken for analysis (ml)

V3 is the final volume to which the aliquot V2 is diluted (ml)

1 is the path length of the spectrophotometer cell used (1 cm)

A is the measured absorbance of the final solution at 510 nm

63

ε is the molar absorptivity of the Fe2+

complex (111 times 104 M

ndash1 cm

ndash1)

The number of quanta absorbed by the actinometer nabs can then be obtained as follows

n Fe

2+

Σ nabs = ф

(33)

where ф is the quantum yield for the Fe2+

formation at the desired wavelength

The number of quanta per second per cell nabs is therefore given by

Σ nabs 6023 times 1020

V1 V3 A nabs =

t =

ф V2 1 ε t (34)

where t is the irradiation time of the actinometer in seconds

The relative spectral energy distribution of the radiation source (Fig 3) shows

100 emission at 254 nm the wavelength used for the photolysis of ascorbic acid (λmax

265 nm at pH 4ndash7) The energy emitted by the radiation source at various wavelengths

can be calculated using the equation

1197 times 105

E (KJ molndash1

) = λ nm

(35)

The quantum efficiency of ferrioxalate actinometer at the wavelength absorbed by

ascorbic acid (ie 254 nm) is high although the sensitivity drops over 450 nm The

average intensity of the TUV tube used in this study was determined as 556 plusmn 012 times

1018

quanta sndash1

429 Viscosity Measurements

The viscosity of the cream formulations was measured with a Brookfield RV

viscometer (Model DV-II + Pro USA) The instrument was calibrated using the

manufacturerrsquos viscosity standard A 200 g quantity of the cream was placed in a beaker

and the spindle (TE) was dipped into the cream It was rotated at a speed of 06 rpm for

64

00

02

04

06

08

10

12

0 2 4 6 8 10 12

Concentration of Fe++

times 105 M

Ab

sorb

an

ce a

t 51

0 n

m

Fig 4 Calibration graph for the determination of K3Fe(C2O4)3

65

one minute and the viscosity was recorded at 25plusmn1 ordmC The test was repeated three times

to account for the experimental variability and the average viscosity was noted

4210 Assay Methods

42101 UV spectrophotometric method for the assay of creams containing ascorbic

acid alone

The creams were thoroughly mixed a quantity of 2 g was accurately weighed and

the assay of ascorbic acid was carried out by the UV method of Zeng et al (2005) In the

case of photodegraded creams (2 g) the material was completely removed from the glass

plate and transferred to a volumetric flask The method involved extraction of ascorbic

acid with methanol (3 times 10 ml) adjustment of the pH of combined methanolic solutions

to 20 (with H3PO4) dilution of the final solution with acidified methanol (pH 20) to 100

ml and measurement of the absorbance at 245 nm using appropriate blank The

concentration of ascorbic acid was calculated using 560 as the value of specific

absorbance [A (1 1 cm)] at the analytical wavelength (Table 5)

The same method was used for the assay of ascorbic acid in creams stored in the

dark and in the presence of individual stabilizing agents (citric tartaric and boric acids)

42102 Iodimetric method for the assay of ascorbic acid in creams containing

riboflavin nicotinamide and alpha-tocopherol

The assay of ascorbic acid in creams in the presence of riboflavin nicotinamide

and alpha-tocopherol was carried out according to the procedure of British

Pharmacopoeia (2009) as follows

The photolysed cream (2 g) was completely scrapped from the glass plate and

transferred to a flask containing 40 ml of distilled water and 10 ml of 1 M sulphuric acid

66

Table 5 Calibration data for ascorbic acid showing linear regression analysisa

λ max 245 nm

Concentration range 01ndash10 times 10ndash4

M (0176ndash1761 mg )

Slope 9920

SE (plusmn) of slope 00114

Intercept 00012

Correlation coefficient 09996

Molar absorptivity (ε) 9920 Mndash1

cmndash1

Specific absorbance [A (1 1 cm)] 560

a Values represent a mean of five determinations

67

was added The solution was titrated with 002 M iodine solution using 1 ml of starch

solution as indicator until a persistent violet-blue color was obtained Each ml of 002 M

iodine solution is equivalent to 352 mg of C6H8O6 The same method was used for the

assay of ascorbic acid in creams stored in the dark

42103 Spectrophotometric method for the assay of ascorbic acid in aqueous and

organic solvents

A 1 ml aliquot of the photolysed solutions of ascorbic acid in water or in an

organic solvent was evaporated to dryness under nitrogen at room temperature and the

residue redissolved in a small volume of methanol The solution was transferred to a 10

ml volumetric flask made up to volume with acidified methanol (pH 20) and the

absorbance measured at 245 nm using an appropriate blank The content of ascorbic acid

in the solutions was determined using 9920 Mndash1

cmndash1

as the value of molar absorptivity at

the analytical wavelength (Table 5)

CHAPTER V

PHOTODEGRADATION OF

ASCORBIC ACID IN

ORGANIC SOLVENTS AND

CREAM FORMULATIONS

69

51 INTRODUCTION

Ascorbic acid (vitamin C) is an essential micronutrient that performs important

metabolic functions (Packer and Fuchs 1999 Davey et al 2000 Johnston et al 2007)

It is an ingredient of anti-aging cosmetic products (Darr et al 1996 Gallarate et al

1999 Traikovich 1999 Zhang et al 1999 Ozer et al 2000 Nusgens et al 2001

Pinnell et al 2001 2003 Lee et al 2004 Raschke et al 2004 Sauermann et al 2004

Elmore 2005 Jentzsch et al 2005 Lin et al 2005 Placzek et al 2005 Carlotti et al

2006 Farahmand et al 2006 Heber et al 2006 Maia et al 2006 Tournas et al 2006)

and exerts several functions on the skin as collagen synthesis depigmentation and

antioxidant activity (Nusgens et al 2001 Spiclin et al 2003) As an antioxidant it

protects skin by neutralizing reactive oxygen species generated on exposure to sunlight

(Shindo et al 1994) In biological systems it reduces both oxygenndash and nitrogenndash based

free radicals (Higdon and Frei 2002) and thus delays the aging process In view of the

instability of ascorbic acid in skin care formulations (Bissett 2006) it is often used in

combination with another redox partner such as alpha-tocopherol (vitamin E) to retard its

oxidation (Wille 2005)

The details of the cream formulations used in this study are given in Table 6 The

results obtained on the photodegradation of ascorbic acid in aqueous organic solvents

and cream formulations are discussed in the following sections

70

Table 6 Composition of cream formulations containing ascorbic acid

Ingredients Cream

No pH

SA PA MA CA AH2 GL PG EG PH DW

1 a 4 + minus minus + + + minus minus + +

b 5 + minus minus + + + minus minus + +

c 6 + minus minus + + + minus minus + +

d 7 + minus minus + + + minus minus + +

2 a 4 minus + minus + + + minus minus + +

b 5 minus + minus + + + minus minus + +

c 6 minus + minus + + + minus minus + +

d 7 minus + minus + + + minus minus + +

3 a 4 minus minus + + + + minus minus + +

b 5 minus minus + + + + minus minus + +

c 6 minus minus + + + + minus minus + +

d 7 minus minus + + + + minus minus + +

4 a 4 + minus minus + + minus + minus + +

b 5 + minus minus + + minus + minus + +

c 6 + minus minus + + minus + minus + +

d 7 + minus minus + + minus + minus + +

5 a 4 minus + minus + + minus + minus + +

b 5 minus + minus + + minus + minus + +

c 6 minus + minus + + minus + minus + +

d 7 minus + minus + + minus + minus + +

6 a 4 minus minus + + + minus + minus + +

b 5 minus minus + + + minus + minus + +

c 6 minus minus + + + minus + minus + +

d 7 minus minus + + + minus + minus + +

7 a 4 + minus minus + + minus minus + + +

b 5 + minus minus + + minus minus + + +

c 6 + minus minus + + minus minus + + +

d 7 + minus minus + + minus minus + + +

8 a 4 minus + minus + + minus minus + + +

b 5 minus + minus + + minus minus + + +

c 6 minus + minus + + minus minus + + +

d 7 minus + minus + + minus minus + + +

9 a 4 minus minus + + + minus minus + + +

b 5 minus minus + + + minus minus + + +

c 6 minus minus + + + minus minus + + +

d 7 minus minus + + + minus minus + + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PG = propylene glycol EG = ethylene glycol

PH = potassium hydroxide DW = distilled water

71

52 PHOTOPRODUCTS OF ASCORBIC ACID

The photolysis of ascorbic acid (AH2) in aqueous and organic solvents and in

cream formulations on UV irradiation leads to the formation of dehydroascorbic acid

(DHA) as detected by TLC along with the undegraded AH2 using the solvent systems A

B and C The identification of DHA was carried out by comparison of the Rf value and

spot color with those of the authentic compound The formation of DHA on

photooxidation of ascorbic acid solutions has previously been reported (Homan and

Gaffron 1964 Sattar et al 1977 Heelis et al 1981 Rozanowska et al 1997 Lavoie et

al 2004) DGA the hydrolysis product of DHA (Homan and Gaffron 1964) could not

be detected under the present experimental conditions

53 SPECTRAL CHARACTERISTICS OF PHOTOLYSED SOLUTIONS

A typical set of the UV absorption spectra of photolysed solutions of AH2 in

methanol is shown in Fig 5 There is a gradual loss of absorbance around 245 nm with

time as a result of the oxidation of the molecule to DHA (Pelletier 1985 Davies et al

1991 Rumsey and Levine 2000) which does not absorb in this region due to the loss of

conjugation Similar absorption changes are observed on the photolysis of AH2 in other

organic solvents and in the methanolic extracts of cream formulations However the

magnitude of these changes varies with the rate of photolysis in a particular solvent or

cream and appears to be a function of the polar character pH and viscosity of the

medium

72

Fig 5 UV absorption spectra of photolysed solutions of ascorbic acid in methanol at

0 40 80 120 160 220 and 300 min

73

54 ASSAY OF ASCORBIC ACID IN CREAMS AND SOLUTIONS

The assay of AH2 in creams and solutions has been carried out in acidified

methanol (pH 20) according to the UV spectrophotometric method of Zeng et al (2005)

Aqueous solutions of AH2 (~pH 2) exhibit absorption maxima at 243 nm (OrsquoNeil 2001

Moffat et al 2004 Sweetman 2009) 244 nm (Ogata and Kosugi 1969) and 245 nm

(Verma et al 1991 Johnston et al 2007) The absorption maxima of AH2 in methanol

and phosphate buffer (pH 25) occur at 245 nm (Zeng et al 2005) Since dilute solutions

of AH2 are highly susceptible to oxidation the pH was adjusted to 20 with phosphoric

acid to convert the molecule to the non-ionized form (99) to minimize degradation

during the assay AH2 in acidified methanol (pH 20) was found to exhibit the absorption

maximum at 245 nm as reported by Zeng et al (2005) The method was also used for the

assay of AH2 in aqueous and organic solvents

The validity of Beerrsquos law relation in the concentration range used was confirmed

prior to the assay The calibration data for AH2 at the analytical wavelength are presented

in Table 5 (Chapter 4) The correlation coefficient (r = 09996) indicates a good linear

relationship over the concentration range employed The values of specific absorbance

and molar absorptivity at 245 nm determined from the slope of the curve are in good

agreement with those reported by previous workers (Davies et al 1991 Johnston et al

2007) The method of Zeng et al (2005) has been found to be satisfactory for the assay of

AH2 in cream formulations and solutions and has been used to study the kinetics of

photolysis reactions The method was validated before its application to the assay of AH2

in photolysed creams The reproducibility of the method was confirmed by the analysis of

known amounts of AH2 in the concentration range likely to be found in photodegraded

74

creams The values of the recoveries of AH2 in creams by the UV spectrophotometric

method are in the range of 90ndash96 The values of RSD for the assays indicate the

precision of the method within plusmn5 (Table 7)

In order to compare the UV spectrophotometric method with the British

Pharmacopoeia iodimetric method (2009) using a dilute iodine solution (002 M) the

creams were simultaneously assayed for AH2 content by the two methods and the results

are reported in Table 8 The statistical evaluation of the accuracy and precision of the two

methods was carried out by the application of the F test and the t test respectively The F

test showed that there is no significant difference between the precision of the two

methods and the calculated value of F is lower than the critical value (F = 639 P = 005)

in each case The t test indicated that the calculated t values are lower than the tabulated t

values (t = 2776 P = 005) suggesting that at 95 confidence level the differences

between the results of the two methods are statistically non-significant Thus the accuracy

and precision of the UV spectrophotometric method is comparable to that of the official

iodimetric method for the assay of AH2 in cream formulations The results of the assays

of AH2 in aqueous organic solvents and cream formulations are reported in Table 9

55 EFFECT OF SOLVENT

The influence of solvent on the rate of degradation of drugs is of considerable

importance to the formulator since the stability of drug species in solution media may be

predicted on the basis of their chemical reactivity The reactivity of drugs in a particular

medium depends to a large extent on solvent characteristics such as the dielectric

constant and viscosity (Connors et al 1986 Yoshioka and Stella 2000 Sinko 2006)

75

Table 7 Recovery of ascorbic acid added to cream formulationsa

Cream

Formulationb

Added

(mg)

Found

(mg)

Recovery

()

RSD

()

1a 400

200

380

183

950

915

21

25

2b 400

200

371

185

928

925

15

25

3c 400

200

375

181

938

905

11

31

4d 400

200

384

189

960

945

13

21

5b 400

200

370

189

925

945

14

26

6c 400

200

369

190

923

950

10

22

7d 400

200

374

182

935

910

17

39

8c 400

200

380

188

950

940

15

33

9d 400

200

367

189

918

945

20

42

a Values expressed as a mean of three to five determinations

b The cream formulations represent combinations of each emulsifier (stearic acid

palmitic acid myristic acid) with each humectant (glycerin propylene glycol ethylene

glycol) to observe the efficiency of methanol to extract AH2 from different creams

(Table 6)

76

Table 8 Assay of ascorbic acid in creams using UV spectrophotometric and iodimetric

methods

Ascorbic acid (mg) Cream

Formulationb Added UV method

a

Iodimetric

methoda

Fcalc tcalc

1a 40

20

380 plusmn 081

183 plusmn 046

375 plusmn 095

185 plusmn 071

138

238

245

104

2b 40

20

371 plusmn 056

185 plusmn 047

373 plusmn 064

193 plusmn 038

130

065

181

200

3c 40

20

375 plusmn 040

181 plusmn 056

374 plusmn 046

183 plusmn 071

132

160

101

223

4d 40

20

384 plusmn 051

189 plusmn 039

381 plusmn 066

190 plusmn 052

167

178

176

231

5b 40

20

370 plusmn 052

189 plusmn 050

372 plusmn 042

185 plusmn 067

065

179

162

125

6c 40

20

369 plusmn 037

190 plusmn 042

371 plusmn 058

188 plusmn 056

245

177

122

197

7d 40

20

374 plusmn 062

182 plusmn 072

370 plusmn 070

184 plusmn 082

127

129

144

168

8c 40

20

380 plusmn 058

188 plusmn 062

375 plusmn 075

192 plusmn 060

167

094

123

162

9d 40

20

367 plusmn 072

189 plusmn 080

365 plusmn 082

187 plusmn 075

149

092

130

203

Theoretical values (P = 005) for F is 639 and for t is 2776

a Mean plusmn SD (n = 5)

b Table 6

77

Table 9 Photodegradation of ascorbic acid in cream formulations

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) pHa 40 50 60 70

0 383 382 384 383

60 374 369 366 361

120 361 354 346 325

180 351 345 325 305

240 345 327 301 284

1

300 336 316 287 264

0 380 383 382 379

60 371 376 362 346

120 359 357 342 320

180 352 345 322 301

240 341 335 299 283

2

300 336 321 291 261

0 384 376 381 385

60 377 367 360 358

120 366 348 334 324

180 356 337 317 305

240 343 320 301 282

3

300 335 307 273 253

78

Table 9 continued

0 377 378 386 372

60 365 361 371 355

120 353 345 347 322

180 344 327 325 298

240 332 320 306 279

4

300 317 303 284 252

0 381 367 372 373

60 372 358 358 353

120 360 337 336 321

180 352 325 320 302

240 341 313 300 284

5

300 327 302 278 256

0 376 386 380 377

60 366 372 350 350

120 353 347 323 316

180 337 334 308 298

240 329 320 291 274

6

300 313 306 267 245

79

Table 9 continued

0 380 372 378 380

60 373 362 350 354

120 358 340 329 321

180 344 328 304 300

240 332 315 292 283

7

300 319 302 272 252

0 380 381 378 361

60 368 364 361 335

120 355 354 340 313

180 342 340 315 281

240 337 331 303 269

8

300 323 314 281 248

0 378 382 370 375

60 370 369 349 342

120 356 347 326 321

180 339 333 298 291

240 326 314 277 271

9

300 313 302 265 242

a The values at pH 40ndash70 represent the formulations a to d of each cream respectively

80

In order to observe the effect of solvent dielectric constant the apparent first-

order rate constants (kobs) for the photolysis of AH2 in alcoholic solvents (Table 10) were

plotted against the dielectric constants of the solvents A linear relationship indicated the

dependence of the rates of photolysis on solvent dielectric constant (Fig 6) This implies

the involvement of a polar intermediate in the reaction to facilitate the formation of the

degradation products as suggested by Ahmad and Tollin (1981) in the case of flavin

electron transfer reactions The effect of solvent polarity has been observed on the

autooxidation of AH2 in organic solvents (Ogata and Kosugi 1969)

Another solvent parameter affecting the rate of a chemical reaction is viscosity

which can greatly influence the stability of oxidisable substances (Wallwork and Grant

1977 Laidler 1987 Fung 1990) A plot of kobs for the photolysis of AH2 against the

reciprocal of solvent viscosity (Table 10) is linear showing that an increase in solvent

viscosity results in a decrease in the rate of photolysis (Fig 7) The viscosity of the liquid

affects the rate at which molecules can diffuse through the solution This in turn may

affect the rate at which a compound can suffer oxidation at the liquid surface This

applies to AH2 and an increase in the viscosity of the medium makes access to air at the

surface more difficult to prevent oxidation (Wallwork and Grant 1977)

56 EFFECT OF CONCENTRATION

In order to observe the effect of concentration (Table 11) on the photostability of

AH2 in a cream using stearic palmitic and myristic acids as emulsifying agents and

glycerin as humectant plots of log concentration versus time were constructed (Fig 8)

and the apparent first-order rate constants were determined (Table 12) A graph of kobs

against concentration of AH2 (Fig 9) exhibited an apparent linear relationship between

81

Table 10 Apparent first-order rate constants (kobs) for the photolysis of ascorbic acid in

water and organic solvents

Solvent Dielectric

Constant (25 ordmC)

Viscosity

(mPas) ndash1

kobs times104

(minndash1

)

Water 785 1000 404

Methanol 326 1838 324

Ethanol 243 0931 316

1-Propanol 201 0514 302

1-Butanol 178 0393 295

82

00

20

40

60

80

0 10 20 30 40 50 60 70 80

Dielectric constant

k (

min

ndash1)

Fig 6 A plot of kobs for photolysis of ascorbic acid against solvent dielectric constant

(times) Water () methanol () ethanol (diams) 1-propanol () 1-butanol

83

00

10

20

30

40

50

00 05 10 15 20

Viscosity (mPas)ndash1

k times

10

4 (m

inndash1)

Fig 7 A plot of kobs for photolysis of ascorbic acid against reciprocal of solvent

viscosity Symbols are as in Fig 6

84

Table 11 Effect of concentration on the photodegradation of ascorbic acid in cream

formulations at pH 60

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) 05 10 15 20 25

0 95 191 290 379 471

60 90 182 277 358 453

120 82 167 260 339 431

180 77 158 239 311 401

240 70 144 225 298 382

1

300 64 134 210 282 363

0 92 186 287 380 472

60 88 175 272 369 453

120 82 160 251 342 429

180 75 152 238 326 405

240 71 144 225 309 392

2

300 65 134 215 289 366

0 94 182 286 376 470

60 87 171 265 352 454

120 78 152 251 337 426

180 69 143 227 315 404

240 62 129 215 290 378

3

300 58 119 195 271 353

85

05

10

15

20

25

06

08

10

12

14

16

18

log

co

nce

ntr

ati

on

(m

g)

a

05

10

15

20

25

06

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

b

05

10

15

20

25

06

08

10

12

14

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

c

Fig 8 Log concentration versus time plots for the photodegradation of ascorbic acid at

various concentrations in creams at pH 60 a) stearic acid b) palmitic acid

c) myristic acid

86

Table 12 Apparent first-order rate constants (kobs) for the photodegradation of various

ascorbic acid concentrations in cream formulations at pH 60

kobs times 103 (min

ndash1)a Cream

Formulationb 05 10 15 20 25

1 133

(0994)

120

(0993)

111

(0995)

101

(0994)

090

(0994)

2 118

(0992)

108

(0994)

098

(0993)

093

(0992)

084

(0994)

3 169

(0994)

144

(0995)

126

(0994)

109

(0993)

097

(0992)

a The values in parenthesis are correlation coefficients

b Table 6

87

Stearic acid

Palmitic acid

Myristic acid

00

05

10

15

20

25

00 05 10 15 20 25

Ascorbic acid concentration ()

kob

s (min

ndash1)

Fig 9 A plot of kobs for photodegradation against ascorbic acid concentrations in cream

formulations

88

the two values Thus the rate of degradation of AH2 is faster at a lower concentration on

exposure to the same intensity of light This may be due to a relatively greater number of

photons available for excitation of the molecule at lower concentration compared to that

at a higher concentration The AH2 concentrations of creams used in this study are within

the range (1ndash15) reported by previous workers for topical applications to skin (Kaplan

et al 1989 Traikovich et al 1999 Nusgens et al 2001 Matsubayashi et al 2003

Espinal-Perez et al 2004 Sauermann et al 2004 Lin et al 2005 Heber et al 2006)

57 EFFECT OF CARBON CHAIN LENGTH OF EMULSIFYING AGENT

The values of kobs for the photodegradation of AH2 (2) in various cream

formulations are reported in Table 13 The first-order plots for the photodegradation of

AH2 at pH 4ndash7 in various cream formulations are shown in Fig 10ndash12 The plots of kobs

against carbon chain length of the emulsifying agents are shown in Fig 13 They indicate

that the photodegradation of AH2 is affected by the emulsifying agent in the order

myristic acid gt stearic acid gt palmitic acid

These acids possess a polar character (Yao et al 2009) and the carbon chain of the acid

may play a part in the photostability of AH2 However the results indicate that in the

presence of palmitic acid AH2 exhibits greater stability as indicated by the plots of kobs

versus the carbon chain length of the emulsifying agents (Fig 13) This could be

predominantly due to the interaction of AH2 with palmitic acid in the cream to impart it

greater stability Ascorbic acid-6-palmitate is known to be an antioxidant in cosmetic

preparations (Lee et al 2009) and food products (Doores 2002)

In view of the above observations it may be suggested that the photodegradation

of AH2 could involve a polar semiquinone intermediate (Johnston et al 2007) which

89

Table 13 First-order rate constants (kobs) for the photodegradation of ascorbic acid in

cream formulations

kobs times 103 (min

ndash1)abc

Cream

Formulation pH 40 50 60 70

1 044

(0992)

064

(0994)

100

(0995)

126

(0995)

2 042

(0992)

060

(0991)

095

(0992)

120

(0995)

3 047

(0993)

069

(0993)

107

(0991)

137

(0995)

4 056

(0993)

072

(0992)

104

(0994)

131

(0993)

5 050

(0991)

067

(0992)

097

(0991)

124

(0992)

6 061

(0992)

079

(0993)

113

(0992)

140

(0994)

7 060

(0992)

071

(0993)

108

(0994)

133

(0992)

8 053

(0991)

062

(0992)

099

(0994)

126

(0993)

9 065

(0991)

081

(0996)

117

(0993)

142

(0995)

a The rate constants at pH 40ndash70 represent the values for formulations a to d of each

cream respectively

b The values in parenthesis are correlation coefficients

c The values of rate constants are relative and depend on specific experimental conditions

including light intensity

The estimated error is plusmn5

90

12

13

14

15

16

17

log

co

nce

ntr

ati

on

(m

g)

1

12

13

14

15

16

17

log

co

nce

ntr

ati

on

(m

g)

2

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

3

Fig 10 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (1ndash3) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

91

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

4

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

5

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

6

Fig 11 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (4ndash6) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

92

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

7

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

8

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

9

Fig 12 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (7ndash9) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

93

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

ko

bs times

10

3 (m

inndash

1)

1-3

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

ko

bs times

10

3 (

min

ndash1)

4-6

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

Carbon chain length

ko

bs times

10

3 (

min

ndash1)

7-9

Fig 13 Plots of kobs for photodegradation of ascorbic acid in creams (1ndash9) against carbon

chain length of emulsifier () Stearic acid () palmitic acid () myristic acid

Humectant used glycerin (1ndash3) propylene glycol (4ndash6) ethylene glycol (7ndash9)

94

depending on the polar character of the medium undergoes oxidation with varying rates

This is similar to the behavior of the photolysis of riboflavin analogs which is dependent

on the polar character of the medium (Ahmad and Tollin 1981) The effect of carbon

chain length on the transdermal delivery of an active ingredient has been discussed (Lu

and Flynn 2009)

58 EFFECT OF VISCOSITY

The plots of rates of AH2 degradation in cream formulations (Table 13) as a

function of carbon chain length (Fig 13) indicate that the rates vary with the humectant

and hence the viscosity of the medium in the order

ethylene glycol gt propylene glycol gt glycerin

This is in agreement with the rate of photolysis of AH2 in organic solvents that

higher the viscosity of the medium lower the rate of photolysis Thus apart from the

carbon chain length of the emulsifier viscosity of the humectant added to the cream

formulation appears to play an important part in the stability of AH2 The stabilizing

effect of viscosity imparting substances on AH2 solutions has been reported (Stone 1969

Kassem et al 1969ab)

59 EFFECT OF pH

The kobsndashpH profiles for the photodegradation of AH2 in various creams (1ndash9) at

pH 4ndash7 (Fig 14) represent a sigmoid type curve indicating the oxidation of the ionized

form (AHndash) of AH2 (pKa 41) (OrsquoNeil 2001) with pH The AH

ndash species appears to be

more susceptible to photooxidation than the non-ionized form (AH2) The behavior of

AH2 on photooxidation in the pH range 4ndash7 is similar to that observed for the chemical

oxidation of AH2 by molecular oxygen (DeRitter 1982) and involves the interaction of

95

Stearic Acid (1)

Palmitic Acid (2)

Myristic Acid (3)

04

06

08

10

12

14

kob

s times

10

3 (m

inndash

1)

Stearic Acid (4)

Palmitic Acid (5)

Myristic Acid (6)

04

06

08

10

12

14

ko

bs times

10

3 (

min

ndash1)

Stearic Acid (7)

Palmitic Acid (8)

Myristic Acid (9)

04

06

08

10

12

14

30 40 50 60 70

pH

ko

bs

times 1

03

(min

ndash1)

Fig 14 The kobsndashpH profiles for the photodegradation of ascorbic acid in creams (1ndash9)

Glycerin

Propylene glycol

Ethylene glycol

96

AH2 with singlet oxygen on UV irradiation (Silva and Quina 2006) The AHndash species

(predominant in the pH range 42ndash70 557ndash999) is more reactive towards singlet

oxygen than its protonated form the AH2 molecule as suggested by Bisby et al (1999)

and therefore the rate of photooxidation is higher in the pH range above 41

corresponding to the pKa1 of AH2 The major goal of a ratendashpH profile is to determine

the optimum pH range for a particular formulation Several workers have studied the

ratendashpH profiles of the chemical oxidation of AH2 in the pH range 2ndash7 (Garrett 1967

Taqui Khan and Martell 1967 Rogers and Yacomeni 1971 Blaugh and Hajratwala

1972 DeRitter 1982 Moura et al 1994) however the kinetics of photooxidation of

AH2 in cream formulations has so far not been reported

510 EFFECT OF REDOX POTENTIAL

The photooxidation of AH2 is also influenced by its redox potential which varies

with pH The greater photostability of AH2 at pH 5ndash6 compared to that at pH 7 and above

is due to its lower rate of oxidationndashreduction in this range (Eordm pH 50 = +0127 V)

(OrsquoNeil 2001) The increase in the rate of photooxidation with pH is due to a

corresponding increase in the redox potential (Eordm pH 70 = +0058 V) (Fasman 1976) of

AH2 and is similar to the photolysis behavior of riboflavin at pH 5ndash6 (Eordm pH 50 = ndash0117

V) (Sinko 2006) compared to that at pH 70 (Eordm pH 70 = ndash 0207 V) (Ahmad et al

2004a Sinko 2006) Since the ionization as well as the redox potentials of AH2 are a

function of pH the rate of photooxidation depends upon the specific species present and

its redox behavior at a particular pH

97

511 PRIMARY PHOTOCHEMICAL REACTIONS IN THE OXIDATION OF

ASCORBIC ACID

A reaction scheme based on general photochemical principles for the important

reactions involved in the photooxidation of ascorbic acid is presented below

0AH2 hv k1

1AH2 (51)

1AH2 k2 Products (52)

1AH2 isc k3

3AH2 (53)

3AH2 k4 Products (54)

0AH

ndash hv k5

1AH

ndash (55)

1AH

ndash k6 Products (56)

1AH

ndash k7

3AH

ndash (57)

3AH

ndash k8 Products (58)

3AH

ndash +

0AH2 k9 AH٠

ndash + AH٠ (59)

2 AH٠ k10 A + AH2 (510)

3AH2 +

3O2 k11

0AH2 +

1O2 (511)

AHndash +

1O2 k12

3AH

ndash +

3O2 (512)

AH٠ + 1O2 k13 AHOO٠ (513)

AHOO٠ k14 A + HO2٠ (514)

AHOO٠ + 0AH2 k15 AH٠ + AHOOH (515)

AHOOH k16 secondary reaction

A + H2O2 (516)

According to this reaction scheme the ground state ascorbic acid species (0AH2

0AH

ndash) each is excited to the lowest singlet state (

1AH2

1AH

ndash) by the absorption of a

quantum of UV light (51 55) These excited states may directly be converted to

98

photoproducts (52 56) or may undergo intersystem crossing (isc) to form the excited

triplet states (53 57) The excited triplet states may then degrade to the photoproducts

(54 58) The monoascorbate triplet (3AH

ndash) may react with the ground state ascorbic

acid to form a monoascorbate radical anion (AH٠ndash) and a monoascorbate radical (AH٠)

(59) Two AH٠ radical species may lead to the formation of an oxidized (A) and a

reduced ascorbic acid molecule (AH2) (510) Ascorbic acid triplet (3AH2) may react with

molecular oxygen (3O2) to yield singlet oxygen (

1O2) (511) which may then react with

monoascorbate anion (AHndash) to form the excited triplet state (

3AH

ndash) (512) or with

monoascorbate radical to form a peroxyl radical (AHOO٠) (513) The peroxyl radical

may yield dehydroascorbic acid (A) (514) or react with ground state ascorbic acid to

give monoascorbate radical and a reduced species AHOOH (515) The reduced species

may give rise to dehydroascorbic acid and hydrogen peroxide (516)

512 DEGRADATION OF ASCORBIC ACID IN THE DARK

In view of the instability of AH2 and to observe its degradation in the dark the

creams were stored in airtight containers at room temperature in a cupboard for a period

of about 3 months and assayed for AH2 content at appropriate intervals The analytical

data (Table 14) were subjected to kinetic treatment (Fig 15ndash17) and the apparent first-

order rate constants for the degradation of AH2 were determined (Table 15) The values

of the rate constants indicate that the degradation of AH2 in the dark is about 70 times

slower than those of the creams exposed to UV irradiation (Table 13) The degradation of

AH2 in creams in the dark is due to chemical oxidation (Section 132) and occurs in the

order of emulsifying agents (Fig 18)

myristic acid gt stearic acid gt palmitic acid

99

Table 14 Degradation of ascorbic acid in the dark in cream formulations

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(days) pHa 40 50 60 70

0 383 382 384 383

10 354 340 313 278

20 309 306 279 245

40 244 209 183 161

60 172 166 131 105

1

80 145 114 81 61

0 380 383 382 379

10 360 343 350 335

20 322 310 301 294

40 266 250 211 186

60 233 211 168 142

2

80 182 153 114 89

0 384 376 381 385

10 368 350 340 318

20 318 273 273 266

40 223 199 172 155

60 174 132 117 84

3

80 122 97 66 54

100

Table 14 continued

0 377 378 386 372

10 350 334 334 318

20 314 268 256 244

40 238 208 182 136

60 179 155 107 94

4

80 128 101 79 59

0 381 367 372 373

10 350 293 300 320

20 299 266 270 263

40 220 191 192 184

60 183 153 139 129

5

80 149 115 87 76

0 376 386 380 377

10 312 320 314 251

20 255 282 226 199

40 175 194 159 131

60 139 128 99 74

6

80 102 81 55 41

101

Table 14 continued

0 380 372 378 380

10 323 330 333 323

20 288 273 276 224

40 212 174 182 146

60 152 133 108 83

7

80 100 82 66 56

0 380 381 378 361

10 333 320 310 310

20 281 266 260 257

40 230 189 171 177

60 156 148 128 111

8

80 123 96 78 66

0 378 382 370 375

10 313 295 281 300

20 256 247 257 203

40 194 178 151 133

60 119 114 88 74

9

80 88 68 49 39

a The values at pH 40ndash70 represent the formulations a to d of each cream respectively

102

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

1

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

2

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

3

Fig 15 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (1ndash3) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

103

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

4

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

5

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

6

Fig 16 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (4ndash6) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

104

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

7

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

8

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

9

Fig 17 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (7ndash9) at pH 40 (times) 50 () 60 () and 70 ()

Palmitic acid

Myristic acid

Stearic acid

105

Table 15 First-order rate constants (kobs) for the degradation of ascorbic acid in cream

formulations in the dark

kobs times 102 (day

ndash1)abc

Cream

Formulation pH 40 50 60 70

1 128

(0991)

152

(0994)

191

(0995)

220

(0994)

2 091

(0992)

110

(0991)

152

(0993)

182

(0992)

3 148

(0991)

176

(0995)

220

(0993)

254

(0995)

4 137

(0992)

161

(0993)

205

(0994)

236

(0995)

5 121

(0991)

141

(0994)

175

(0993)

195

(0993)

6 162

(0992)

194

(0995)

237

(0994)

265

(0994)

7 164

(0994)

189

(0994)

222

(0993)

246

(0996)

8 143

(0994)

167

(0995)

193

(0996)

212

(0993)

9 184

(0995)

208

(0994)

251

(0992)

280

(0996)

a The rate constants at pH 40ndash70 represent the values for formulations a to d of each

cream respectively

b The values in parenthesis are correlation coefficients

c The values of rate constants are relative and depend on specific experimental

conditions

The estimated error is plusmn5

106

pH 4

pH 5

pH 6

pH 7

00

10

20

30

k times

10

2 (d

ayndash

1)

1-3

pH 4

pH 5

pH 6

pH 7

00

10

20

30

k times

10

2 (

da

yndash1)

4-6

pH 4

pH 5

pH 6

pH 7

00

10

20

30

12 14 16 18

Carbon chain length

k times

10

2 (

da

yndash1)

7-9

Fig 18 Plots of kobs for degradation of ascorbic acid in the dark in creams (1ndash9) against

carbon chain length of emulsifier () Stearic acid () palmitic acid

() myristic acid Humectant used glycerin (1ndash3) propylene glycol (4ndash6)

ethylene glycol (7ndash9)

107

Although it is logical to expect a linear relationship between the rate of degradation and

the carbon chain length of the emulsifier due to its polar character (Yao et al 2009) it

has not been observed in the present case The reason for the slowest rate of degradation

of AH2 in the presence of palmitic acid appears to be due to the interaction of AH2 with

palmitic acid (Lee et al 2009) as explained in Section 57

The degradation of AH2 also appears to be affected by the viscosity of the cream

in the order of humectant (Fig 19)

ethylene glycol gt propylene glycol gt glycerin

Thus the presence of glycerin imparts the most stabilizing effect on the degradation of

AH2 This is the same order as observed in the case of photodegradation of AH2 in the

creams The airtight containers used for the storage of creams make the access of air to

the creams difficult to cause chemical oxidation of AH2 However it has been observed

that the degradation of AH2 is highest in the upper layer of the creams compared to that

of the middle and the bottom layers Therefore the creams were thoroughly mixed before

sampling for the assay of AH2 However the scattering in kinetic plots (Fig 15ndash17) is

due to non-uniform distribution of AH2 in degraded creams

The effect of pH on the degradation of AH2 in the creams (Fig 19) shows that the

degradation increases with an increase in pH as observed in the case of photodegradation

of AH2 in the creams This is due to an increase in the ionization and redox potential of

AH2 with pH causing greater oxidation of the molecule and has been discussed in

Sections 59 and 510

108

Stearic Acid (1)

Palmitic Acid (2)

Myristic Acid (3)

00

10

20

30

k times

10

2 (d

ayndash

1)

Glycerin

Stearic Acid (4)

Palmitic Acid (5)

Myristic Acid (6)

00

10

20

30

k times

10

2 (

da

yndash1)

Propylene glycol

Stearic Acid (7)

Palmitic Acid (8)

Myristic Acid (9)

00

10

20

30

30 40 50 60 70

pH

k times

10

2 (d

ayndash

1)

Ethylene glycol

Fig 19 The kobsndashpH profiles for the degradation of ascorbic acid in the dark in creams

(1ndash9)

CHAPTER VI

PHOTOCHEMICAL INTERACTION

OF ASCORBIC ACID WITH

RIBOFLAVIN NICOTINAMIDE

AND ALPHA-TOCOPHEROL IN

CREAM FORMULATIONS

110

61 INTRODUCTION

It is now medically recognized that sagging skin and other signs of degenerative

skin conditions such as wrinkles and age spots are caused primarily by oxy-radical

damage Ascorbic acid can accelerate wound healing protect fatty tissues from oxidative

damage and play an integral role collagen synthesis (Zhang et al 1999) It is used in

cosmetic preparations for its anti-aging depigmentation and antioxidant properties

(Spiclin 2003 Ehrlich et al 2006) It is also used in combination with other vitamins

such as alpha-tocopherol as a co-antioxidant to stabilize cosmetic preparations (Eberlein-

Koumlnig and Ring 2005 Bissett 2006 Murray 2008) Ascorbic acid in the presence of air

or light is known to interact with alpha-tocopherol (Packer et al 2002 Johnston et al

2007) riboflavin (Homann and Gaffron 1964 Sattar et al 1977 Heelis et al 1981

Kim et al 1993 Jung et al 1995 De La Rochette et al 2000 2003 Lavoie et al

2004 Vaid et al 2005 Ahmad and Vaid 2006 Silva and Quina 2006) and

nicotinamide (Bailey et al 1945 Werner et al 1949 Guttman and Brooke 1963

DeRitter 1982) The present work involves a study of the effect of alpha-tocopherol

riboflavin and nicotinamide on the photostability of ascorbic acid in cream formulations

to observe whether the interaction in these formulations leads to the stabilization of

ascorbic acid The chemical structures of nicotinamide (NA) alpha-tocopherol (TP)

riboflavin (RF) formylmethylflavin (FMF) and lumichrome (LC) are shown in Fig 20

The details of the cream formulations used in this study are given in Table 16

The results obtained on the photodegradation of ascorbic acid in cream formulations are

discussed in the following sections

111

Riboflavin

N

N

NH

N

CH2

CH

C OHH

CH OH

CH2OH

N

N

NH

N

CH2

CHO

Formylmethylflavin

N

N

NH

HN

Lumichrome

OH

N

NH2

O

Nicotinamide

O CH3

CH3

CH3

HO

H3C

CH3 CH3 CH3

CH3

Alpha-Tocopherol

O

O

H3C

H3C

H3C

H3C

O

O

H3C

H3C

O

O

Fig 20 Chemical structures of alpha-tocopherol nicotinamide riboflavin

formylmethylflavin and lumichrome

112

Table 16 Composition of cream formulations containing ascorbic acid (2) and other

vitamins

Ingredients Cream

No SA PA MA CA GL AH2 RFa NA

b TP

c PH DW

10 a + minus minus + + + a minus minus + +

b + minus minus + + + b minus minus + +

c + minus minus + + + c minus minus + +

d + minus minus + + + d minus minus + +

e + minus minus + + + e minus minus + +

11 a minus + minus + + + a minus minus + +

b minus + minus + + + b minus minus + +

c minus + minus + + + c minus minus + +

d minus + minus + + + d minus minus + +

e minus + minus + + + e minus minus + +

12 a minus minus + + + + a minus minus + +

b minus minus + + + + b minus minus + +

c minus minus + + + + c minus minus + +

d minus minus + + + + d minus minus + +

e minus minus + + + + e minus minus + +

13 a + minus minus + + + minus a minus + +

b + minus minus + + + minus b minus + +

c + minus minus + + + minus c minus + +

d + minus minus + + + minus d minus + +

e + minus minus + + + minus e minus + +

14 a minus + minus + + + minus a minus + +

b minus + minus + + + minus b minus + +

c minus + minus + + + minus c minus + +

d minus + minus + + + minus d minus + +

e minus + minus + + + minus e minus + +

113

Table 16 continued

15 a minus minus + + + + minus a minus + +

b minus minus + + + + minus b minus + +

c minus minus + + + + minus c minus + +

d minus minus + + + + minus d minus + +

e minus minus + + + + minus e minus + +

16 a + minus minus + + + minus minus a + +

b + minus minus + + + minus minus b + +

c + minus minus + + + minus minus c + +

d + minus minus + + + minus minus d + +

e + minus minus + + + minus minus e + +

17 a minus + minus + + + minus minus a + +

b minus + minus + + + minus minus b + +

c minus + minus + + + minus minus c + +

d minus + minus + + + minus minus d + +

e minus + minus + + + minus minus e + +

18 a minus minus + + + + minus minus a + +

b minus minus + + + + minus minus b + +

c minus minus + + + + minus minus c + +

d minus minus + + + + minus minus d + +

e minus minus + + + + minus minus e + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PH = potassium hydroxide DW = distilled water

RF = riboflavin NA = nicotinamide TP = alpha-tocopherol

a RF(g ) a = 0002 b = 0004 c = 0006 d = 0008 e = 0010

b NA (g ) a = 028 b = 056 c = 084 d = 112 e = 140

c TP (g ) a = 017 b = 034 c = 052 d = 069 e = 086

The molar concentrations of these vitamins are given in Section 421

114

62 ABSORPTION CHARACTERISTICS OF PHOTOLYSED CREAMS

A typical set of the absorption spectra of the methanolic extracts (pH 20) of the

freshly prepared and photolysed creams containing AH2 and TP is shown in Fig 21 AH2

in acidified methanol exhibits absorption maximum at 245 nm (Zeng et al 2005) as

observed in Fig 21 The absorption due to TP at 284 nm (Moffat et al 2004) was

cancelled by using an appropriate blank containing an equivalent concentration of TP

The gradual decrease in absorption at around 245 nm during UV irradiation indicates the

transformation of AH2 to DHA which does not absorb in this region (Davies et al 1991)

as a result of the loss of C3=C2 chromophore Similar spectral changes around 245 nm are

observed in the presence of RF and NA which also strongly absorb in this region A

decrease in the absorption of AH2 around 266 nm in aqueous solution (pH 60) in the

presence of RF has been reported (Vaid et al 2005) The spectral changes and loss of

absorbance in methanolic extracts of creams depends on the rate of photolysis of AH2 in

the presence of these vitamins

63 PHOTOPRODUCTS OF ASCORBIC ACID AND OTHER VITAMINS

The UV irradiation of AH2 in cream formulations (pH 60) in the presence of RF

NA and TP results in the degradation of AH2 and RF and the following photoproducts

have been identified on comparison of their RF values and spot color fluorescence with

those of the authentic compounds

AH2 DHA

RF FMF LC CMF

In the TLC systems used NA and TP did not show the formation of any

degradation product in creams

115

Fig 21 UV absorption spectra of methanolic extracts of photodegraded ascorbic acid in

cream at 0 60 120 180 300 and 480 min

116

The formation of DHA in the photooxidation of AH2 has previously been reported by

many workers (Homann and Gaffron 1964 Sattar et al 1977 Heelis et al 1981

Rozanowska et al 1997 Lavoie et al 2004 Vaid et al 2006) RF is sensitive to light in

aqueous solutions (DeRitter 1982 British Pharmacopoeia 2009 Sweetman 2009) and is

known to form a number of products under aerobic conditions (Treadwell et al 1968

Cairns and Metzler 1971 Schuman Jorns et al 1975 Ahmad and Rapson 1990 Ahmad

and Vaid 2006 Ahmad et al 2004ab 2005 2008 Vaid et al 2006) It has been found

to degrade on UV irradiation in cream formulations to give FMF LC and CMF and these

products have been reported in the photolysis of RF by the workers cited above The

formation of these products may be affected by the interaction of AH2 and RF in creams

(Section 66) NA and TP individually did not appear to form any photoproduct of their

own directly or on interaction with AH2 in creams and may influence the degradation of

AH2 on UV irradiation

64 ASSAY METHOD

In view of the presence of RF (absorption maxima 223 267 373 and 444 nm)

(British Pharmacopoeia 2009) NA (absorption maximum 261 nm) (Moffat et al 2004)

and TP (absorption maximum 284 nm) (Moffat et al 2004) in the cream formulations

and the interference of these vitamins with the absorption of AH2 (absorption maximum

265 nm) (Davies et al 1991) the direct spectrophotometric method cannot be applied for

the determination of AH2 Therefore the iodimetric method (British Pharmacopoeia

2009) was used to determine AH2 in cream formulations The method was validated in

the presence of RF NA and TP before its application to the determination of AH2 in

photodegraded creams The reproducibility of the method has been confirmed by the

117

assay of known concentrations of AH2 in the range present in photodegraded creams The

recovery of AH2 in the creams has been found to be in the range 90ndash96 The values of

RSD indicate that the precision of the method is within plusmn5 (Table 17) and it can be

applied to study the kinetics of AH2 photolysis in cream formulations The assay data on

AH2 in various cream formulations are given in Table 18

65 KINETICS OF PHOTODEGRADATION OF ASCORBIC ACID

Several chemical and physical factors play a role in the photodegradation of AH2

in the presence of other vitamins (RF NA TP) and affect the rate of its degradation in

cream formulations The present work involves the study of photodegradation of AH2 in

cream formulations containing glycerin as humectant as AH2 has been found to be most

stable in these creams (Chapter 5) The apparent first-order rate constants (kobs) for the

photodegradation of AH2 in the presence of other vitamins in cream formulations

derived from the kinetic plots (Fig 22ndash24) are reported in Table 19 The second-order

rate constants (correlation coefficients 0991ndash0996) determined from the slopes of the

graphs of kobs versus vitamin concentration for the individual vitamins (Fig 25) and the

values of k0 determined from the intercept on the vertical axis at zero concentration are

reported in Table 20 The values of k0 give an indication of the effect of other vitamins on

the rate of degradation of AH2 These values are about 13 times lower than the values of

kobs obtained at the highest concentrations of RF and NA indicating that RF and NA both

accelerate the photodegradation of AH2 in creams RF is known to act as a

photosensitizer for the degradation of AH2 (Section 66) and therefore its presence in

creams would accelerate the degradation of AH2 The increase in the rate of

photodegradation of AH2 in the presence of NA has not previously been reported NA

118

Table 17 Recovery of ascorbic acid in cream formulations in the presence of other

vitamins by iodimetric methoda

Cream

Formulationb

Added

(mg )

Found

(mg )

Recovery

()

RSD

()

10e (RF) 400

200

373

187

933

935

29

22

11e (RF) 400

200

379

187

948

935

25

31

12e (RF) 400

200

375

188

938

940

29

28

13e (NA) 400

200

382

191

955

955

23

27

14e (NA) 400

200

380

185

950

925

19

26

15e (NA) 400

200

379

191

948

955

21

17

16e (TP) 400

200

368

183

920

915

29

44

17e (TP) 400

200

391

195

978

975

11

13

18e (TP) 400

200

377

182

943

910

32

37

a Values expressed as a mean of three to five determinations

b The cream formulations represent all the emulsifiers (stearic acid palmitic acid

myristic acid) to observe the efficiency of iodimetric method for the recovery of

ascorbic acid in presence of the highest concentration of vitamins (Table 16)

119

Table 18 Photodegradation of ascorbic acid in cream formulations in the presence of

other vitamins

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) a b C d e

0 373 372 374 372 375

60 362 354 354 360 359

150 342 336 336 332 334

240 315 314 308 310 302

10 (RF)

330 301 291 288 281 282

0 380 379 376 374 374

60 370 366 362 362 361

150 343 337 340 332 328

240 329 323 320 313 310

11 (RF)

330 307 301 294 288 282

0 379 380 375 372 376

60 362 366 361 351 342

150 341 335 319 307 312

240 310 306 295 284 282

12 (RF)

330 285 278 263 254 243

120

Table 18 continued

0 372 370 371 368 365

60 361 358 348 350 349

120 342 343 329 326 330

180 327 325 319 312 308

240 317 309 299 289 285

13 (NA)

300 299 291 283 278 273

0 386 380 375 378 370

60 371 362 365 362 355

120 359 351 343 339 336

200 341 332 325 316 311

14 (NA)

300 313 303 296 294 280

0 375 371 374 370 366

60 362 356 352 352 345

120 343 332 336 326 314

200 323 315 311 295 293

15 (NA)

300 293 283 275 270 259

121

Table 18 continued

0 380 378 380 377 377

60 362 365 369 369 371

120 351 352 360 360 364

180 340 346 349 353 355

240 331 334 343 343 346

16 (TP)

300 320 323 330 332 337

0 383 380 378 380 377

60 372 371 372 373 370

120 363 360 361 366 365

180 348 348 350 356 355

240 341 343 343 348 348

17 (TP)

300 330 332 336 339 341

0 380 383 377 375 373

60 364 370 366 367 366

120 352 356 351 352 351

180 334 338 339 343 342

240 324 328 324 332 330

18 (TP)

300 307 315 317 318 322

122

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

10

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

11

ab

c

de

13

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

12

Fig 22 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing riboflavin (a) 0002 (b) 0004 (c) 0006 (d) 0008

(e) 0010

Stearic acid

Palmitic acid

Myristic acid

123

abcde

13

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

13

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

14

abcde

13

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

15

Fig 23 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing nicotinamide (a) 028 (b) 056 (c) 084 (d) 112 (e) 140

Stearic acid

Palmitic acid

Myristic acid

124

abcde

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

16

abcde

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

17

abcde

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

18

Fig 24 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing alpha-tocopherol (a) 017 (b) 034 (c) 052 (d) 069

(e) 086

Stearic acid

Myristic acid

Palmitic acid

125

Table 19 First-order rate constants (kobs) for the photodegradation of ascorbic acid in the

presence of other vitamins in cream formulations

kobs times 103 (min

ndash1)ab

Cream

formulationd

Other

vitaminc

a b C d e

10 RF 068

(0991)

073

(0996)

079

(0995)

085

(0992)

089

(0995)

11 RF 065

(0992)

070

(0992)

073

(0994)

080

(0995)

086

(0993)

12 RF 087

(0993)

096

(0995)

109

(0993)

116

(0994)

127

(0992)

13 NA 073

(0993)

081

(0992)

088

(0994)

096

(0994)

101

(0993)

14 NA 069

(0992)

074

(0992)

080

(0991)

086

(0995)

094

(0995)

15 NA 083

(0994)

090

(0993)

101

(0993)

109

(0994)

115

(0995)

16 TP 055

(0991)

051

(0994)

046

(0994)

042

(0993)

038

(0991)

17 TP 050

(0995)

045

(0993)

041

(0992)

038

(0995)

034

(0994)

18 TP 070

(0996)

066

(0996)

060

(0994)

055

(0993)

051

(0993)

a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental conditions

including the light intensity

c Vitamin concentrations (andashe) are as given in Table 16

d All the creams contain glycerin as humectant

The estimated error is plusmn5

126

00

05

10

15

00 10 20 30

Riboflavin concentration (M times 104)

kob

s times

10

3 (

min

ndash1)

10-12

00

05

10

15

00 20 40 60 80 100 120

Nicotinamide concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

13-15

00

02

04

06

08

00 04 08 12 16 20

Alpha-Tocopherol concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

16-18

Fig 25 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against individual vitamin concentration in cream formulations (10ndash18)

127

Table 20 First-order rate constants (k0)a for the photodegradation of ascorbic acid in the

absence of other vitamins and second-order rate constants (k) for the

photochemical interaction of ascorbic acid with RF NA and TP

Cream

formulation

Other

vitamin

k0 times 103

(minndash1

)

k

(Mndash1

minndash1

)

Correlation

coefficient

10 RF 062 102 0994

11 RF 059 097 0992

12 RF 077 189 0995

13 NA 066 032 times 10ndash2

0995

14 NA 062 027 times 10ndash2

0993

15 NA 074 037 times 10ndash2

0994

16 TP 059 110 times 10ndash2b

0996

17 TP 053 096 times 10ndash2b

0992

18 TP 075 123 times 10ndash2b

0994

a

The variations in the values of k0 are due to the degradation of AH2 in the presence of

different emulsifying agents in cream formulations

b Values for the inhibition of photodegradation of AH2

128

forms a complex with AH2 (Section 67) and may also act as a photosensitizer for AH2 by

energy transfer in the excited state on UV irradiation The absorption maximum of NA

(261 nm) (Moffat et al 2004) is very close to that of AH2 (265 nm) (Davies et al 1991)

and the possibility of energy transfer in the excited state (Moore 2004) is greater leading

to the photodegradation of AH2

The value of k0 is about 13 times greater than the values of kobs obtained for the

degradation of AH2 in the presence of the highest concentrations of TP in the creams

This indicates that TP has a stabilising effect on the photodegradation of AH2 in the

cream formulations This is in agreement with the view that the TP acts as a redox partner

with AH2 to retard its oxidation (Wille 2005) Thus among the three vitamins studied

only TP appears to have a stabilising effect on photodegradation of AH2 The

photochemical interaction of individual vitamins with AH2 is discussed below

66 INTERACTION OF RIBOFLAVIN WITH ASCORBIC ACID

The interaction of RF with the ascorbate ion (AHndash) may be represented by the

following reactions proposed by Silva and Quina (2006)

RF rarr 1RF (61)

1RF rarr

3RF (62)

3RF + AH

ndash rarr RF

ndashmiddot + AHmiddot (63)

AHmiddot + O2 rarr A + HO2middot (64)

HO2middot + AHndash rarr H2O2 + AHmiddot (65)

RF on the absorption of a quantum of light is promoted to the excited singlet state (1RF)

(61) 1RF may undergo intersystem crossing (isc) to form the excited triplet state (

3RF)

(62) The excited triplet state may react with the ascorbate ion to generate the ascorbyl

hv

isc

129

radical (AH) (63) (Kim et al 1993) The ascorbyl radical reacts with oxygen to give

dehydroascorbic acid (A) and peroxyl radical (HO2) (64) This radical may interact with

ascorbate ion to generate further ascorbyl radicals (65) These radicals may again take

part in the sequence of reactions to form A The role of RF in this reaction is to act as a

photosensitiser in the oxidation of ascorbic acid to A Ascorbic acid is reported to protect

riboflavin in milk under the influence of light by reacting with singlet oxygen (Hall et al

2009) (Section 511)

67 INTERACTION OF NICOTINAMIDE WITH ASCORBIC ACID

NA is known to form a 11 complex with ascorbic acid (Guttman and Brooke

1963 OrsquoNeil 2001 Doores 2002) The complexation of NA and AH2 may result from

the donor-acceptor interaction between AH2 (donor) and NA (acceptor) as observed in

the case of tryptophan and NA (Florence and Attwood 2006) In the presence of light the

interaction may cause reduction of NA (NAH) to form the ascorbyl radical (AH) ((66)-

(68)) which is oxidized to dehydroascorbic acid (A) (69) The NAH may be oxidized to

NA and H2O2 (610)

NA rarr 1NA (66)

1NA rarr

3NA (67)

3NA + AH2 rarr NAH + AHmiddot (68)

2 AH٠ rarr A + AH2 (69)

NAH + O2 rarr NA + H2O2 (610)

The proposed reactions suggest that on photochemical interaction AH2 undergoes

photosensitised oxidation in the presence of NA indicating that the photostability of

ascorbic acid is affected by NA

isc

130

68 INTERACTION OF ΑLPHA-TOCOPHEROL WITH ASCORBIC ACID

TP is an unstable compound and its oxidation by air results in the formation of an

epoxide which than produces a quinone that is inactive (Connors et al 1986) TP is

destroyed by sun light and artificial light containing the wavelengths in the UV region

(Ball 2006) It interacts with other antioxidants such as ascorbic acid (AH2) according to

the following reactions

TPndashO + AH2 rarr TP + AHmiddot (611)

2 AHmiddot rarr A + AH2 (612)

TP + AHmiddot rarr TPndashO + AH2 (613)

The tocopheroxyl radical (TPndashO) reacts with AH2 to regenerate TP and form the

ascorbyl radical (AHmiddot) (611) This radical undergoes further reactions as described in

equations (64) and (65) (Traber 2007) It may also disproportionate back to A and AH2

(612) TP reacts with AHmiddot to produce again the TPndashO radical and AH2 Thus in the

presence of light TP leads to the oxidation of AH2 which is ultimately regenerated in the

reaction (Davies et al 1991) Ascorbic acid has the ability to act as a co-antioxidant with

the TPndashO to regenerate TP (Packer et al 1979 2002) In this manner AH2 and TP act

synergistically to function in a redox cycle and AH2 is stabilized in the cream

formulations and microemulsions (Rozman and Gasperlin 2007 Rozman et al 2009)

69 EFFECT OF CARBON CHAIN LENGTH OF EMULSIFYING AGENT

The graphs of kobs for the photodegradation of AH2 in the presence of RF NA and

TP versus the carbon chain length of emulsifying agents are shown in Fig 26 It appears

that the photodegradation of AH2 in the presence of all the three vitamins in the creams

lies in the order

131

Fig 26 Plots of k for photodegradation of ascorbic acid in creams (10ndash18) against

carbon chain length of emulsifier () Stearic acid () palmitic acid

() myristic acid

00

05

10

15

20

25

k

(Mndash

1 m

inndash

1)

00

05

10

15

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash

1 m

inndash

1)

132

myristic acid gt stearic acid gt palmitic acid

The same order of emulsifying agents has been observed in the absence of the

added vitamins (Section 57) The polar character of these acids (Yao et al 2009) on the

basis of their carbon chain length may play a part in the photostability of AH2 The

greater stability of AH2 in creams in the presence of palmitic acid (Fig 26) may be due to

the interaction of AH2 with palmitic acid as discussed in Section 57 Ascorbic acid-6-

palmitate is known to be an antioxidant in cosmetic preparations (Lee et al 2009) and

food products (Doores 2002)

610 EFFECT OF VISCOSITY OF CREAMS

The plots of kobs for the degradation of AH2 in the presence of the highest

concentration of vitamins versus reciprocal of the viscosity of creams (Table 21) are

linear (Fig 27) and indicate that the increase in cream viscosity leads to a decrease in the

rate of degradation of AH2 The slopes of the plots indicate the effect of viscosity on the

interaction of AH2 with other vitamins in the order

riboflavin gt nicotinamide gt alpha-tocopherol

The relatively slow rate of degradation of AH2 in creams containing palmitic acid may be

due to the interaction of AH2 with the vitamins as well as palmitic acid (Lee et al 2009)

Thus viscosity is an important factor in the stability of AH2 in cream formulations and

may affect its rate of interaction with other vitamins It has been suggested that an

increase in the viscosity of the medium makes access to air at the surface more difficult to

prevent the oxidation of a drug (Wallwork and Grant 1977) This is in agreement with

the photolysis of AH2 in aqueous and organic solvents cream formulations (Chapter 5)

and aerobic oxidation of Ah2 in syrups (Blaug and Hajratwala 1972)

133

Table 21 Average viscosity of cream formulations containing different emulsifying

agents and glycerin as humectant (25 plusmn 1 ordmC) and the photodegradation rate

constants of AH2

Cream No Emulsifying

agent

Viscosityab

(mPa s)

kobs times 103c

10 (RF)

13 (NA)

16 (TP)

Stearic acid 9000 089

101

038

11 (RF)

14 (NA)

17 (TP)

Palmitic acid 8600 086

094

034

12 (RF)

15 (NA)

18 (TP)

Myristic acid 7200 127

115

051

a plusmn10

b Average viscosity of creams containing the individual vitamins (RF NA TP)

c The values have been obtained in the presence of highest concentration of the

vitamins

134

00

05

10

15

20

25

30

100 110 120 130 140

Viscosity (mPa s)ndash1

times 103

kob

s (m

inndash1)

Fig 27 Plots of kobs in the presence of highest concentration of vitamins versus

reciprocal of the viscosity of creams () riboflavin

( ) nicotinamide (- - -- - -) alpha-tocopherol

135

611 DEGRADATION OF ASCORBIC ACID IN THE PRESENCE OF OTHER

VITAMINS IN THE DARK

In order to observe the effect of riboflavin nicotinamide and alpha-tocopherol on

the degradation of AH2 in the creams stored in the dark the AH2 contents of the creams

were assayed at appropriate intervals (Table 22) The apparent first-order rate constants

determined from the kinetic plots (Fig 28) for the degradation of AH2 in the presence of

the highest concentrations of the individual vitamins in cream formulations (10ndash18) are

reported in Table 23 These rate constants indicate that the overall degradation of AH2 in

the presence of the highest concentration of the individual vitamins (RF NA and TP) is

about 70 times slower than that obtained on the exposure of creams to UV irradiation

This decrease in the rate of degradation of AH2 in the creams is the same as observed in

the case of AH2 alone In the absence of light the degradation of AH2 occurs due to

chemical oxidation (Section 132) and does not appear to be affected by the presence of

riboflavin and nicotinamide as indicated by the comparisons of the values of kobs in the

presence and absence of these vitamins (Table 15 and 23) In the presence of alpha-

tocopherol the degradation is slower than that in the presence of riboflavin and

nicotinamide This may be due to some interaction of AH2 and alpha-tocopherol causing

stabilisation of AH2 in the creams

As observed in the case of AH2 degradation alone in creams in the dark the AH2

degradation in the presence of the highest concentrations of other vitamins also occurs in

the same order of emulsifying agents (Fig 29)

myristic acid gt stearic acid gt palmitic acid

136

Table 22 Degradation of ascorbic acid in cream formulations in the dark in presence of

highest concentration of other vitamins

Concentration of ascorbic acid (mg 2g) Cream

No Time (days) 0 10 20 40 60 80

10e (RF) 375 285 233 171 110 69

11e (RF) 374 341 281 221 148 113

12e (RF) 372 259 203 130 89 59

13e (NA) 365 330 255 187 126 81

14e (NA) 370 321 289 219 159 109

15e (NA) 366 289 249 159 110 63

16e (TP) 377 359 321 261 211 159

17e (TP) 377 366 333 275 228 191

18e (TP) 373 361 304 252 200 167

137

02

07

12

17lo

g c

on

cen

tra

tio

n (

mg

)

10-12Riboflavin

02

07

12

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

13-15Nicotinamide

10

12

14

16

18

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

16-18Alpha-Tocopherol

Fig 28 First-order plots for the degradation of ascorbic acid in the dark in presence of

other vitamins using the emulsifying agents (minusminusminusminus) Stearic acid

(minus minusminus minus) palmitic acid (----) myristic acid

138

Table 23 First-order rate constants (kobs) for the degradation of ascorbic acid in presence

of other vitamins in cream formulations in the dark

Cream

formulation

Other

vitaminc

kobs times 102

(dayndash1

)ab

10e RF 204

(0995)

11e RF 156

(0992)

12e RF 222

(0992)

13e NA 189

(0995)

14e NA 151

(0993)

15e NA 214

(0995)

16e TP 100

(0994)

17e TP 088

(0995)

18e TP 105

(0993)

a The values in parenthesis are correlation coefficients and range from 0991ndash0996 due to

some variations in AH2 distribution in the creams

b The values of rate constants are relative and depend on specific experimental

conditions

c Vitamin concentrations (andashe) are as given in Table 16

The estimated error is plusmn5

139

Riboflavin

Nicotinamide

Alpha-Tocopherol

00

10

20

30

12 14 16 18Carbon chain length

ko

bs times

10

2 (

da

yndash1)

Fig 29 Plots of kobs for degradation of ascorbic acid in the dark in creams (10ndash18)

against carbon chain length of the emulsifier () Stearic acid () palmitic acid

() myristic acid

140

This indicates that the rate of degradation of AH2 is slowest in the creams containing

palmitic acid as the emulsifying agent The reason for AH2 degradation in the dark in this

order has already been explained in section 512

CHAPTER VII

STABILIZATION OF

ASCORBIC ACID WITH

CITRIC ACID TARTARIC

ACID AND BORIC ACID IN

CREAM FORMULATIONS

142

71 INTRODUCTION

Ascorbic acid is an ingredient of cosmetic preparations (Section 51) and is

sensitive to light (Rowe et al 2009 Sweetman 2009 British Pharmacopoeia 2009)

degrading to dehydroascorbic acid on UV irradiation by photooxidation (Kitagawa

1968) The photosensitivity of ascorbic acid makes it unstable in pharmaceutical and

cosmetic preparations (DeRitter 1982) The present work is an attempt to study the

photodegradation of ascorbic acid in cream formulations in the presence of certain

compounds (eg citric acid tartaric acid and boric acid) to investigate their role in the

stabilization of the vitamin on exposure to light and in the dark Citric acid and tartaric

acid are used as an antioxidant synergist (Rowe et al 2009) and boric acid is a

complexing agent for hydroxy compounds (Ahmad et al 2009cd)

72 CREAM FORMULATIONS

The details of the various cream formulations used in this study are given in Table

24 and the results obtained on the photodegradation of ascorbic acid in the presence of

stabilizing agents in these formulations are discussed in the following sections

143

Table 24 Composition of cream formulations containing ascorbic acid (2) and

stabilizers

Ingredients Cream

No SA PA MA CA GL PG EG AH2 CTa TA

b BA

c PH DW

19 a + minus minus + + minus minus + a minus minus + +

b + minus minus + + minus minus + b minus minus + +

c + minus minus + + minus minus + c minus minus + +

20 a minus + minus + + minus minus + a minus minus + +

b minus + minus + + minus minus + b minus minus + +

c minus + minus + + minus minus + c minus minus + +

21 a minus minus + + + minus minus + a minus minus + +

b minus minus + + + minus minus + b minus minus + +

c minus minus + + + minus minus + c minus minus + +

22 a + minus minus + + minus minus + minus a minus + +

b + minus minus + + minus minus + minus b minus + +

c + minus minus + + minus minus + minus c minus + +

23 a minus + minus + + minus minus + minus a minus + +

b minus + minus + + minus minus + minus b minus + +

c minus + minus + + minus minus + minus c minus + +

24 a minus minus + + + minus minus + minus a minus + +

b minus minus + + + minus minus + minus b minus + +

c minus minus + + + minus minus + minus c minus + +

25 a + minus minus + + minus minus + minus minus a + +

b + minus minus + + minus minus + minus minus b + +

c + minus minus + + minus minus + minus minus c + +

26 a minus + minus + + minus minus + minus minus a + +

b minus + minus + + minus minus + minus minus b + +

c minus + minus + + minus minus + minus minus c + +

27 a minus minus + + + minus minus + minus minus a + +

b minus minus + + + minus minus + minus minus b + +

c minus minus + + + minus minus + minus minus c + +

144

Table 24 continued

28 a + minus minus + minus + minus + minus minus a + +

b + minus minus + minus + minus + minus minus b + +

c + minus minus + minus + minus + minus minus c + +

29 a minus + minus + minus + minus + minus minus a + +

b minus + minus + minus + minus + minus minus b + +

c minus + minus + minus + minus + minus minus c + +

30 a minus minus + + minus + minus + minus minus a + +

b minus minus + + minus + minus + minus minus b + +

c minus minus + + minus + minus + minus minus c + +

31 a + minus minus + minus minus + + minus minus a + +

b + minus minus + minus minus + + minus minus b + +

c + minus minus + minus minus + + minus minus c + +

32 a minus + minus + minus minus + + minus minus a + +

b minus + minus + minus minus + + minus minus b + +

c minus + minus + minus minus + + minus minus c + +

33 a minus minus + + minus minus + + minus minus a + +

b minus minus + + minus minus + + minus minus b + +

c minus minus + + minus minus + + minus minus c + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PG = propylene glycol EG = ethylene glycol

PH = potassium hydroxide DW = distilled water CT = citric acid TA = tartaric acid

BA = boric acid

a CT (g ) a = 01 b = 02 c = 04

b TA (g ) a = 01 b = 02 c = 04

c BA (g ) a = 01 b = 02 c = 04

145

73 PRODUCTS OF ASCORBIC ACID PHOTODEGRADATION

The photodegradation of AH2 in cream formulations leads to the formation of

DHA as detected by TLC and reported earlier in the photolysis of AH2 in aqueous

solutions (Vaid et al 2006) and cream formulations (Sections 52 and 63) AH2 and

DHA in the methanolic extracts of the degraded creams were identified by comparison of

their Rf and color of the spots with those of the reference standards DHA is also

biologically active (Gardner 1972 Doores 2002) but its further degradation to 23-

diketo-gulonic acid (DGA) results in the loss of vitamin activity (Section 132)

However this product has not been detected in the present cream formulations

Therefore the creams may still possess their biological efficacy

74 SPECTRAL CHANGES IN PHOTODEGRADED CREAMS

In order to observe the spectral changes in photodegraded creams in the presence

of stabilizing agents the absorption spectra of the methanolic extracts of a degraded

cream were determined The spectra show a gradual loss of absorbance around 245 nm

due to the oxidation of AH2 to DHA on UV irradiation and similar to that shown for the

photodegradation of AH2 alone in Fig 5 DHA has negligible absorbance around 245 nm

(Davies et al 1991) and therefore it does not interfere with the absorbance of AH2 in

methanolic solutions The spectral changes and loss of absorbance around 245 nm in

methanolic solution depend on the extent of photooxidation of AH2 in a particular cream

75 ASSAY OF ASCORBIC ACID IN CREAMS

The UV spectrophotometric method (Zeng et al 2005) has previously been

applied to the determination of AH2 in cream formulations (Section 54) The absorbance

of the methanolic extracts of creams containing AH2 during photodegradation was used

146

to determine the concentration of AH2 The method was validated in the presence of citric

acid (CT) tartaric acid (TA) and boric acid (BA) before its application to the evaluation

of the kinetics of AH2 degradation in cream formulations The recovery of AH2 in creams

has been found to be in the range of 90ndash96 and is similar to that reported in Table 7

The reproducibility of the method lies within plusmn5 The assay data on the degradation of

AH2 in various creams in the presence of the stabilizing agents are reported in Table 25

76 KINETICS OF PHOTODEGRADATION

The effect of CT TA and BA as stabilizing agents on the photodegradation of

AH2 was studied by adding 01ndash04 of each compound to the cream formulations (19ndash

33) at pH 60 This concentration range is normally used for the stabilization of drugs in

pharmaceutical preparations (Im-Emsap et al 2002) The apparent first-order rate

constants (kobs) determined from the plots of log concentration versus time (Fig 30ndash34)

are reported in Table 26 The second-order rate constants (k) determined from the plots

of kobs versus concentration of the individual compounds (Fig 35ndash36) are given in Table

27 The values of k indicate the rate of inhibition of photodegradation of AH2 by each

compound

77 EFFECT OF STABILIZING AGENTS

In order to compare the effectiveness of CT TA and BA as stabilizing agents for

AH2 plots of k versus carbon chain length of the emulsifying agents were constructed

(Fig 37) The k values for the interaction of these compounds with AH2 are in the order

citric acid gt tartaric acid gt boric acid

The curves indicate that the highest interaction of these compounds with AH2 is in the

order

147

Table 25 Photodegradation of ascorbic acid in cream formulations in the presence of

stabilizers

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) a b c

0 374 378 379

60 362 362 372

120 349 355 367

210 333 335 349

19 (CT)

300 319 322 336

400 296 309 324

0 381 378 380

60 368 370 369

120 355 363 364

210 344 345 355

20 (CT)

300 328 335 341

400 312 319 331

21 (CT) 0 368 370 374

60 355 356 360

120 340 344 343

210 321 322 333

300 296 299 315

400 272 285 299

148

Table 25 continued

0 375 374 378

60 363 363 368

120 352 354 362

210 329 335 345

22 (TA)

300 307 314 333

400 292 299 313

0 370 377 374

60 364 365 368

120 352 357 357

210 332 344 349

23 (TA)

300 317 330 335

400 301 310 322

24 (TA) 0 376 379 377

60 367 369 368

120 351 348 352

210 325 330 344

300 306 317 326

400 284 294 310

149

Table 25 continued

0 370 375 380

60 356 362 359

120 331 339 344

210 311 318 330

25 (BA)

300 279 288 305

400 260 269 283

0 377 375 370

60 364 363 361

120 351 353 351

210 331 332 337

26 (BA)

300 323 324 325

400 301 307 313

27 (BA) 0 380 377 375

60 369 368 366

120 333 338 341

210 305 313 318

300 292 294 304

400 262 266 281

150

Table 25 continued

0 373 376 378

60 348 349 360

120 329 336 339

210 315 312 323

28 (BA)

300 282 283 299

400 249 264 280

0 370 373 380

60 358 355 367

120 343 346 356

210 325 329 347

29 (BA)

300 307 312 325

400 287 295 315

30 (BA) 0 369 375 372

60 353 358 362

120 321 330 335

210 283 294 303

300 265 281 293

400 242 254 270

151

Table 25 continued

0 374 376 379

60 348 366 352

120 324 340 337

210 303 319 322

31 (BA)

300 275 289 293

400 243 260 275

0 370 374 375

60 355 354 366

120 339 344 345

210 313 319 330

32 (BA)

300 288 297 308

400 261 271 290

33 (BA) 0 377 380 377

60 357 361 367

120 324 335 339

210 288 294 307

300 270 280 293

400 233 248 265

Creams 19ndash27 contain glycerin 28ndash30 contain propylene glycol and 31ndash33 contain

ethylene glycol as humectants

152

a

b

c

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

19

ab

c

14

15

16

log

co

nce

ntr

ati

on

(m

g)

20

a

b

c

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

21

Fig 30 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and citric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

153

a

b

c

14

15

16lo

g c

on

cen

tra

tio

n (

mg

)

22

a

b

c

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

23

a

b

c

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

24

Fig 31 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and tartaric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

154

ab

c

13

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

25

abc

13

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

26

ab

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

27

Fig 32 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and boric acid (a) 01 (b) 02 (c) 04

Palmitic acid

Stearic acid

Myristic acid

155

a

b

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

28

ab

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

29

a

b

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

30

Fig 33 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing propylene glycol and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

156

a

b

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

31

ab

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

32

a

b

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

33

Fig 34 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing ethylene glycol and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

157

Table 26 Apparent first-order rate constants (kobs) for the photodegradation of ascorbic

acid in presence of different stabilizers in cream formulations

kobs times 103 (min

ndash1)ab

Cream

formulation Stabilizer

c

a b c

19 CT 057

(0995)

050

(0992)

041

(0991)

20 CT 049

(0996)

043

(0995)

034

(0993)

21 CT 076

(0995)

067

(0995)

055

(0992)

22 TA 065

(0995)

058

(0995)

046

(0991)

23 TA 054

(0994)

047

(0993)

038

(0994)

24 TA 072

(0996)

063

(0992)

049

(0991)

25 BA 091

(0994)

086

(0995)

071

(0993)

26 BA 055

(0994)

050

(0993)

042

(0993)

27 BA 095

(0995)

089

(0992)

074

(0996)

28 BA 097

(0995)

088

(0992)

075

(0993)

29 BA 064

(0994)

057

(0991)

047

(0993)

30 BA 110

(0994)

100

(0996)

084

(0992)

31 BA 105

(0995)

094

(0994)

078

(0992)

32 BA 088

(0994)

079

(0993)

066

(0993)

33 BA 120

(0995)

108

(0993)

091

(0993) a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental conditions

including the light intensity

c Stabilizer concentrations (andashc) are as given in Table 24

The estimated error is plusmn5

158

00

04

08

12

00 04 08 12 16 20

Citric acid concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

19-21

00

04

08

12

00 05 10 15 20 25

Tartaric acid concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

22-24

Fig 35 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against citric acid (19ndash21) and tartaric acid concentrations (22ndash24) in cream

formulations

159

00

04

08

12k

ob

s times

10

3 (

min

ndash1)

25-27

00

04

08

12

00 20 40 60

ko

bs times

10

3 (

min

ndash1)

28-30

00

04

08

12

00 20 40 60

Boric acid concentration (M times 102)

kob

s times

10

3 (

min

ndash1)

30-33

Fig 36 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against boric acid concentrations in cream formulations (25ndash33)

Propylene glycol

Glycerin

Ethylene glycol

160

Table 27 First-order rate constants (k0)a for the photodegradation of ascorbic acid in the

absence of stabilizers and second-order rate constants (k) for the interaction of

ascorbic acid with CT TA and BA

Cream

formulation Stabilizers

k0 times 103

(minndash1

)

k times 102

(Mndash1

minndash1

)

Correlation

coefficient

19 CT 062 111 0991

20 CT 053 103 0994

21 CT 082 145 0995

22 TA 071 092 0995

23 TA 059 080 0993

24 TA 080 118 0996

25 BA 098 041 0994

26 BA 059 026 0994

27 BA 102 044 0995

28 BA 104 046 0992

29 BA 069 033 0995

30 BA 118 054 0994

31 BA 113 053 0995

32 BA 095 045 0995

33 BA 129 060 0993

a The variations in the values of k0 are due to the presence of different emulsifying agents

in cream formulations

161

00

04

08

12

16

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash1

min

ndash1)

18-33

a

b

e

cd

Fig 37 Plots of k for photodegradation of ascorbic acid in creams (19ndash33) against

carbon chain length of emulsifier () Stearic acid () palmitic acid ()

myristic acid (a) CT (b) TA (c) BA with glycerin (d) BA with propylene

glycol (e) BA with ethylene glycol

162

myristic acid gt stearic acid gt palmitic acid

In the case of myristic acid and stearic acid it may be explained on the basis of the

decreasing polarity (Yao et al 2009) It is interesting to observe the lowest rates of

interaction of these compounds in the creams containing palmitic acid This could be due

to the interaction of AH2 with palmitic acid to form a palmitate derivative in addition to

its interaction with the individual stabilizing agents CT and TA are known to act as

antioxidant synergists (Rowe et al 2009 Sweetman 2009) and in this capacity may

inhibit the photooxidation of AH2 as indicated by the values of the degradation rate

constants in the presence of these compounds The addition of CT to nutritional

supplements is known to inhibit the oxidation of AH2 (Doores 2002) Boric acid forms a

complex with AH2 (Rivlin 2007) and there by may inhibit its degradation Boric acid

may also interact with glycerin added to the creams as a humectant and form a complex

(Rowe et al 2009) This may influence its interaction and stabilizing effect on AH2 in

creams as indicated by the lower k values compared to those in the presence of CT and

TA It has further been observed that the k values for BA are greater in propylene glycol

and ethylene glycol compared to those in glycerin (Table 27) Again this may be due to

greater interaction of BA with glycerin compared to other humectants in the creams

78 DEGRADATION OF ASCORBIC ACID IN PRESENCE OF STABILIZING

AGENTS IN THE DARK

An important factor in the formulation of cosmetic preparations is to ensure the

chemical and photostability of the active ingredient by the use of appropriate stabilizing

agents The choice of these agents would largely depend on the nature and

physicochemical characteristics of the active ingredient AH2 possesses a redox system

163

and can be easily oxidized by air or light In order to observe the effect of CT TA and

BA on the stability of AH2 the cream formulations containing the individual compounds

were stored in the dark for a period of about three months and the rate of degradation of

AH2 was determined The assay data are reported in Table 28 and the kinetic plots are

shown in Fig 38ndash42 The values of apparent first-order rate constants for the degradation

of AH2 in the presence of the stabilizing agents are reported in Table 29 The second

order-rate constants for the interaction of CT TA and BA with AH2 are reported in Table

30 (Fig 43ndash44) The plots of k against the carbon chain length of the emulsifiers are

shown in Fig 45 The kinetic data indicate the same pattern of rates of degradation and

interaction of AH2 with these compounds as observed in the presence of light except that

the rates are much slower in the dark Thus the stabilizing agents are equally effective in

inhibiting the rate of degradation of AH2 in the dark The effect of emulsifying agents and

the humectants on the rate of degradation of AH2 in the presence of the stabilizers has

been discussed in the above Section 77

79 EFFECT OF ADDITIVES ON TRANSMISSION OF ASCORBIC ACID

In order to observe the effect of additives (citric tartaric and boric acids) on the

transmission characteristics of ascorbic acid (0002 mg100 ml) in methanol containing

the highest concentration of the additives (004) used in this study the transmission

spectra were measured It has been found that these additives produce a hypsochromic

shift in the absorption maximum of ascorbic acid This may result in the reduction of the

fraction of light absorbed by ascorbic acid to the extent of about 10 and thus influence

the rate of photodegradation reactions However since all the additives produce similar

effects the rate constants can be considered on a comparative basis

164

Table 28 Degradation of ascorbic acid in cream formulations in the presence of

stabilizers in the dark

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(days) a b c

0 374 378 379

10 355 346 362

20 326 328 342

40 293 297 322

19 (CT)

60 264 269 295

80 241 245 262

0 381 378 380

10 361 364 372

20 339 350 348

40 309 312 330

20 (CT)

60 279 286 301

80 260 266 282

21 (CT) 0 368 370 374

10 342 346 364

20 310 321 348

40 278 282 313

60 249 251 278

80 217 228 249

165

Table 28 continued

0 375 374 378

10 339 344 351

20 317 326 336

40 282 288 306

22 (TA)

60 251 258 280

80 222 235 252

0 370 377 374

10 340 354 355

20 332 336 343

40 297 303 310

23 (TA)

60 266 282 294

80 238 248 267

24 (TA) 0 376 379 377

10 341 339 350

20 306 319 323

40 263 284 279

60 223 241 249

80 196 202 223

166

Table 28 continued

0 370 375 380

10 331 341 334

20 287 289 301

40 225 247 245

25 (BA)

60 189 185 214

80 141 154 170

0 377 375 370

10 355 357 349

20 326 314 324

40 264 267 286

26 (BA)

60 232 238 254

80 189 199 211

27 (BA) 0 380 377 375

10 346 339 337

20 309 288 301

40 233 241 260

60 192 196 211

80 140 147 163

167

Table 28 continued

0 373 376 378

10 314 322 333

20 267 281 305

40 217 233 253

28 (BA)

60 167 177 204

80 122 135 151

0 370 373 380

10 336 329 343

20 283 277 306

40 233 243 267

29 (BA)

60 189 190 217

80 144 154 173

30 (BA) 0 369 375 372

10 308 319 329

20 255 275 310

40 210 226 244

60 158 163 191

80 113 131 147

168

Table 28 continued

0 374 376 379

10 303 311 329

20 266 260 289

40 211 219 239

31 (BA)

60 155 158 178

80 112 121 149

0 370 374 375

10 314 323 339

20 276 280 305

40 222 233 258

32 (BA)

60 172 187 193

80 126 136 162

33 (BA) 0 377 380 377

10 308 306 320

20 254 265 280

40 205 214 237

60 144 155 175

80 107 118 138

169

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

19

abc

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

20

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

21

Fig 38 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing citric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

170

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

22

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

23

ab

c

12

13

14

15

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

24

Fig 39 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing tartaric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

171

a

b

c

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

25

abc

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

26

ab

c

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

27

Fig 40 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing glycerin and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

172

a

b

c

10

12

14

16

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

28

ab

c

10

12

14

16

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

29

a

b

c

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

30

Fig 41 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing propylene glycol and boric acid (a) 01 (b) 02 (c)

04

Palmitic acid

Stearic acid

Myristic acid

173

ab

c

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

31

ab

c

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

32

a

b

c

08

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

33

Fig 42 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing ethylene glycol and boric acid (a) 01 (b) 02 (c)

04

Myristic acid

Palmitic acid

Stearic acid

174

Table 29 Apparent first-order rate constants (kobs) for the degradation of ascorbic acid in

presence of different stabilizers in cream formulations in the dark

kobs times 102 (day

ndash1)ab

Cream

formulation Stabilizer

c

a b c

19 CT 055

(0994)

052

(0992)

044

(0991)

20 CT 048

(0995)

046

(0995)

038

(0992)

21 CT 064

(0994)

061

(0995)

052

(0994)

22 TA 063

(0994)

058

(0995)

049

(0996)

23 TA 054

(0995)

050

(0995)

041

(0994)

24 TA 081

(0995)

075

(0993)

066

(0995)

25 BA 118

(0996)

113

(0994)

097

(0994)

26 BA 087

(0995)

079

(0993)

068

(0994)

27 BA 124

(0995)

114

(0994)

101

(0993)

28 BA 134

(0995)

124

(0996)

110

(0992)

29 BA 116

(0996)

108

(0992)

096

(0995)

30 BA 142

(0993)

131

(0995)

115

(0995)

31 BA 145

(0995)

137

(0992)

117

(0995)

32 BA 130

(0996)

120

(0993)

107

(0994)

33 BA 153

(0995)

141

(0994)

122

(0994) a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental

conditions

c Stabilizer concentrations (andashc) are as given in Table 24

The estimated error is plusmn5

175

176

Table 30 First-order rate constants (k0)a for the degradation of ascorbic acid in the

absence of stabilizers and second-order rate constants (k) for the chemical

interaction of ascorbic acid with CT TA and BA in the dark

Cream

formulation Stabilizers

k0 times 102

(dayndash1

)

k times 102

(Mndash1

dayndash1

)

Correlation

coefficient

19 CT 060 797 0996

20 CT 052 723 0995

21 CT 069 850 0994

22 TA 068 710 0996

23 TA 058 636 0994

24 TA 086 758 0994

25 BA 126 444 0993

26 BA 092 375 0992

27 BA 131 480 0991

28 BA 141 488 0993

29 BA 122 418 0994

30 BA 149 531 0991

31 BA 155 578 0996

32 BA 137 472 0994

33 BA 163 627 0996

a The variations in the values of k0 are due to the presence of different emulsifying agents

in cream formulations

177

00

04

08

12

00 04 08 12 16 20

Citric acid concentration (M times 102)

kob

s times

10

2 (

da

yndash1)

19-21

00

04

08

12

00 05 10 15 20 25

Tartaric acid concentration (M times 102)

kob

s times

10

2 (

da

yndash1)

22-24

Fig 35 Plots of first-order rate constants (kobs) for the degradation of ascorbic acid in the

dark against citric acid (19ndash21) and tartaric acid (22ndash24) concentrations in

cream formulations

178

00

10

20

00 20 40 60

kob

s times

10

3 (

min

ndash1)

25-27

00

10

20

00 20 40 60

kob

s times

10

3 (

min

ndash1)

28-30

00

10

20

00 20 40 60

Boric acid concentration (M times 102)

kob

s times

10

3 (

min

ndash1)

30-33

Fig 44 Plots of first-order rate constants (kobs) for the degradation of ascorbic acid in the

dark against boric acid concentrations in cream formulations (25ndash33)

Glycerin

Propylene glycol

Ethylene glycol

179

00

04

08

12

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash

1 d

ayndash

1)

18-33

b

a

e

dc

Fig 45 Plots of k for degradation of ascorbic acid in the dark in creams (19ndash33) against

carbon chain length of emulsifier () Stearic acid () palmitic acid ()

myristic acid (a) CT (b) TA (c) BA with glycerin (d) BA with propylene

glycol (e) BA with ethylene glycol

CONCLUSIONS

AND

SUGGESTIONS

180

CONCLUSIONS

The main conclusions of the present study on the photodegradation of the ascorbic

acid in organic solvents and cream formulations are as follows

1 Identification of Photodegradation Products

The photodegradation of ascorbic acid in aqueous organic solvents and

laboratory prepared oil-in-water cream preparations on UV irradiation leads to the

formation of dehydroascorbic acid No further degradation products of dehydroascorbic

acid have been detected under the present experimental conditions The product was

identified by comparison of its Rf value and color of the spot with those of the authentic

compound by thin-layer chromatography and spectral changes

2 Assay of Ascorbic Acid

Ascorbic acid in aqueous organic solvents and cream preparations was assayed

in acidified methanolic solutions (pH 20) at 245 nm using a UV spectrophotometric

method Ascorbic acid in combination with other vitamins (riboflavin nicotinamide and

alpha-tocopherol) was assayed by the official iodimetric method due to interference by

these vitamins at the analytical wavelength Both analytical methods were validated

under the experimental conditions employed before their application to the assay of

ascorbic acid The recoveries of ascorbic acid in cream preparations are in the range of

90ndash96 and the reproducibility of both methods are within plusmn5 The F test and the t test

show that there is no significant difference between the precision of the two methods and

therefore these methods can be applied to the assay of ascorbic acid in cream

preparations with comparable results

181

3 Kinetics of Photodegradation

a) Photodegradation of ascorbic acid in organic solvents

Ascorbic acid degradation follows apparent first-order kinetics in aqueous

organic solvents A plot of the first-order rate constants (log kobs) versus solvent dielectric

constant is linear with positive slope indicating an increase in the rate with dielectric

constant On the contrary a plot of kobs verses reciprocal of solvent viscosity is linear with

a positive slope showing a decrease in the rate with solvent viscosity Thus the rate of

photodegradation of ascorbic acid (an oxidizable drug) depends on the solvent

characteristics

b) Photodegradation of ascorbic acid in cream preparations

Ascorbic acid has been found to follow apparent first-order kinetics in cream

preparations and the rate of degradation is affected by the following factors

i Effect of concentration

An apparent linear relationship has been observed between log kobs and

concentration (05ndash25) of ascorbic acid in a cream preparation Thus the rate of

degradation of ascorbic acid appears to be faster at a lower concentration

compared to that of a higher concentration on exposure to the same intensity of

light

ii Effect of carbon chain length of the emulsifying agent

The plots of kobs verses carbon chain length of the emulsifying agent show that the

photodegradation of ascorbic acid is affected in the order myristic acid gt stearic

acid gt palmitic acid This is predominantly due to the interaction of ascorbic acid

with palmitic acid and the carbon chain length (measure of relative polar

182

character) of the emulsifying acid probably does not play a part in the

photodegradation kinetics of ascorbic acid in creams This is evident from the

non-linear relationship between the rate constants for ascorbic acid degradation

and the carbon chain length of the emulsifying acids

iii Effect of viscosity

The values of kobs for the photodegradation of ascorbic acid in cream preparations

are in the order of humectant ethylene glycol gt propylene glycol gt glycerin

showing that the rates of degradation are influenced by the viscosity of the

humectant and decrease with an increase in the viscosity as observed in the case

of organic solvents

iv Effect of pH

The log kndashpH profiles for the photodegradation of ascorbic acid in creams

represent sigmoid type curves indicating an increase in the rate of oxidation of the

molecule with ionization (pH 42ndash70 557ndash999) The AHndash species appears to

be more susceptible to oxidation than the non-ionized molecule in the pH range

studied

v Effect of redox potential

The values of kobs show that the rate of photooxidation of ascorbic acid is

influenced by its redox potential which varies with pH The greater photostability

of ascorbic acid at pH 5ndash6 compared to that at pH 7 and above is due to its lower

rate of oxidation-reduction in the lower range The increase in the rate of

photooxidation with pH is due to a corresponding increase in the redox potential

of ascorbic acid

183

c) Photodegradation of ascorbic acid in the presence of other vitamins (riboflavin

nicotinamide alpha-tocopherol) in cream preparations

The photodegradation of ascorbic acid is affected by the presence of other

vitamins in creams The kinetic data on the photochemical interactions indicate that

riboflavin and nicotinamide act as photosensitizers in the degradation of ascorbic acid

and have an adverse effect on the photostability of the vitamin in creams Whereas

alpha-tocopherol exerts an inhibitory effect on the degradation of ascorbic acid by acting

as a redox partner in the creams Thus a combination of ascorbic acid and alpha-

tocopherol has a synergistic effect on the stabilization of ascorbic acid in creams These

vitamins do not appear to influence the rate of degradation of ascorbic acid in the dark

d) Photodegradation of ascorbic acid in the presence of citric acid tartaric acid and

boric acid in cream preparations

The rate of photodegradation of ascorbic acid in creams has been found to be

inhibited by the addition of compounds such as citric acid tartaric acid and boric acid in

creams These compounds show a stabilizing effect on the photodegradation of ascorbic

acid in the order citric acid gt tartaric acid gt boric acid The lower effect of boric acid

may be due to its interaction with the emulsifying agents and humectants Boric acid

exerts this effect by complex formation with ascorbic acid Citric acid and tartaric acid

are antioxidant synergists and in combination with ascorbic acid may exert a stabilizing

effect on its degradation

184

Salient Features of the Work

In the present work an attempt has been made to study the effects of solvent

characteristics formulation factors particularly the emulsifying agents in terms of the

carbon chain length and humectants in terms of viscosity medium pH drug

concentration redox potential and interactions with other vitamins and stabilizers on the

kinetics of photodegradation of ascorbic acid in cream preparations The study may

provide useful information to improve the photostability and efficacy of ascorbic acid in

cream preparations

SUGGESTIONS

The present work may provide guidelines for a systematic study of the stability of

drug substances in cream ointment preparations and the evaluation of the influence of

formulation variables such as emulsifying agents and humectants concentration pH

polarity viscosity redox potential on the rate of degradation and stabilization of drug

substances This may enable the formulator in the judicious design of formulations that

have improved stability and efficacy for therapeutic use The kinetic parameters may

throw light on the comparative stability of the preparations and help in the choice of

appropriate formulation ingredients

REFERENCES

186

Abe Y Horii H Taniguchi S (1986) A theoretical study of the oxidation mechanism

of triose reductone in reference to L-ascorbic acid Can J Chem 64 360-365

Ahmad I (1968) A Study of the Degradation of Riboflavin and Related Compounds

PhD Thesis University of London London

Ahmad I Rapson HDC (1990) Multicomponent spectrophotometric assay of

riboflavin and photoproducts J Pharm Biomed Anal 8 217-223

Ahmad I Vaid FHM (2006) Photochemistry of flavins in aqueous and organic

solvents In Silva E Edwards AM Eds Flavins Photochemistry and

Photobiology Royal Society of Chemistry Cambridge Chap 2

Ahmad I Tollin G (1981) Solvent effect on flavin electron transfer reactions

Biochemistry 20 5925-5928

Ahmad I Jamil N Zoha SMS (1973) Statistical evaluation of antioxidants used in

the manufacture of ascorbic acid tablets J Sci Univ Kar 2 269-287

Ahmad I Fasihullah Q Vaid FHM (2004b) A study of simultaneous photolysis and

photoaddition reactions of riboflavin in aqueous solution J Photochem Photobiol B

Biol 75 13-20

Ahmad I Fasihullah Q Vaid FHM (2005) Effect of phosphate buffer on

photodegradation reactions of riboflavin in aqueous solution J Photochem

Photobiol B Biol 78 229-234

Ahmad I Fasihullah Q Vaid FHM (2006a) Effect of light intensity and wavelength

on photodegradation reactions of riboflavin in aqueous solution J Photochem

Photobiol B Biol 82 21-27

187

Ahmad I Fasihullah Q Vaid F H M (2006b) Photolysis of formylmethylflavin in

aqueous and organic solvents Photochem Photobiol Sci 5 680-685

Ahmad I Ahmed S Sheraz MA Vaid FHM (2008) Effect of borate buffer on the

photolysis of riboflavin in aqueous solution J Photochem Photobiol B Biol 93 82-

87

Ahmad I Ahmed S Sheraz MA Vaid FHM (2009c) Borate toxicity effect on

drug stability and analytical applications In Chung MP Ed Handbook on

Borates Chemistry Production and Applications Nova Science Publishers Inc

New York in press

Ahmad I Sheraz MA Ahmed S Vaid FHM (2009d) Analytical applications of

borates Mater Sci Res J in press

Ahmad I Fasihullah Q Noor A Ansari IA Ali QNM (2004a) Photolysis of

riboflavin in aqueous solution a kinetic study Int J Pharm 280 199-208

Ahmad I Ahmed S Sheraz MA Aminuddin M Vaid FHM (2009a) Effect of

caffeine complexation on the photolysis of riboflavin in aqueous solution a kinetic

study Chem Pharm Bull in press

Ahmad I Ahmed S Sheraz MA Vaid FHM Ansari IA (2009b) Effect of

divalent ions on photodegradation kinetics and pathways of riboflavin in aqueous

solution Photochem Photobiol Sci accepted

Ahmed S (2009) Effect of Complexing Agents on the Photodegradation of Riboflavin

in Aqueous Solution PhD Thesis Baqai Medical University Karachi

Ahmad T (1978) Studies on the Stability of Sulphacetamide and Degradation Products

PhD Thesis University of Karachi Karachi

188

Ahmad T Ahmad I (1983) The effect of antioxidants on the photolysis of aqueous

sulfacetamide and sulfanilamide solutions Pol J Pharmacol Pharm 35 69-75

Albini A Fasani E Eds (1998) Drugs Photochemistry and Photostability The Royal

Society of Chemistry Cambridge

Allwood MC (1984a) Factors influencing the stability of ascorbic acid in total

parenteral nutrition infusions J Clin Hosp Pharm 9 75-85

Allwood MC (1984b) Compatibility and stability of TPN mixtures in big bags J Clin

Pharm Ther 9 181-198

Allwood MC Kearney MCJ (1998) Compatibility and stability of additives in

parenteral nutrition admixtures Nutrition 14 697-706

Al-Meshal IA Hassan MMA (1982) Ascorbic Acid In Florey K Ed Analytical

Profiles of Drug Substances Vol 11 Academic Press New York pp 45-78

Anderson NH Johnston D McLelland MA Munden P (1991) Photostability

testing of drug substances and drug products in UK pharmaceutical laboratories J

Pharm Biomed Anal 9 443-449

Ansari IA (2002) Photolysis and Interactions of Cyanocobalamin with some B-

Vitamins and Ascorbic Acid in Parenteral Solutions PhD Thesis University of

Karachi Karachi

Ansari IA Vaid Vaid HM Ahmad I (2004) Spectral Study of Photolysis of

Aqueous Cyanocobalamin Solutions in Presence of Vitamins B and C Pak J Pharm

Sci 17 83-93

Arcus CL Zilva SS (1940) The photochemical decomposition of L-ascorbic acid II

Biochem J 34 61-66

189

Arger CB Rupp D Lo P (1996) Preservation of dispersed systems In Liberman

HA Rieger MM Banker GS Eds Pharmaceutical Dosage Forms Disperse

Systems 2nd ed vol1 Marcel Dekker New York Chap9

Asard H May J Smirnoff N Eds (2004) Vitamin C Function and Biochemistry in

Animals and Plants BIOS Scientific Publishers New York

Asker AF Canady D Cobb C (1985) Influence of DL-methionine on the

photostability of ascorbic acid solutions Drug Dev Ind Pharm 11 2109-2125

Ault RG Baird DK Carrington HC Haworth WN Herbert R Hirst EL

Percival EGV Smith F Stacey M (1933) Synthesis of d- and of l-ascorbic acid

and of analogous substances J Chem Soc 1419-1423

Austria R Semenzato A Bettero A (1997) Stability of vitamin C derivatives in

solution and topical formulations J Pharm Biomed Anal 15 795-801

Baertschi SW Alsante KM (2005) Stress testing The chemistry of drug degradation

In Baertschi SW Ed Pharmaceutical Stress testing Predicting Drug Degradation

Taylor amp Francis London Chap 3

Bailey CW Bright JR Jasper JJ (1945) A study of the binary system nicotinamide-

ascorbic acid J Am Chem Soc 67 1184-1186

Ball GFM (2006) Vitamins in Foods Analysis Bioavailability and Stability CRC

Press Boca Raton FL Chap 15

Banhegyi G Marcolongo P Puskas F Fulceri R Mandl J Benedetti A (1998)

Dehydroascorbate and ascorbate transport in rat liver microsomal vesicles J Biol

Chem 273 2758-2762

190

Bardolph EM Taylor RH (1997) Sailors scurvy science and authority J R Soc

Med 90 238

Barel AO Paye M Maibach HI Eds (2001) Handbook of Cosmetic Science and

Technology Marcel Dekker New York

Barry BW (1983) Dermatological Formulations Percutaneous Absorption Marcel

Dekker New York

Barry BW (2002) Transdermal drug delivery In Aulton ME Ed Pharmaceutics

The Science of Dosage Form Design Churchill Livingstone London Chap 33

Basu TK Temple NJ Garg ML (1999) Antioxidants in Human Health and

Disease CABI Publishing Oxford

Becher P (1965) Emulsions Theory and Practice Reinhold New York p 1

Beissenhirtz M Scheller F Lisdat F (2003) Immobilized cytochrome c sensor in

organicaqueous media for the characterization of hydrophilic and hydrophobic

antioxidants Electroanalysis 15 1425-1435

Berlett BS Stadtman ER (1997) Protein oxidation in aging disease and oxidative

stress J Biol Chem 272 20313-20316

Betageri G Prabhu S (2002) Semisolid preparations In Swarbrick J Boylan JC

Eds Encyclopedia of Pharmaceutical Technology 2nd ed Marcel Dekker New

York pp 2436-2457

Bielski BHJ (1982) Chemistry of ascorbic acid radicals In Seib PA Tolbert BM

Eds Ascorbic Acid Chemistry Metabolism and Uses Advances in Chemistry

Series American Chemical Society Washington DC pp 81-100

191

Bielski BHJ Allen AO Schwartz HA (1981) Mechanism of disproportionation of

ascorbate radicals J Am Chem Soc 103 3516-3518

Billany M (2002) Suspensions and emulsions In Aulton ME Ed Pharmaceutics

The Science of Dosage Form Design 2nd ed Churchill Livingstone London Chap

23

Bisby RH Morgan CG Hamblett I Gorman AA (1999) Quenching of singlet

oxygen by trolox C ascorbate and amino acids effect of pH and temperature J Phys

Chem 103 7454-7459

Bissett DL (2006) Anti-aging skin care formulations In Draelos ZD Thaman LA

Eds Cosmetic Formulation of Skin Care Products Taylor amp Francis Group New

York Chap 11

Blaugh SM Hajratwala B (1972) Kinetics of aerobic oxidation of ascorbic acid J

Pharm Sci 61 556-562

Block LH (1996) Pharmaceutical emulsions and microemulsions In Lieberman HA

Rieger MM Banker GS Eds Pharmaceutical Dosage Forms Disperse Systems

Vol 2 2nd ed Chap 2

Bolliger HR Konig A (1969) Water-soluble vitamins In Stahl E Ed Thin-layer

Chromatography Springer-Verlag Berlin pp 304-306

Bonwstra JA De Graaff A Gooris GS Nijsse J Wiechers JW Van Aelst AC

(2003) Water distribution and related morphology in human stratum corneum at

different hydration levels J Invest Dermatol 120 750-758

Bors SR (2005) Scurvy Thomas Dunne Books St Martinrsquos Griffen New York

192

Bors W Buettner GR (1997) The vitamin C radical and its reactions In Packer L

Fuchs J Eds Vitamin C in Health and Disease Marcel Dekker New York Chap

4

Braun L Puskas F Csala M Meszaros G Mandl J Banhegyi G (1997)

Ascorbate as a substrate for glycolysis or gluconeogenesis evidence for an interorgan

ascorbate cycle Free Radic Biol Med 23 804-808

British Pharmacopoeia (2009) Her Majestyrsquos Stationary Office London Electronic

version

Buettner G (1993) The pecking order of free radicals and antioxidants lipid

peroxidation α-tocopherol and ascorbate Arch Biochem Biphys 300 535-543

Buettner GR Schafer FQ (2004) Ascorbate as an antioxidant In Asard H May

JM Smirnoff N Eds Vitamin C Function and Biochemistry in Animals and

Plants BIOS Scientific Publishers Oxford Chap 10

Burke KE (2006) Topical nutritional antioxidants In Draelos ZD Thaman LA

Eds Cosmetic Formulation of Skin Care Products Taylor amp Francis Group New

York Chap 21

Burns JJ Ed (1961) Vitamin C Ann NY Acad Sci 92 1

Byrom P Turnbull JH (1967) Excited states of flavin coenzymes II Anaerobic

oxidation of amino acids by excited riboflavin derivatives Photochem Photobiol 6

125-131

Cadenas E Packer L (2002) Handbook of Antioxidants 2nd ed Marcel Dekker New

York Chap 7-9

193

Capellmann M Bolt HM (1992) Simultaneous determination of ascorbic acid and

dehydroascorbic acid by HPLC with postcolumn derivatisation and fluorometric

detection Fresenius J Anal Chem 342 462-466

Cairns WL Metzler DE (1971) Photochemical degradation of flavins VI A new

photoproduct and its use in studying photolytic mechanism J Am Chem Soc 93

2772-2777

Carlotti ME Ugazio E Sapino S Peira E Gallarate M (2006) Photodegradation

of retinol and anti-aging effectiveness of two commercial emulsions J Cosmet Sci

57 261-277

Carpenter K (1986) The History of Scurvy and Vitamin C Cambridge University Press

Cambridge

Carr AC Zhu BZ Frei B (2000) Potential antiatherogenic mechanisms of ascorbate

(vitamin C) and alpha-tocopherol (vitamin E) Circ Res 87 349-354

Carstensen JT (1972) Theory of Pharmaceutical Systems Academic Press New York

pp 211-214

Chaudhri SK Jain NK (2009) History of cosmetics Asian J Pharm 3 164-167

Chessex P Lovoic JC Roulean T Brochu P St Louis P Levy E Al Varez F

(2002) Photooxidation of parenteral multivitamins induced hepatic steatosis in a

neonatal guinea pig model of intravenous infusion Pediatr Res 52 958-963

Connors KA Amidon GL Stella VJ (1986) Chemical Stability of Pharmaceuticals

A Handbook for Pharmacists 2nd ed Wiley New York pp 105-106 142 208-220

194

Dabrowski K Huiterleitner S (1989) Application of a simultaneous assay of ascorbic

acid dehydroascorbic acid and ascorbic sulfate in biological matter Analyst

(London) 114 83-87

Dale JK Booth RE (1976) In Hoover JE Ed Dispensing of Medication 8th ed

Mack Easton Pennsylvania pp 479-481

Darr D Dunston S Faust H Pinnell S (1996) Effectiveness of antioxidants (vitamin

C and E) with and without sunscreens as topical photoprotectants Acta Derm

Venereol 76 264-268

Davey MW Montagu MV Inze D Sanmartin M Kanellis A Smirnoff N

Benzie IJJ Strain JJ Favell D Fletcher J (2000) Plant L-ascorbic acid

chemistry function metabolism bioavailability and effect of processing J Sci Food

Agr 80 825-860

Davies MB Austin J Partridge DA (1991) Vitamin C Its Chemistry and

Biochemistry The Royal Society of Chemistry Cambridge

De La Rochette A Birlouez-Aragon I Silva E Morliere P (2003) Advanced

glycation endproducts as UVA photosensitizers of tryptophan and ascorbic acid

consequences for the lens Biochem Biophys Acta 1621 235-241

De La Rochette A Silva E Birlouez-Aragon I Mancini M Edwards AM

Morliere P (2000) Riboflavin photodegradation and photosensitization effects are

highly dependent on oxygen and ascorbate concentration Photochem Photobiol 72

815-820

195

De Vries H Beijersbergen Van Henegouwen GMJ Huff FA (1984) Photochemical

decomposition of chloramphenicol in a 025 eye drop and in a therapeutic

intraocular concentration Int J Pharm 20 265-271

Dean RT Fu S Stocker R Davies MJ (1997) Biochemistry and pathology of

radicalmediated protein oxidation Biochem J 324 1-18

DeRitter E (1982) Vitamins in pharmaceutical formulations J Pharm Sci 71 1073-

1096

Deutsch JC (1998a) Ascorbic acid oxidation by hydrogen peroxide Anal Biochem

255 1-7

Deutsch JC (1998b)Spontaneous hydrolysis and dehydration of dehydroascorbic acid

in aqueous solution Anal Biochem 260 223-229

Deutsch JC (1998c)oxygen-accepting antioxidants which arise during ascorbate

oxidation Anal Biochem 265 238-245

Dodsun KY Young ER Soliman AGM (1992) Determination of total vitamin C

in various food matrices by liquid chromatography and fluorescence detection J

AOAC Int 75 887-891

Dollery C Ed (1999) Therapeutic Drugs 2nd ed Vol 1 Churchill Livingstone

London pp A213-A216

Doores S (2002) pH control agents and acidulants In Branen LA Davidson PM

Salminen S Thorngate JH Eds Food Additives Marcel Dekker New York p

637

Eberlein-Koumlnig B Ring J (2005) Relevance of vitamins C and E in cutaneous

photoprotection J Cosmet Dermatol 4 4-9

196

Ehrlich M Rao J Pabby A Goldman MP (2006) Improvement in the appearance

of wrinkles with topical transforming growth factor beta(1) and l-ascorbic acid

Dermatol Surg 32 618-625

EIRI Board (2007) Profitable Small Scale Manufacture of Cosmetics Engineers India

Research Institute Nai Sarak Delhi

Eitenmiller RR Ye L Landen WOJr (2008) Vitamin Analysis for the Health and

Food Sciences 2nd ed CRC Press Boca Raton FL Chap 5

Elia S (2009) Nutrition In Kumar P Clark M Eds Kumar and Clarkrsquos Clinical

Medicine 7th ed Elsevier Limited Chap 5

Elmore AR (2005) Final report of the safety assessment of L-ascorbic acid calcium

ascorbate magnesium ascorbate magnesium ascorbyl phosphate sodium ascorbate

and sodium ascorbyl phosphate as used in cosmetics Int J Toxicol 24 51-111

Erb C Nau-Staudt K Flammer J Nau W (2004) Ascorbic acid as a free radical

scavenger in porcine and bovine aqueous humour Ophthal Res 36 38-42

Espinal-Perez LE Moncada B Castanedo-Cazares JP (2004) A double-blind

randomized trial of 5 ascorbic acid vs 4 hydroquinone in melasma Int J

Dermatol 43 604-607

Fall HH Petering HG (1956) Metabolic inhibitors 1 67-Dimethyl-9-

formylmethylisoalloxazine67-dimethyl-9-(2-hydroxyethyl)-isoalloxazine and

derivatives J Am Chem Soc 78 377-381

Farahmand S Tajerzadeh H Farboud ES (2006) Formulation and evaluation of a

vitamin C multiple emulsion Pharm Dev Technol 11 255-261

197

Fasani E Albini A (2005) Photostability Stress Testing Predicting Drug Degradation

Taylor amp Francis London Chap 10

Fasihullah Q (1988) Photolysis of Flavins in Aqueous and Organic Solvents PhD

Thesis University of Karachi Karachi

Fasman GD Ed (1976) CRC Handbook of Biochemistry and Molecular Biology 3rd

ed Physical Chemical Data Vol 1 CRC Press Cleveland Ohio pp 122-130

Ferreira SLC Bandeira MLSF Lemos VA Dos Santos HC Spinola Costa

AC De Jesus DS (1997) Sensitive spectrophotometric determination of ascorbic

acid in fruit juices and pharmaceutical formulations using 2-(5-bromo-2-pyridylazo)-

5-diethylaminophenol (Br-PADAP) Anal Bioanal Chem 357 1174-1178

Fessenden RW Verma NC (1978) A time-resolved electron spin resonance study of

the oxidation of ascorbic acid by hydroxyl radical Biophys J 24 413-430

Finholt P Paulssen RB Higuchi T (1963) Rate of anaerobic degradation of ascorbic

acid in aqueous solution J Pharm Sci 52 948-954

Florence AT Attwood D (2006) Drug Stability Physicochemical Principles of

Pharmacy 4th ed Pharmaceutical Press London p 411

Flynn GL (2002) Cutaneous and transdermal delivery-processes and systems of

delivery In Banker GS and Rhodes CT Eds Modern Pharmaceutics Marcel

Dekker New York Chap 8

Foote CS (1991) Definition of Type I and Type II Photosensitized oxidation

Photochem Photobiol 54 659

Fornaro A Coicher N (1998) L-ascorbic acid complexation and redox reactions with

some transition metal ions Quni Nova 21 642-650

198

Fraga CG Motchink PA Wyrobek AJ Rempel DM Ames BN (1996)

Smoking and low antioxidant levels increase oxidative damage to sperm DNA

Mutat Res 351 199-203

Fraga CG Motchnik PA Shigenaga MK Helbock HJ Jacob RA Ames BN

(1991) Ascorbic acid protects against endogenous oxidative DNA damage in human

sperm Proc Natl Acad Sci USA 88 11003-11006

Frenich AG Torres ME Vega AB Vidal JL Bolanos PP (2005) Determination

of ascorbic acid and carotenoids in food commodities by liquid chromatography with

mass spectrometry detection J Agric Food Chem 53 7371-7376

Fung HL (1990) Drug stability In Banker GS Rhodes CT Eds Modern

Pharmaceutics 2nd ed Marcel Dekker New York Chap 6

Gallarate M Carlotti ME Trotta M Bovo S (1999) On the stability of ascorbic

acid in emulsified systems for topical and cosmetic use Int J Pharm 188 233-241

Ganshirt H Malzacher A (1960) Separation of several vitamins of the B group and C

by chromatography Naturwiss 47 279-280

Gardner WH (1972) Acidulants in food processing In Furia TE Ed Handbook of

Food Additives 2nd ed Vol 1 CRC Press Cleveland OH pp 225ndash270

Garrett ER (1967) Kinetics and mechanisms in stability of drugs In Bean HS

Beckett AH Carless JE Eds Advances in Pharmaceutical Sciences Vol 2

Academic Press London Chap 1

Garti N Aserin A (1996) Pharmaceutical emulsions double emulsions and

microemulsions In Benita S Ed Microencapsulation Methods and Industrial

Applications Marcel Dekker New York pp 411-534

199

Gensler M Rossmann A Schmidt HL (1995) Detection of added L-ascorbic acid in

fruit juice by isotope ratio mass spectrometry J Agric Food Chem 43 2662-2666

Gibbons E Allwood MC Neal T Hardy G (2001) Degradation of dehydroascorbic

acid in parenteral nutrition mixtures J Pharm Biomed Anal 25 605-611

Guitton C Gros P Comtat M (2007) Direct electrochemical measurements in dermo-

cosmetic creams In Alegret Merkoci Eds Comprehensive Analytical Chemistry

Elsevier Amsterdam 49 e59-e68

Guttman DE Brooke D (1963) Solution phase interaction of nicotinamide with

ascorbic acid J Pharm Sci 52 941-945

Gyorgy P Pearson WN Eds (1967) Vitamin Methods Vol VII Academic Press

New York pp 27-49

Hagg M Ylikoski S Kumpulainen J (1994) Vitamin C and α- and β-carotene in

vegetables consumed in Finland during 1988-1989 and 1992-1993 J Food Compos

Anal 7 252-259

Hagg M Ylikoski S Kumpulainen A (1995) Vitamin C content in fruits and berries

consumed in Finland J Food Compos Anal 8 12-20

Hall NK Chapman TM Kim HJ Min DB (2009) Antioxidant mechanisms of

Trolox and ascorbic acid on the oxidation of riboflavin in milk under light Food

Chem in press doi 101016jfoodchem200905017

Halliwell B (1996) Vitamin C antioxidant or pro-oxidant in vivo Free Radic Res 25

439-454

200

Halliwell B Whiteman M (1997) Antioxidant and prooxidant properties of vitamin C

In Packer L Fuchs J Eds Vitamin C in Health and Disease Marcel Dekker New

York pp 25-94

Halver JE Felton SP (2001) Analytical enigmas in assaying for vitamers C In

Dabrowski K Ed Ascorbic Acid in Aquatic Organisms CRC Press Boca Raton

Chap 3

Hanck A Ed (1982) Vitamin C New Clinical Applications in Immunology Lipid

Metabolism and Cancer Hans Huber Bern

Hashmi MH (1973) Assay of Vitamins in Pharmaceutical Preparations John Wiley

New York Chap 13

Hatchard CG Parker CA (1956) A new sensitive chemical actinometer II Potassium

ferrioxalate as a standard chemical actinometer Proc R Soc London Ser A A235

518-536

Heber GK Markovic B Hayes A (2006) An immunohistological study of anhydrous

topical ascorbic acid compositions on ex vivo human skin J Cosmet Dermatol 5

150-156

Heelis PF Parsons BJ Phillips GO McKellar JF (1981) The flavin sensitized

photooxidation of ascorbic acid a continuous and flash photolysis study Photochem

Photobiol 37 7-13

Henry JA Ed (1997) The New Guide to Medicines and Drugs The British Medical

Association 4th ed Dorling Kindersley London p 434

Hickey S Roberts H (2004) Ascorbate-The Science of Vitamin C Electronic Version

(ISBN 1-4116-0724-4)

201

Higdon JV Frei B (2002) The Antioxidant Vitamin C and E AOAC Press Arlington

VA pp 1-16

Hirst EL Zilva SS (1933) Ascorbic acid as the anti-scorbutic factor Biochem J 27

1271-1278

Ho AHL Puri A Sugden JK (1994) Effect of sweetening agents on the light

stability of aqueous solutions of L-ascorbic acid Int J Pharm 107 199-203

Holmstrom B Oster G (1961) Riboflavin as an electron donor in photochemical

reactions J Am Chem Soc 83 1867-1871

Homann P Gaffron H (1964) Photochemistry and metal catalyzes studies in a flavin

sensitized oxidation of ascorbic acid Photochem Photobiol 3 499-515

Hughes DE (1985) Irreversible reaction kinetics of the aerobic oxidation of ascorbic

acid Anal Chem 57 555-558

Humbert PG Haftek M Creidi P Lapiegravere C Nusgens B Richard A Schmitt D

Rougier A Zahouani H (2003) Topical ascorbic acid on photoaged skin Clinical

topographical and ultrastructural evaluation double-blind study vs placebo Exp

Dermatol 12 237-244

ICH Harmonized Tripartite Guideline (1997) QIB Photostability Testing of New Drug

Substances and Products Federal Register 62 27115-27122

Ichikawa M Ide N Shiraishi S Ono K (2005) Effect of various halide salts on the

incompatibility of cyanocobalamin and ascorbic acid in aqueous solution Chem

Pharm Bull 53 688-690

202

Ihara H Shino Y Aoki Y Hashizume N Minegishi N (2000) A simple and rapid

method for the routine assay of total ascorbic acid in serum and plasma using

ascorbate oxidase and o-phenylenediamine J Nutr Sci Vitaminol 46 321-324

Im-Emsap W Paeratakul O Siepmann J (2002) Disperse Systems In Banker GS

Rhodes CT Ed Modern Pharmaceutics Maecel Dekker New York Chap 9

Irache JM Ezpeleta I Vega FA (1993) HPLC determination of antioxidant

synergists and ascorbic acid in some fatty pharmaceuticals cosmetics and food

Chromatographia 35 232-23

Iwase H Ono I (1994) Determination of ascorbic acid in human plasma by high-

performance liquid chromatography with electrochemical detection using a

hydroxyapatite cartridge for precolumn deproteinization J Chromatogr B Biomed

Appl 655 195-200

Jain S Tiwari AK Jain NK (2006) Topical products In Jain NK Ed

Pharmaceutical Product Development CBS Publishers New Delhi Chap 7

Jamil N Ahmad I (1984) Moisture content of ascorbic acid tablets stored at various

temperatures and humidities Pak J Sci Ind Res 27 252

Jamil N Ahmad I Beg AE (1980a) The effect of UV and tungsten light on physical

characteristics of ascorbic acid tablets Laboratory prepared samples IV Kar Univ

J Sci 8 77-87

Jamil N Ahmad I Beg AE (1980b) The effect of UV and tungsten light on physical

characteristics of ascorbic acid tablets Commercial samples III Kar Univ J Sci 8

117-126

203

Jentzsch A Aikens P (2005) RetiSTARTM

for cosmetic formulations-stabilized retinol

In Rosen MR Ed Delivery System Handbook for Personal Care and Cosmetic

Products Technology Applications and Formulations William Andrew Publishing

Norwich NY pp 862-866

Johnston CS Steinberg FM Rucker RB (2007) Ascorbic Acid In Zempleni J

Rucker RB McCormick DB Suttie JW (Eds) Handbook of Vitamins 4th ed

CRC Press Boca Raton FL Chap 15

Jung MY Kim SM Kim SY (1995) Riboflavin-sensitized photooxidation of

ascorbic acid kinetics and amino acid effects Food Chem 53 397-403

Kall MA Anderson CJ (1999) Improved method for simultaneous determination of

ascorbic acid and dehydroascorbic acid isoascorbic acid and dehydroisoascorbic acid

in food and biological samples J Chromatogr B Biomed Sci Appl 730 101-111

Kampfenkel K Van Montagu M Inze D (1995) Extraction and determination of

ascorbate and dehydroascorbate from plant tissue Anal Biochem 225 165-167

Kaplan DL Moloney SJ Troy WR Dickens MS Pinnell SR (1989) A new

stabilized ascorbic acid solution percutaneous absorption and effect on relative

collagen synthesis J Cutan Aging Cosmet Derm I 115-121

Karatepe M (2004) Simultaneous determination of ascorbic acid and free

malondialdehyde in human serum by HPLC-UV LC GC (North America) 22 362-

365

Karlsen J (1996) Light-activated drugs and drug formulations in drug targeting In

Tonneson HH Ed The Photostability of Drugs and Formulations Taylor amp

Francis London Chap 8

204

Kassem MA Kassem AA Ammar HO (1969a) Studies on the stability of

injectable L-ascorbic acid solutions I Effect of pH solvent light and container

Pharm Acta Helv 44 611-623

Kassem MA Kassem AA Ammar HO (1969b) Studies on the stability of

injectable L-ascorbic acid solutions II Effect of metal ions and oxygen content of

solvent water Pharm Acta Helv 44 667-675

Kassem MA Kassem AA Ammar HO (1972) Studies on the stability of injectable

L-ascorbic acid solutions 3 Effect of metal-complexing agents Pharm Acta Helv

47 89-97

Kaushansky K Kipps TJ (2006) Hematopoietic agents growth factors minerals and

vitamins In Brunton L Ed Goodman amp Gilmans The Pharmacologic Basis of

Therapeutics 11th ed digital ed McGraw-Hill Companies USA Chap 53

Kearney MCJ Allwood MC Martin H Neal T Hardy G (1998) The influence

of amino acid source on the stability of ascorbic acid in TPN mixtures Nutrition 14

173-178

Kim H Kirshenbaum LJ Rosenithol I Riesz P (1993) Photosensitized formation

of ascorbate radicals by riboflavin an ESR study Photochem Photobiol 57 777-

784

Kimoto K Terada S Yamaguchi T (1997) Analysis of ascorbic acid

dehydroascorbic acid and transformation products by ion-pairing high-performance

liquid chromatography with multi-wavelength ultraviolet and electrochemical

detection Methods Enzymol 279 3-12

205

King CG Burns JJ Ed (1975) Second Conference on Vitamin C Ann N Y Acad

Sci 258

Kitagawa Y (1968) The effects of ultraviolet rays on vitamin C Eiyo To Shokuryo 20

416-421 through CA (1968) 68 111560f

Klick S Muijselaar PG Waterval J EichingerT Korn C Gerding TK Debets

AJ de Griend CS van den Beld C Somsen GW De Jong GJ (2005)Toward

a generic approach for stress testing of drug substances and drug products Pharm

Tech 29 48-66

Konings EJ (2006) Water-soluble vitamins J AOAC Int 89 285-288

Kwon BM Foote CS (1988) Chemistry of singlet oxygen 50 Hydroperoxide

intermediates in the photooxygenation of ascorbic acid J Am Chem Soc 110

6582-6583

Kwon BM Foote CS Khan SI (1989) Photooxygenation of ascorbic acid

derivatives and model compounds J Am Chem Soc 111 1854-1860

Laborie S Lavoie JC Chessex P (1998) Paradoxical role of ascorbic acid and

riboflavin in solutions of total parenteral nutrition implication in photoinduced

peroxide generation Pediatr Res 43 601-601

Laborie S Lavoie JC Pineault M Chessex P (1999) Protecting solutions of

parenteral nutrition from peroxidation JPEN 23 104-108

Laborie S Lavoie JC Pineault M Chessex P (2000) Contribution of multivitamin

air and light in the generation of peroxides in adult and neonatal parenteral nutrition

solutions Ann Pharmacother 34440-445

206

Laborie S Lavoie JC Rouleau T Chessex P (2002) Multivitamin solutions for

enteral supplementation a source of peroxides Nutrition 18 470-473s

Lachman L DeLuca P Akers MJ (1986) Kinetic principles and stability testing In

Lachman L Liberman HA Kanig JL Eds The Theory and Practice of

Industrial Pharmacy 3rd ed Lea amp Febiger Philadelphia Chap 26

Laidler KJ (1987) Chemical Kinetics 3rd ed Harper and Row New York pp 183-

185

Lambert WE de Leenheer A (1992) In Nollet LML Ed Food Analysis by HPLC

Marcel Dekker New York Chap 10

Lavoie JC Chessex P Rouleau T Migneault D Comte B (2004) Light-induced

byproducts of vitamin C in multivitamin solutions Clin Chem 50 135-140

Lee JS Kim JW Han SH Chang IS Kang HH Lee OS Oh SG Suh KD

(2004) The stabilization of L-ascorbic acid in aqueous solution and water-in-oil-in-

water double emulsion by controlling pH and electrolyte concentration J Cosmet

Sci 55 1-12

Lee KM Kwon JY Lee KW Lee HJ (2009) Ascorbic acid 6-palmitate

suppresses gap-junctional intercellular communication through phosphorylation of

connexin 43 via activation of the MEK-ERK pathway Mutat Res 660 51-56

Leacutevecircque N Mac-Mary S Muret P Makki S Aubin F Kantelip JP Heusegravele C

SS Humbert P (2005) Evaluation of a sunscreen photoprotective effect by ascorbic

acid assessment in human dermis using microdialysis and gas chromatography mass

spectrometry Exp Dermatol 14 176-181

207

Levine M (1986) New concepts in the biology and biochemistry of ascorbic acid N

Engl J Med 314 892-902

Lewin S (1976) Vitamin C Its Molecular Biology and Medical Potential Academic

Press New York

Liau LS Lee BL New AL Ong CN (1993) Determination of plasma ascorbic

acid by high-performance liquid chromatography with ultraviolet and electrochemical

detection J Chromatogr 612 63-70

Lin JY Selim MA Shea CR Grichnik JM Omar MM Monteiro-Riviere NA

Pinnell SR (2003) UV photoprotection by combination topical antioxidants vitamin

C and vitamin E J Am Acad Dermatol 48 866-874

Lin FH Lin JY Gupta RD Tournas JA Burch JA Selim MA Monteiro-

Riviere NA Grichnik JM Zielinski J Pinnell SR (2005) Ferulic acid

stabilizes a solution of vitamins C and E and doubles its photoprotection of skin J

Invest Dermatol 125 826-832

Lindahl T (1993) Instability and decay of the primary structure of DNA Nature 362

709-715

Litner CJ (1973) Drug Product Stability In Cooper MS Ed Quality Control in

Pharmaceutical Industry Vol 2 Academic Press New York p 185

Liu TZ Chin N Kiser MD Bigler WN (1982) Specific spectrophotometry of

ascorbic acid in serum or plasma by use of ascorbate oxidase Clin Chem 28 2225-

2228

Lu GW Flynn GL (2009) Cutaneous and transdermal delivery-processes and

systems of delivery In Florence AT Siepmann J Eds Modern Pharmaceutics-

208

Applications and Advances Vol 2 5th ed Informa Healthcare Inc New York

Chap 3

Lykkesfeldt J (2000) Determination of ascorbic acid and dehydroascorbic acid in

biological samples by high-performance liquid chromatography using subtraction

methods reliable reduction with tris [2-carboxyethyl] phosphine hydrochloride Anal

Biochem 282 89-93

Macek TJ (1960) Stability problems with some vitamins in pharmaceuticals Am J

Pharm 132 433-455

Mader WJ (1961) Vitamins In Higuchi T Brochmann-Hanssen E Eds

Pharmaceutical Analysis Interscience New York pp 689-693

Maggi N Pasqualucci CR Ballotta R Sensi P (1966) Rifampicin a new orally

active rifamycin Chemotherapy 11 285-292

Maia AM Baby AR Pinto CA Yasaka WJ Suenaga E Kaneko TM Velasco

MV (2006) Influence of sodium metabisulfite and glutathione on the stability of

vitamin C in OW emulsion and extemporaneous aqueous gel Int J Pharm 322

130-135

Mancini M Edwards AM Becker MI De Ioannes A Silva E (2000) Reactivity

of monoclonal antibodies against a tryptophan-riboflavin adduct toward irradiated

and non-irradiated bovine-eye-lens protein fractions an indicator of in vivo visible

light mediated phototransformations J Photochem Photobiol B Biol 55 9-15

Manoharan M Schwille PO (1994) Measurement of ascorbic acid in human plasma

and urine by high performance liquid chromatography Results in healthy subjects

209

and patients with idiopathic calcium urolithiasis J Chromatogr B Biomed Appl

654 134-139

Margolis SA Schmidt HL (1996) Measurement of ascorbic acid in human plasma

and serum stability intralaboratory repeatability and interlaboratory reproducibility

Clin Chem 42 1257-1262

Mason P (2007) Dietary Supplements 3rd ed Pharmaceutical Press pp 330-339

Massad WA Bertolotti S Garcia N (2004) Kinetics and mechanism of the vitamin

B2-sensitized photooxidation of isoproterenol Photochem Photobiol 79 428-433

Matsubayashi T Sakaeda T Kita T Kurimoto Y Nakamura T Nishiguchi K

Fujita T Kamiyama F Yamamoto A Okumura K (2003) Intradermal

concentration of hydroquinone after application of hydroquinone ointments is higher

than its cytotoxic concentration Biol Pharm Bull 26 1365-1367

McBride MM Moore WM (1967) The photochemistry of riboflavin II

Polarographic studies on the hydroxyethyl and formylmethyl analogues of riboflavin

Photochem Photobiol 6 103-113

McDowell LR (2000) Vitamins in Animal and Human Nutrition 2nd ed Iowa State

University Press Chap 15

Meister A (1994) Glutathione-ascorbic acid antioxidant system in animals J Biol

Chem 269 9397-9400

Meister A (1995) Mitochondrial changes associated with glutathione deficiency

Biochim Biophys Acta 1271 35-42

Min DB Boff JM (2002) Chemistry and reaction of singlet oxygen in foods

CRFSFS 1 58-72

210

Moeslinger T Brunner M Volf I Spieckermann PG (1995) Spectrophotometric

determination of ascorbic acid and dehydroascorbic acid Clin Chem 41 1177-1181

Moffat AC Osselton MD Widdop B (2004) Clarkersquos Analysis of Drugs and

Poisons 3rd ed Pharmaceutical Press London pp 649-650

Moore DE (2004) Standardization of kinetic studies of photodegradation reactions In

Tonnesen HH Ed Photostability of Drugs and Drug Formulations 2nd ed CRC

Press Boca Raton FL Chap 3

Moura T Gaudy D Jacob M Cassanas G (1994) pH influence on the stability of

ascorbic acid spray-drying solutions Pharm Acta Helv 69 77-80

Murray JC Burch JA Streilein RD Iannacchione MA Hall RP Pinnell SR

(2008) A topical antioxidant solution containing vitamins C and E stabilized by

ferulic acid provides protection for human skin against damage caused by ultraviolet

irradiation J Am Acad Dermatol 59 418-425

Mushran SP Agarwal MC (1977) Mechanistic studies on the oxidation of ascorbic

acid 5 J Sci Res (India) 36 274-283

Nerlo H Palak W Tomaszek J (1968) Stabilization of vitamin preparations II

Syrup of vitamin D3 and its stabilization Acta Polon Pharm 25 183-188

Niki E Noguchi N (1997) Protection of human low-density lipoprotein from oxidative

modification by vitamin C In Packer L Fuchs J Eds Vitamin C in Health and

Disease Marcel Dekker New York pp 183-192

Nishikimi M Fukuyama R Minoshima S Shimizu N Yagi K (1994) Cloning and

chromosomal mapping of the human nonfunctional gene for L-gulono-gamma-

211

lactone oxidase the enzyme for L-ascorbic acid biosynthesis missing in man J Biol

Chem 269 13685-13688

Njus D Wigle M Kelly PM Kipp BH Schlegel HB (2001) Mechanism of

ascorbic acid oxidation by Cytochrome B561 Biochemistry 40 11905-11911

Nusgens BV Humbert P Rougier A Colige AC Haftek M Lambert CA

Richard A Creidi P Lapiegravere CM (2001) Topically applied vitamin C enhances

the mRNA level of collagens I and III their processing enzymes and tissue inhibitor

of matrix metalloproteinase 1 in the human dermis J Invest Dermatol 116 853-859

Ogata Y Kosugi Y (1969) Solvent effect on the autooxidation of L-ascorbic acid

Tetrahedron 25 1055-1062

OrsquoKeefe T (2001) Ascorbic acid and stable ascorbate esters as sources of vitamin C in

aquaculture feeds ASA Technical Bulletin AQ48 wwwsoyaquaorg

OrsquoNeil MJ Ed (2001) The Merck Index 13th ed Merck amp Co Inc Rahway NJ

USA Electronic version

Ortega GR Delgado JN (1998) Vitamins and related compounds In Wilson and

Gisvoldrsquos Textbook of Organic Medicinal and Pharmaceutical Chemistry 10th ed

Lippincott-Raven Philadelphia Chap 27

Ortwerth BJ Monnier VM (1997) Protein glycation by the oxidation products of

ascorbic acid In Packer L Fuchs J Eds Vitamin C in Health and Disease Marcel

Dekker New York pp 123ndash142

Ozer O Muguet V Roy E Grossiord JL Seiller M (2000) Stability study of

WOW viscosified multiple emulsions Drug Dev Ind Pharm 26 1185-1189

Packer I Fuchs J (1999) Vitamin C in Health and Disease Marcel Dekker New York

212

Packer JE Slater TF Wilson RL (1979) Direct observation of a free radical

interaction between vitamin E and vitamin C Nature 278 737-738

Packer L Traber MG Kraemer K Frei B (2002) The Antioxidant Vitamins C and

E AOCS Press Illinois

Padayatty SJ Katz A Wang Y Eck P Kwon O Lee JH Chen S Corpe C

Dutta A Dutta SK Levine M (2003) Vitamin C as an antioxidant Evaluation of

its role in disease prevention J Am Coll Nutr 22 18-35

Parker CA (1953) A new sensitive chemical actinometer I Some trials with potassium

ferrioxalate Proc Roy Soc (Lond) A220 104-116

Parker JN Parker PM (2003) Antioxidants ICON Group International

Pastore P Rizzetto T Curcuruto O Cin MD Zaramella A Martin D (2001)

Characterization of dehydroascorbic acid solutions by liquid chromatography mass

spectrometry Rapid Commun Mass Spectrom 15 2051-2057

Pelletier O (1985) In Augustin J Klein BP Becker D Venugopal PB Eds

Methods of Vitamins Assay Wiley New York pp 303-347

Peyrat-Maillard MN Bonnely S Rondini L Berset C (2001) Effect of vitamin E

and vitamin C on the antioxidant activity of malt rootlets extracts Lebensmittel-

Wissenschaft Und-Technological 34 176-182

Pinnell SR (2003) Cutaneous photodamage oxidative stress and topical antioxidant

protection J Am Acad Dermatol 48 1-19

Pinnell SR Madey DL (1998) Topical vitamin C in skin care Aesthet Surg J 18

468-470

213

Pinnell SR Yang H Omar M Monteiro-Riviere N DeBuys HV Walker LC

Wang Y Levine M (2001) Topical L-ascorbic acid percutaneous absorption

studies Dermatol Surg 27 137-142

Placzek M Gaube S Kerkmann U Gilbertz KP Herzinger T Haen E Przybilla

B (2005) Ultraviolet B-induced DNA damage in human epidermis is modified by

the antioxidants ascorbic acid and D-alpha-tocopherol J Invest Dermatol 124 304-

307

Puskas F Gergely PJr Banki K Perl A (2000) Stimulation of the pentose

phosphate pathway and glutathione levels by dehydroascorbate the oxidized form of

vitamin C FASEB J 14 1352-1361

Puskas F Gergely P Niland B Banki K Perl A (2002) Differential regulation of

hydrogen peroxide and Fas-dependent apoptosis pathways by dehydroascorbate the

oxidized form of vitamin C Antioxid Redox Signal 4 357-369

Racz I (1989) Drug Formulation Wiley New York pp 121-122

Ramu A Mehta MM (2000) The riboflavin-mediated photooxidation of doxorubicin

Cancer Chemoth Pharm 46 449-458

Raschke T Koop U Duumlsing HJ Filbry A Sauermann K Jaspers S Wenck H

Wittern KP (2004) Topical activity of ascorbic acid from in vitro optimization to in

vivo efficacy Skin Pharmacol Physiol 17 200-206

Reed RA Harmon P Manas D Wasylaschuk W Galli C Biddell R Bergquist

PA Hunke W Templeton AC Ip D (2003) The role of excipients and package

components in the photostability of liquid formulations PDA J Pharm Sci Tech 57

351-368

214

Reichstein T Griissner A Oppcnuuer R (1933) Synthese der d- und l-Ascorbinsaumlure

(C-Vitamin) Helv Chim Acta 16 1019-1033

Riehl J Maupin C Layloff T (1995) On the choice of a photolysis source for the

photostability testing of pharmaceuticals Pharmacop Forum 21 1654-1663

Rivlin RS (2007) Riboflavin (vitamin B2) In Zempleni J Rucker RB McCormick

DB Suttie JW Eds Handbook of Vitamins 4th ed Taylor amp Francis CRC

Press Boca Raton FL p 241

Rogers AR Yacomeni JA (1971) The effect of pH on the aerobic degradation of

ascorbic acid solutions J Pharm Pharmacol 23 218S

Rosen MR Ed (2005) Delivery System Handbook for Personal Care and Cosmetic

Products-Technology Applications and Formulations William Andrew Publishing

Norwich NY

Rosenberg HR (1945) Chemistry and Physiology of the Vitamins Interscience New

York pp 289-338

Roth HJ Eger K Troschutz R (1991) Pharmaceutical Chemistry Vol 2 Drug

Analysis Ellis Horwood London pp 282-288

Rowe RC Sheskey PJ Quinn ME (2009) Handbook of Pharmaceutical Excipients

6th ed Pharmaceutical Press London pp 43-47 181-183 731-732

Roacutezanowska M Bober A Burke JM Sarna T (1997) The role of retinal pigment

epithelium melanin in photoinduced oxidation of ascorbate Photochem Photobiol

65 472-479

Rozman B Gasperlin M (2007) Stability of vitamins C and E in topical

microemulsions for combined antioxidant therapy Drug Deliv 14 235-245

215

Rozman B Gasperlin M Tinois-Tessoneaud E Pirot F Falson F (2009)

Simultaneous absorption of vitamins C and E from topical microemulsions using

reconstructed human epidermis as a skin model Eur J Pharm Biopharm 72 69-75

Ruumlmelin A Fauth U Halmagyi M (1999) Determination of ascorbic acid in plasma

and urine by high performance liquid chromatography with ultraviolet detection Clin

Chem Lab Med 37 533-536

Rumsey SC Levine M (2000) Vitamin C In Song WO Beecher GR

Eitenmiller RR Eds Modern Analytical Methodologies in Fat- and Water-Soluble

Vitamins Wiley New York Chap 13

Saari JC Baker EM Sauberlich HE (1967) Thin-layer chromatographic separation

of the oxidative degradation products of ascorbic acid Anal Biochem 18 173-177

Salkić M Keran H Jašić M (2007) Direct spectrophotometric determination of l-

ascorbic acid in the presence of potassium cyanide Agric Conspec Sci 72 371-375

Salvador A Chisvert A (2007) Analysis of Cosmetic Products Elsevier Amsterdam

Sattar A DeMan JM Alexander JC (1977) Light-induced degradation of vitamins

II Kinetic studies on ascorbic acid decomposition in solution Can Inst Food Sci

Technol J 10 65-68

Satoh K Sakagami H Terasaka H Ida Y Fujisawa S (2000) Interaction between

hydroquinone and ascorbic acid derivatives quenching effects of organic solvents

Antican Res 20 1577-1581

Sauberlich HE (1994a) Pharmacology of vitamin C Annu Rev Nutr 14 371-391

Sauberlich HE (1994b) Vitamin C and Cancer In Kritchevsky D Carroll K Eds

Nutrition and Disease Update Cancer AOCS Press Champaign IL pp 111-172

216

Sauermann K Jaspers S Koop U Wenck H (2004) Topically applied vitamin C

increases the density of dermal papillae in aged human skin BMC Dermatol 4 13

Sawicka J (1991) The influence of excipients and technological process on

cholecalciferol stability and its liberation from tablets Pharmazie 46 519-521

Scheuplein RJ Blank IH (1973) Mechanism of percutaneous absorption IV

Penetration of non-electrolytes (alcohols) from aqueous solutions and from pure

liquids J Invest Dermatol 60 286-296

Schick B (1943) Scurvy and pine needles Science 98 241-242

Schuman Jorns M Schollnhammer G Hemmerich P (1975) Intramolecular addition

of the riboflavin side chain Anion-catalysed neutral photochemistry Eur J

Biochem 57 35-48

Seib PA Tolbert BM (1982) Ascorbic Acid Chemistry Metabolism and Uses

Advances in Chemistry Series 200 American Chemical Society Washington DC

Sequeira F Vozone C (2000) Photostability studies of drug substances and products

Practical implications of the ICH guideline Pharm Tech 24 30-35

Shacter E (2000) Quantification and significance of protein oxidation in biological

samples Drug Metab Rev 32 307-326

Shah VP (1997) FDA Guidance for Industry Document Nonsterile Semisolid Dosage

Forms Centre for Drug Evaluation and Research Rockville MD pp 1-40

Sheraz MA Ahmed S Ahmad S (2007) Developments in the clinical and food

analysis of vitamin C J Baqai Med Univ 10 19-24

217

Shindo Y Witt E Han D Packer L (1994) Dose-response effect of acute ultraviolet

irradiation on antioxidants and molecular markers of oxidation in murine epidermis

and dermis J Invest Dermatol 102 470-475

Sibi M Batcu A Gheorghiu I (1953) Vitamin oxidation-reduction effect II Effect of

light on the oxidation-reduction reactions of lactoflavin Studii cercetari stiinf

(Romania) 4 291-305 through CA (1956) 50 14816g

Sidhu DS Sugden JK (1992) Effect of food dyes in the photostability of aqueous

solutions of L-ascorbic acid Int J Pharm 83 263-266

Silva E Quina FH (2006) Photoinduced processes in the eye lens do flavins really

play a role In Silva E Edwards AM Eds Flavins Photochemisry and

Photobiology The Royal Society of Chemistry Cambridge Chap 7

Silva E Ugarte P Andrade A Edwards AM (1994) Riboflavin-sensitized photo-

processes of tryptophan J Photochem Photobiol B Biol 23 43-48

Sim AK (1972) Ascorbic acid-A scurvy past and present Chem Indust 8 160-165

Singh S (2006) Stability testing during product development In Jain NK ED

Pharmaceutical Product Development CBS Publishers New Delhi Chap 9

Singh S Bakshi M (2000) Guidance on conduct of stress testing to determine inherent

stability of drugs Pharm Tech 24 1-14

Sinko PJ (2006) Chemical Kinetics and Stability Martinrsquos Physical Pharmacy and

Pharmaceutical Sciences 5th ed Lippincott Williams amp Wilkins Baltimore Chap

8 15 18

218

Smith JL Canham JE Kirkland WD Wells PA (1988) Effect of intralipid amino

acids container temperature and duration of storage on vitamin stability in total

parenteral nutrition admixtures J Parenter Enteral Nutr 12 478-483

Speek AJ Schrijver J Schreurs WHP (1984) Fluorimetric determination of total

vitamin C and total isovitamin C in food stuffs and beverages by high-performance

liquid chromatography with precolumn derivatization J Agric Food Chem 32 352-

355Spratley JE Hellstroumlm SO Mattsson CK Pais-Clemente M (2001)

Topical ascorbic acid reduces myringosclerosis in perforated tympanic membranes A

study in the rat Ann Otol Rhinol Laryngol 110 585-591

Spiclin P Homer M Zupancic A Gasperlin M (2003) Sodium ascorbyl phosphate

in topical microemulsions Int J Pharm 256 65-73

Stadtman ER (1991) Ascorbic acid and oxidative inactivation of proteins Am J Clin

Nutr 54 1125S-1128S

Stone I (1969) The Healing Factor Vitamin C Against Disease Grosset and Dunlap

New York

Sugden JK (1985) Photostability of cosmetic materials Int J Cosmet Sci 7 165-173

Swarbrick J Rubino JT Rubino OP (2006) Coarse Dispersions In Hendrickson

R Ed Remington The Science and Practice of Pharmacy 21st ed Lippincott

Williams amp Wilkins Baltimore Chap22

Sweetman SC Ed (2009) Martindale The Complete Drug Reference 36th ed

Pharmaceutical Press London Electronic version

219

Szent-Gyoumlrgi A (1928) Observation on function of peroxidase systems and chemistry

of adrenal cortex description of new carbohydrate derivative Biochem J 22 1387-

1389

Szymula M (2005) The influence of ascorbic acid on the rheological properties of the

microemulsion region of the SDSpentanolwater system J Cosmet Sci 56 267-

277

Taqui Khan MM Martell AE (1967) Metal ion and metal chelate catalyzed oxidation

of ascorbic acid by molecular oxygen 1 Cupric acid and ferric ion catalyzed

oxidation J Am Chem Soc 89 4176-4185

Tangney CC (1988) Analysis of vitamin C in biological samples with an emphasis on

recent chromatographic techniques Prog Clin Biol Res 259 331-362

Tatsumi K Ichikawa H Wada S (1992) Flavin-sensitized photooxidation of

substituted phenols in natural water J Contam Hydrol 9 207-219

Tessier F Birlouez-Aragon I Tjani C Guilland JC (1996) Validation of a

micromethod for determining oxidized and reduced vitamin C in plasma by HPLC-

fluorescence Int J Vitam Nutr Res 66 166-170

Thatcher SR Mansfield RK Miller RB Davis CW Baertschi SW (2001a)

Pharmaceutical photostability a technical and practical interpretation of the ICH

guideline and its application to pharmaceutical stability Part 1 Pharm Tech 25 98-

110

Thatcher SR Mansfield RK Miller RB Davis CW Baertschi SW (2001b)

Pharmaceutical photostability a technical and practical interpretation of the ICH

220

guideline and its application to pharmaceutical stability Part 2 Pharm Tech 25 50-

62

Thiele JJ Dreher F Packer L (2000) Antioxidant defense systems in skin In

Elsner P Maibach HI Eds Cosmeceuticals- Drugs vs Cosmetics Marcel

Dekker New York Chap 11

Thomas DP (1997) Sailors scurvy and science J R Soc Med 90 50-54

Thomson CO Trenerry VC (1995) A rapid method for the determination of total L-

ascorbic acid in fruits and vegetables by micellar electrokinetic capillary

chromatography Food Chem 53 43-50

Tonnesen HH (1991) Photochemical degradation of components in drug formulations

1 An approach to the standardization of degradation studies Pharmazie 46 263-265

Tonnesen HH (2002) Photodecomposition of Drugs In Swarbrick J Boylan JC

Eds Encyclopedia of Pharmaceutical Technology Vol 3 Marcel Dekker New

York pp 2197-2203

Tonnesen HH Ed (2004) The Photostability of Drugs and Drug Formulations

Culinary and Hospitality Industry Publications Services Weimex TX

Tonnesen HH Karlsen J (1997) A comment on photostability testing according to

ICH guideline calibration of light sources PharmEuropa 9 735-736

Tonnesen HH Moore DE (1993) Photochemical degradation of components in drug

formulations Pharm Technol Int 5 27-33

Tournas JA Lin FH Burch JA Selim MA Monteiro-Riviere NA Zielinski

JE Pinnell SR (2006) Ubiquinone idebenone and kinetin provide ineffective

221

photoprotection to skin when compared to a topical antioxidant combination of

vitamins C and E with ferulic acid J Invest Dermatol 126 1185-1187

Traber MG (2007) Vitamin E In Zempleni J Rucker RB McCormick DB

Suttie JW Eds Handbook of Vitamins 4th ed Taylor amp Francis CRC Press

Boca Raton FL Chap 4

Traikovich SS (1999) Use of topical ascorbic acid and its effect on photodamaged skin

topography Arch Otolaryngol Head Neck Surg 125 1091-1098

Treadwell GE Cairns WL Metzler DE (1968) Photochemical degradation of

flavins V Chromatographic studies of the products of photolysis of riboflavin J

Chromatogr 35 376-388

Truswell AS (2003) ABC of Nutrition 4th ed BMJ Publishing Group Chap 10

Tsao CS Young M (1985) Analysis of ascorbic acid derivatives by high-performance

liquid chromatography with electrochemical detection J Chromatogr 13 855-856

Underberg WJM (1978) Oxidative degradation of pharmaceutically important

phenothiazines III Kinetics and mechanism of promethazine oxidation J Pharm Sci

67 1133-1138

Urso ML Clarkson PM (2003) Oxidative stress exercise and antioxidant

supplementation Toxicology 189 41-54

Vaid FHM (1998) Photolysis and Interaction of Thiamine Hydrochloride with

Riboflavin in Aqueous Solution PhD Thesis University of Karachi Karachi

Vaid FHM Shaikh RH Ansari IA Ahmad I (2005) Spectral study of the

photolysis of aqueous thiamine hydrochloride and ascorbic acid solution in the

presence and absence of riboflavin J Chem Soc Pak 27 227-232

222

Vaid F H M Shaikh RH Ansari I A Ahmad I (2006) Chromatographic study of

the photolysis of aqueous thiamine hydrochloride and ascorbic acid solutions in the

presence and absence of riboflavin J Chem Soc Pak 28 481-464

Valvani SC (2000) Industrial stability testing in the United States and computerization

of stability data In Carstensen JT Rhodes CT Eds Drug Stability Principles

and Practices 3rd

ed Marcel Dekker New York pp 534-536

Vanderslice JT Higgs DJ (1988) Chromatographic separation of ascorbic acid

isoascorbic acid dehydroascorbic acid and dehydroisoascorbic acid and their

quantitation in food products J Micronutr Anal 4 109-118

Vanderslice JT Higgs DJ Hayes JM Block G (1990) Ascorbic acid and

dehydroascorbic acid content of foods-as-eaten J Food Comp Anal 3 105-118

Vanderslice JT Higgs DJ (1993) Quantitative determination of ascorbic acid

dehydroascorbic acid by HPLC in foods and other matrices J Nutr Biochem 4 184-

190

Varvaresou A Tsirivas E Iakovou K Gikas E Papathomas Z Vonaparti A

Panderi I (2006) Development and validation of a reversed-phase ion-pair liquid

chromatography method for the determination of magnesium ascorbyl phosphate and

melatonin in cosmetic creams Anal Chim Acta 573-574 284-290

Verma KK Jain A Verma A Chaurasia A (1991) Spectrophotometric

determination of ascorbic acid in pharmaceuticals by background correction and flow

injection Analyst (London) 116 641-645

Vimaladevi M (2005) Textbook of Cosmetics CBS Publishers New Dehli Chap 2

and 17

223

Wallwork SC Grant DJW (1977) Physical Chemistry for Students of Pharmacy and

Biology 3rd ed Longmann New York pp 502-505

Waugh WA King CG (1932) Isolation and identification of vitamin C J Biol

Chem 97 325-331

Wechtersbach L Cigic B (2007) Reduction of dehydroascorbic acid at low pH J

Biochem Biophys Methods 70 767-772

Weiner N (1996) Introduction In Liberman HA Rieger MM Banker GS Eds

Pharmaceutical Dosage Forms Disperse Systems 2nd ed vol1 Marcel Dekker

New York Chap1

Werner W (1949) The reaction of l-ascorbic and d-isoascorbic acid with nicotinic acid

and its amide J Org Chem 14 22-26

Wiechers JW (2003) Formulating for Efficacy Proceedings of the 2003 IFSCC

Conference Seoul Korea

Wiechers JW (2005) Optimizing skin delivery of active ingredients from emulsions

from theory to practice In Rosen MR Ed Delivery System Handbook for

Personal Care and Cosmetic Products-Technology Applications and Formulations

William Andrew Inc Norwich NY Chap 20

Wille JJ (2005) Thixogel-Novel topical delivery systems for hydrophobic plant actives

In Rosen MR Ed Delivery System Handbook for Personal Care and Cosmetic

Products-Technology Applications and Formulations William Andrew Inc

Norwich NY Chap 36

224

Wright JL (1986) Vitamin A monograph In Connors KA Amidon GL Stella

VJ Eds Chemical Stability of Pharmaceuticals A Handbook for Pharmacists 2nd

ed Wiley New York pp 791-803

Yao AA Wathelet B Thonart P (2009) Effect of protective compounds on the

survival electrolyte leakage and lipid degradation of freeze-dried Weissella

paramesenteroides LC11 during storage J Microbiol Biotechnol 19 810-817

Yeh SY Lach JL (1961) Stability of morphine in aqueous solutions III Kinetics of

morphine degradation in aqueous solution J Pharm Sci 50 35-42

Yoshimura A Ohno T (1988) Lumiflavin-sensitized photooxygenation of indole

Photochem Photobiol 48 561-565

Yoshioka S Stella VJ (2000) Stability of Drugs and Dosage Forms Kluwer

Academic Plenum Publishers New York Chap 2

Zaeslein C (1982) Vitamins in the Field of Medicine Hoffman La Roche Basle pp

89-96

Zeng W Martinuzzi F MacGregor A (2005) Development and application of a novel

UV method for the analysis of ascorbic acid J Pharm Biomed Anal 36 1107-1111

Zhang GF Chen HY (2000) Chemiluminescence studies of the oxidation of ascorbic

acid with copper (II) catalyzed by halide anions and its applications to the

determination of halide anions and ascorbic acid Anal Sci 16 1317-1321

Zhang L Lerner S Rustrum WV Hofmann GA (1999) Electroporation-mediated

topical delivery of vitamin C for cosmetic applications Bioelectrochem Bioenerg

48 453-461

225

Zilva SS (1932) The non-specificity of the phenolindophenol reducing capacity of

lemon juice and its fractions as a measure of their antiscorbutic activity Biochem J

26 1624-1627

226

AUTHORrsquoS PUBLICATIONS

The author obtained his B Pharm degree in 2003 and joined the post graduate

program securing an M Phil degree in Pharmaceutics in 2006 from Baqai Medical

University He is a co-author of following publications

CHAPTER IN BOOK

1 Chapter on ldquoBorate Toxicity Effect on Drug Stability and Analytical

Applicationsrdquo by Iqbal Ahmad Sofia Ahmed MA Sheraz and Faiyaz H M

Vaid In Handbook on Borates Chemistry Production and Applications (MP

Chung Ed) Nova Science Publishers Inc NY USA (in press)

PAPERS PUBLISHED

INTERNATIONAL

2 Iqbal Ahmad Sofia Ahmed MA Sheraz and Faiyaz HM Vaid ldquoEffect of Borate

Buffer on the Photolysis of Riboflavin in Aqueous Solutionrdquo Journal of

Photochemistry and Photobiology B Biology 93 82-87 (2008)

3 Iqbal Ahmad Sofia Ahmed MA Sheraz M Aminuddin and Faiyaz HM Vaid

ldquoEffect of Caffeine Complexation on the Photolysis of Riboflavin in Aqueous

Solution A Kinetic Studyrdquo Chemical and Pharmaceutical Bulletin 57 (2009)

published online September 14 2009

4 Iqbal Ahmad MA Sheraz Sofia Ahmed and Faiyaz HM Vaid ldquoAnalytical

Applications of Boratesrdquo Materials Science Research Journal (in press)

5 Iqbal Ahmad Sofia Ahmed MA Sheraz Kefi Iqbal and Faiyaz HM Vaid

ldquoPharmacological Aspects of Boratesrdquo International Journal of Medical and

Biological Frontiers (in press)

6 Iqbal Ahmad Sofia Ahmed MA Sheraz Faiyaz HM Vaid and Izhar A Ansari

ldquoEffect of Divalent Ions on Photodegradation Kinetics and Pathways of

Riboflavin in Aqueous Solutionrdquo Photochemical and Photobiological Sciences

accepted

227

NATIONAL

7 Sofia Ahmed MA Sheraz and Iqbal Ahmad ldquoAdvances in Antioxidant Activity of

Vitamin Erdquo Journal of Baqai Medical University 10 13-18 (2007)

8 MA Sheraz Sofia Ahmed and Iqbal Ahmad ldquoDevelopments in the Clinical and

Food Analysis of Vitamin Crdquo Journal of Baqai Medical University 10 19-24

(2007)

9 A Azmi SNH Naqvi M Usman MA Sheraz and Sofia Ahmed ldquoPancreatic

Glucagon in Certain Ungulates Comparative Study of Extraction and

Bioassayrdquo Pakistan Journal of Entomology 20 23-28 (2005)

10 Iqbal Ahmad Sofia Ahmed MA Sheraz Faiyaz HM Vaid and S Hasan

ldquoAdvances in Biochemical Functions and the Photochemistry of Flavins and

Flavoproteinsrdquo Pakistan Journal of Pharmaceutical Sciences in press

11 MA Sheraz Sofia Ahmed and Iqbal Ahmad ldquoEffect of Borates on the Stability of

Chemical and Pharmaceutical Compoundsrdquo Journal of Baqai Medical University

accepted

PAPERS SUBMITTED

12 Iqbal Ahmad MA Sheraz Sofia Ahmed Riaz H Shaikh and Faiyaz HM Vaid

ldquoPhotostability of Ascorbic Acid in Organic Solvents and Cream Formulationsrdquo

Chemical and Pharmaceutical Bulletin

13 Iqbal Ahmad MA Sheraz Sofia Ahmed Riaz H Shaikh and Faiyaz HM Vaid

ldquoPhotochemical Interaction of Ascorbic Acid with Riboflavin Nicotinamide and

Alpha-Tocopherol in Cream Formulationsrdquo Journal of Cosmetic Science

14 Iqbal Ahmad Kefi Iqbal Sofia Ahmed MA Sheraz ldquoApplications of Laser Flash

Photolysis Spectroscopy and Electron Microscopy in Photopolymerization and

Development of Glass Ionomer Dental Cementsrdquo Materials Science Research

Journal

15 Sofia Ahmed MA Sheraz M Aminuddin I Ahmad and Faiyaz HM Vaid ldquoA

Rapid Titrimetric Assay for Quantitation of Vitamin B1 in Neat and

Pharmaceutical Preparationsrdquo Pakistan Journal of Pharmaceutical Sciences

• 01 SZ-786
• 02 SZ-title
• 03 SZ-Certificate
• 04 SZ-Abstract
• 05 SZ-Acknowledgement
• 06 SZ-Dedication
• 07 SZ-Contents
• 08 SZ-Chapter 1
• 09 SZ-Chapter 2
• 10 SZ-Chapter 3
• 11 SZ-Object of Present Investigation
• 12 SZ-Chapter 4
• 13 SZ-Chapter 5
• 14 SZ-Chapter 6
• 15 SZ-Chapter 7
• 16 SZ-Conclusion
• 17 SZ-References
• 18 SZ-Authors Publications

CERTIFICATE

This is to certify that the work presented in this thesis entitled ldquoFormulation and

Stability of Ascorbic Acid in Liquid and Semisolid Preparationsrdquo is original and has

been conducted by Mr Muhammad Ali Sheraz under my supervision as fulfilment of

the requirement of PhD degree from the Faculty of Pharmaceutical Sciences Baqai

Medical University Karachi

Professor Dr Iqbal Ahmad

Faculty of Pharmaceutical Sciences

Baqai Medical University Karachi

iv

ABSTRACT

The present investigation is based on a study of the photodegradation of ascorbic

acid (vitamin C) in organic solvents and in oil-in-water cream preparations containing a

combination of emulsifying agents and humectants It also involves the study of the effect

of other vitamins (riboflavin nicotinamide and alpha-tocopherol) and certain compounds

acting as stabilizing agents (citric acid tartaric acid and boric acid) on the rate of

photodegradation of ascorbic acid in cream preparations The photodegradation of

ascorbic acid in organic solvents and cream preparations (pH 40ndash70) leads to the

formation of dehydroascorbic acid which is also biologically active The kinetics of

photodegradation of ascorbic acid alone and in combination with other vitamins in

creams has been studied using a UV spectrophotometric method and the official

iodimetric method respectively These methods were validated in the presence and

absence of other vitamins stabilizing agents under the experimental conditions

employed The recoveries of ascorbic acid in creams are in the range of 90ndash96 and the

reproducibility of the analytical methods is within plusmn 5

The apparent first-order rate constants (kobs) for the photodegradation of ascorbic

acid in aqueous organic solvents (029ndash040 times 10ndash3

minndash1

) and in creams (044ndash142 times

10ndash3

minndash1

) have been determined A linear relationship has been observed between kobs

and solvent dielectric constant reciprocal of solvent viscosity indicating the dependence

of the rate of photodegradation on solvent characteristics

In the creams the photodegradation of ascorbic acid appears to be affected by the

concentration of other vitamins pH of the medium carbon chain length of the

emulsifying agents (myristic acid palmitic acid and stearic acid) viscosity of the

v

humectant (ethylene glycol propylene glycol and glycerin) and redox potentials of

ascorbic acid The study indicates that the relative polar character of the emulsifying

agent and the ionized state and redox potential of ascorbic acid at a particular pH are

important factors in the photodegradation of ascorbic acid in creams

The second-order rate constants (kprime) (320 times 10ndash2

ndash 189 Mndash1

minndash1

) for the

photochemical interaction of ascorbic acid and the individual vitamins (riboflavin

nicotinamide alpha-tocopherol) along with the values of k0 obtained from the intercepts

of the plots of kobs versus vitamin concentration are also reported The values of k0

indicate that riboflavin and nicotinamide act as photosensitizing agents and alpha-

tocopherol acts as a stabilizing agent in the photodegradation of ascorbic acid in the

creams The kobs verses pH profiles for the photodegradation of ascorbic acid in creams

represents sigmoid type curves indicating the oxidation of the ionized form (AHndash) of

ascorbic acid (pKa1 41) with pH The AHndash species appears to be more susceptible to

photooxidation than the non-ionized form of ascorbic acid The effect of stabilizing

agents on the photodegradation of ascorbic acid has been found to be in the order of citric

acid gt tartaric acid gt boric acid The low activity of boric acid may be to some extent due

to its interaction with the emulsifying agents and humectants The polarity of the

emulsifying acids also plays a part in the rate of degradation of ascorbic acid Reaction

schemes for the photodegradation of ascorbic acid and its photochemical interaction with

riboflavin nicotinamide and alpha-tocopherol have been presented

vi

ACKNOWLEDGMENTS

I am highly grateful to All Mighty Allah who guided me in all difficulties and

provided me strength to overcome the problems during this work

Words are confined and inefficacious to express my immense gratitude to my

respectable supervisor Prof Dr Iqbal Ahmad Department of Pharmaceutical

Chemistry for his guidance encouragement keen interest and above all giving his

valuable time suggestions and attention His personality has been a source of constant

inspiration through out my research work

I would like to extend my sincere thanks to Prof Lt Gen (R) Dr Syed Azhar

Ahmed Vice Chancellor Baqai Medical University for his personal interest and

constant encouragement through out the study

It is my great desire to express my gratitude to Prof Dr Syed Fazal Hussain

CEO Baqai Institute of Pharmaceutical Sciences for his cooperation and attention and

providing all the facilities of the Institute at my disposal during the research work

I am also thankful to Mrs Shaukat Khalid Dean Faculty of Pharmaceutical

Sciences for her support during the study

I feel honored to express my sincere thanks and indebtedness to Prof Dr

Khursheed Ali Khan Department of Pharmaceutics Prof Dr Aminuddin Department

of Pharmaceutical Chemistry and Dr Faiyaz H M Vaid Chairman Department of

Pharmaceutical Chemistry Faculty of Pharmacy University of Karachi who helped me

selflessly with their invaluable suggestions through out the research work

vii

I feel immense pleasure to pay my sincere and special thanks to Ms Sofia

Ahmed Assistant Professor and In charge Department of Pharmaceutics who lent all

sort of cooperation and spared no effort in helping me during this work

Special thanks are due to Mr Saif-ur-Rehman Khatak Deputy Drug Controller

for his cooperation and help during this study

I acknowledge with sincere thanks the contribution of Tabros Pharmaceutical

Industry Karachi for providing me the opportunity to use their facilities for certain

measurements without which the completion of this work would not have been possible

I highly appreciate the technical services rendered by Mr Anees Mr Wajahat

and Mr Sajjad in pursuance of this study

I am very grateful to Mrs Prof Dr Iqbal Ahmad for her kindness and generous

hospitality during my innumerable visits to their residence

Last but not the least I would like to express my immense indebtedness to My

Gracious Parents Beloved Brothers and Sisters for their moral support kindness and

encouragement throughout my career

I am also thankful to all my students for their affectionate feelings

M A S

viii

To

My Beloved Parents amp

Late Prof Dr S Sabir Ali for their interest and endless support

ix

CONTENTS

Chapter Page

ABSTRACT iv

ACKNOWLEDGEMENTS vi

I INTRODUCTION 1

11 HISTORICAL BACKGROUND 2

12 PHYSICOCHEMICAL CHARACTERISTICS OF

ASCORBIC ACID

2

13 CHEMISTRY OF ASCORBIC ACID 3

131 Nomenclature and Structure 3

132 Chemical Stability 3

14 BIOCHEMICAL FUNCTIONS 7

15 ANTIOXIDANT ACTIVITY 8

16 PHOTOSTABILITY OF DRUGS 9

17 KINETIC TREATMENTS OF PHOTOCHEMICAL

REACTIONS

12

18 LITERATURE ON ASCORBIC ACID 15

II PHOTODEGRADATION REACTIONS AND ASSAY OF

ASCORBIC ACID

17

21 PHOTODEGRADATION REACTIONS 18

211 Photodegradation of Ascorbic Acid 18

212 Effect of Various Substances on Photodegradation of Ascorbic

Acid

20

213 Photosensitized Oxidation of Ascorbic Acid 22

214 Ascorbic Acid Interaction with Other Water-Soluble Vitamins 25

22 ASSAY OF ASCORBIC ACID 26

221 Spectrophotometric Methods 26

222 Fluorimetric Methods 28

x

223 Mass spectrometric Methods 28

224 Chromatographic Methods 28

225 Enzymatic Methods 29

226 Commercial Kits for Clinical Analysis 30

227 Analysis in Creams 30

III FORMULATION AND STABILITY OF CREAM

PREPARATIONS

31

31 FORMULATION OF CREAM PREPARATIONS 32

311 Choice of Emulsion Type 32

312 Choice of Oil Phase 33

313 Emulsion Consistency 33

314 Choice of Emulsifying Agent 34

315 Formulation by the HLB Method 34

316 Concept of Relative Polarity Index 35

32 FORMULATION OF ASCORBIC ACID CREAMS 37

33 STABILITY OF CREAMS 39

331 Physical Stability 39

332 Chemical Stability 39

333 Microbial Stability 40

334 Stability of Ascorbic Acid in Liquid Formulations 41

335 Stability of Ascorbic Acid in Emulsions and Creams 41

336 Stability Testing of Emulsions 45

3361 Macroscopic examination 46

3362 Globule size analysis 46

3363 Change in viscosity 46

3364 Accelerated stability tests 46

337 FDA Guidelines for Semisolid Preparations 46

xi

OBJECT OF PRESENT INVESTIGATION 48

IV MATERIALS AND METHODS 51

41 MATERIALS 52

42 METHODS 55

421 Cream Formulations 55

422 Preparation of Creams 56

423 Thin-Layer Chromatography 57

424 pH Measurements 57

425 Ultraviolet and Visible Spectrometry 58

426 Photolysis of Ascorbic Acid 59

4261 Creams 59

4262 Aqueous and organic solvents 59

4263 Storage of creams in dark 59

427 Measurement of Light Intensity 59

428 Procedure 60

4281 Calculation 62

429 Viscosity Measurements 63

4210 Assay method 65

42101 UV spectrophotometric method for the assay of creams

containing ascorbic acid alone

65

42102 Iodimetric method for the assay of ascorbic acid in creams

containing riboflavin nicotinamide and alpha-tocopherol 65

42103 Spectrophotometric method for the assay of ascorbic acid in

aqueous and organic solvents

67

V PHOTODEGRADATION OF ASCORBIC ACID IN

ORGANIC SOLVENTS AND CREAM FORMULATIONS

68

51 INTRODUCTION 69

52 PHOTOPRODUCTS OF ASCORBIC ACID 71

xii

53 SPECTRAL CHARACTERISTICS OF PHOTOLYSED

SOLUTIONS

71

54 ASSAY OF ASCORBIC ACID IN CREAMS AND

SOLUTIONS

73

55 EFFECT OF SOLVENT 74

56 EFFECT OF CONCENTRATION 80

57 EFFECT OF CARBON CHAIN LENGTH OF

EMULSIFYING AGENT

88

58 EFFECT OF VISCOSITY 94

59 EFFECT OF pH 94

510 EFFECT OF REDOX POTENTIAL 96

511 PRIMARY PHOTOCHEMICAL REACTIONS IN THE

OXIDATION OF ASCORBIC ACID

97

512 DEGRADATION OF ASCORBIC ACID IN THE DARK 98

VI PHOTOCHEMICAL INTERACTION OF ASCORBIC

ACID WITH RIBOFLAVIN NICOTINAMIDE AND

ALPHA-TOCOPHEROL IN CREAM FORMULATIONS

109

61 INTRODUCTION 110

62 ABSORPTION CHARACTERISTICS OF PHOTOLYSED

CREAMS

114

63 PHOTOPRODUCTS OF ASCORBIC ACID AND OTHER

VITAMINS

114

64 ASSAY METHOD 116

65 KINETICS OF PHOTODEGRADATION OF ASCORBIC

ACID

117

66 INTERACTION OF RIBOFLAVIN WITH ASCORBIC ACID 128

67 INTERACTION OF NICOTINAMIDE WITH ASCORBIC

ACID

129

68 INTERACTION OF ΑLPHA-TOCOPHEROL WITH

ASCORBIC ACID

130

69 EFFECT OF CARBON CHAIN LENGTH OF

EMULSIFYING AGENT

130

xiii

610 EFFECT OF VISCOSITY OF CREAMS 132

611 DEGRADATION OF ASCORBIC ACID IN THE PRESENCE

OF OTHER VITAMINS IN THE DARK

135

VII STABILIZATION OF ASCORBIC ACID WITH CITRIC

ACID TARTARIC ACID AND BORIC ACID IN CREAM

FORMULATIONS

141

71 INTRODUCTION 142

72 CREAM FORMULATIONS 142

73 PRODUCTS OF ASCORBIC ACID

PHOTODEGRADATION

145

74 SPECTRAL CHANGES IN PHOTODEGRADED CREAMS 145

75 ASSAY OF ASCORBIC ACID IN CREAMS 145

76 KINETICS OF PHOTODEGRADATION 146

77 EFFECT OF STABILIZING AGENTS 146

78 DEGRADATION OF ASCORBIC ACID IN PRESENCE OF

STABILIZING AGENTS IN THE DARK

162

79 EFFECT OF ADDITIVES ON TRANSMISSION OF

ASCORBIC ACID

163

CONCLUSIONS AND SUGGESTIONS 179

CONCLUSIONS 180

SUGGESTIONS 184

REFERENCES 185

AUTHORrsquoS PUBLICATIONS 226

CHAPTER I

INTRODUCTION

2

11 HISTORICAL BACKGROUND

The disease scurvy which now is known as a condition due to a deficiency of

ascorbic acid in the diet has considerable historical significance (Schick 1943

Carpenter 1986 Bardolph and Taylor 1997 Thomas 1997 Bors 2005) Zilva (1932)

isolated the antiscorbutic activity factor from a crude fraction of lemon and showed that

the activity was destroyed by oxidation and protected by reducing agents Waugh and

King (1932) isolated crystalline vitamin C from lemon juice and showed it to be the

antiscorbutic factor Szent-Gyorgyi (1928) had isolated the same factor from pepper in

connection with his biological oxidation-reduction studies Hirst and Zilva (1933)

identified the antiscorbutic factor as ascorbic acid Early work on the chemical

identification and elucidation of the structure of ascorbic acid has been well documented

(Carpenter 1986) The first synthesis of L-ascorbic acid was achieved almost

simultaneously by Ault et al (1933) and Reichstein et al (1933)

Plants and most animals synthesize their own vitamin C but humans lack this

ability due to the deficiency in an enzyme L-gulono-gamma-lactone oxidase that

catalyzes the terminal step in ascorbic acid biosynthesis (Nishikimi et al 1994)

Therefore humans obtain this vitamin from diet and or vitamin supplements to not only

avoid the development of scurvy but also for overall well being (Stone 1969 Lewin

1976 Davies et al 1991) The minimal daily requirement for ascorbic acid in healthy

adults is 40ndash60 mg (Truswell 2003 Mason 2007 Eitenmiller et al 2008 Elia 2009)

12 PHYSICOCHEMICAL CHARACTERISTICS OF ASCORBIC ACID

The important physicochemical characteristics of ascorbic acid (Table 1) involved

in its identification and degradation are described by many authors (Connors et al 1986

3

OrsquoNeil 2001 Moffat et al 2004 Sinko 2006 Johnston et al 2007) The most

important chemical property of ascorbic acid is the reversible oxidation to semidehydro-

L-ascorbic acid and further oxidation to dehydro-L-ascorbic acid This property is the

basis for its physiological activity In addition the proton on oxygenndash3 is acidic (pKa1 =

417) which contributes to the acidic nature of ascorbic acid (1)

13 CHEMISTRY OF ASCORBIC ACID

131 Nomenclature and Structure

The IUPAC-IUB Commission on Biochemical Nomenclature changed the name

vitamin C (2-oxo-L-theo-hexono-4-lactone-23-enediol) to ascorbic acid or L-ascorbic

acid in 1965 (Johnston et al 2007) The chemical structure of ascorbic acid (1) is

HO OH

O

OHHO

H

(1)

O

The molecule has a near planar five-membered ring with two chiral centers

which contain four stereoisomers

132 Chemical Stability

Ascorbic acid is sensitive to air and light and is kept in a well-closed container

protected from light (British Pharmacopoeia 2009) The degradation reactions of

ascorbic acid in aqueous solution depend on a number of factors such as pH temperature

presence of oxygen or metal It is not very stable in aqueous media at room

temperature and undergoes oxidative degradation to dehydroascorbic acid and

4

Table 1 Physicochemical characteristics of ascorbic acid

Empirical formula C6H8O6

Molar mass 17613

Crystalline form Monoclinic mix of platelets and needles

Melting point 190 to 192 degC

[α]25

+205deg to +215deg

pH

5 mg ml

50 mg ml

~3

~2

pKa 417 1157 (20deg)

Redox potential

(dehydroascorbic acid ascorbate)

(H+ ascorbate

ndash)

ndash174 mV

+282 mV

Solubility g ml

Water

Ethanol absolute

Ether chloroform benzene

033

002

Insoluble

UV spectrum

Absorption maximum [A(1 1 cm)]

pH 20

pH 70

245 nm [695]

265 nm [940]

Infrared spectrum

Principal peaks (Nujol mull)

1026 (CminusOH str) 1111(CminusOminusC str) 1312

(minusCminusOminus str) 1653 (C=O str) 990 (C=C str)

cmndash1

Mass spectrum

Principal ions at mz

29 41 39 42 69 116 167 168

D

5

23-diketogulonic acid The stability of ascorbic acid and dehydroascorbic acid can be

improved by lowering the pH below 2 (Wechtersbach and Cigic 2007) Above pH 7

alkali-catalyzed degradation by cleavage at Cndash1 or Cndash2 results in a number of

compounds mainly monondash dindash and tricarboxylic acids (Connors et al 1986 Bors and

Buettner 1997 Halliwell and Whiteman 1997) The oxidative degradation of ascorbic

acid and dehydroascorbic acid in parenteral nutrition mixtures is catalyzed by trace

elements particularly copper (Allwood 1984ab Allwood et al 1992 Allwood and

Kearney 1998 Kearney et al 1998 Gibbons et al 2001) Stabilized ascorbic acid

preparations in hydroalcoholic vehicle (Kaplan et al 1989) and aquaculture feeds

(OrsquoKeefe 2001) have been reported The various oxidation products of ascorbic acid are

shown in Fig 1

It is interesting to note that in addition to redox and acid-base properties ascorbic

acid can exist as a free radical (Bielski et al 1981 Bielski 1982 Halliwell 1996 Bors

and Buettner 1997) The ascorbate radical anion is an important intermediate in the

reactions involving oxidants and ascorbic acidrsquos antioxidant activity Rate constants for

the generation of ascorbate radicals are in the range of 104ndash10

8 s

ndash1 When ascorbate

radicals are generated by oxyanions the rate constants are of the order of 104ndash10

7 s

ndash1

when generated by halide radicals 106ndash10

8 s

ndash1 and when generated by tocopherols and

flavonoids radicals 106ndash10

8 s

ndash1 (Bielski 1982 Halliwell and Whiteman 1997) The

ascorbate radicals decay usually by disproportionation However a change in ionic

strength or pH can influence the rate of dismutation of ascorbic acid Certain oxyanions

such as phosphates accelerate dismutation (Bielski et al 1981) The acceleration is

attributed to the activity of various protonated forms of phosphate to donate a proton

6

Fig 1 Oxidation products of ascorbic acid

O

OHOH

H

OO

OHOH

H

OO

OHOH

H

O

Ascorbyl radical anion

(interm ediate)

Ascorbic acid

(1)

-e- -2H

+

+e- +2H

+

-e-

+e-

Dehydroascorbic acid

(2)

23-diketo-L-gulonic acid

O xalic acid

+

L-Threonic acid

L-Xylose

+

C O 2

CO 2

L-Xylonic acid

+

L-Lyxonic acid CO 2

HO OH O O-

O O

7

efficiently to the ascorbate radical particularly the dimer form of ascorbate

The unusual stability of the ascorbate radical in biological systems dictates that

accessory enzymatic systems be made available to reduce the potential transient

accumulation of ascorbate radical The excess ascorbate radicals may initiate a chain of

free-radicals reactions In plants NADHmonodehydroascorbate reductase maintains

ascorbic acid in its reduced form NADHmonodehydroascorbate reductase plays a major

role in stress related responses in plants Glutathione dehydroascorbate reductase serves

this purpose in animal tissues Such enzymes keep ascorbic acid operating at maximum

efficiency so that other enzyme systems may take advantage of the univalent redox

cycling capacity of ascorbate (Asard et al 2004 Johnston et al 2007)

The anaerobic degradation of ascorbic acid has been studied by Finholt et al

(1963) Under these conditions the molecule is dehydrated and hydrolyzed in aqueous

solution to give furfural and carbon dioxide The rate of degradation is maximum at pH

41 corresponding to the pKa of ascorbic acid This has been suggested due to the

formation of a saltndashacid complex in solution The reaction is dependent on buffer

concentration but has relatively small effect of ionic strength

14 BIOCHEMICAL FUNCTIONS

Ascorbic acid plays an essential role in the activities of several enzymes It is vital

for the growth and maintenance of healthy bones teeth gums ligaments and blood

vessels It is important for the manufacture of certain neurotransmitters and adrenal

hormones Ascorbic acid is required for the utilization of folic acid and the absorption of

iron It is also necessary for normal immune responses to infection and for wound healing

(Henry 1997)

8

Ascorbic acid deprivation and scurvy include a range of signs and symptoms that

involves defects in specific enzymatic processes (Johnston et al 2007) The

administration of ascorbic acid improves most of the signs of chemically induced

glutathione (L-γ-glutamyl-L-cysteine-glycine GSH) deficiency (Meister 1994) The

effect is very pronounced in newborn rats which do not efficiently synthesize ascorbic

acid in contrast to adult rats and guinea pigs When L-buthionine-(SR)-sulphoxime is

administered in addition to the loss in GSH there is a marked increase in

dehydroascorbic acid This has led to the hypothesis that GSH is very important to

dehydroascorbic acid reduction and as a sequence to ascorbic acid recycling (Meister

1995)

Ascorbic acid also possesses pro-oxidant properties and may cause apoptosis

lymphoid and myeloid cells It has been shown that dehydroascorbic acid also stimulates

the antioxidant defenses in some cells by preferentially importing dehydroascorbate over

ascorbate (Braun et al 1997 Banhegyi et al 1998 Puskas et al 2000 2002)

15 ANTIOXIDANT ACTIVITY

Ascorbic acid is known to readily scavenge reactive oxygen and nitrogen species

such as superoxide and hydroperoxyl radicals aqueous peroxyl radicals singlet oxygen

ozone peroxynitrite nitrogen dioxide nitroxide radicals and hypochlorous acid Excess

of such products has been associated with lipids (Niki and Noguchi 1997 Carr et al

2000 Urso and Clarkson 2003) DNA (Fraga et al 1991 1996 Lindahl 1993) and

protein oxidation (Stadtman 1991 Berlett and Stadtman 1997 Dean et al 1997

Ortwerth and Monnier 1997 Padayatty et al 2003)

9

The electron donor character of ascorbate may be responsible for many of its

known biological functions Inspite of the availability of ascorbic acid to influence the

production of hydroxyl and alkoxyl radicals it remains uncertain whether this is the

principal effect or mechanism that occurs in vivo There is a good evidence that ascorbic

acid protects lipids in biological fluids as an antioxidant (Johnston et al 2007) A

detailed account of the function of ascorbate as an antioxidant and its reactions with

reactive nitrogen species and singlet oxygen has been reported by Packer et al (2002)

and Buettner and Schafer (2004)

Ascorbic acid (Eordm ndash0115 V pH 52 Sinko 2006) has been used as an antioxidant

for the stabilization of drugs with a higher oxidation potential These drugs include

morphine (Yeh and Lach 1961) vitamin A (Wright 1986) rifampin (Maggi et al

1966) cholecalciferol (Nerlo et al 1968 Sawicka 1991) promethazine (Underberg

1978) and sulphacetamide and sulphanilamide (Ahmad and Ahmad 1983)

16 PHOTOSTABILITY OF DRUGS

Many drug substances are sensitive to light (British Pharmacopoeia 2009) and

may degrade in pharmaceutical formulations to inactive or toxic compounds This could

make a product therapeutically inactive while in use by the patients The

photodegradation (photolysis) of drug substances may occur not only during storage but

also during the use of the product It may involve several mechanisms including

oxidation reduction hydrolysis decarboxylation isomerization rearrangement and other

reactions Normal sunlight or room light may cause substantial degradation of drug

molecules The study of degradation of drug substances under the action of UVvisible

light is relevant to the process of drug development for several reasons such as

10

Exposure to light can influence the stability of a drug formulation resulting in the

loss of potency

Inappropriate exposure to light of the raw material or the final product can lead to

the formation of toxic photoproducts that are dangerous to health

Information about the stability of drug substances and formulations is needed to

predict the shelf-life of the final product (Tonnesen and Moore 1993)

The development of light-activated drugs involves activation of the compound

through photochemical reactions (Tonnesen 1991)

Adverse effects due to the formation of minor degradation products during

storage and administration have been reported (de Vries et al 1984) The drugs

substances may also cause light-induced side effects after administration to the patient by

interaction with endogenous substances The study of the photochemical properties of

drug substances and formulated products is an integral part of formulation development

to ensure the safety and efficacy of the product

The photodegradation of drug substances occurs as a result of the absorption of

radiation energy by a molecule (A) to produce an excited state species (A) (11) The

absorbed energy can be lost either by a radiative process involving fluorescence or

phosphorescence (12) or by a physical or chemical radiationless process The physical

process results in the loss of energy as heat (13) or by collisional quenching (14) The

chemical decay leads to the formation of a new species (15) The whole process is

represented as

11

A A (11)

A A + hυprime (12)

A A + heat (13)

A + A 2A (14)

A product (s) (15)

According to the Stark-Einstein law the absorption of one quantum of radiation

results in the formation of one excited molecule which may take part in several

photochemical processes [Eqs (11)ndash(15)] The quantum yield φ for any one of these

processes is defined by

Number of molecules undergoing the photochemical process φ =

Number of quanta absorbed

Considering a pure photochemical reaction the quantum yield has a value of 0ndash1

however if A is a radical that can take part in a free-radical chain reaction so that the

absorption of energy simply initiates the reaction then each quantum of energy may

result in the decomposition of molecules and φ may appear to be greater than 1 (Connors

et al 1986)

Detailed information on the photostability and photodegradation of drug

substances including vitamins alone or in solid or liquid formulations is available in the

reviews published by DeRitter (1982) Albini and Fasani (1998) Sequeira and Vozone

(2000) Tonnesen (2002 2004) Yoshioka and Stella (2002) Min and Boff (2002) Reed

et al (2003) Fasani and Albini (2005) and Sinko (2006) The photostability of cosmetic

materials has been reviewed by Sugden (1985) Important aspects dealing with the

photostability testing of drug substances have been dealt by Anderson et al (1991)

k1

k2

k3

k4

hυ

12

Tonnesen and Moore (1993) Tonnesen and Karlsen (1997) Riehl et al (1995) ICH

(1997) Singh and Bakshi (2000) Valvani (2000) Thatcher et al (2001ab) Fasani and

Albini (2005) Klick et al (2005) Singh (2006) and Ahmad and Vaid (2006)

17 KINETIC TREATMENT OF PHOTOCHEMICAL REACTIONS

The kinetic treatment of photochemical reactions with reference to the

photostability of drug substances has been considered by Moore (2004) and is presented

in this section

The photostability testing of a drug substance at the preformulation stage involves

a study of the drugrsquos rate of degradation in solution on exposure to light for a period of

time The value of the degradation rate constant depends very much on the design of the

experimental conditions (eg concentration solvent pH irradiation source oxygen

content) The factors that determine the rate of a photochemical reaction are simply the

rate at which the radiation is absorbed by the test sample (ie the number N of photons

absorbed per second) and the efficiency of the photochemical process (ie the quantum

yield of the reaction φ) For a monochromatic photon source the number of photons

absorbed depends upon the intensity of the photon source and the absorbance at that

wavelength of the absorbing species The rate of a photochemical reaction is defined as

Rate = number of molecules transformed per second = N φ (16)

In the first instance the rate can be determined for a homogeneous liquid sample

in which the only photon absorption is due to the drug molecule undergoing

transformation with the restriction that the concentration is low so that the drug does not

absorb all of the available radiation in the wavelength range corresponding to its

13

absorption spectrum The value of N can be derived at a particular wavelength λ and is

given by

Nλ = Iλ ndash It = Iλ (1 ndash 10ndashA

) (17)

where Iλ and It are the incident and transmitted radiation intensities respectively and A is

the absorbance of the sample at the wavelength of irradiation This expression can be

expanded as a power series

Nλ = 2303 Iλ (A + A22 + A

36 + hellip) (18)

When the absorbance is low (Alt 002) the second- and higher-order terms are negligible

and the expression simplifies to the first term in Eq 18 Given the Beerrsquos law relation

between absorbance and concentration N can be seen to be directly proportional to

concentration

Nλ = 2303 Iλ A = 2303 Iλ ελ b C (19)

where ελ is the molar absorptivity at wavelength λ C the molar concentration of the

absorbing species and b the optical path length of the reaction vessel Now Iλ and ελ vary

with wavelength so the expression must be integrated over the relevant wavelength range

where each has a non-zero value

N = 2303 b C int (Iλ ελ) dλ integrated from λ1 to λ2 (110)

Thus

Rate = 2303 b C φ int (Iλ ελ) dλ (111)

Now the overlap integral (int Iλ ελ dλ) is a constant for a particular combination of photon

source and absorbing substance b is determined by the reaction vessel chosen and φ is a

characteristic of the reaction Thus by grouping the constant terms into an overall

constant k1 the expression is simplified to a first-order kinetic equation

14

Rate = ndashd [Drug] dt = k1C (112)

The integrated form of Eq 112 can be expressed in exponential form (Eq 113) or

logarithmic form (Eq 114)

[Drug]t = [Drug]0 endashk1t

(113)

ln [Drug]t = ln [Drug]0 ndash k1t (114)

Verification of first-order kinetics is obtained when a plot of the logarithm of the

concentration of drug remaining is linear with slope equal to (ndashk1)

Eq 112 predicts that a photodegradation reaction studied at low concentrations in

solution will follow first-order kinetics however the rate constant derived from a study

performed in one laboratory will not be the same as that found in another The reason for

this is the inherent difficulty in reproducing exactly the experimental arrangement of

photon source and sample irradiation geometry Therefore the relative values of the rate

constants are useful in a given experimental arrangement for making comparisons of

degradation of the absorbing substance in different formulations eg those containing

ingredients designed to inhibit the photoreaction The use of rate constants is helpful for

comparative purposes when studying a number of different reaction mixtures under the

same irradiation conditions such as the effect of pH on the degradation of a drug

However the reaction order and numerical values of the rate constants are relative to the

specific conditions used

15

18 LITERATURE ON ASCORBIC ACID

A large number of reviews have been published on various aspects of ascorbic

acid A list of important reviews is given below

Chemistry biochemical functions and related aspects

Rosenberg (1945) Burns (1961) King and Burns (1975) Sim (1972) Hanck

(1982) Zaeslein (1982) Seib and Tolbert (1982) Carpenter (1986) Levine

(1986) Davies et al (1991) Halliwell and Whiteman (1997) Ortega and Delgado

(1998) Asard et al (2004) Hickey and Roberts (2004) Johnston et al (2007)

Eitenmiller (2008)

Chemical and pharmaceutical stability

Macek (1960) Garrett (1967) Carstensen (1972) Dale and Booth (1976) Hashmi

(1973) Litner (1973) DeRitter (1982) Allwood (1984ab) Allwood and Kearney

(1998) Connors et al (1986) Smith et al (1988) Racz (1989) Roth et al 1991

Ball (2006) Eitenmiller et al (2008) Sweetman (2009)

Methods of assay and chromatography

Mader (1961) Gyorgy and Pearson (1967) Bolliger and Konig (1969) Hashmi

(1973) Al-Meshal and Hassan (1982) Pelletier (1985) Lambert and deLeenheer

(1992) Halver and Felton (2001) Moffat et al (2004) Ball (2006) Eitenmiller et

al (2008)

Pharmacology and related aspects

Levine (1986) Dollery (1999) Sauberlich (1994ab) McDowell (2000)

Kaushansky and Kipps (2006) Sweetman (2009)

16

Antioxidant activity

Basu et al (1999) Shacter (2000) Thiele et al (2000) Cadenas and Packer

(2002) Packer et al (2002) Padayathy et al (2003) Parker and Parker (2003)

Burke (2006) Johnston et al (2007)

Cosmetic Preparations

Barel et al (2001) Salvador and Chisvert (2007) Rosen (2005) Bissett (2006)

Chaudhri and Jain (2009)

CHAPTER II

PHOTODEGRADATION

REACTIONS AND ASSAY

OF ASCORBIC ACID

18

21 PHOTODEGRADATION REACTIONS

211 Photodegradation of Ascorbic Acid

Aqueous ascorbic acid (1) solutions are degraded by UV light to give

dehydroascorbic acid (2) (Arcus and Zilva 1940) Ascorbic acid degradation at a

concentration of 52 and 50 mg on UV irradiation for 2 hours gave a loss of 43 and 8

respectively Dehydroascorbic acid solutions are more stable to UV light than the

ascorbic acid (Kitagawa 1968) In many natural products the vitamin is oxidized on

exposure to air and light (OrsquoNeil 2001) When solutions of multivitamin preparations are

exposed to light H2O2 as well as organic peroxides are generated and specific

byproducts that differ from dehydroascorbic acid and 23-diketogulonic acid (3) are

produced (Lavoie et al 2004)

In aqueous neutral or alkaline solution ascorbic acid (1) undergoes chemical or

photochemical oxidation to dehydroascorbic acid (2) which upon saponification of the

lactone ring under the influence of the base water produces 23-diketo-L-gulonic acid (an

α szlig- diketogulonic acid) (3) This acid undergoes further oxidation to oxalic acid (4) and

L-threonic acid (5) (Racz 1989) (Fig 2a) At room temperature oxalic acid (4) is also

formed along with threonolactone (6) by photochemical degradation of ascorbic acid (1)

in the presence of singlet oxygen (1O2) (Silva and Quina 2006) (Fig 2a) The low-

temperature photooxygenation of ascorbic acid (1) gives a mixture of unstable

hydroperoxide ketones (7) and (8) which on standing interconvert and cyclize to

hydroperoxyhemiketal (9) The hydroperoxyhemiketal breaks down on warming to

produce the oxalate esters of threonic acid (10) (Fig 2b) (Kwon and Foote 1988)

19

COOH

COOH

O

OHHO

O

HOH2C

HO2

O

O

HO

OO

O O2H

OHHO

O

HOH2C

OH

O

O

OH

O2H

OO

HO O2CCO2H

(1)hv

room temperature

(4)(6)

(1)hv

85 oC

(7)

(a)

(8)

+

cyclization

(9)

ring cleavage

(b)

(10)

(2)

OH O

OHHO

OH O O

(3)

OH OH

OH

OH O

O

OH

1O2 [O]

+

(5)

COOH

COOH

(4)

+

OH

Fig 2 Photooxidation of ascorbic acid at room and low temperature

20

An important consideration in the stability of ascorbic acid in total parenteral

nutrition (TPN) solutions is the generation of hydrogen peroxide in the presence of light

(Laborie et al 1998 1999 2000 2002 Chessex et al 2002) This may result from the

oxidation of ascorbate anion by molecular oxygen (Homann and Gaffron 1964 Taqui

Khan and Martell 1967 Mushran and Agarwal 1977 Hughes 1985 De La Rochette et

al 2000) leading to further degradation of ascorbic acid (Deutsch 1998a 1998b

1998c) The kinetics and mechanism of oxidation reactions of ascorbic acid have been

studied by several workers (Taqui Khan and Martell 1967 Ogata and Kosugi 1969

Blaugh and Hajratwala 1972 Fessenden and Verma 1978 Abe et al 1986 Kwon et al

1989 Fornaro and Coicher 1998 Njus et al 2001)

The photostability of various ascorbic acid tablets on exposure to UV light has

been studied and the influence of antioxidants and moisture on the potency loss of

ascorbic acid has been evaluated The physical characteristics of ascorbic acid tablets are

also affected on UV irradiation (Ahmad et al 1973 Jamil et al 1980ab Jamil and

Ahmad 1984)

212 Effect of Various Substances on Photodegradation of Ascorbic Acid

The oxidation-reduction reactions of ascorbic acid in the presence of riboflavin at

pH 8ndash9 under the influence of light have been studied Under these conditions ascorbic

acid is a more active H donor to riboflavin than phenolphthalein (Sibi et al 1953)

Riboflavin has been found to catalyze the photodegradation of ascorbic acid solutions

during exposure to light and air The losses of ascorbic acid are markedly increased by

the presence of Cu2+

and Fe3+

ions under light exposed and unexposed conditions (Sattar

et al 1977) A spectral study of the UV photolysis of ascorbic acid solutions in the

21

presence of riboflavin has shown that the degradation of ascorbic acid is enhanced to the

extent of about 15 (Vaid et al 2005) The influence of DL- methionine on the

photostability of ascorbic acid solutions has also been studied DL- methionine (10 mg

) enhances the photostability of ascorbic acid (40 mg ) in acetate and phosphate

buffers but not in citrate buffer at pH 45 The photoprotective action of DL-methionine

on ascorbic acid appears to be influenced by its concentration pH of the medium and the

buffer species (Asker et al 1985)

The degradation of ascorbic acid solutions on irradiation with simulated sunlight

in the presence of the food dye quinolone yellow (E 104) is enhanced However this

effect is reversed by the addition of mannitol indicating that this dye facilitates the

photogeneration of hydroxyl radicals which may cause degradation of the vitamin The

incorporation of triplet quenchers enhances the stability of substrate solutions suggesting

that the dye acts as a triplet sensitizer to facilitate the reaction (Sidhu and Sugden 1992)

The photostability of ascorbic acid solutions is enhanced by sweetening agents (mannitol

sorbitol sucrose dextrose and Canderal) at 5 wv concentration However the addition

of stoichiometric amounts of hydrogen peroxide as a source of hydroxyl radicals and 2

2rsquo-azobis (2-amidopropane) as a source of hydroperoxyl radicals results in diminished

stability of ascorbic acid solutions The diminished activity may be due to the action of

hydroperoxyl radicals in the presence of hydroxyl radical scavengers (Ho et al 1994)

Metal-complexing agents (eg disodium ethylenediaminetetraacetic acid N-

hydroxylethyl ethylenediaminetetraacetic acid 8-hydroxyquinoline) have a stabilizing

effect on the photolysis of ascorbic acid injectable solutions (Kassem et al 1969ab

22

1972) This may be due to the interaction of these agents with metal ions and other

impurities

213 Photosensitized Oxidation of Ascorbic Acid

In the presence of visible light a photosensitizer such as riboflavin can exhibit

photosensitizing properties through a mixed Type IndashType II mechanism (Yoshimura and

Ohno 1988 Foote 1991 Silva et al 1994 Silva and Quina 2006) as presented below

Type I mechanism (low oxygen concentration)

RF + hυ rarr 1RF rarr

3RF (21)

3RF + SH rarr RF

middot ndash + SH

middot + rarr RFH

middot + S

middot (22)

RFmiddot ndash

+ O2 rarr RF + O2middot ndash

(23)

2RFHmiddot rarr RF + RFH2 (24)

RFH2 + O2 rarr RF + H2O2 (25)

H2O2 + O2middot ndashrarr

ndashOH +

middotOH + O2 (26)

Smiddot and or SH

middot +

+ H2O2 O2middot ndash

rarr Soxid (27)

Type II mechanism (high oxygen concentration)

RF + hυ rarr 1RF rarr

3RF (28)

3RF + O2 rarr RF +

1O2 (29)

SH + 1O2 rarr Soxid (210)

In these reactions RF 1RF and

3RF represent RF in the ground state and in the excited

singlet and triplet states respectively RFmiddot ndash

RFHmiddot and RFH2 are the radical anion the

radical and the reduced form of RF SH is the reduced substrate and SHmiddot

+ S

middot and Soxid

23

represent the intermediate radical cation the radical and the oxidized form of the

substrate respectively

An early study of the riboflavin-sensitized photooxidation of ascorbic acid has

been carried out by flash photolysis (Heelis et al 1981) ESR spectrometry has been

used to investigate the photosensitized formation of ascorbate radicals by riboflavin (Kim

et al 1993) The photochemical behavior of a system consisting of ascorbate ion (AHndash)

and riboflavin has been studied by Mancini et al (2000) and De La Rochette et al (2000

2003) The photosensitized processes were examined as a function of oxygen pressure

and the efficiency of RF induced degradation of AHndash

at various oxygen concentrations

was compared on the basis of the respective initial photosensitization quantum yields

(Table 2)

In this reaction a Type I photosensitization mechanism (Karlsen 1996) implies a

direct electron transfer between AHndash and the RF triplet-excited state followed by the

oxidation of semioxidized ascorbyl radical (AHmiddot) by molecular oxygen or some other

reactive species On the contrary in a Type II photosensitization mechanism singlet

oxygen is produced directly by energy transfer from the RF triplet-excited state to

molecular oxygen and the singlet oxygen then oxidizes the AHndash Thus by irradiating

under increasing oxygen pressure it is possible to control the relative prevalence and

efficiency of Type I or Type II mechanisms The absence of a linear relationship between

the quantum yields of ascorbate degradation and oxygen concentration indicates that the

photosensitization mechanism involved in not exclusively Type II

24

Table 2 Initial quantum yield (φ) for ascorbate (AHndash) degradation during

photosensitization by RF (35 microM) in solutions irradiated at 365 nm and

37ordmC

O2 103 times φ (AH

ndash)a

0

5

20

14 plusmn 06

1670 plusmn 220

1940 plusmn 200

a Data are the mean plusmn SD of three independent experiments

25

In the presence of RF and O2 the quantum yields for degradation of ascorbate ion

have been found to be greater than one suggesting the participation of chain reactions

initiated by the ascorbyl radical as given by the following reactions

3RF + AH

ndash rarr RFmiddot

ndash + AHmiddot (211)

AHmiddot + O2 rarr A + HO2middot (212)

HO2middot + AHndash rarr H2O2 + AHmiddot (213)

The generation of the ascorbyl radical by the reaction between the RF excited-

triplet state and the ascorbate ion (Eq 211) is the only step that requires the absorption of

photons (to form the excited-triplet state of RF) The subsequent reactions (Eqs 212 and

213) are independent of light and lead to further degradation of the ascorbate ion In the

presence of transition metal ions such as Fe3+

in trace amounts in the buffer solution

containing RF and ascorbate ions further oxidation of ascorbate ion could also occur As

a result the reduced form of the metal ion (ie Fe2+

) can be generated by the metal

catalyzed oxidation of ascorbate ion This has been confirmed by the significant decrease

in the AHndash photooxidation quantum yield in the presence of the metal chelator EDTA

which inactivates the trace amounts of iron present in the buffer solution The quantum

yields for the photosensitized oxidation of ascorbate ion are decreased twofold at 20 O2

and fourfold at 5 O2 concentration in the presence of EDTA (Silva and Quina 2006)

Amino acids have been found to affect the photosensitized oxidation of ascorbic acid

(Jung et al 1995)

214 Ascorbic Acid Interaction with Other Water-Soluble Vitamins

The stability of ascorbic acid is reported to be enhanced in syrups containing B-

complex vitamins (Connors et al 1986) This may be due to the increased viscosity of

the syrups inhibiting the oxidation of ascorbic acid The rate of photolysis in solution

26

containing cyanocobalamin and ascorbic acid is reported to decrease with an increase in

pH (Ansari et al 2004) where as use of certain halide salts has been reported to be

beneficial in stabilizing pharmaceutical products and dietary supplements when vitamin

B12 and vitamin C are combined in solution (Ichikawa et al 2005) When a solution of

multivitamins is exposed to light it is reported that organic peroxidases are generated and

the concentration of ascorbic acid decreases (Lavoie et al 2004)

22 ASSAY OF ASCORBIC ACID

Recent accounts of the development and application of analytical methods to the

determination of ascorbic acid in pharmaceuticals biological samples and food materials

are reported in the literature (Rumsey and Levine 2000 Halver and Felton 2001 Moffat

et al 2004 Ball 2006 Sheraz et al 2007 Eitenmiller et al 2008 Salkic and Kubicek

2008) Most of these methods are based on the application of spectrophotometric

fluorimetric and chromatographic techniques to suit the requirements of a particular assay

and are summarized below

221 Spectrophotometric Methods

Spectrophotometric methods are the most widely used methods for the assay of

ascorbic acid in aqueous solution Ascorbic acid exhibits strong absorption in the

ultraviolet region (absorption maxima 243 nm at pH 2 and 265 nm at pH 4ndash10 OrsquoNeil

2001 Moffat et al 2004 British Pharmacopoeia 2009) This is the basis of

spectrophotometric methods for the determination of the vitamins in pure solutions and in

sample preparations where no interference is observed from UV absorbing impurities

The value of A (1 1 cm) at the analytical wavelength of 245 nm (pH 20) is high (695)

which makes the method very sensitive for the determination of mg quantities of the

27

vitamin Treatment of the material to be analyzed with ascorbic acid oxidase is often used

as a blank to correct for the presence of interfering substances in biological samples (Liu

et al 1982) A spectrophotometric method for the determination of ascorbic acid in

pharmaceuticals by background correction (245 nm) has been reported (Verma et al

1991) The direct determination of ascorbic acid in mixtures involves the use of 22prime-

dipyridyl as a colorimetric reagent The method is based on the reduction of Fe (III) by

ascorbic acid to Fe (II) which reacts with 2 2prime-dipyridyl to form a colored complex

(absorption maximum 510 nm) that can be used for quantitative determination (Margolis

and Schmidt 1996) A spectrophotometric method has been developed for the

determination of ascorbic acid and its oxidation product dehydroascorbic acid in

biological samples (Moeslinger et al 1995) A sensitive method has been reported for

the determination of ascorbic acid in pharmaceutical formulations and fruit juices by

interaction with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (Br-PADAP) (Ferreira

et al 1997) A novel UV method has been developed for the analysis of ascorbic acid in

methanol at 245 nm in various formulations (Zeng et al 2005)

Ascorbic acid in aqueous solutions has been assayed at 244 nm (pH ~2) (Ogata

and Kosugi 1969) 245 nm (pH 35) (Blaugh and Hajratwala 1972) 264 nm (pH 7)

(Salkic et al 2007) 265 nm (pH 7) (Hashmi 1973) 275 nm (pH 41 and 70) (Heelis et

al 1981) 265 nm (pH 7) (Al-Meshal and Hassan 1982) 245 nm (pH ~2) (Verma et al

1991) and 265 nm (pH ~7) (Erb et al 2004) Dehydroascorbic acid and 23-

diketogulonic acid do not significantly absorb in this region (Pelletier 1985 Davies et

al 1991 Rumsey and Levine 2000) and therefore do not interfere with the assay of

ascorbic acid in degraded solutions

28

222 Fluorimetric Methods

Fluorimetry is a highly sensitive technique for the determination of fluorescent

compounds or fluorescent derivatives of non-fluorescent compounds The technique has

been used for the detection of microg quantities of ascorbic acid Methods based on

fluorimetric (Kampfenkel et al 1995) and chemiluminescence detection (Zhang and

Chen 2000) provide highly sensitive methods for the determination of ascorbic acid in

plant and other materials

223 Mass Spectrometric Methods

Conventional and isotope mass spectrometric techniques have also been used for

the analysis of ascorbic acid Isotope ratio mass spectrometry is particularly useful and

sensitive when 13

C ascorbic acid is used as a standard in the analysis of complex matrices

(Gensler et al 1995)

224 Chromatographic Methods

High-performance liquid chromatographic (HPLC) methods have extensively

been employed for the determination of ascorbic acid in biological samples These

methods include ion exchange reversed phase and ion-pairing HPLC protocols

Spectrophotometric fluorimetric and electrochemical detection has been used in the

HPLC analysis of ascorbic acid The electrochemical detection is used for the

simultaneous determination of ascorbic acid dehydroascorbic acid and their isomers and

derivatives A number of HPLC methods have been developed for the detection and

determination of ascorbic acid and its oxidation products and derivatives in biological

samples and plant materials (Tsao and Young 1985 Tangney 1988 Dabrowski and

Huiterleitner 1989 Thomson and Trenerry 1995 Kimoto et al 1997 Kall and

29

Anderson 1999 Rumelin et al 1999 Lykkesfeldt 2000 Zhang et al 2000 Pastore et

al 2001 Frenich et al 2005) The limit of detection of ascorbic acid in plasma or urine

with UV detection lies in the range of 100-120 microg (Liau et al 1993 Manoharan and

Schwille 1994) Fluorescence detection of ascorbic acid and dehydroascorbic acid in

plasma and its comparison with coulometric detection has been reported (Tessier et al

1996) A liquid chromatography-diode-array detection (LCndashDAD) method has been

reported for the determination of 10 water-soluble and 10 fat-soluble vitamins including

ascorbic acid in pharmaceutical preparations with a coefficient of variation lt 65

(Konings 2006)

Liquid chromatography methods based on precolumn and o-phenylenediamine

(OPD) derivatization have been used for the determination of total vitamin C and total

isovitamin C in foods and dehydro forms of the vitamin Isoascorbic acid has been used

as an internal standard in the analysis (Speek et al 1985 Vanderslice et al 1990

Dodsun et al 1992 Vanderslice and Higgs 1988 1993 Hagg et al 1994 1995) The

limits of detection of ascorbic acid by HPLC using different detectors are in the range of

16ndash400 microgl (Capellmann and Bolt 1992 Iwase and Ono 1994 Karatepe 2004)

225 Enzymatic Methods

Enzymatic methods using ascorbate oxidase are specific and have the advantage

of selectively measuring the biological activity of ascorbic acid in serum or plasma (Liu

et al 1982) Ascorbate oxidase and OPD derivatization has been used to develop a rapid

automated method for the routine assay of ascorbic acid in serum and plasma The

method has a sample throughput of 100h (Ihara et al 2000)

30

226 Commercial Kits for Clinical Analysis

Commercial kits (eg Immunodiagnostic Germany Biovision USA) are also

used for the determination of ascorbic acid in biological samples (serum or plasma) in

clinical laboratories

227 Analysis in Creams

The general methods for the analysis of active ingredients and excipients in

cosmetic products including creams are described by Salvador and Chisvert (2007)

Ascorbic acid and derivatives in creams have been determined by liquid chromatography

(Irache et al 1993 Varvaresou et al 2006) gas chromatography-mass spectrometry

(Leveque et al 2005) and electrochemical methods (Beissenhirtz et al 2003 Guitton et

al 2007)

CHAPTER III

FORMULATION AND

STABILITY OF CREAM

PREPARATIONS

32

31 FORMULATION OF CREAM PREPARATIONS

Traditionally emulsions have been defined as dispersions of macroscopic droplets

of one liquid in another liquid with a droplet diameter approximately in the range of 05-

100 microm (Becher 1965) According to the definition of International Union of Pure and

Applied Chemistry (IUPAC) (1971) ldquoIn an emulsion liquid droplets and or liquid

crystals are dispersed in a liquidrdquo

Creams are semisolid emulsions intended for external applications They are often

composed of two phases Oil-in-water (ow) emulsions are most useful as water-washable

bases whereas water-in-oil (wo) emulsions are emollient and cleansing agents The

active ingredient is often dissolved in one or both phases thus creating a three-phase

system Patients often prefer a wo cream to an ointment because the cream spreads more

readily is less greasy and the evaporating water soothes the inflamed tissue OW creams

(vanishing creams) rub into the skin the continuous phase evaporates and increases the

concentration of a water-soluble drug in the adhering film The concentration gradient for

drug across the stratum corneum therefore increases promoting percutaneous absorption

(Barry 2002 Betageri and Prabhu 2002)

The various factors involved in the formulation of emulsions and topical products

have been discussed by Block (1996) Barry (2002) and Jain et al (2006) and are briefly

presented in the following sections

311 Choice of Emulsion Type

Oil-in-water emulsions are used for the topical application of water-soluble drugs

mainly for local effect They do not have the greasy texture associated with oily bases

and are therefore pleasant to use and easily washed from skin surfaces Moisturizing

33

creams designed to prevent moisture loss from the skin and thus inhibit drying of the

stratum corneum are more efficient if formulated as ow emulsions which produce a

coherent water-repellent film

312 Choice of Oil Phase

Many emulsions for external use contain oils that are present as carriers for the

active ingredient It must be realized that the type of oil used may also have an effect both

on the viscosity of the product and on the transport of the drug into the skin (Barry

2002) One of the most widely used oils for this type of preparation is liquid paraffin

This is one of a series of hydrocarbons which also includes hard paraffin soft paraffin

and light liquid paraffin They can be used individually or in combination with each other

to control emulsion consistency This will ensure that the product can be spread easily but

will be sufficiently viscous to form a coherent film over the skin The film-forming

capabilities of the emulsion can be further modified by the inclusion of various waxes

such as bees wax carnauba wax or higher fatty alcohols

313 Emulsion Consistency

A consideration of the texture or feel of a product intended for external use is

important A wo preparation will have a greasy texture and often exhibits a higher

apparent viscosity than ow emulsions This fact imparts a feeling of richness to many

cosmetic formulations Oil-in-water emulsions will however feel less greasy or sticky on

application to the skin will be absorbed more readily because of their lower oil content

and can be more easily washed from skin surface Ideally emulsions should exhibit the

rheological properties of plasticity pseudoplasticity and thixotropy Emulsions of high

apparent viscosity for external use (cream) are of a semisolid consistency There are

34

several methods by which the rheological properties of an emulsion can be controlled

(Billany 2002)

314 Choice of Emulsifying Agent

The choice of emulgent to be used would depend on factors such as its

emulsifying ability route of administration and toxicity Most of the non-ionic emulgents

are less irritant and less toxic than their anionic and cationic counter parts Some

emulgents such as the ionic alkali soaps often have a high pH and are thus unsuitable for

application to broken skin Even in normal intact skin with a pH of 55 the application of

such alkaline materials can cause irritation Some emulsifiers in particular wool fat can

cause sensitizing reactions in susceptible people The details of various types of

emulsifying agents are available in the literature (Betageri and Prabhu 2002 Billany

2002 Swarbrick et al 2006)

315 Formulation by the HLB Method

The physically stable emulsions are best achieved by the presence of a condensed

layer of emulgent at the oil water interface and the complex interfacial films formed by a

blend of an oil-soluble emulsifying agent with a water-soluble one produces the most

satisfactory emulsions

It is possible to calculate the relative quantities of the emulgents necessary to

produce the most physically stable emulsions for a particular formulation with water

combination This approach is called the hydrophilic-lipophilic balance (HLB) method

Each surfactant is allocated an HLB number representing the relative properties of the

lipophilic and hydrophilic parts of the molecule High numbers (up to a theoretical

number of 20) therefore indicates a surfactant exhibiting mainly hydrophilic or polar

35

properties whereas low numbers represent lipophilic or non-polar characteristics Each

type of oil requires an emulgent of a particular HLB number in order to ensure a stable

product For an ow emulsion the more polar the oil phase the more polar must be the

emulgent system (Billany 2002 Im-Emsap et al 2002 Swarbrick et al 2006)

316 Concept of Relative Polarity Index

In the ingredient selection in cosmetic formulations a new concept of relative

polarity index (RPI) has been presented (Wiechers 2005) The physicochemical

characteristics of the ingredients determine their skin delivery to a greater extent than the

formulation type The cosmetic formulation cannot change the chemistry of the active

molecule that needs to penetrate to a specific site within the skin However the

formulation type can be selected based on the polarity of the active ingredient and the

desired site of action for the active ingredient For optimum skin delivery the solubility of

the active ingredient needs to be as high as possible (to create a large concentration

gradient) and as small as possible (to create a large partition coefficient) To achieve this

it is necessary to determine the following parameters

The total amount dissolved in the formulation that is available for skin penetration

the higher this amount the more will penetrate until a solution concentration is

reached in the skin therefore a high absolute solubility in the formulation is required

The polarity of the formulation relative to that of the stratum corneum if an active

ingredient dissolves better in the stratum corneum than in the formulation then the

partition of the active ingredient will favour the stratum corneum therefore a low

(relative to that in the stratum corneum) solubility in the formulation is required

(Wiechers 2005)

36

These requirements can be met by considering the concept of RPI (Wiechers

2003 2005) In this systematic approach it is essential to consider the stratum corneum

as another solvent with its own polarity The stratum corneum appears to behave very

similarly to and in a more polar fashion than butanol with respect to its solubilizing

ability for active ingredients (Scheuplein and Blank 1973) The polarity of stratum

corneum as expressed by its octanol water partition coefficient is 63

The relative polarity index may be used to compare the polarity of an active

ingredient with both that of the skin and that of the oil phase of a cosmetic formulation

predominantly consisting of emollients It may be visualized as a vertical line with a high

polarity at the top and a high lipophilicity at the bottom The polarity is expressed as the

log10 of the octanol water coefficient For example the relative polarity index values of

glycerin and isostearyl isostearate are -176 and 2698 respectively (Wiechers 2005) In

order to use the concept of the relative polarity index three numbers (on log10 scale) are

required

The polarity of the stratum corneum is set at 08 However in reality this value will

change with the hydration state of the stratum corneum that is determined in part by

the external relative humidity (Bonwstra et al 2003)

The polarity of the active molecule

The polarity of the formulation

For multiphase or multipolarity systems like emulsions the active ingredient is dissolved

in the phase For example in an ow emulsion where a lipophilic active ingredient is

dissolved in the oil phase it is the polarity of the homogenous mixture of the lipophilic

active ingredient and internal oil For the same lipophilic active in a wo emulsion it is

37

the polarity of the homogenous mixture of the lipophilic active ingredients and external

oil For water-soluble active ingredients it is the polarity of the homogenous mixture of

the hydrophilic active ingredient and the aqueous phase regardless whether it is internal

(wo emulsions) or external (ow emulsions)

Once the active ingredient and the formulation type have been chosen it is

necessary to create the delivery system that will effectively deliver the molecule The

concept of relative polarity index allows the formulator to select the polarity of the phase

in which the active ingredient is incorporated on the basis of its own properties and those

of the stratum corneum In order to achieve maximum delivery the polarity of the active

ingredient and the stratum corneum need to be considered In order to improve the skin

delivery of active ingredients the first step involves selecting a primary emollient with a

polarity close to that of the active ingredient in which it will have a high solubility The

second step is to reduce the solubility of the active ingredient in the primary emollient via

the addition of a secondary emollient with a different polarity and therefore lower

solubility for the active ingredient This approach has shown a 3-4 fold increase in skin

penetration with out increasing the amount of active ingredients in the formulation

(Wiechers 2005)

32 FORMULATION OF ASCORBIC ACID CREAMS

Ascorbic acid is a water-soluble material and is included frequently in skin care

formulations to restore skin health It is very unstable and is easily oxidized in aqueous

solution This vitamin is known to be a reducing agent in biological systems and causes

the reduction of both oxygen- and nitrogen- based free radicals (Higdon and Frei 2002)

It can also act as a co-antioxidant with the tocopheroxyl radical to regenerate alpha-

38

tocopherol (Packer et al 1979 Buettner 1993 Peyrat-Maillard et al 2001) In this

reaction the two vitamins act synergistically Alpha-tocopherol first functions as the

primary antioxidant that reacts with an organic free radical Thereafter ascorbic acid

reacts with the free radical tocopheroxyl to general alpha-tocopherol In physiological

conditions the ascorbyl radical formed by regenerating tocopherol is then converted back

to ascorbate by the redox cycle (Davies et al 1991) The interaction of ascorbic acid

with a redox partner such as alpha-tocopherol has been found useful to slow its oxidation

and prolong its action

The instability of ascorbic acid makes this antioxidant active ingredient a

formulation challenge to deliver to the skin and retain its effective form In addition to its

use in combination with alpha-tocopherol in cream formulations the stability of ascorbic

acid may be improved by its use in the form of a fatty acid ester such as ascorbyl

palmitate Ascorbyl palmitate has been used in thixogel formulations and is typically

incorporated into the mineral oil phase Preliminary experiments have shown that it could

be slowly released from the starch-oil emulsion matrix and act as an antioxidant (Wille

2005)

Various physical and chemical factors involved in the formulation of cream

preparations have been discussed in the above sections For polar and air light sensitive

compounds such as ascorbic acid it is important to consider factors such as the choice of

formulation ingredients polar character of formulation HLB value pH viscosity etc to

achieve stability

39

33 STABILITY OF CREAMS

331 Physical Stability

The most important consideration with respect to pharmaceutical and cosmetic

emulsions (creams) is the stability of the finished product The stability of a

pharmaceutical emulsion is characterized by the absence of coalescence of the internal

phase absence of creaming and maintenance of elegance with respect to appearance

odor color and other physical properties An emulsion is a dynamic system however

any flocculation and resultant creaming represent potential steps towards complete

coalescence of the internal phase In pharmaceutical emulsions creaming results as a lack

of uniformity of drug distribution and poses a problem to the pharmaceutical

compounder Another important factor in the stabilization of emulsions is phase inversion

which involves the change of emulsion type from ow to wo or vice versa and is

considered as a case of instability The four major phenomena associated with the

physical instability of emulsions are flocculation creaming coalescence and breaking

These have been discussed by Garti and Aserin (1996) Im-Emsap et al (2002) and Sinko

(2006)

332 Chemical Stability

The instability of a drug may lead to the loss of its concentration through a

chemical reaction under normal or stress conditions This results in a reduction of the

potency and is a well-recognized cause of poor product quality The degradation of the

drug may make the product esthetically unacceptable if significant changes in color or

odor have occurred The degradation product may also be a toxic substance The various

pathways of chemical degradation of a drug depend on the structural characteristics of the

40

drug and may involve hydrolysis dehydration isomerization and racemization

decarboxylation and elimination oxidation photodegradation drug-excipients and drug-

drug interactions Factors determining the chemical stability of drug substances include

intrinsic factors such as molecular structure of the drug itself and environmental factors

such as temperature light pH buffer species ionic strength oxygen moisture additives

and excipients The application of well-established kinetic principles may throw light on

the role of each variable in altering the kinetics of degradation and to provide valuable

insight into the mechanism of degradation (Baertschi and Alsante 2005 Yoshioka and

Stella 2002 Lachman et al 1986) The chemical stability of individual components

within an emulsion system may be very different from their stability after incorporation

into other formulation types For example many unsaturated oils are prone to oxidation

and their degree of exposure to oxygen may be influenced by factors that affect the extent

of molecular dispersion (eg droplet size) This could be particularly troublesome in

emulsions because emulsification may introduce air into the product and because of the

high interfacial contact area between the phases (Barry 2002) The use of antioxidants

retards oxidation of unsaturated oils minimizes changes in color and texture and prevents

rancidity in the formulation Moreover they can retard the degradation of certain active

ingredients such as vitamin C (Vimaladevi 2005) The stability problems of dispersed

systems and the factors leading to these stability problems have been discussed by

Weiner (1996) and Lu and Flynn (2009)

333 Microbial Stability

Topical bases often contain aqueous and oily phases together with carbohydrates

and proteins and are susceptible to bacterial and fungal attack Microbial growth spoils

41

the formulation and is a potential toxic hazard Therefore topical formulations need

appropriate preservatives to prevent microbial growth and to maintain their quality and

shelf-life (Barry 2002 Arger et al 1996) Cream formulations may contain fats and oils

with high percentage of unsaturated linkages that are susceptible to oxidation degradation

and development of rancidity The addition of antioxidants retards oxidation of fats and

oils minimizes changes in color and texture and prevents rancidity in the formulation

Moreover they can retard the degradation of certain active ingredients such as vitamin C

These aspects in relation to dermatological formulations have been discussed by Barry

(1983 2002) and Vimaladevi 2005)

334 Stability of Ascorbic Acid in Liquid Formulations

Ascorbic acid is very unstable in aqueous solution Different workers have studied

the stability of ascorbic acid in liquid formulations (Connors et al 1986 Austria et al

1997) Its shelf-life can be prolonged by appropriate choice of vehicle and control of

other variables such as pH stabilizers temperature light and oxygen (Table 3)

Similarly the stability of various concentrations of ascorbic acid in solution form may

vary depending upon the type of solvent used (Table 4) (Connors et al 1986 Satoh et

al 2000 Lee et al 2004 Zeng et al 2005)

335 Stability of Ascorbic Acid in Emulsions and Creams

Ascorbic acid exerts several functions on skin such as collagen synthesis

depigmentation and antioxidant activity Ultraviolet radiation generates reactive oxygen

species (ROS) which produce some harmful effects on the skin including photocarcinoma

and photoaging In order to combat these problems ascorbic acid as an antioxidant has

42

Table 3 Effect of vehicles on the stability of ascorbic acid ( ascorbic acid remaining in

solutions after storage at room temperature) (Connors et al 1986)

Storage Time (days) No Vehicle

30 60 90 120 180 240 360

1 Corn Syrup 965 925 920 920 900 860 760

2 Sorbitol 990 990 990 970 960 925 890

3 4 Carboxymethyl

Cellulose

840 680 565 380 ndash ndash ndash

4 Glycerin 100 100 990 990 970 935 920

5 Propylene glycol 995 990 980 945 920 900 900

6 Syrup USP 100 100 980 980 930 900 880

7 Syrup 212 gL 880 810 775 745 645 590 440

8 25 Tragacanth 785 620 510 320 ndash ndash ndash

9 Saturated solution of

Dextrose

990 935 875 800 640 580 510

10 Distilled Water 900 815 745 675 405 185 ndash

11 50 Propylene glycol +

50 Glycerin

980 ndash 960 ndash 933 ndash ndash

12 25 Distilled Water +

75 Sorbo (70 solution

of Sorbitol)

955 954 ndash 942 930 ndash ndash

13 50 Glycerin + 50

Sorbo

982 984 978 ndash ndash 914 ndash

43

Table 4 Stability of various concentrations of ascorbic acid in water propylene glycol

and USP syrup at room temperature ( of ascorbic acid remaining in solution)

(Connors et al 1986)

Storage Time (days) Concentration

(mg ml)

Solvent

30 60 90 120 180 240 360

10 Water 930 840 820 670 515 410 ndash

50 Water 940 920 880 795 605 590 300

100 Water 970 930 910 835 705 680 590

10 Propylene glycol 100 985 980 975 960 920 860

50 Propylene glycol 100 970 980 980 980 965 935

100 Propylene glycol 100 100 100 100 990 100 925

10 Syrup 100 100 980 990 970 960 840

50 Syrup 100 100 100 100 990 100 960

100 Syrup 100 100 100 100 100 100 995

44

been used in various dosage forms and in different concentrations (Darr et al 1996

Gallarate et al 1999 Zhang et al 1999 Pinnell et al 2001 Lee et al 2004 Raschke

et al 2004 Elmore 2005 Farahmand et al 2006 Maia et al 2006) Ascorbic acid has

good photoprotective ability against UVA-mediated phototoxicity (Darr et al 1996) A

variety of formulations containing ascorbic acid or its derivatives have been studied in

order to evaluate their stability and delivery through the skin (Gallarate et al 1999

Zhang et al 1999 Ozer et al 2000 Pinnell et al 2001 Lee et al 2004 Raschke et al

2004 Farahmand et al 2006) Formulations containing derivatives of ascorbic acid are

found to be more stable than ascorbic acid but they do not produce the same effect as that

of the parent compound probably due to the lack of redox properties (Heber et al 2006)

Effective delivery of ascorbic acid through topical preparations is a major factor that

should be critically evaluated as it may be dependent upon the nature or type of the

formulation (Gallarate et al 1999 Pinnell et al 2001) The pH of the formulation

should be on the acidic side (~ pH 35) for effective penetration of the vitamin in the skin

(Pinnell et al 2001) and for its stabilization in the formulation (Gallarate et al 1999)

Some other antioxidants such as alpha-tocopherol ferulic acid and sodium metabisulphite

have also been used in combination with ascorbic acid for the purpose of its stabilization

in topical formulations and to produce some synergistic effects (Darr et al 1996 Lin et

al 2005 Maia et al 2006 Tournas et al 2006) Effect of some rheological properties

such as viscosity and dielectric constant on the stability of ascorbic acid in emulsions has

also been investigated (Connors et al 1986) Viscosity of the medium is an important

factor that should be considered for the purpose of ascorbic acid stability as higher

viscosity formulations have shown some degree of protection (Ozer et al 2000

45

Szymula 2005) Along with other factors formulation type also plays an important role in

the stability of ascorbic acid It is reported that ascorbic acid is more stable in emulsified

system as compared to aqueous solutions (Gallarate et al 1999 Lee et al 2004) In

multiemulsions ascorbic acid is reported to be more stable as compared to simple

emulsions (Gallarate et al 1999 Ozer et al 2000 Lee et al 2004 Farahmand et al

2006)

Ascorbic acid and its derivatives have been used in a variety of cosmetic

formulations as an antioxidant pH adjuster anti-aging and photoprotectant (Elmore

2005) The control of instability of ascorbic acid poses a significant challenge in the

development of cosmetic formulations It is also reported that certain metal ions or

enzyme systems effectively convert ascorbic acidrsquos antioxidant action to pro-oxidant

activity (Elmore 2005) Therefore utilization of an effective antioxidant system is

required to maintain the stability of vitamin C in various preparations (Zhang et al 1999

Pinnell et al 2001 Maia et al 2006) The chemical stability of ascorbic acid has been

studied in emulsions and creams by several workers (Darr et al 1996 Gallarate et al

1999 Lee et al 2004 Raschke et al 2004 Elmore 2005 Farahmand et al 2006)

however there is a lack of information on the photostability of ascorbic acid in cream

formulations

336 Stability Testing of Emulsions

The stability testing of emulsions (creams) may be carried out by performing the

following tests (Billany 2002)

46

3361 Macroscopic examination

The assessment of the physical stability of an emulsion is made by an

examination of the degree of creaming or coalescence occurring over a period of time

This involves the calculation of the ratio of the volume of the creamed or separated part

of the emulsion and the total volume A comparison of these values can be made for

different products

3362 Globule size analysis

An increase in mean globule size with time (coupled with a decrease in globule

numbers) indicates that coalescence is the cause of this behavior This can be used to

compare the rates of coalescence for a variety of emulsion formulations For this purpose

microscopic examination or electronic particle counting devices (coulter counter) or

laser diffraction sizing are widely used

3363 Change in viscosity

Many factors may influence the viscosity of emulsions A change in apparent

viscosity may result from any variation in globule size or number or in the orientation or

migration of emulsifier over a period of time

3264 Accelerated stability tests

In order to compare the relative stabilities of a range of similar products it is

necessary to speed up the processes of creaming and coalescence by storage at elevated

temperatures and then carrying out the tests described in the above sections

337 FDA guidelines for semisolid preparations

According to FDA draft guidelines to the industry (Shah 1997) semisolid

preparations (eg creams) should be evaluated for appearance clarity color

47

homogencity odour pH consistency viscosity particle size distribution (when feasible)

assay degradation products preservative and antioxidant content (if present) microbial

limits sterility and weight loss when appropriate Additionally samples from

production lot or approved products are retained for stability testing in case of product

failure in the field Retained samples can be tested along with returned samples to

ascertain if the problem was manufacturing or storage related Appropriate stability data

should be provided for products supplied in closed-end tubes to support the maximum

anticipated use period during patient use and after the seal is punctured allowing product

contact with the cap cap lever Creams in large containers including tubes should be

assayed by sampling at the surface top middle and bottom of the container In addition

tubes should be sampled near the crimp The objective of stability testing is to determine

whether the product has adequate shelf-life under market and use conditions

48

OBJECT OF PRESENT INVESTIGATION

Ascorbic acid (vitamin C) is extensively used as a single ingredient or in

combination with vitamin B complex and other vitamins in the form of drops injectables

lotions and syrups It is an ingredient of anti-aging cosmetic products alone or along with

alpha-tocopherol (vitamin E) Ascorbic acid exerts several functions on the skin as

collagen synthesis depigmentation and antioxidant activity It protects the signs of

degenerative skin conditions caused by oxy-radical damage In solutions and creams

ascorbic acid is susceptible to air and light and undergoes oxidative degradation to

dehydroascorbic acid and inactive products The degradation is influenced by

temperature viscosity and polarity of the medium and is catalysed by metal ions

particularly Cu+2

Fe+2

and Zn+2

One of the major problems faced in cream preparations is the instability of

ascorbic acid as it may be exposed to light during formulation manufacturing and

storage and the possibility of photochemical degradation can not be neglected The

behaviour of ascorbic acid in light is of particular interest since no systematic kinetic

studies have been conducted on its photodegradation in these preparations under various

conditions The study of the formulation variables such as emulsifier humectants and pH

may throw light on the photostabilization of ascorbic acid in creams

The main object of this investigation is to study the behaviour of ascorbic acid in

cream preparations on exposure to UV light in the pharmaceutically useful pH range An

important aspect of the work is to study the interaction of ascorbic acid with other

vitamins such as riboflavin nicotinamide and alpha-tocopherol and the effect of certain

stabilizers such as citric acid tartaric acid and boric acid on its photodegradation In

49

addition it is intended to study the photolysis of ascorbic acid in organic solvents to

evaluate the effect of solvent characteristics (eg dielectric constant and viscosity) on the

stability of the vitamin The study of all these aspects may provide useful information to

improve the photostability and efficacy of ascorbic acid in cream preparations

An outline of the proposed plan of work is presented as follows

1 To prepare a number of oil-in-water cream formulations based on different

emulsifying agents and humectants containing ascorbic acid alone and in

combination with other vitamins and stabilizing agents

2 To perform photodegradation studies on ascorbic acid in creams using a UV

irradiation source with emission corresponding to the absorption maximum of

ascorbic acid

3 To identify the photoproducts of ascorbic acid in creams using chromatographic

and spectrophotometric methods

4 To apply appropriate and validated analytical methods for the assay of ascorbic

acid alone and in combination with other vitamins and stabilizing agents

5 To study the effect of solvent characteristics such as dielectric constant and

viscosity on the photolysis of ascorbic acid in aqueous and organic solvents

6 To evaluate the kinetics of photodegradation of ascorbic acid and its interactions

with other vitamins (riboflavin nicotinamide and alpha-tocopherol) in creams

7 To evaluate the effect of carbon chain length of the emulsifying agent and the

viscosity of the humectant on the photodegradation of ascorbic acid

50

8 To develop relationships between the rate of photodegradation of ascorbic acid

and the concentration pH carbon chain length of emulsifier viscosity of the

creams

9 To determine the effect of compounds such as citric acid tartaric acid and boric

acid used as stabilizing agents on the rate of photodegradation and stabilization

of ascorbic acid in creams

10 To present reaction schemes for the photodegradation of ascorbic acid and its

interactions with other vitamins

CHAPTER IV

MATERIALS

AND

METHODS

52

41 MATERIALS

Vitamins and Related Compounds

L-Ascorbic Acid vitamin C (5R)-5-[(1S)-12-dihydroxyethyl]-34-dihydroxyfuran-2(5H)-

one Merck

C6H8O6 Mr 1761

Dehydroascorbic Acid L-threo-23-hexodiulosonic acid γ-lactone Sigma

C6H6O6 Mr 1741

23-Diketogulonic Acid

C6H8O7 Mr 192

It was prepared according to the method of Homann and Gaffron (1964) by the

hydrolysis of dehydroascorbic acid

Riboflavin vitamin B2 (310-dihydro-78-dimethyl-10-[(2S3S4R)-2345-

tetrahydroxypentyl] benzopteridine-24-dione) Merck

C17H20N4O6 Mr 3764

Nicotinamide vitamin B3 (pyridine-3-carboxamide) Merck

C6H6N2O Mr 1221

Alpha-Tocopherol vitamin E ((2R)-2578-tetramethyl-2-[(4R8R)-4812-

trimethyltridecyl]-34-dihydro-2H-1-benzopyran-6-ol) Merck

C29H50O2 Mr 4307

Formylmethylflavin (78-dimethyl-10-formylmethylisoalloxazine)

C14H12N4O3 Mr 2843

53

Formylmethylflavin was synthesized according to the method of Fall and Petering

(1956) by the periodic acid oxidation of riboflavin It was recrystallized from absolute

methanol dried in vacuo and stored in the dark in a refrigerator

Lumichrome (78-dimethylalloxazine) Sigma

C12H10N4O2 Mr 2423

It was stored in the dark in a desiccator

Stabilizers

Boric Acid orthoboric acid Merck

H3BO3 Mr 618

Citric Acid 2-hydroxypropane-123-tricarboxylic acid Merck

C6H8O7H2O Mr 2101

L-Tartaric acid [(2R3R)-23-dihydroxybutanedioic acid] Merck

C4H6O6 Mr 1501

Emulsifying Agents

Stearic Acid (95) octadecanoic acid Merck

C18H36O2 Mr 2845

Palmitic Acid hexadecanoic acid Merck

C16H32O2 Mr 2564

Myristic Acid tetradecanoic acid Merck

C14H28O2 Mr 2284

Cetyl alcohol hexadecan-1-ol Merck

C16H34O Mr 2424

54

Humectants

Glycerin glycerol (propane-123-triol) Merck

C3H8O3 Mr 921

Propylene glycol (RS)-propane-12-diol Merck

C3H8O2 Mr 7610

Ethylene glycol ethane-12-diol Merck

C2H6O2 Mr 6207

Potassium Ferrioxalate Actinometry

Potassium Ferrioxalate

K3Fe(C2O4)3 3H2O Mr 4912

Potassium Ferrioxalate was prepared according to the method of Hatchard and

Parker (1956) Three volumes of 15 M potassium oxalate was mixed with one volume of

15 M ferric chloride with vigorous stirring The yellow green precipitate of potassium

ferrioxalate was recrystallized twice from water dried at 45 ordmC and stored in the dark in a

desiccator

Reagents

All the reagents and solvents used were of analytical grade obtained from BDH

Merck

Water

Freshly boiled distilled water was used throughout the work

55

42 METHODS

421 Cream Formulations

On the basis of the various cream formulations reported in the literature (Block

1996 Flynn 2002 Betageri and Prabhu 2002 Vimaladevi 2005 EIRI Board Lu and

Flynn 2009) the following basic formula was used for the preparation of oil-in-water

creams containing ascorbic acid

Oil phase Percentage (ww)

Emulsifier

Myristic palmitic stearic acid

Cetyl alcohol

120

30

Aqueous phase

Humectant

Ethylene glycol propylene glycol glycerin

50

Active ingredient

Ascorbic acid

20 (0114 M)

Neutralizer

Potassium hydroxide

10

Continuous phase

Distilled water

QS

Additional ingredientsa

Vitamins

Riboflavin (Vitamin B2)

Nicotinamide (Vitamin B3)

Alpha-Tocopherol (Vitamin E)

0002ndash001 (053ndash266times10ndash4

M)

028ndash140 (0023ndash0115 M)

017ndash086 (0395ndash200times10ndash2

M)

Stabilizers

Citric acid

Tartaric acid

Boric acid

010ndash040 (0476ndash190times10ndash2

M)

010ndash040 (067ndash266times10ndash2

M)

010ndash040 (0016ndash0065 M)

a The vitamin stabilizer concentrations used were found to be effective in promotion

inhibition of photodegradation of ascorbic acid in cream formulations

56

422 Preparation of Creams

The emulsifiers were melted at 70ndash80 ordmC in a glass jar immersed in a water bath

Ascorbic acid was separately dissolved in a small portion of distilled water Potassium

hydroxide and humectant were dissolved in the remaining portion of water and mixed

with the oily phase with constant stirring until the formation of a thick white mass It was

cooled to ~40 ordmC and the ascorbic acid solution was added The thick mass was mixed

using a mechanical mixer with a glass stirrer at 1000 rpm for 5 minutes The pH of the

cream was adjusted to the desired value and the contents again mixed for 10 minutes at

500 rpm All the creams were prepared under uniform conditions to maintain their

individual physical characteristics and stored at room temperature in airtight glass

containers protected from light

In the case of other vitamins nicotinamide was dissolved along with ascorbic acid

in water and added to the cream Riboflavin or alpha-tocopherol were directly added to

the cream and mixed thoroughly according to the procedure described above

In the case of stabilizing agents (citric tartaric and boric acids) the individual

compounds were dissolved in the ascorbic acid solution and added to the cream followed

by the procedure described above

The details of the various cream formulations used in this study are given in

chapters 5ndash7

57

423 Thin-Layer Chromatography (TLC)

The following TLC systems were used for the separation and identification of

ascorbic acid and photodegradation products

Adsorbent a) Silica gel GF 254 (250-microm) precoated plates

(Merck)

Solvent systems S1 acetic acid-acetone-methanol-benzene

(552070 vv) (Ganshirt and Malzacher 1960)

S2 ethanol-10 acetic acid-water (9010 vv)

(Bolliger and Konig 1969)

S3 acetonitrile-butylnitrile-water (66332 vv)

(Saari et al 1967)

Temperature 25ndash27 ordmC

Location of spots Ascorbic acid UV light 254 nm (Uvitec lamp

UK)

Dehydroascorbic acid Spray with a 3 aqueous

phenylhydrazine hydrochloride solution

424 pH Measurements

The measurements of pH of aqueous solutions and cream formulations were

carried out using an Elmetron LCD display pH meter (modelndashCP501 sensitivity plusmn 001

pH units) (Poland) with a combination electrode The electrode was calibrated

automatically in the desired pH range (25 ordmC) using the following buffer solutions

58

Phthalate pH 4008

Phosphate pH 6865

Disodium tetraborate pH 9180

The electrode was immersed directly into the cream (British Pharmacopoeia

2009) kept for few seconds to equilibrate and the pH adjusted in the range of 40ndash70

with phosphoric acid sodium hydroxide solution

425 Ultraviolet and Visible Spectrometry

The absorbance measurements and spectral determinations were performed on

Shimadzu UVndashVisible recording spectrophotometer (model UVndash1601) using matched

silica cells of 10 mm path length The cells were employed always in the same orientation

using appropriate control solutions in the reference beam The baseline was automatically

corrected by the built-in baseline memory at the initializing period Auto-zero adjustment

was made by a one-touch operation The instrument checked the wavelength calibration

(6561 nm) using the deuterium lamp at the initializing period The absorbance scale was

periodically checked using the following calibration standard (British Pharmacopoeia

2009)

0057ndash0063 gl of potassium dichromate in 0005 M sulphuric acid

The specific absorbance [A(1 1 cm)] of the solution should match the

following values with the stated limit of tolerance

Wavelength

(nm)

Specific absorbance

A (1 1 cm)

Maximum

tolerance

235 1245 1229ndash1262

257 1445 1428ndash1462

313 486 470ndash503

350 1073 1056ndash109

430 159 157ndash161

59

426 Photolysis of Ascorbic Acid

4261 Creams

A 2 g quantity of the cream was uniformly spread on several rectangular glass

plates (5 times 15 cm) covered with a 1 cm tape on each side to give a 1 mm thick layer The

plates were irradiated in a dark chamber using a Philips 30 watt TUV tube (100

emission at 254 nm the wavelength absorbed by ascorbic acid at pH 4ndash7) fixed

horizontally at a distance of 30 cm from the centre of the plates Each plate was removed

at appropriate interval and the cream was subjected to spectrophotometric assay and

chromatographic examination

4262 Aqueous and organic solvents

A 10ndash3

M solution of ascorbic acid (50 ml) prepared in water (pH 70 005 M

phosphate buffer) or in an organic solvent in a 100 ml beaker (Pyrex) was placed in a

water bath maintained at 20 plusmn 1 ordmC The solution was irradiated with the Philips 30 watt

TUV tube in a dark chamber as stated above Samples were withdrawn at appropriate

intervals for assay and chromatography

4263 Storage of creams in dark

In order to determine the stability of various cream formulations in the dark

samples were stored at room temperature in a cupboard protected from light for a period

of three months The samples were analyzed periodically for the content of ascorbic acid

and the presence of any degradation product

427 Measurement of Light Intensity

The potassium ferrioxalate actinometry was used for the measurement of light

intensity of the radiation source employed in this work This actinometer has been

60

developed by Parker (1953) and Hatchard and Parker (1956) and is considered as the

most useful actinometer over a wide range of wavelengths (254ndash577 nm) It has been

used by Holmstrom and Oster (1961) Byrom and Turnbull (1967) McBride and Moore

(1967) Ahmad (1968) Ahmad (1978) Ahmad et al (2004a 2004b 2005 2006a

2006b 2008 2009ab) Fasihullah (1988) Vaid (1998) Ansari (2002) and Ahmad (2009)

for the measurement of light intensity

The irradiation of potassium ferrioxalate solutions in sulphuric acid results in the

reduction of ferric ion to ferrous ion according to the following reaction

2Fe [(C2O4)3]3ndash

rarr 2 Fe (C2O4) + 3 (C2O4)2ndash

+ 2CO2 (31)

The amount of Fe2+

ions formed in the reaction may be determined by

complexation with 110-phenanthroline to give a red colored complex The absorbance of

the complex is measured at 510 nm

428 Procedure

An oxygen free 0006 M solution of potassium ferrioxalate (2947 gl) in 01 N

H2SO4 was placed in the reaction vessel and irradiated with the lamp used for the

photolysis of riboflavin The irradiation was carried out under nitrogen (90ndash120

bubblesminute) which also caused stirring of the solution The temperature of the

reaction vessel was maintained at 25 plusmn 1 ordmC during the reaction

An aliquot of the photolysed solution (1ndash2 ml) was pipetted out at suitable

intervals (up to 30 minutes) into a 10 ml volumetric flask to which was then added 09

ml of N H2SO4 + 1 ml (01) 110-phenanthroline + 05 ml buffer (60 ml N CH3COONa

+ 36 ml N H2SO4 made up to 100 ml with distilled water) The flask was made up

to the mark with distilled water (final pH 35) thoroughly shaken to mix the contents and

61

Fig 3 Spectral power distribution of TUV 30 W tube (Philips)

62

allowed to stand for one hour in the dark to develop the colorndashcomplex The absorbance

of the phenanthrolinendashferrous complex was measured in a 1 cm cell at 510 nm using the

appropriate solution as blank The amount of Fe2+

ions formed was determined from the

calibration graph The calibration graph was constructed in a similar manner using

several dilutions of 1 times 10ndash6

mole ml Fe2+

in 01 N H2SO4 (freshly prepared by dilution

from standardized 01 M FeSO4 in 01 N H2SO4) (Fig 8) The experimental value of the

molar absorptivity of Fe2+

complex as determined from the slope of the calibration graph

is equal to 111 times 104 M

ndash1 cm

ndash1 and is in agreement with the value reported by Parker

(1953) Using the values of the known quantum yield for ferrioxalate actinometer at

different wavelengths (Hatchard and Parker 1956) the number of Fe2+

ions formed

during photolysis the time of exposure and the fraction of the light absorbed by the

length of the actinometer solution employed the light intensity incident just inside the

front window of the photolysis cell can be calculated In the present case total absorption

of the light has been assumed

4281 Calculation

The number of Fe2+

ions formed during photolysis (nFe

2+) is given by the

equation

6023 times 1020

V1 V3 A Σ

n Fe

2+ =

V2 1 ε (32)

where V1 is the volume of the actinometer solution irradiated (ml)

V2 is the volume of the aliquot taken for analysis (ml)

V3 is the final volume to which the aliquot V2 is diluted (ml)

1 is the path length of the spectrophotometer cell used (1 cm)

A is the measured absorbance of the final solution at 510 nm

63

ε is the molar absorptivity of the Fe2+

complex (111 times 104 M

ndash1 cm

ndash1)

The number of quanta absorbed by the actinometer nabs can then be obtained as follows

n Fe

2+

Σ nabs = ф

(33)

where ф is the quantum yield for the Fe2+

formation at the desired wavelength

The number of quanta per second per cell nabs is therefore given by

Σ nabs 6023 times 1020

V1 V3 A nabs =

t =

ф V2 1 ε t (34)

where t is the irradiation time of the actinometer in seconds

The relative spectral energy distribution of the radiation source (Fig 3) shows

100 emission at 254 nm the wavelength used for the photolysis of ascorbic acid (λmax

265 nm at pH 4ndash7) The energy emitted by the radiation source at various wavelengths

can be calculated using the equation

1197 times 105

E (KJ molndash1

) = λ nm

(35)

The quantum efficiency of ferrioxalate actinometer at the wavelength absorbed by

ascorbic acid (ie 254 nm) is high although the sensitivity drops over 450 nm The

average intensity of the TUV tube used in this study was determined as 556 plusmn 012 times

1018

quanta sndash1

429 Viscosity Measurements

The viscosity of the cream formulations was measured with a Brookfield RV

viscometer (Model DV-II + Pro USA) The instrument was calibrated using the

manufacturerrsquos viscosity standard A 200 g quantity of the cream was placed in a beaker

and the spindle (TE) was dipped into the cream It was rotated at a speed of 06 rpm for

64

00

02

04

06

08

10

12

0 2 4 6 8 10 12

Concentration of Fe++

times 105 M

Ab

sorb

an

ce a

t 51

0 n

m

Fig 4 Calibration graph for the determination of K3Fe(C2O4)3

65

one minute and the viscosity was recorded at 25plusmn1 ordmC The test was repeated three times

to account for the experimental variability and the average viscosity was noted

4210 Assay Methods

42101 UV spectrophotometric method for the assay of creams containing ascorbic

acid alone

The creams were thoroughly mixed a quantity of 2 g was accurately weighed and

the assay of ascorbic acid was carried out by the UV method of Zeng et al (2005) In the

case of photodegraded creams (2 g) the material was completely removed from the glass

plate and transferred to a volumetric flask The method involved extraction of ascorbic

acid with methanol (3 times 10 ml) adjustment of the pH of combined methanolic solutions

to 20 (with H3PO4) dilution of the final solution with acidified methanol (pH 20) to 100

ml and measurement of the absorbance at 245 nm using appropriate blank The

concentration of ascorbic acid was calculated using 560 as the value of specific

absorbance [A (1 1 cm)] at the analytical wavelength (Table 5)

The same method was used for the assay of ascorbic acid in creams stored in the

dark and in the presence of individual stabilizing agents (citric tartaric and boric acids)

42102 Iodimetric method for the assay of ascorbic acid in creams containing

riboflavin nicotinamide and alpha-tocopherol

The assay of ascorbic acid in creams in the presence of riboflavin nicotinamide

and alpha-tocopherol was carried out according to the procedure of British

Pharmacopoeia (2009) as follows

The photolysed cream (2 g) was completely scrapped from the glass plate and

transferred to a flask containing 40 ml of distilled water and 10 ml of 1 M sulphuric acid

66

Table 5 Calibration data for ascorbic acid showing linear regression analysisa

λ max 245 nm

Concentration range 01ndash10 times 10ndash4

M (0176ndash1761 mg )

Slope 9920

SE (plusmn) of slope 00114

Intercept 00012

Correlation coefficient 09996

Molar absorptivity (ε) 9920 Mndash1

cmndash1

Specific absorbance [A (1 1 cm)] 560

a Values represent a mean of five determinations

67

was added The solution was titrated with 002 M iodine solution using 1 ml of starch

solution as indicator until a persistent violet-blue color was obtained Each ml of 002 M

iodine solution is equivalent to 352 mg of C6H8O6 The same method was used for the

assay of ascorbic acid in creams stored in the dark

42103 Spectrophotometric method for the assay of ascorbic acid in aqueous and

organic solvents

A 1 ml aliquot of the photolysed solutions of ascorbic acid in water or in an

organic solvent was evaporated to dryness under nitrogen at room temperature and the

residue redissolved in a small volume of methanol The solution was transferred to a 10

ml volumetric flask made up to volume with acidified methanol (pH 20) and the

absorbance measured at 245 nm using an appropriate blank The content of ascorbic acid

in the solutions was determined using 9920 Mndash1

cmndash1

as the value of molar absorptivity at

the analytical wavelength (Table 5)

CHAPTER V

PHOTODEGRADATION OF

ASCORBIC ACID IN

ORGANIC SOLVENTS AND

CREAM FORMULATIONS

69

51 INTRODUCTION

Ascorbic acid (vitamin C) is an essential micronutrient that performs important

metabolic functions (Packer and Fuchs 1999 Davey et al 2000 Johnston et al 2007)

It is an ingredient of anti-aging cosmetic products (Darr et al 1996 Gallarate et al

1999 Traikovich 1999 Zhang et al 1999 Ozer et al 2000 Nusgens et al 2001

Pinnell et al 2001 2003 Lee et al 2004 Raschke et al 2004 Sauermann et al 2004

Elmore 2005 Jentzsch et al 2005 Lin et al 2005 Placzek et al 2005 Carlotti et al

2006 Farahmand et al 2006 Heber et al 2006 Maia et al 2006 Tournas et al 2006)

and exerts several functions on the skin as collagen synthesis depigmentation and

antioxidant activity (Nusgens et al 2001 Spiclin et al 2003) As an antioxidant it

protects skin by neutralizing reactive oxygen species generated on exposure to sunlight

(Shindo et al 1994) In biological systems it reduces both oxygenndash and nitrogenndash based

free radicals (Higdon and Frei 2002) and thus delays the aging process In view of the

instability of ascorbic acid in skin care formulations (Bissett 2006) it is often used in

combination with another redox partner such as alpha-tocopherol (vitamin E) to retard its

oxidation (Wille 2005)

The details of the cream formulations used in this study are given in Table 6 The

results obtained on the photodegradation of ascorbic acid in aqueous organic solvents

and cream formulations are discussed in the following sections

70

Table 6 Composition of cream formulations containing ascorbic acid

Ingredients Cream

No pH

SA PA MA CA AH2 GL PG EG PH DW

1 a 4 + minus minus + + + minus minus + +

b 5 + minus minus + + + minus minus + +

c 6 + minus minus + + + minus minus + +

d 7 + minus minus + + + minus minus + +

2 a 4 minus + minus + + + minus minus + +

b 5 minus + minus + + + minus minus + +

c 6 minus + minus + + + minus minus + +

d 7 minus + minus + + + minus minus + +

3 a 4 minus minus + + + + minus minus + +

b 5 minus minus + + + + minus minus + +

c 6 minus minus + + + + minus minus + +

d 7 minus minus + + + + minus minus + +

4 a 4 + minus minus + + minus + minus + +

b 5 + minus minus + + minus + minus + +

c 6 + minus minus + + minus + minus + +

d 7 + minus minus + + minus + minus + +

5 a 4 minus + minus + + minus + minus + +

b 5 minus + minus + + minus + minus + +

c 6 minus + minus + + minus + minus + +

d 7 minus + minus + + minus + minus + +

6 a 4 minus minus + + + minus + minus + +

b 5 minus minus + + + minus + minus + +

c 6 minus minus + + + minus + minus + +

d 7 minus minus + + + minus + minus + +

7 a 4 + minus minus + + minus minus + + +

b 5 + minus minus + + minus minus + + +

c 6 + minus minus + + minus minus + + +

d 7 + minus minus + + minus minus + + +

8 a 4 minus + minus + + minus minus + + +

b 5 minus + minus + + minus minus + + +

c 6 minus + minus + + minus minus + + +

d 7 minus + minus + + minus minus + + +

9 a 4 minus minus + + + minus minus + + +

b 5 minus minus + + + minus minus + + +

c 6 minus minus + + + minus minus + + +

d 7 minus minus + + + minus minus + + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PG = propylene glycol EG = ethylene glycol

PH = potassium hydroxide DW = distilled water

71

52 PHOTOPRODUCTS OF ASCORBIC ACID

The photolysis of ascorbic acid (AH2) in aqueous and organic solvents and in

cream formulations on UV irradiation leads to the formation of dehydroascorbic acid

(DHA) as detected by TLC along with the undegraded AH2 using the solvent systems A

B and C The identification of DHA was carried out by comparison of the Rf value and

spot color with those of the authentic compound The formation of DHA on

photooxidation of ascorbic acid solutions has previously been reported (Homan and

Gaffron 1964 Sattar et al 1977 Heelis et al 1981 Rozanowska et al 1997 Lavoie et

al 2004) DGA the hydrolysis product of DHA (Homan and Gaffron 1964) could not

be detected under the present experimental conditions

53 SPECTRAL CHARACTERISTICS OF PHOTOLYSED SOLUTIONS

A typical set of the UV absorption spectra of photolysed solutions of AH2 in

methanol is shown in Fig 5 There is a gradual loss of absorbance around 245 nm with

time as a result of the oxidation of the molecule to DHA (Pelletier 1985 Davies et al

1991 Rumsey and Levine 2000) which does not absorb in this region due to the loss of

conjugation Similar absorption changes are observed on the photolysis of AH2 in other

organic solvents and in the methanolic extracts of cream formulations However the

magnitude of these changes varies with the rate of photolysis in a particular solvent or

cream and appears to be a function of the polar character pH and viscosity of the

medium

72

Fig 5 UV absorption spectra of photolysed solutions of ascorbic acid in methanol at

0 40 80 120 160 220 and 300 min

73

54 ASSAY OF ASCORBIC ACID IN CREAMS AND SOLUTIONS

The assay of AH2 in creams and solutions has been carried out in acidified

methanol (pH 20) according to the UV spectrophotometric method of Zeng et al (2005)

Aqueous solutions of AH2 (~pH 2) exhibit absorption maxima at 243 nm (OrsquoNeil 2001

Moffat et al 2004 Sweetman 2009) 244 nm (Ogata and Kosugi 1969) and 245 nm

(Verma et al 1991 Johnston et al 2007) The absorption maxima of AH2 in methanol

and phosphate buffer (pH 25) occur at 245 nm (Zeng et al 2005) Since dilute solutions

of AH2 are highly susceptible to oxidation the pH was adjusted to 20 with phosphoric

acid to convert the molecule to the non-ionized form (99) to minimize degradation

during the assay AH2 in acidified methanol (pH 20) was found to exhibit the absorption

maximum at 245 nm as reported by Zeng et al (2005) The method was also used for the

assay of AH2 in aqueous and organic solvents

The validity of Beerrsquos law relation in the concentration range used was confirmed

prior to the assay The calibration data for AH2 at the analytical wavelength are presented

in Table 5 (Chapter 4) The correlation coefficient (r = 09996) indicates a good linear

relationship over the concentration range employed The values of specific absorbance

and molar absorptivity at 245 nm determined from the slope of the curve are in good

agreement with those reported by previous workers (Davies et al 1991 Johnston et al

2007) The method of Zeng et al (2005) has been found to be satisfactory for the assay of

AH2 in cream formulations and solutions and has been used to study the kinetics of

photolysis reactions The method was validated before its application to the assay of AH2

in photolysed creams The reproducibility of the method was confirmed by the analysis of

known amounts of AH2 in the concentration range likely to be found in photodegraded

74

creams The values of the recoveries of AH2 in creams by the UV spectrophotometric

method are in the range of 90ndash96 The values of RSD for the assays indicate the

precision of the method within plusmn5 (Table 7)

In order to compare the UV spectrophotometric method with the British

Pharmacopoeia iodimetric method (2009) using a dilute iodine solution (002 M) the

creams were simultaneously assayed for AH2 content by the two methods and the results

are reported in Table 8 The statistical evaluation of the accuracy and precision of the two

methods was carried out by the application of the F test and the t test respectively The F

test showed that there is no significant difference between the precision of the two

methods and the calculated value of F is lower than the critical value (F = 639 P = 005)

in each case The t test indicated that the calculated t values are lower than the tabulated t

values (t = 2776 P = 005) suggesting that at 95 confidence level the differences

between the results of the two methods are statistically non-significant Thus the accuracy

and precision of the UV spectrophotometric method is comparable to that of the official

iodimetric method for the assay of AH2 in cream formulations The results of the assays

of AH2 in aqueous organic solvents and cream formulations are reported in Table 9

55 EFFECT OF SOLVENT

The influence of solvent on the rate of degradation of drugs is of considerable

importance to the formulator since the stability of drug species in solution media may be

predicted on the basis of their chemical reactivity The reactivity of drugs in a particular

medium depends to a large extent on solvent characteristics such as the dielectric

constant and viscosity (Connors et al 1986 Yoshioka and Stella 2000 Sinko 2006)

75

Table 7 Recovery of ascorbic acid added to cream formulationsa

Cream

Formulationb

Added

(mg)

Found

(mg)

Recovery

()

RSD

()

1a 400

200

380

183

950

915

21

25

2b 400

200

371

185

928

925

15

25

3c 400

200

375

181

938

905

11

31

4d 400

200

384

189

960

945

13

21

5b 400

200

370

189

925

945

14

26

6c 400

200

369

190

923

950

10

22

7d 400

200

374

182

935

910

17

39

8c 400

200

380

188

950

940

15

33

9d 400

200

367

189

918

945

20

42

a Values expressed as a mean of three to five determinations

b The cream formulations represent combinations of each emulsifier (stearic acid

palmitic acid myristic acid) with each humectant (glycerin propylene glycol ethylene

glycol) to observe the efficiency of methanol to extract AH2 from different creams

(Table 6)

76

Table 8 Assay of ascorbic acid in creams using UV spectrophotometric and iodimetric

methods

Ascorbic acid (mg) Cream

Formulationb Added UV method

a

Iodimetric

methoda

Fcalc tcalc

1a 40

20

380 plusmn 081

183 plusmn 046

375 plusmn 095

185 plusmn 071

138

238

245

104

2b 40

20

371 plusmn 056

185 plusmn 047

373 plusmn 064

193 plusmn 038

130

065

181

200

3c 40

20

375 plusmn 040

181 plusmn 056

374 plusmn 046

183 plusmn 071

132

160

101

223

4d 40

20

384 plusmn 051

189 plusmn 039

381 plusmn 066

190 plusmn 052

167

178

176

231

5b 40

20

370 plusmn 052

189 plusmn 050

372 plusmn 042

185 plusmn 067

065

179

162

125

6c 40

20

369 plusmn 037

190 plusmn 042

371 plusmn 058

188 plusmn 056

245

177

122

197

7d 40

20

374 plusmn 062

182 plusmn 072

370 plusmn 070

184 plusmn 082

127

129

144

168

8c 40

20

380 plusmn 058

188 plusmn 062

375 plusmn 075

192 plusmn 060

167

094

123

162

9d 40

20

367 plusmn 072

189 plusmn 080

365 plusmn 082

187 plusmn 075

149

092

130

203

Theoretical values (P = 005) for F is 639 and for t is 2776

a Mean plusmn SD (n = 5)

b Table 6

77

Table 9 Photodegradation of ascorbic acid in cream formulations

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) pHa 40 50 60 70

0 383 382 384 383

60 374 369 366 361

120 361 354 346 325

180 351 345 325 305

240 345 327 301 284

1

300 336 316 287 264

0 380 383 382 379

60 371 376 362 346

120 359 357 342 320

180 352 345 322 301

240 341 335 299 283

2

300 336 321 291 261

0 384 376 381 385

60 377 367 360 358

120 366 348 334 324

180 356 337 317 305

240 343 320 301 282

3

300 335 307 273 253

78

Table 9 continued

0 377 378 386 372

60 365 361 371 355

120 353 345 347 322

180 344 327 325 298

240 332 320 306 279

4

300 317 303 284 252

0 381 367 372 373

60 372 358 358 353

120 360 337 336 321

180 352 325 320 302

240 341 313 300 284

5

300 327 302 278 256

0 376 386 380 377

60 366 372 350 350

120 353 347 323 316

180 337 334 308 298

240 329 320 291 274

6

300 313 306 267 245

79

Table 9 continued

0 380 372 378 380

60 373 362 350 354

120 358 340 329 321

180 344 328 304 300

240 332 315 292 283

7

300 319 302 272 252

0 380 381 378 361

60 368 364 361 335

120 355 354 340 313

180 342 340 315 281

240 337 331 303 269

8

300 323 314 281 248

0 378 382 370 375

60 370 369 349 342

120 356 347 326 321

180 339 333 298 291

240 326 314 277 271

9

300 313 302 265 242

a The values at pH 40ndash70 represent the formulations a to d of each cream respectively

80

In order to observe the effect of solvent dielectric constant the apparent first-

order rate constants (kobs) for the photolysis of AH2 in alcoholic solvents (Table 10) were

plotted against the dielectric constants of the solvents A linear relationship indicated the

dependence of the rates of photolysis on solvent dielectric constant (Fig 6) This implies

the involvement of a polar intermediate in the reaction to facilitate the formation of the

degradation products as suggested by Ahmad and Tollin (1981) in the case of flavin

electron transfer reactions The effect of solvent polarity has been observed on the

autooxidation of AH2 in organic solvents (Ogata and Kosugi 1969)

Another solvent parameter affecting the rate of a chemical reaction is viscosity

which can greatly influence the stability of oxidisable substances (Wallwork and Grant

1977 Laidler 1987 Fung 1990) A plot of kobs for the photolysis of AH2 against the

reciprocal of solvent viscosity (Table 10) is linear showing that an increase in solvent

viscosity results in a decrease in the rate of photolysis (Fig 7) The viscosity of the liquid

affects the rate at which molecules can diffuse through the solution This in turn may

affect the rate at which a compound can suffer oxidation at the liquid surface This

applies to AH2 and an increase in the viscosity of the medium makes access to air at the

surface more difficult to prevent oxidation (Wallwork and Grant 1977)

56 EFFECT OF CONCENTRATION

In order to observe the effect of concentration (Table 11) on the photostability of

AH2 in a cream using stearic palmitic and myristic acids as emulsifying agents and

glycerin as humectant plots of log concentration versus time were constructed (Fig 8)

and the apparent first-order rate constants were determined (Table 12) A graph of kobs

against concentration of AH2 (Fig 9) exhibited an apparent linear relationship between

81

Table 10 Apparent first-order rate constants (kobs) for the photolysis of ascorbic acid in

water and organic solvents

Solvent Dielectric

Constant (25 ordmC)

Viscosity

(mPas) ndash1

kobs times104

(minndash1

)

Water 785 1000 404

Methanol 326 1838 324

Ethanol 243 0931 316

1-Propanol 201 0514 302

1-Butanol 178 0393 295

82

00

20

40

60

80

0 10 20 30 40 50 60 70 80

Dielectric constant

k (

min

ndash1)

Fig 6 A plot of kobs for photolysis of ascorbic acid against solvent dielectric constant

(times) Water () methanol () ethanol (diams) 1-propanol () 1-butanol

83

00

10

20

30

40

50

00 05 10 15 20

Viscosity (mPas)ndash1

k times

10

4 (m

inndash1)

Fig 7 A plot of kobs for photolysis of ascorbic acid against reciprocal of solvent

viscosity Symbols are as in Fig 6

84

Table 11 Effect of concentration on the photodegradation of ascorbic acid in cream

formulations at pH 60

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) 05 10 15 20 25

0 95 191 290 379 471

60 90 182 277 358 453

120 82 167 260 339 431

180 77 158 239 311 401

240 70 144 225 298 382

1

300 64 134 210 282 363

0 92 186 287 380 472

60 88 175 272 369 453

120 82 160 251 342 429

180 75 152 238 326 405

240 71 144 225 309 392

2

300 65 134 215 289 366

0 94 182 286 376 470

60 87 171 265 352 454

120 78 152 251 337 426

180 69 143 227 315 404

240 62 129 215 290 378

3

300 58 119 195 271 353

85

05

10

15

20

25

06

08

10

12

14

16

18

log

co

nce

ntr

ati

on

(m

g)

a

05

10

15

20

25

06

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

b

05

10

15

20

25

06

08

10

12

14

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

c

Fig 8 Log concentration versus time plots for the photodegradation of ascorbic acid at

various concentrations in creams at pH 60 a) stearic acid b) palmitic acid

c) myristic acid

86

Table 12 Apparent first-order rate constants (kobs) for the photodegradation of various

ascorbic acid concentrations in cream formulations at pH 60

kobs times 103 (min

ndash1)a Cream

Formulationb 05 10 15 20 25

1 133

(0994)

120

(0993)

111

(0995)

101

(0994)

090

(0994)

2 118

(0992)

108

(0994)

098

(0993)

093

(0992)

084

(0994)

3 169

(0994)

144

(0995)

126

(0994)

109

(0993)

097

(0992)

a The values in parenthesis are correlation coefficients

b Table 6

87

Stearic acid

Palmitic acid

Myristic acid

00

05

10

15

20

25

00 05 10 15 20 25

Ascorbic acid concentration ()

kob

s (min

ndash1)

Fig 9 A plot of kobs for photodegradation against ascorbic acid concentrations in cream

formulations

88

the two values Thus the rate of degradation of AH2 is faster at a lower concentration on

exposure to the same intensity of light This may be due to a relatively greater number of

photons available for excitation of the molecule at lower concentration compared to that

at a higher concentration The AH2 concentrations of creams used in this study are within

the range (1ndash15) reported by previous workers for topical applications to skin (Kaplan

et al 1989 Traikovich et al 1999 Nusgens et al 2001 Matsubayashi et al 2003

Espinal-Perez et al 2004 Sauermann et al 2004 Lin et al 2005 Heber et al 2006)

57 EFFECT OF CARBON CHAIN LENGTH OF EMULSIFYING AGENT

The values of kobs for the photodegradation of AH2 (2) in various cream

formulations are reported in Table 13 The first-order plots for the photodegradation of

AH2 at pH 4ndash7 in various cream formulations are shown in Fig 10ndash12 The plots of kobs

against carbon chain length of the emulsifying agents are shown in Fig 13 They indicate

that the photodegradation of AH2 is affected by the emulsifying agent in the order

myristic acid gt stearic acid gt palmitic acid

These acids possess a polar character (Yao et al 2009) and the carbon chain of the acid

may play a part in the photostability of AH2 However the results indicate that in the

presence of palmitic acid AH2 exhibits greater stability as indicated by the plots of kobs

versus the carbon chain length of the emulsifying agents (Fig 13) This could be

predominantly due to the interaction of AH2 with palmitic acid in the cream to impart it

greater stability Ascorbic acid-6-palmitate is known to be an antioxidant in cosmetic

preparations (Lee et al 2009) and food products (Doores 2002)

In view of the above observations it may be suggested that the photodegradation

of AH2 could involve a polar semiquinone intermediate (Johnston et al 2007) which

89

Table 13 First-order rate constants (kobs) for the photodegradation of ascorbic acid in

cream formulations

kobs times 103 (min

ndash1)abc

Cream

Formulation pH 40 50 60 70

1 044

(0992)

064

(0994)

100

(0995)

126

(0995)

2 042

(0992)

060

(0991)

095

(0992)

120

(0995)

3 047

(0993)

069

(0993)

107

(0991)

137

(0995)

4 056

(0993)

072

(0992)

104

(0994)

131

(0993)

5 050

(0991)

067

(0992)

097

(0991)

124

(0992)

6 061

(0992)

079

(0993)

113

(0992)

140

(0994)

7 060

(0992)

071

(0993)

108

(0994)

133

(0992)

8 053

(0991)

062

(0992)

099

(0994)

126

(0993)

9 065

(0991)

081

(0996)

117

(0993)

142

(0995)

a The rate constants at pH 40ndash70 represent the values for formulations a to d of each

cream respectively

b The values in parenthesis are correlation coefficients

c The values of rate constants are relative and depend on specific experimental conditions

including light intensity

The estimated error is plusmn5

90

12

13

14

15

16

17

log

co

nce

ntr

ati

on

(m

g)

1

12

13

14

15

16

17

log

co

nce

ntr

ati

on

(m

g)

2

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

3

Fig 10 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (1ndash3) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

91

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

4

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

5

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

6

Fig 11 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (4ndash6) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

92

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

7

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

8

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

9

Fig 12 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (7ndash9) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

93

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

ko

bs times

10

3 (m

inndash

1)

1-3

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

ko

bs times

10

3 (

min

ndash1)

4-6

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

Carbon chain length

ko

bs times

10

3 (

min

ndash1)

7-9

Fig 13 Plots of kobs for photodegradation of ascorbic acid in creams (1ndash9) against carbon

chain length of emulsifier () Stearic acid () palmitic acid () myristic acid

Humectant used glycerin (1ndash3) propylene glycol (4ndash6) ethylene glycol (7ndash9)

94

depending on the polar character of the medium undergoes oxidation with varying rates

This is similar to the behavior of the photolysis of riboflavin analogs which is dependent

on the polar character of the medium (Ahmad and Tollin 1981) The effect of carbon

chain length on the transdermal delivery of an active ingredient has been discussed (Lu

and Flynn 2009)

58 EFFECT OF VISCOSITY

The plots of rates of AH2 degradation in cream formulations (Table 13) as a

function of carbon chain length (Fig 13) indicate that the rates vary with the humectant

and hence the viscosity of the medium in the order

ethylene glycol gt propylene glycol gt glycerin

This is in agreement with the rate of photolysis of AH2 in organic solvents that

higher the viscosity of the medium lower the rate of photolysis Thus apart from the

carbon chain length of the emulsifier viscosity of the humectant added to the cream

formulation appears to play an important part in the stability of AH2 The stabilizing

effect of viscosity imparting substances on AH2 solutions has been reported (Stone 1969

Kassem et al 1969ab)

59 EFFECT OF pH

The kobsndashpH profiles for the photodegradation of AH2 in various creams (1ndash9) at

pH 4ndash7 (Fig 14) represent a sigmoid type curve indicating the oxidation of the ionized

form (AHndash) of AH2 (pKa 41) (OrsquoNeil 2001) with pH The AH

ndash species appears to be

more susceptible to photooxidation than the non-ionized form (AH2) The behavior of

AH2 on photooxidation in the pH range 4ndash7 is similar to that observed for the chemical

oxidation of AH2 by molecular oxygen (DeRitter 1982) and involves the interaction of

95

Stearic Acid (1)

Palmitic Acid (2)

Myristic Acid (3)

04

06

08

10

12

14

kob

s times

10

3 (m

inndash

1)

Stearic Acid (4)

Palmitic Acid (5)

Myristic Acid (6)

04

06

08

10

12

14

ko

bs times

10

3 (

min

ndash1)

Stearic Acid (7)

Palmitic Acid (8)

Myristic Acid (9)

04

06

08

10

12

14

30 40 50 60 70

pH

ko

bs

times 1

03

(min

ndash1)

Fig 14 The kobsndashpH profiles for the photodegradation of ascorbic acid in creams (1ndash9)

Glycerin

Propylene glycol

Ethylene glycol

96

AH2 with singlet oxygen on UV irradiation (Silva and Quina 2006) The AHndash species

(predominant in the pH range 42ndash70 557ndash999) is more reactive towards singlet

oxygen than its protonated form the AH2 molecule as suggested by Bisby et al (1999)

and therefore the rate of photooxidation is higher in the pH range above 41

corresponding to the pKa1 of AH2 The major goal of a ratendashpH profile is to determine

the optimum pH range for a particular formulation Several workers have studied the

ratendashpH profiles of the chemical oxidation of AH2 in the pH range 2ndash7 (Garrett 1967

Taqui Khan and Martell 1967 Rogers and Yacomeni 1971 Blaugh and Hajratwala

1972 DeRitter 1982 Moura et al 1994) however the kinetics of photooxidation of

AH2 in cream formulations has so far not been reported

510 EFFECT OF REDOX POTENTIAL

The photooxidation of AH2 is also influenced by its redox potential which varies

with pH The greater photostability of AH2 at pH 5ndash6 compared to that at pH 7 and above

is due to its lower rate of oxidationndashreduction in this range (Eordm pH 50 = +0127 V)

(OrsquoNeil 2001) The increase in the rate of photooxidation with pH is due to a

corresponding increase in the redox potential (Eordm pH 70 = +0058 V) (Fasman 1976) of

AH2 and is similar to the photolysis behavior of riboflavin at pH 5ndash6 (Eordm pH 50 = ndash0117

V) (Sinko 2006) compared to that at pH 70 (Eordm pH 70 = ndash 0207 V) (Ahmad et al

2004a Sinko 2006) Since the ionization as well as the redox potentials of AH2 are a

function of pH the rate of photooxidation depends upon the specific species present and

its redox behavior at a particular pH

97

511 PRIMARY PHOTOCHEMICAL REACTIONS IN THE OXIDATION OF

ASCORBIC ACID

A reaction scheme based on general photochemical principles for the important

reactions involved in the photooxidation of ascorbic acid is presented below

0AH2 hv k1

1AH2 (51)

1AH2 k2 Products (52)

1AH2 isc k3

3AH2 (53)

3AH2 k4 Products (54)

0AH

ndash hv k5

1AH

ndash (55)

1AH

ndash k6 Products (56)

1AH

ndash k7

3AH

ndash (57)

3AH

ndash k8 Products (58)

3AH

ndash +

0AH2 k9 AH٠

ndash + AH٠ (59)

2 AH٠ k10 A + AH2 (510)

3AH2 +

3O2 k11

0AH2 +

1O2 (511)

AHndash +

1O2 k12

3AH

ndash +

3O2 (512)

AH٠ + 1O2 k13 AHOO٠ (513)

AHOO٠ k14 A + HO2٠ (514)

AHOO٠ + 0AH2 k15 AH٠ + AHOOH (515)

AHOOH k16 secondary reaction

A + H2O2 (516)

According to this reaction scheme the ground state ascorbic acid species (0AH2

0AH

ndash) each is excited to the lowest singlet state (

1AH2

1AH

ndash) by the absorption of a

quantum of UV light (51 55) These excited states may directly be converted to

98

photoproducts (52 56) or may undergo intersystem crossing (isc) to form the excited

triplet states (53 57) The excited triplet states may then degrade to the photoproducts

(54 58) The monoascorbate triplet (3AH

ndash) may react with the ground state ascorbic

acid to form a monoascorbate radical anion (AH٠ndash) and a monoascorbate radical (AH٠)

(59) Two AH٠ radical species may lead to the formation of an oxidized (A) and a

reduced ascorbic acid molecule (AH2) (510) Ascorbic acid triplet (3AH2) may react with

molecular oxygen (3O2) to yield singlet oxygen (

1O2) (511) which may then react with

monoascorbate anion (AHndash) to form the excited triplet state (

3AH

ndash) (512) or with

monoascorbate radical to form a peroxyl radical (AHOO٠) (513) The peroxyl radical

may yield dehydroascorbic acid (A) (514) or react with ground state ascorbic acid to

give monoascorbate radical and a reduced species AHOOH (515) The reduced species

may give rise to dehydroascorbic acid and hydrogen peroxide (516)

512 DEGRADATION OF ASCORBIC ACID IN THE DARK

In view of the instability of AH2 and to observe its degradation in the dark the

creams were stored in airtight containers at room temperature in a cupboard for a period

of about 3 months and assayed for AH2 content at appropriate intervals The analytical

data (Table 14) were subjected to kinetic treatment (Fig 15ndash17) and the apparent first-

order rate constants for the degradation of AH2 were determined (Table 15) The values

of the rate constants indicate that the degradation of AH2 in the dark is about 70 times

slower than those of the creams exposed to UV irradiation (Table 13) The degradation of

AH2 in creams in the dark is due to chemical oxidation (Section 132) and occurs in the

order of emulsifying agents (Fig 18)

myristic acid gt stearic acid gt palmitic acid

99

Table 14 Degradation of ascorbic acid in the dark in cream formulations

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(days) pHa 40 50 60 70

0 383 382 384 383

10 354 340 313 278

20 309 306 279 245

40 244 209 183 161

60 172 166 131 105

1

80 145 114 81 61

0 380 383 382 379

10 360 343 350 335

20 322 310 301 294

40 266 250 211 186

60 233 211 168 142

2

80 182 153 114 89

0 384 376 381 385

10 368 350 340 318

20 318 273 273 266

40 223 199 172 155

60 174 132 117 84

3

80 122 97 66 54

100

Table 14 continued

0 377 378 386 372

10 350 334 334 318

20 314 268 256 244

40 238 208 182 136

60 179 155 107 94

4

80 128 101 79 59

0 381 367 372 373

10 350 293 300 320

20 299 266 270 263

40 220 191 192 184

60 183 153 139 129

5

80 149 115 87 76

0 376 386 380 377

10 312 320 314 251

20 255 282 226 199

40 175 194 159 131

60 139 128 99 74

6

80 102 81 55 41

101

Table 14 continued

0 380 372 378 380

10 323 330 333 323

20 288 273 276 224

40 212 174 182 146

60 152 133 108 83

7

80 100 82 66 56

0 380 381 378 361

10 333 320 310 310

20 281 266 260 257

40 230 189 171 177

60 156 148 128 111

8

80 123 96 78 66

0 378 382 370 375

10 313 295 281 300

20 256 247 257 203

40 194 178 151 133

60 119 114 88 74

9

80 88 68 49 39

a The values at pH 40ndash70 represent the formulations a to d of each cream respectively

102

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

1

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

2

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

3

Fig 15 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (1ndash3) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

103

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

4

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

5

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

6

Fig 16 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (4ndash6) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

104

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

7

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

8

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

9

Fig 17 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (7ndash9) at pH 40 (times) 50 () 60 () and 70 ()

Palmitic acid

Myristic acid

Stearic acid

105

Table 15 First-order rate constants (kobs) for the degradation of ascorbic acid in cream

formulations in the dark

kobs times 102 (day

ndash1)abc

Cream

Formulation pH 40 50 60 70

1 128

(0991)

152

(0994)

191

(0995)

220

(0994)

2 091

(0992)

110

(0991)

152

(0993)

182

(0992)

3 148

(0991)

176

(0995)

220

(0993)

254

(0995)

4 137

(0992)

161

(0993)

205

(0994)

236

(0995)

5 121

(0991)

141

(0994)

175

(0993)

195

(0993)

6 162

(0992)

194

(0995)

237

(0994)

265

(0994)

7 164

(0994)

189

(0994)

222

(0993)

246

(0996)

8 143

(0994)

167

(0995)

193

(0996)

212

(0993)

9 184

(0995)

208

(0994)

251

(0992)

280

(0996)

a The rate constants at pH 40ndash70 represent the values for formulations a to d of each

cream respectively

b The values in parenthesis are correlation coefficients

c The values of rate constants are relative and depend on specific experimental

conditions

The estimated error is plusmn5

106

pH 4

pH 5

pH 6

pH 7

00

10

20

30

k times

10

2 (d

ayndash

1)

1-3

pH 4

pH 5

pH 6

pH 7

00

10

20

30

k times

10

2 (

da

yndash1)

4-6

pH 4

pH 5

pH 6

pH 7

00

10

20

30

12 14 16 18

Carbon chain length

k times

10

2 (

da

yndash1)

7-9

Fig 18 Plots of kobs for degradation of ascorbic acid in the dark in creams (1ndash9) against

carbon chain length of emulsifier () Stearic acid () palmitic acid

() myristic acid Humectant used glycerin (1ndash3) propylene glycol (4ndash6)

ethylene glycol (7ndash9)

107

Although it is logical to expect a linear relationship between the rate of degradation and

the carbon chain length of the emulsifier due to its polar character (Yao et al 2009) it

has not been observed in the present case The reason for the slowest rate of degradation

of AH2 in the presence of palmitic acid appears to be due to the interaction of AH2 with

palmitic acid (Lee et al 2009) as explained in Section 57

The degradation of AH2 also appears to be affected by the viscosity of the cream

in the order of humectant (Fig 19)

ethylene glycol gt propylene glycol gt glycerin

Thus the presence of glycerin imparts the most stabilizing effect on the degradation of

AH2 This is the same order as observed in the case of photodegradation of AH2 in the

creams The airtight containers used for the storage of creams make the access of air to

the creams difficult to cause chemical oxidation of AH2 However it has been observed

that the degradation of AH2 is highest in the upper layer of the creams compared to that

of the middle and the bottom layers Therefore the creams were thoroughly mixed before

sampling for the assay of AH2 However the scattering in kinetic plots (Fig 15ndash17) is

due to non-uniform distribution of AH2 in degraded creams

The effect of pH on the degradation of AH2 in the creams (Fig 19) shows that the

degradation increases with an increase in pH as observed in the case of photodegradation

of AH2 in the creams This is due to an increase in the ionization and redox potential of

AH2 with pH causing greater oxidation of the molecule and has been discussed in

Sections 59 and 510

108

Stearic Acid (1)

Palmitic Acid (2)

Myristic Acid (3)

00

10

20

30

k times

10

2 (d

ayndash

1)

Glycerin

Stearic Acid (4)

Palmitic Acid (5)

Myristic Acid (6)

00

10

20

30

k times

10

2 (

da

yndash1)

Propylene glycol

Stearic Acid (7)

Palmitic Acid (8)

Myristic Acid (9)

00

10

20

30

30 40 50 60 70

pH

k times

10

2 (d

ayndash

1)

Ethylene glycol

Fig 19 The kobsndashpH profiles for the degradation of ascorbic acid in the dark in creams

(1ndash9)

CHAPTER VI

PHOTOCHEMICAL INTERACTION

OF ASCORBIC ACID WITH

RIBOFLAVIN NICOTINAMIDE

AND ALPHA-TOCOPHEROL IN

CREAM FORMULATIONS

110

61 INTRODUCTION

It is now medically recognized that sagging skin and other signs of degenerative

skin conditions such as wrinkles and age spots are caused primarily by oxy-radical

damage Ascorbic acid can accelerate wound healing protect fatty tissues from oxidative

damage and play an integral role collagen synthesis (Zhang et al 1999) It is used in

cosmetic preparations for its anti-aging depigmentation and antioxidant properties

(Spiclin 2003 Ehrlich et al 2006) It is also used in combination with other vitamins

such as alpha-tocopherol as a co-antioxidant to stabilize cosmetic preparations (Eberlein-

Koumlnig and Ring 2005 Bissett 2006 Murray 2008) Ascorbic acid in the presence of air

or light is known to interact with alpha-tocopherol (Packer et al 2002 Johnston et al

2007) riboflavin (Homann and Gaffron 1964 Sattar et al 1977 Heelis et al 1981

Kim et al 1993 Jung et al 1995 De La Rochette et al 2000 2003 Lavoie et al

2004 Vaid et al 2005 Ahmad and Vaid 2006 Silva and Quina 2006) and

nicotinamide (Bailey et al 1945 Werner et al 1949 Guttman and Brooke 1963

DeRitter 1982) The present work involves a study of the effect of alpha-tocopherol

riboflavin and nicotinamide on the photostability of ascorbic acid in cream formulations

to observe whether the interaction in these formulations leads to the stabilization of

ascorbic acid The chemical structures of nicotinamide (NA) alpha-tocopherol (TP)

riboflavin (RF) formylmethylflavin (FMF) and lumichrome (LC) are shown in Fig 20

The details of the cream formulations used in this study are given in Table 16

The results obtained on the photodegradation of ascorbic acid in cream formulations are

discussed in the following sections

111

Riboflavin

N

N

NH

N

CH2

CH

C OHH

CH OH

CH2OH

N

N

NH

N

CH2

CHO

Formylmethylflavin

N

N

NH

HN

Lumichrome

OH

N

NH2

O

Nicotinamide

O CH3

CH3

CH3

HO

H3C

CH3 CH3 CH3

CH3

Alpha-Tocopherol

O

O

H3C

H3C

H3C

H3C

O

O

H3C

H3C

O

O

Fig 20 Chemical structures of alpha-tocopherol nicotinamide riboflavin

formylmethylflavin and lumichrome

112

Table 16 Composition of cream formulations containing ascorbic acid (2) and other

vitamins

Ingredients Cream

No SA PA MA CA GL AH2 RFa NA

b TP

c PH DW

10 a + minus minus + + + a minus minus + +

b + minus minus + + + b minus minus + +

c + minus minus + + + c minus minus + +

d + minus minus + + + d minus minus + +

e + minus minus + + + e minus minus + +

11 a minus + minus + + + a minus minus + +

b minus + minus + + + b minus minus + +

c minus + minus + + + c minus minus + +

d minus + minus + + + d minus minus + +

e minus + minus + + + e minus minus + +

12 a minus minus + + + + a minus minus + +

b minus minus + + + + b minus minus + +

c minus minus + + + + c minus minus + +

d minus minus + + + + d minus minus + +

e minus minus + + + + e minus minus + +

13 a + minus minus + + + minus a minus + +

b + minus minus + + + minus b minus + +

c + minus minus + + + minus c minus + +

d + minus minus + + + minus d minus + +

e + minus minus + + + minus e minus + +

14 a minus + minus + + + minus a minus + +

b minus + minus + + + minus b minus + +

c minus + minus + + + minus c minus + +

d minus + minus + + + minus d minus + +

e minus + minus + + + minus e minus + +

113

Table 16 continued

15 a minus minus + + + + minus a minus + +

b minus minus + + + + minus b minus + +

c minus minus + + + + minus c minus + +

d minus minus + + + + minus d minus + +

e minus minus + + + + minus e minus + +

16 a + minus minus + + + minus minus a + +

b + minus minus + + + minus minus b + +

c + minus minus + + + minus minus c + +

d + minus minus + + + minus minus d + +

e + minus minus + + + minus minus e + +

17 a minus + minus + + + minus minus a + +

b minus + minus + + + minus minus b + +

c minus + minus + + + minus minus c + +

d minus + minus + + + minus minus d + +

e minus + minus + + + minus minus e + +

18 a minus minus + + + + minus minus a + +

b minus minus + + + + minus minus b + +

c minus minus + + + + minus minus c + +

d minus minus + + + + minus minus d + +

e minus minus + + + + minus minus e + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PH = potassium hydroxide DW = distilled water

RF = riboflavin NA = nicotinamide TP = alpha-tocopherol

a RF(g ) a = 0002 b = 0004 c = 0006 d = 0008 e = 0010

b NA (g ) a = 028 b = 056 c = 084 d = 112 e = 140

c TP (g ) a = 017 b = 034 c = 052 d = 069 e = 086

The molar concentrations of these vitamins are given in Section 421

114

62 ABSORPTION CHARACTERISTICS OF PHOTOLYSED CREAMS

A typical set of the absorption spectra of the methanolic extracts (pH 20) of the

freshly prepared and photolysed creams containing AH2 and TP is shown in Fig 21 AH2

in acidified methanol exhibits absorption maximum at 245 nm (Zeng et al 2005) as

observed in Fig 21 The absorption due to TP at 284 nm (Moffat et al 2004) was

cancelled by using an appropriate blank containing an equivalent concentration of TP

The gradual decrease in absorption at around 245 nm during UV irradiation indicates the

transformation of AH2 to DHA which does not absorb in this region (Davies et al 1991)

as a result of the loss of C3=C2 chromophore Similar spectral changes around 245 nm are

observed in the presence of RF and NA which also strongly absorb in this region A

decrease in the absorption of AH2 around 266 nm in aqueous solution (pH 60) in the

presence of RF has been reported (Vaid et al 2005) The spectral changes and loss of

absorbance in methanolic extracts of creams depends on the rate of photolysis of AH2 in

the presence of these vitamins

63 PHOTOPRODUCTS OF ASCORBIC ACID AND OTHER VITAMINS

The UV irradiation of AH2 in cream formulations (pH 60) in the presence of RF

NA and TP results in the degradation of AH2 and RF and the following photoproducts

have been identified on comparison of their RF values and spot color fluorescence with

those of the authentic compounds

AH2 DHA

RF FMF LC CMF

In the TLC systems used NA and TP did not show the formation of any

degradation product in creams

115

Fig 21 UV absorption spectra of methanolic extracts of photodegraded ascorbic acid in

cream at 0 60 120 180 300 and 480 min

116

The formation of DHA in the photooxidation of AH2 has previously been reported by

many workers (Homann and Gaffron 1964 Sattar et al 1977 Heelis et al 1981

Rozanowska et al 1997 Lavoie et al 2004 Vaid et al 2006) RF is sensitive to light in

aqueous solutions (DeRitter 1982 British Pharmacopoeia 2009 Sweetman 2009) and is

known to form a number of products under aerobic conditions (Treadwell et al 1968

Cairns and Metzler 1971 Schuman Jorns et al 1975 Ahmad and Rapson 1990 Ahmad

and Vaid 2006 Ahmad et al 2004ab 2005 2008 Vaid et al 2006) It has been found

to degrade on UV irradiation in cream formulations to give FMF LC and CMF and these

products have been reported in the photolysis of RF by the workers cited above The

formation of these products may be affected by the interaction of AH2 and RF in creams

(Section 66) NA and TP individually did not appear to form any photoproduct of their

own directly or on interaction with AH2 in creams and may influence the degradation of

AH2 on UV irradiation

64 ASSAY METHOD

In view of the presence of RF (absorption maxima 223 267 373 and 444 nm)

(British Pharmacopoeia 2009) NA (absorption maximum 261 nm) (Moffat et al 2004)

and TP (absorption maximum 284 nm) (Moffat et al 2004) in the cream formulations

and the interference of these vitamins with the absorption of AH2 (absorption maximum

265 nm) (Davies et al 1991) the direct spectrophotometric method cannot be applied for

the determination of AH2 Therefore the iodimetric method (British Pharmacopoeia

2009) was used to determine AH2 in cream formulations The method was validated in

the presence of RF NA and TP before its application to the determination of AH2 in

photodegraded creams The reproducibility of the method has been confirmed by the

117

assay of known concentrations of AH2 in the range present in photodegraded creams The

recovery of AH2 in the creams has been found to be in the range 90ndash96 The values of

RSD indicate that the precision of the method is within plusmn5 (Table 17) and it can be

applied to study the kinetics of AH2 photolysis in cream formulations The assay data on

AH2 in various cream formulations are given in Table 18

65 KINETICS OF PHOTODEGRADATION OF ASCORBIC ACID

Several chemical and physical factors play a role in the photodegradation of AH2

in the presence of other vitamins (RF NA TP) and affect the rate of its degradation in

cream formulations The present work involves the study of photodegradation of AH2 in

cream formulations containing glycerin as humectant as AH2 has been found to be most

stable in these creams (Chapter 5) The apparent first-order rate constants (kobs) for the

photodegradation of AH2 in the presence of other vitamins in cream formulations

derived from the kinetic plots (Fig 22ndash24) are reported in Table 19 The second-order

rate constants (correlation coefficients 0991ndash0996) determined from the slopes of the

graphs of kobs versus vitamin concentration for the individual vitamins (Fig 25) and the

values of k0 determined from the intercept on the vertical axis at zero concentration are

reported in Table 20 The values of k0 give an indication of the effect of other vitamins on

the rate of degradation of AH2 These values are about 13 times lower than the values of

kobs obtained at the highest concentrations of RF and NA indicating that RF and NA both

accelerate the photodegradation of AH2 in creams RF is known to act as a

photosensitizer for the degradation of AH2 (Section 66) and therefore its presence in

creams would accelerate the degradation of AH2 The increase in the rate of

photodegradation of AH2 in the presence of NA has not previously been reported NA

118

Table 17 Recovery of ascorbic acid in cream formulations in the presence of other

vitamins by iodimetric methoda

Cream

Formulationb

Added

(mg )

Found

(mg )

Recovery

()

RSD

()

10e (RF) 400

200

373

187

933

935

29

22

11e (RF) 400

200

379

187

948

935

25

31

12e (RF) 400

200

375

188

938

940

29

28

13e (NA) 400

200

382

191

955

955

23

27

14e (NA) 400

200

380

185

950

925

19

26

15e (NA) 400

200

379

191

948

955

21

17

16e (TP) 400

200

368

183

920

915

29

44

17e (TP) 400

200

391

195

978

975

11

13

18e (TP) 400

200

377

182

943

910

32

37

a Values expressed as a mean of three to five determinations

b The cream formulations represent all the emulsifiers (stearic acid palmitic acid

myristic acid) to observe the efficiency of iodimetric method for the recovery of

ascorbic acid in presence of the highest concentration of vitamins (Table 16)

119

Table 18 Photodegradation of ascorbic acid in cream formulations in the presence of

other vitamins

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) a b C d e

0 373 372 374 372 375

60 362 354 354 360 359

150 342 336 336 332 334

240 315 314 308 310 302

10 (RF)

330 301 291 288 281 282

0 380 379 376 374 374

60 370 366 362 362 361

150 343 337 340 332 328

240 329 323 320 313 310

11 (RF)

330 307 301 294 288 282

0 379 380 375 372 376

60 362 366 361 351 342

150 341 335 319 307 312

240 310 306 295 284 282

12 (RF)

330 285 278 263 254 243

120

Table 18 continued

0 372 370 371 368 365

60 361 358 348 350 349

120 342 343 329 326 330

180 327 325 319 312 308

240 317 309 299 289 285

13 (NA)

300 299 291 283 278 273

0 386 380 375 378 370

60 371 362 365 362 355

120 359 351 343 339 336

200 341 332 325 316 311

14 (NA)

300 313 303 296 294 280

0 375 371 374 370 366

60 362 356 352 352 345

120 343 332 336 326 314

200 323 315 311 295 293

15 (NA)

300 293 283 275 270 259

121

Table 18 continued

0 380 378 380 377 377

60 362 365 369 369 371

120 351 352 360 360 364

180 340 346 349 353 355

240 331 334 343 343 346

16 (TP)

300 320 323 330 332 337

0 383 380 378 380 377

60 372 371 372 373 370

120 363 360 361 366 365

180 348 348 350 356 355

240 341 343 343 348 348

17 (TP)

300 330 332 336 339 341

0 380 383 377 375 373

60 364 370 366 367 366

120 352 356 351 352 351

180 334 338 339 343 342

240 324 328 324 332 330

18 (TP)

300 307 315 317 318 322

122

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

10

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

11

ab

c

de

13

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

12

Fig 22 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing riboflavin (a) 0002 (b) 0004 (c) 0006 (d) 0008

(e) 0010

Stearic acid

Palmitic acid

Myristic acid

123

abcde

13

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

13

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

14

abcde

13

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

15

Fig 23 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing nicotinamide (a) 028 (b) 056 (c) 084 (d) 112 (e) 140

Stearic acid

Palmitic acid

Myristic acid

124

abcde

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

16

abcde

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

17

abcde

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

18

Fig 24 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing alpha-tocopherol (a) 017 (b) 034 (c) 052 (d) 069

(e) 086

Stearic acid

Myristic acid

Palmitic acid

125

Table 19 First-order rate constants (kobs) for the photodegradation of ascorbic acid in the

presence of other vitamins in cream formulations

kobs times 103 (min

ndash1)ab

Cream

formulationd

Other

vitaminc

a b C d e

10 RF 068

(0991)

073

(0996)

079

(0995)

085

(0992)

089

(0995)

11 RF 065

(0992)

070

(0992)

073

(0994)

080

(0995)

086

(0993)

12 RF 087

(0993)

096

(0995)

109

(0993)

116

(0994)

127

(0992)

13 NA 073

(0993)

081

(0992)

088

(0994)

096

(0994)

101

(0993)

14 NA 069

(0992)

074

(0992)

080

(0991)

086

(0995)

094

(0995)

15 NA 083

(0994)

090

(0993)

101

(0993)

109

(0994)

115

(0995)

16 TP 055

(0991)

051

(0994)

046

(0994)

042

(0993)

038

(0991)

17 TP 050

(0995)

045

(0993)

041

(0992)

038

(0995)

034

(0994)

18 TP 070

(0996)

066

(0996)

060

(0994)

055

(0993)

051

(0993)

a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental conditions

including the light intensity

c Vitamin concentrations (andashe) are as given in Table 16

d All the creams contain glycerin as humectant

The estimated error is plusmn5

126

00

05

10

15

00 10 20 30

Riboflavin concentration (M times 104)

kob

s times

10

3 (

min

ndash1)

10-12

00

05

10

15

00 20 40 60 80 100 120

Nicotinamide concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

13-15

00

02

04

06

08

00 04 08 12 16 20

Alpha-Tocopherol concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

16-18

Fig 25 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against individual vitamin concentration in cream formulations (10ndash18)

127

Table 20 First-order rate constants (k0)a for the photodegradation of ascorbic acid in the

absence of other vitamins and second-order rate constants (k) for the

photochemical interaction of ascorbic acid with RF NA and TP

Cream

formulation

Other

vitamin

k0 times 103

(minndash1

)

k

(Mndash1

minndash1

)

Correlation

coefficient

10 RF 062 102 0994

11 RF 059 097 0992

12 RF 077 189 0995

13 NA 066 032 times 10ndash2

0995

14 NA 062 027 times 10ndash2

0993

15 NA 074 037 times 10ndash2

0994

16 TP 059 110 times 10ndash2b

0996

17 TP 053 096 times 10ndash2b

0992

18 TP 075 123 times 10ndash2b

0994

a

The variations in the values of k0 are due to the degradation of AH2 in the presence of

different emulsifying agents in cream formulations

b Values for the inhibition of photodegradation of AH2

128

forms a complex with AH2 (Section 67) and may also act as a photosensitizer for AH2 by

energy transfer in the excited state on UV irradiation The absorption maximum of NA

(261 nm) (Moffat et al 2004) is very close to that of AH2 (265 nm) (Davies et al 1991)

and the possibility of energy transfer in the excited state (Moore 2004) is greater leading

to the photodegradation of AH2

The value of k0 is about 13 times greater than the values of kobs obtained for the

degradation of AH2 in the presence of the highest concentrations of TP in the creams

This indicates that TP has a stabilising effect on the photodegradation of AH2 in the

cream formulations This is in agreement with the view that the TP acts as a redox partner

with AH2 to retard its oxidation (Wille 2005) Thus among the three vitamins studied

only TP appears to have a stabilising effect on photodegradation of AH2 The

photochemical interaction of individual vitamins with AH2 is discussed below

66 INTERACTION OF RIBOFLAVIN WITH ASCORBIC ACID

The interaction of RF with the ascorbate ion (AHndash) may be represented by the

following reactions proposed by Silva and Quina (2006)

RF rarr 1RF (61)

1RF rarr

3RF (62)

3RF + AH

ndash rarr RF

ndashmiddot + AHmiddot (63)

AHmiddot + O2 rarr A + HO2middot (64)

HO2middot + AHndash rarr H2O2 + AHmiddot (65)

RF on the absorption of a quantum of light is promoted to the excited singlet state (1RF)

(61) 1RF may undergo intersystem crossing (isc) to form the excited triplet state (

3RF)

(62) The excited triplet state may react with the ascorbate ion to generate the ascorbyl

hv

isc

129

radical (AH) (63) (Kim et al 1993) The ascorbyl radical reacts with oxygen to give

dehydroascorbic acid (A) and peroxyl radical (HO2) (64) This radical may interact with

ascorbate ion to generate further ascorbyl radicals (65) These radicals may again take

part in the sequence of reactions to form A The role of RF in this reaction is to act as a

photosensitiser in the oxidation of ascorbic acid to A Ascorbic acid is reported to protect

riboflavin in milk under the influence of light by reacting with singlet oxygen (Hall et al

2009) (Section 511)

67 INTERACTION OF NICOTINAMIDE WITH ASCORBIC ACID

NA is known to form a 11 complex with ascorbic acid (Guttman and Brooke

1963 OrsquoNeil 2001 Doores 2002) The complexation of NA and AH2 may result from

the donor-acceptor interaction between AH2 (donor) and NA (acceptor) as observed in

the case of tryptophan and NA (Florence and Attwood 2006) In the presence of light the

interaction may cause reduction of NA (NAH) to form the ascorbyl radical (AH) ((66)-

(68)) which is oxidized to dehydroascorbic acid (A) (69) The NAH may be oxidized to

NA and H2O2 (610)

NA rarr 1NA (66)

1NA rarr

3NA (67)

3NA + AH2 rarr NAH + AHmiddot (68)

2 AH٠ rarr A + AH2 (69)

NAH + O2 rarr NA + H2O2 (610)

The proposed reactions suggest that on photochemical interaction AH2 undergoes

photosensitised oxidation in the presence of NA indicating that the photostability of

ascorbic acid is affected by NA

isc

130

68 INTERACTION OF ΑLPHA-TOCOPHEROL WITH ASCORBIC ACID

TP is an unstable compound and its oxidation by air results in the formation of an

epoxide which than produces a quinone that is inactive (Connors et al 1986) TP is

destroyed by sun light and artificial light containing the wavelengths in the UV region

(Ball 2006) It interacts with other antioxidants such as ascorbic acid (AH2) according to

the following reactions

TPndashO + AH2 rarr TP + AHmiddot (611)

2 AHmiddot rarr A + AH2 (612)

TP + AHmiddot rarr TPndashO + AH2 (613)

The tocopheroxyl radical (TPndashO) reacts with AH2 to regenerate TP and form the

ascorbyl radical (AHmiddot) (611) This radical undergoes further reactions as described in

equations (64) and (65) (Traber 2007) It may also disproportionate back to A and AH2

(612) TP reacts with AHmiddot to produce again the TPndashO radical and AH2 Thus in the

presence of light TP leads to the oxidation of AH2 which is ultimately regenerated in the

reaction (Davies et al 1991) Ascorbic acid has the ability to act as a co-antioxidant with

the TPndashO to regenerate TP (Packer et al 1979 2002) In this manner AH2 and TP act

synergistically to function in a redox cycle and AH2 is stabilized in the cream

formulations and microemulsions (Rozman and Gasperlin 2007 Rozman et al 2009)

69 EFFECT OF CARBON CHAIN LENGTH OF EMULSIFYING AGENT

The graphs of kobs for the photodegradation of AH2 in the presence of RF NA and

TP versus the carbon chain length of emulsifying agents are shown in Fig 26 It appears

that the photodegradation of AH2 in the presence of all the three vitamins in the creams

lies in the order

131

Fig 26 Plots of k for photodegradation of ascorbic acid in creams (10ndash18) against

carbon chain length of emulsifier () Stearic acid () palmitic acid

() myristic acid

00

05

10

15

20

25

k

(Mndash

1 m

inndash

1)

00

05

10

15

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash

1 m

inndash

1)

132

myristic acid gt stearic acid gt palmitic acid

The same order of emulsifying agents has been observed in the absence of the

added vitamins (Section 57) The polar character of these acids (Yao et al 2009) on the

basis of their carbon chain length may play a part in the photostability of AH2 The

greater stability of AH2 in creams in the presence of palmitic acid (Fig 26) may be due to

the interaction of AH2 with palmitic acid as discussed in Section 57 Ascorbic acid-6-

palmitate is known to be an antioxidant in cosmetic preparations (Lee et al 2009) and

food products (Doores 2002)

610 EFFECT OF VISCOSITY OF CREAMS

The plots of kobs for the degradation of AH2 in the presence of the highest

concentration of vitamins versus reciprocal of the viscosity of creams (Table 21) are

linear (Fig 27) and indicate that the increase in cream viscosity leads to a decrease in the

rate of degradation of AH2 The slopes of the plots indicate the effect of viscosity on the

interaction of AH2 with other vitamins in the order

riboflavin gt nicotinamide gt alpha-tocopherol

The relatively slow rate of degradation of AH2 in creams containing palmitic acid may be

due to the interaction of AH2 with the vitamins as well as palmitic acid (Lee et al 2009)

Thus viscosity is an important factor in the stability of AH2 in cream formulations and

may affect its rate of interaction with other vitamins It has been suggested that an

increase in the viscosity of the medium makes access to air at the surface more difficult to

prevent the oxidation of a drug (Wallwork and Grant 1977) This is in agreement with

the photolysis of AH2 in aqueous and organic solvents cream formulations (Chapter 5)

and aerobic oxidation of Ah2 in syrups (Blaug and Hajratwala 1972)

133

Table 21 Average viscosity of cream formulations containing different emulsifying

agents and glycerin as humectant (25 plusmn 1 ordmC) and the photodegradation rate

constants of AH2

Cream No Emulsifying

agent

Viscosityab

(mPa s)

kobs times 103c

10 (RF)

13 (NA)

16 (TP)

Stearic acid 9000 089

101

038

11 (RF)

14 (NA)

17 (TP)

Palmitic acid 8600 086

094

034

12 (RF)

15 (NA)

18 (TP)

Myristic acid 7200 127

115

051

a plusmn10

b Average viscosity of creams containing the individual vitamins (RF NA TP)

c The values have been obtained in the presence of highest concentration of the

vitamins

134

00

05

10

15

20

25

30

100 110 120 130 140

Viscosity (mPa s)ndash1

times 103

kob

s (m

inndash1)

Fig 27 Plots of kobs in the presence of highest concentration of vitamins versus

reciprocal of the viscosity of creams () riboflavin

( ) nicotinamide (- - -- - -) alpha-tocopherol

135

611 DEGRADATION OF ASCORBIC ACID IN THE PRESENCE OF OTHER

VITAMINS IN THE DARK

In order to observe the effect of riboflavin nicotinamide and alpha-tocopherol on

the degradation of AH2 in the creams stored in the dark the AH2 contents of the creams

were assayed at appropriate intervals (Table 22) The apparent first-order rate constants

determined from the kinetic plots (Fig 28) for the degradation of AH2 in the presence of

the highest concentrations of the individual vitamins in cream formulations (10ndash18) are

reported in Table 23 These rate constants indicate that the overall degradation of AH2 in

the presence of the highest concentration of the individual vitamins (RF NA and TP) is

about 70 times slower than that obtained on the exposure of creams to UV irradiation

This decrease in the rate of degradation of AH2 in the creams is the same as observed in

the case of AH2 alone In the absence of light the degradation of AH2 occurs due to

chemical oxidation (Section 132) and does not appear to be affected by the presence of

riboflavin and nicotinamide as indicated by the comparisons of the values of kobs in the

presence and absence of these vitamins (Table 15 and 23) In the presence of alpha-

tocopherol the degradation is slower than that in the presence of riboflavin and

nicotinamide This may be due to some interaction of AH2 and alpha-tocopherol causing

stabilisation of AH2 in the creams

As observed in the case of AH2 degradation alone in creams in the dark the AH2

degradation in the presence of the highest concentrations of other vitamins also occurs in

the same order of emulsifying agents (Fig 29)

myristic acid gt stearic acid gt palmitic acid

136

Table 22 Degradation of ascorbic acid in cream formulations in the dark in presence of

highest concentration of other vitamins

Concentration of ascorbic acid (mg 2g) Cream

No Time (days) 0 10 20 40 60 80

10e (RF) 375 285 233 171 110 69

11e (RF) 374 341 281 221 148 113

12e (RF) 372 259 203 130 89 59

13e (NA) 365 330 255 187 126 81

14e (NA) 370 321 289 219 159 109

15e (NA) 366 289 249 159 110 63

16e (TP) 377 359 321 261 211 159

17e (TP) 377 366 333 275 228 191

18e (TP) 373 361 304 252 200 167

137

02

07

12

17lo

g c

on

cen

tra

tio

n (

mg

)

10-12Riboflavin

02

07

12

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

13-15Nicotinamide

10

12

14

16

18

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

16-18Alpha-Tocopherol

Fig 28 First-order plots for the degradation of ascorbic acid in the dark in presence of

other vitamins using the emulsifying agents (minusminusminusminus) Stearic acid

(minus minusminus minus) palmitic acid (----) myristic acid

138

Table 23 First-order rate constants (kobs) for the degradation of ascorbic acid in presence

of other vitamins in cream formulations in the dark

Cream

formulation

Other

vitaminc

kobs times 102

(dayndash1

)ab

10e RF 204

(0995)

11e RF 156

(0992)

12e RF 222

(0992)

13e NA 189

(0995)

14e NA 151

(0993)

15e NA 214

(0995)

16e TP 100

(0994)

17e TP 088

(0995)

18e TP 105

(0993)

a The values in parenthesis are correlation coefficients and range from 0991ndash0996 due to

some variations in AH2 distribution in the creams

b The values of rate constants are relative and depend on specific experimental

conditions

c Vitamin concentrations (andashe) are as given in Table 16

The estimated error is plusmn5

139

Riboflavin

Nicotinamide

Alpha-Tocopherol

00

10

20

30

12 14 16 18Carbon chain length

ko

bs times

10

2 (

da

yndash1)

Fig 29 Plots of kobs for degradation of ascorbic acid in the dark in creams (10ndash18)

against carbon chain length of the emulsifier () Stearic acid () palmitic acid

() myristic acid

140

This indicates that the rate of degradation of AH2 is slowest in the creams containing

palmitic acid as the emulsifying agent The reason for AH2 degradation in the dark in this

order has already been explained in section 512

CHAPTER VII

STABILIZATION OF

ASCORBIC ACID WITH

CITRIC ACID TARTARIC

ACID AND BORIC ACID IN

CREAM FORMULATIONS

142

71 INTRODUCTION

Ascorbic acid is an ingredient of cosmetic preparations (Section 51) and is

sensitive to light (Rowe et al 2009 Sweetman 2009 British Pharmacopoeia 2009)

degrading to dehydroascorbic acid on UV irradiation by photooxidation (Kitagawa

1968) The photosensitivity of ascorbic acid makes it unstable in pharmaceutical and

cosmetic preparations (DeRitter 1982) The present work is an attempt to study the

photodegradation of ascorbic acid in cream formulations in the presence of certain

compounds (eg citric acid tartaric acid and boric acid) to investigate their role in the

stabilization of the vitamin on exposure to light and in the dark Citric acid and tartaric

acid are used as an antioxidant synergist (Rowe et al 2009) and boric acid is a

complexing agent for hydroxy compounds (Ahmad et al 2009cd)

72 CREAM FORMULATIONS

The details of the various cream formulations used in this study are given in Table

24 and the results obtained on the photodegradation of ascorbic acid in the presence of

stabilizing agents in these formulations are discussed in the following sections

143

Table 24 Composition of cream formulations containing ascorbic acid (2) and

stabilizers

Ingredients Cream

No SA PA MA CA GL PG EG AH2 CTa TA

b BA

c PH DW

19 a + minus minus + + minus minus + a minus minus + +

b + minus minus + + minus minus + b minus minus + +

c + minus minus + + minus minus + c minus minus + +

20 a minus + minus + + minus minus + a minus minus + +

b minus + minus + + minus minus + b minus minus + +

c minus + minus + + minus minus + c minus minus + +

21 a minus minus + + + minus minus + a minus minus + +

b minus minus + + + minus minus + b minus minus + +

c minus minus + + + minus minus + c minus minus + +

22 a + minus minus + + minus minus + minus a minus + +

b + minus minus + + minus minus + minus b minus + +

c + minus minus + + minus minus + minus c minus + +

23 a minus + minus + + minus minus + minus a minus + +

b minus + minus + + minus minus + minus b minus + +

c minus + minus + + minus minus + minus c minus + +

24 a minus minus + + + minus minus + minus a minus + +

b minus minus + + + minus minus + minus b minus + +

c minus minus + + + minus minus + minus c minus + +

25 a + minus minus + + minus minus + minus minus a + +

b + minus minus + + minus minus + minus minus b + +

c + minus minus + + minus minus + minus minus c + +

26 a minus + minus + + minus minus + minus minus a + +

b minus + minus + + minus minus + minus minus b + +

c minus + minus + + minus minus + minus minus c + +

27 a minus minus + + + minus minus + minus minus a + +

b minus minus + + + minus minus + minus minus b + +

c minus minus + + + minus minus + minus minus c + +

144

Table 24 continued

28 a + minus minus + minus + minus + minus minus a + +

b + minus minus + minus + minus + minus minus b + +

c + minus minus + minus + minus + minus minus c + +

29 a minus + minus + minus + minus + minus minus a + +

b minus + minus + minus + minus + minus minus b + +

c minus + minus + minus + minus + minus minus c + +

30 a minus minus + + minus + minus + minus minus a + +

b minus minus + + minus + minus + minus minus b + +

c minus minus + + minus + minus + minus minus c + +

31 a + minus minus + minus minus + + minus minus a + +

b + minus minus + minus minus + + minus minus b + +

c + minus minus + minus minus + + minus minus c + +

32 a minus + minus + minus minus + + minus minus a + +

b minus + minus + minus minus + + minus minus b + +

c minus + minus + minus minus + + minus minus c + +

33 a minus minus + + minus minus + + minus minus a + +

b minus minus + + minus minus + + minus minus b + +

c minus minus + + minus minus + + minus minus c + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PG = propylene glycol EG = ethylene glycol

PH = potassium hydroxide DW = distilled water CT = citric acid TA = tartaric acid

BA = boric acid

a CT (g ) a = 01 b = 02 c = 04

b TA (g ) a = 01 b = 02 c = 04

c BA (g ) a = 01 b = 02 c = 04

145

73 PRODUCTS OF ASCORBIC ACID PHOTODEGRADATION

The photodegradation of AH2 in cream formulations leads to the formation of

DHA as detected by TLC and reported earlier in the photolysis of AH2 in aqueous

solutions (Vaid et al 2006) and cream formulations (Sections 52 and 63) AH2 and

DHA in the methanolic extracts of the degraded creams were identified by comparison of

their Rf and color of the spots with those of the reference standards DHA is also

biologically active (Gardner 1972 Doores 2002) but its further degradation to 23-

diketo-gulonic acid (DGA) results in the loss of vitamin activity (Section 132)

However this product has not been detected in the present cream formulations

Therefore the creams may still possess their biological efficacy

74 SPECTRAL CHANGES IN PHOTODEGRADED CREAMS

In order to observe the spectral changes in photodegraded creams in the presence

of stabilizing agents the absorption spectra of the methanolic extracts of a degraded

cream were determined The spectra show a gradual loss of absorbance around 245 nm

due to the oxidation of AH2 to DHA on UV irradiation and similar to that shown for the

photodegradation of AH2 alone in Fig 5 DHA has negligible absorbance around 245 nm

(Davies et al 1991) and therefore it does not interfere with the absorbance of AH2 in

methanolic solutions The spectral changes and loss of absorbance around 245 nm in

methanolic solution depend on the extent of photooxidation of AH2 in a particular cream

75 ASSAY OF ASCORBIC ACID IN CREAMS

The UV spectrophotometric method (Zeng et al 2005) has previously been

applied to the determination of AH2 in cream formulations (Section 54) The absorbance

of the methanolic extracts of creams containing AH2 during photodegradation was used

146

to determine the concentration of AH2 The method was validated in the presence of citric

acid (CT) tartaric acid (TA) and boric acid (BA) before its application to the evaluation

of the kinetics of AH2 degradation in cream formulations The recovery of AH2 in creams

has been found to be in the range of 90ndash96 and is similar to that reported in Table 7

The reproducibility of the method lies within plusmn5 The assay data on the degradation of

AH2 in various creams in the presence of the stabilizing agents are reported in Table 25

76 KINETICS OF PHOTODEGRADATION

The effect of CT TA and BA as stabilizing agents on the photodegradation of

AH2 was studied by adding 01ndash04 of each compound to the cream formulations (19ndash

33) at pH 60 This concentration range is normally used for the stabilization of drugs in

pharmaceutical preparations (Im-Emsap et al 2002) The apparent first-order rate

constants (kobs) determined from the plots of log concentration versus time (Fig 30ndash34)

are reported in Table 26 The second-order rate constants (k) determined from the plots

of kobs versus concentration of the individual compounds (Fig 35ndash36) are given in Table

27 The values of k indicate the rate of inhibition of photodegradation of AH2 by each

compound

77 EFFECT OF STABILIZING AGENTS

In order to compare the effectiveness of CT TA and BA as stabilizing agents for

AH2 plots of k versus carbon chain length of the emulsifying agents were constructed

(Fig 37) The k values for the interaction of these compounds with AH2 are in the order

citric acid gt tartaric acid gt boric acid

The curves indicate that the highest interaction of these compounds with AH2 is in the

order

147

Table 25 Photodegradation of ascorbic acid in cream formulations in the presence of

stabilizers

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) a b c

0 374 378 379

60 362 362 372

120 349 355 367

210 333 335 349

19 (CT)

300 319 322 336

400 296 309 324

0 381 378 380

60 368 370 369

120 355 363 364

210 344 345 355

20 (CT)

300 328 335 341

400 312 319 331

21 (CT) 0 368 370 374

60 355 356 360

120 340 344 343

210 321 322 333

300 296 299 315

400 272 285 299

148

Table 25 continued

0 375 374 378

60 363 363 368

120 352 354 362

210 329 335 345

22 (TA)

300 307 314 333

400 292 299 313

0 370 377 374

60 364 365 368

120 352 357 357

210 332 344 349

23 (TA)

300 317 330 335

400 301 310 322

24 (TA) 0 376 379 377

60 367 369 368

120 351 348 352

210 325 330 344

300 306 317 326

400 284 294 310

149

Table 25 continued

0 370 375 380

60 356 362 359

120 331 339 344

210 311 318 330

25 (BA)

300 279 288 305

400 260 269 283

0 377 375 370

60 364 363 361

120 351 353 351

210 331 332 337

26 (BA)

300 323 324 325

400 301 307 313

27 (BA) 0 380 377 375

60 369 368 366

120 333 338 341

210 305 313 318

300 292 294 304

400 262 266 281

150

Table 25 continued

0 373 376 378

60 348 349 360

120 329 336 339

210 315 312 323

28 (BA)

300 282 283 299

400 249 264 280

0 370 373 380

60 358 355 367

120 343 346 356

210 325 329 347

29 (BA)

300 307 312 325

400 287 295 315

30 (BA) 0 369 375 372

60 353 358 362

120 321 330 335

210 283 294 303

300 265 281 293

400 242 254 270

151

Table 25 continued

0 374 376 379

60 348 366 352

120 324 340 337

210 303 319 322

31 (BA)

300 275 289 293

400 243 260 275

0 370 374 375

60 355 354 366

120 339 344 345

210 313 319 330

32 (BA)

300 288 297 308

400 261 271 290

33 (BA) 0 377 380 377

60 357 361 367

120 324 335 339

210 288 294 307

300 270 280 293

400 233 248 265

Creams 19ndash27 contain glycerin 28ndash30 contain propylene glycol and 31ndash33 contain

ethylene glycol as humectants

152

a

b

c

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

19

ab

c

14

15

16

log

co

nce

ntr

ati

on

(m

g)

20

a

b

c

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

21

Fig 30 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and citric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

153

a

b

c

14

15

16lo

g c

on

cen

tra

tio

n (

mg

)

22

a

b

c

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

23

a

b

c

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

24

Fig 31 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and tartaric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

154

ab

c

13

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

25

abc

13

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

26

ab

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

27

Fig 32 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and boric acid (a) 01 (b) 02 (c) 04

Palmitic acid

Stearic acid

Myristic acid

155

a

b

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

28

ab

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

29

a

b

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

30

Fig 33 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing propylene glycol and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

156

a

b

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

31

ab

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

32

a

b

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

33

Fig 34 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing ethylene glycol and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

157

Table 26 Apparent first-order rate constants (kobs) for the photodegradation of ascorbic

acid in presence of different stabilizers in cream formulations

kobs times 103 (min

ndash1)ab

Cream

formulation Stabilizer

c

a b c

19 CT 057

(0995)

050

(0992)

041

(0991)

20 CT 049

(0996)

043

(0995)

034

(0993)

21 CT 076

(0995)

067

(0995)

055

(0992)

22 TA 065

(0995)

058

(0995)

046

(0991)

23 TA 054

(0994)

047

(0993)

038

(0994)

24 TA 072

(0996)

063

(0992)

049

(0991)

25 BA 091

(0994)

086

(0995)

071

(0993)

26 BA 055

(0994)

050

(0993)

042

(0993)

27 BA 095

(0995)

089

(0992)

074

(0996)

28 BA 097

(0995)

088

(0992)

075

(0993)

29 BA 064

(0994)

057

(0991)

047

(0993)

30 BA 110

(0994)

100

(0996)

084

(0992)

31 BA 105

(0995)

094

(0994)

078

(0992)

32 BA 088

(0994)

079

(0993)

066

(0993)

33 BA 120

(0995)

108

(0993)

091

(0993) a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental conditions

including the light intensity

c Stabilizer concentrations (andashc) are as given in Table 24

The estimated error is plusmn5

158

00

04

08

12

00 04 08 12 16 20

Citric acid concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

19-21

00

04

08

12

00 05 10 15 20 25

Tartaric acid concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

22-24

Fig 35 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against citric acid (19ndash21) and tartaric acid concentrations (22ndash24) in cream

formulations

159

00

04

08

12k

ob

s times

10

3 (

min

ndash1)

25-27

00

04

08

12

00 20 40 60

ko

bs times

10

3 (

min

ndash1)

28-30

00

04

08

12

00 20 40 60

Boric acid concentration (M times 102)

kob

s times

10

3 (

min

ndash1)

30-33

Fig 36 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against boric acid concentrations in cream formulations (25ndash33)

Propylene glycol

Glycerin

Ethylene glycol

160

Table 27 First-order rate constants (k0)a for the photodegradation of ascorbic acid in the

absence of stabilizers and second-order rate constants (k) for the interaction of

ascorbic acid with CT TA and BA

Cream

formulation Stabilizers

k0 times 103

(minndash1

)

k times 102

(Mndash1

minndash1

)

Correlation

coefficient

19 CT 062 111 0991

20 CT 053 103 0994

21 CT 082 145 0995

22 TA 071 092 0995

23 TA 059 080 0993

24 TA 080 118 0996

25 BA 098 041 0994

26 BA 059 026 0994

27 BA 102 044 0995

28 BA 104 046 0992

29 BA 069 033 0995

30 BA 118 054 0994

31 BA 113 053 0995

32 BA 095 045 0995

33 BA 129 060 0993

a The variations in the values of k0 are due to the presence of different emulsifying agents

in cream formulations

161

00

04

08

12

16

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash1

min

ndash1)

18-33

a

b

e

cd

Fig 37 Plots of k for photodegradation of ascorbic acid in creams (19ndash33) against

carbon chain length of emulsifier () Stearic acid () palmitic acid ()

myristic acid (a) CT (b) TA (c) BA with glycerin (d) BA with propylene

glycol (e) BA with ethylene glycol

162

myristic acid gt stearic acid gt palmitic acid

In the case of myristic acid and stearic acid it may be explained on the basis of the

decreasing polarity (Yao et al 2009) It is interesting to observe the lowest rates of

interaction of these compounds in the creams containing palmitic acid This could be due

to the interaction of AH2 with palmitic acid to form a palmitate derivative in addition to

its interaction with the individual stabilizing agents CT and TA are known to act as

antioxidant synergists (Rowe et al 2009 Sweetman 2009) and in this capacity may

inhibit the photooxidation of AH2 as indicated by the values of the degradation rate

constants in the presence of these compounds The addition of CT to nutritional

supplements is known to inhibit the oxidation of AH2 (Doores 2002) Boric acid forms a

complex with AH2 (Rivlin 2007) and there by may inhibit its degradation Boric acid

may also interact with glycerin added to the creams as a humectant and form a complex

(Rowe et al 2009) This may influence its interaction and stabilizing effect on AH2 in

creams as indicated by the lower k values compared to those in the presence of CT and

TA It has further been observed that the k values for BA are greater in propylene glycol

and ethylene glycol compared to those in glycerin (Table 27) Again this may be due to

greater interaction of BA with glycerin compared to other humectants in the creams

78 DEGRADATION OF ASCORBIC ACID IN PRESENCE OF STABILIZING

AGENTS IN THE DARK

An important factor in the formulation of cosmetic preparations is to ensure the

chemical and photostability of the active ingredient by the use of appropriate stabilizing

agents The choice of these agents would largely depend on the nature and

physicochemical characteristics of the active ingredient AH2 possesses a redox system

163

and can be easily oxidized by air or light In order to observe the effect of CT TA and

BA on the stability of AH2 the cream formulations containing the individual compounds

were stored in the dark for a period of about three months and the rate of degradation of

AH2 was determined The assay data are reported in Table 28 and the kinetic plots are

shown in Fig 38ndash42 The values of apparent first-order rate constants for the degradation

of AH2 in the presence of the stabilizing agents are reported in Table 29 The second

order-rate constants for the interaction of CT TA and BA with AH2 are reported in Table

30 (Fig 43ndash44) The plots of k against the carbon chain length of the emulsifiers are

shown in Fig 45 The kinetic data indicate the same pattern of rates of degradation and

interaction of AH2 with these compounds as observed in the presence of light except that

the rates are much slower in the dark Thus the stabilizing agents are equally effective in

inhibiting the rate of degradation of AH2 in the dark The effect of emulsifying agents and

the humectants on the rate of degradation of AH2 in the presence of the stabilizers has

been discussed in the above Section 77

79 EFFECT OF ADDITIVES ON TRANSMISSION OF ASCORBIC ACID

In order to observe the effect of additives (citric tartaric and boric acids) on the

transmission characteristics of ascorbic acid (0002 mg100 ml) in methanol containing

the highest concentration of the additives (004) used in this study the transmission

spectra were measured It has been found that these additives produce a hypsochromic

shift in the absorption maximum of ascorbic acid This may result in the reduction of the

fraction of light absorbed by ascorbic acid to the extent of about 10 and thus influence

the rate of photodegradation reactions However since all the additives produce similar

effects the rate constants can be considered on a comparative basis

164

Table 28 Degradation of ascorbic acid in cream formulations in the presence of

stabilizers in the dark

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(days) a b c

0 374 378 379

10 355 346 362

20 326 328 342

40 293 297 322

19 (CT)

60 264 269 295

80 241 245 262

0 381 378 380

10 361 364 372

20 339 350 348

40 309 312 330

20 (CT)

60 279 286 301

80 260 266 282

21 (CT) 0 368 370 374

10 342 346 364

20 310 321 348

40 278 282 313

60 249 251 278

80 217 228 249

165

Table 28 continued

0 375 374 378

10 339 344 351

20 317 326 336

40 282 288 306

22 (TA)

60 251 258 280

80 222 235 252

0 370 377 374

10 340 354 355

20 332 336 343

40 297 303 310

23 (TA)

60 266 282 294

80 238 248 267

24 (TA) 0 376 379 377

10 341 339 350

20 306 319 323

40 263 284 279

60 223 241 249

80 196 202 223

166

Table 28 continued

0 370 375 380

10 331 341 334

20 287 289 301

40 225 247 245

25 (BA)

60 189 185 214

80 141 154 170

0 377 375 370

10 355 357 349

20 326 314 324

40 264 267 286

26 (BA)

60 232 238 254

80 189 199 211

27 (BA) 0 380 377 375

10 346 339 337

20 309 288 301

40 233 241 260

60 192 196 211

80 140 147 163

167

Table 28 continued

0 373 376 378

10 314 322 333

20 267 281 305

40 217 233 253

28 (BA)

60 167 177 204

80 122 135 151

0 370 373 380

10 336 329 343

20 283 277 306

40 233 243 267

29 (BA)

60 189 190 217

80 144 154 173

30 (BA) 0 369 375 372

10 308 319 329

20 255 275 310

40 210 226 244

60 158 163 191

80 113 131 147

168

Table 28 continued

0 374 376 379

10 303 311 329

20 266 260 289

40 211 219 239

31 (BA)

60 155 158 178

80 112 121 149

0 370 374 375

10 314 323 339

20 276 280 305

40 222 233 258

32 (BA)

60 172 187 193

80 126 136 162

33 (BA) 0 377 380 377

10 308 306 320

20 254 265 280

40 205 214 237

60 144 155 175

80 107 118 138

169

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

19

abc

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

20

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

21

Fig 38 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing citric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

170

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

22

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

23

ab

c

12

13

14

15

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

24

Fig 39 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing tartaric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

171

a

b

c

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

25

abc

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

26

ab

c

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

27

Fig 40 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing glycerin and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

172

a

b

c

10

12

14

16

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

28

ab

c

10

12

14

16

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

29

a

b

c

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

30

Fig 41 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing propylene glycol and boric acid (a) 01 (b) 02 (c)

04

Palmitic acid

Stearic acid

Myristic acid

173

ab

c

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

31

ab

c

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

32

a

b

c

08

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

33

Fig 42 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing ethylene glycol and boric acid (a) 01 (b) 02 (c)

04

Myristic acid

Palmitic acid

Stearic acid

174

Table 29 Apparent first-order rate constants (kobs) for the degradation of ascorbic acid in

presence of different stabilizers in cream formulations in the dark

kobs times 102 (day

ndash1)ab

Cream

formulation Stabilizer

c

a b c

19 CT 055

(0994)

052

(0992)

044

(0991)

20 CT 048

(0995)

046

(0995)

038

(0992)

21 CT 064

(0994)

061

(0995)

052

(0994)

22 TA 063

(0994)

058

(0995)

049

(0996)

23 TA 054

(0995)

050

(0995)

041

(0994)

24 TA 081

(0995)

075

(0993)

066

(0995)

25 BA 118

(0996)

113

(0994)

097

(0994)

26 BA 087

(0995)

079

(0993)

068

(0994)

27 BA 124

(0995)

114

(0994)

101

(0993)

28 BA 134

(0995)

124

(0996)

110

(0992)

29 BA 116

(0996)

108

(0992)

096

(0995)

30 BA 142

(0993)

131

(0995)

115

(0995)

31 BA 145

(0995)

137

(0992)

117

(0995)

32 BA 130

(0996)

120

(0993)

107

(0994)

33 BA 153

(0995)

141

(0994)

122

(0994) a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental

conditions

c Stabilizer concentrations (andashc) are as given in Table 24

The estimated error is plusmn5

175

176

Table 30 First-order rate constants (k0)a for the degradation of ascorbic acid in the

absence of stabilizers and second-order rate constants (k) for the chemical

interaction of ascorbic acid with CT TA and BA in the dark

Cream

formulation Stabilizers

k0 times 102

(dayndash1

)

k times 102

(Mndash1

dayndash1

)

Correlation

coefficient

19 CT 060 797 0996

20 CT 052 723 0995

21 CT 069 850 0994

22 TA 068 710 0996

23 TA 058 636 0994

24 TA 086 758 0994

25 BA 126 444 0993

26 BA 092 375 0992

27 BA 131 480 0991

28 BA 141 488 0993

29 BA 122 418 0994

30 BA 149 531 0991

31 BA 155 578 0996

32 BA 137 472 0994

33 BA 163 627 0996

a The variations in the values of k0 are due to the presence of different emulsifying agents

in cream formulations

177

00

04

08

12

00 04 08 12 16 20

Citric acid concentration (M times 102)

kob

s times

10

2 (

da

yndash1)

19-21

00

04

08

12

00 05 10 15 20 25

Tartaric acid concentration (M times 102)

kob

s times

10

2 (

da

yndash1)

22-24

Fig 35 Plots of first-order rate constants (kobs) for the degradation of ascorbic acid in the

dark against citric acid (19ndash21) and tartaric acid (22ndash24) concentrations in

cream formulations

178

00

10

20

00 20 40 60

kob

s times

10

3 (

min

ndash1)

25-27

00

10

20

00 20 40 60

kob

s times

10

3 (

min

ndash1)

28-30

00

10

20

00 20 40 60

Boric acid concentration (M times 102)

kob

s times

10

3 (

min

ndash1)

30-33

Fig 44 Plots of first-order rate constants (kobs) for the degradation of ascorbic acid in the

dark against boric acid concentrations in cream formulations (25ndash33)

Glycerin

Propylene glycol

Ethylene glycol

179

00

04

08

12

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash

1 d

ayndash

1)

18-33

b

a

e

dc

Fig 45 Plots of k for degradation of ascorbic acid in the dark in creams (19ndash33) against

carbon chain length of emulsifier () Stearic acid () palmitic acid ()

myristic acid (a) CT (b) TA (c) BA with glycerin (d) BA with propylene

glycol (e) BA with ethylene glycol

CONCLUSIONS

AND

SUGGESTIONS

180

CONCLUSIONS

The main conclusions of the present study on the photodegradation of the ascorbic

acid in organic solvents and cream formulations are as follows

1 Identification of Photodegradation Products

The photodegradation of ascorbic acid in aqueous organic solvents and

laboratory prepared oil-in-water cream preparations on UV irradiation leads to the

formation of dehydroascorbic acid No further degradation products of dehydroascorbic

acid have been detected under the present experimental conditions The product was

identified by comparison of its Rf value and color of the spot with those of the authentic

compound by thin-layer chromatography and spectral changes

2 Assay of Ascorbic Acid

Ascorbic acid in aqueous organic solvents and cream preparations was assayed

in acidified methanolic solutions (pH 20) at 245 nm using a UV spectrophotometric

method Ascorbic acid in combination with other vitamins (riboflavin nicotinamide and

alpha-tocopherol) was assayed by the official iodimetric method due to interference by

these vitamins at the analytical wavelength Both analytical methods were validated

under the experimental conditions employed before their application to the assay of

ascorbic acid The recoveries of ascorbic acid in cream preparations are in the range of

90ndash96 and the reproducibility of both methods are within plusmn5 The F test and the t test

show that there is no significant difference between the precision of the two methods and

therefore these methods can be applied to the assay of ascorbic acid in cream

preparations with comparable results

181

3 Kinetics of Photodegradation

a) Photodegradation of ascorbic acid in organic solvents

Ascorbic acid degradation follows apparent first-order kinetics in aqueous

organic solvents A plot of the first-order rate constants (log kobs) versus solvent dielectric

constant is linear with positive slope indicating an increase in the rate with dielectric

constant On the contrary a plot of kobs verses reciprocal of solvent viscosity is linear with

a positive slope showing a decrease in the rate with solvent viscosity Thus the rate of

photodegradation of ascorbic acid (an oxidizable drug) depends on the solvent

characteristics

b) Photodegradation of ascorbic acid in cream preparations

Ascorbic acid has been found to follow apparent first-order kinetics in cream

preparations and the rate of degradation is affected by the following factors

i Effect of concentration

An apparent linear relationship has been observed between log kobs and

concentration (05ndash25) of ascorbic acid in a cream preparation Thus the rate of

degradation of ascorbic acid appears to be faster at a lower concentration

compared to that of a higher concentration on exposure to the same intensity of

light

ii Effect of carbon chain length of the emulsifying agent

The plots of kobs verses carbon chain length of the emulsifying agent show that the

photodegradation of ascorbic acid is affected in the order myristic acid gt stearic

acid gt palmitic acid This is predominantly due to the interaction of ascorbic acid

with palmitic acid and the carbon chain length (measure of relative polar

182

character) of the emulsifying acid probably does not play a part in the

photodegradation kinetics of ascorbic acid in creams This is evident from the

non-linear relationship between the rate constants for ascorbic acid degradation

and the carbon chain length of the emulsifying acids

iii Effect of viscosity

The values of kobs for the photodegradation of ascorbic acid in cream preparations

are in the order of humectant ethylene glycol gt propylene glycol gt glycerin

showing that the rates of degradation are influenced by the viscosity of the

humectant and decrease with an increase in the viscosity as observed in the case

of organic solvents

iv Effect of pH

The log kndashpH profiles for the photodegradation of ascorbic acid in creams

represent sigmoid type curves indicating an increase in the rate of oxidation of the

molecule with ionization (pH 42ndash70 557ndash999) The AHndash species appears to

be more susceptible to oxidation than the non-ionized molecule in the pH range

studied

v Effect of redox potential

The values of kobs show that the rate of photooxidation of ascorbic acid is

influenced by its redox potential which varies with pH The greater photostability

of ascorbic acid at pH 5ndash6 compared to that at pH 7 and above is due to its lower

rate of oxidation-reduction in the lower range The increase in the rate of

photooxidation with pH is due to a corresponding increase in the redox potential

of ascorbic acid

183

c) Photodegradation of ascorbic acid in the presence of other vitamins (riboflavin

nicotinamide alpha-tocopherol) in cream preparations

The photodegradation of ascorbic acid is affected by the presence of other

vitamins in creams The kinetic data on the photochemical interactions indicate that

riboflavin and nicotinamide act as photosensitizers in the degradation of ascorbic acid

and have an adverse effect on the photostability of the vitamin in creams Whereas

alpha-tocopherol exerts an inhibitory effect on the degradation of ascorbic acid by acting

as a redox partner in the creams Thus a combination of ascorbic acid and alpha-

tocopherol has a synergistic effect on the stabilization of ascorbic acid in creams These

vitamins do not appear to influence the rate of degradation of ascorbic acid in the dark

d) Photodegradation of ascorbic acid in the presence of citric acid tartaric acid and

boric acid in cream preparations

The rate of photodegradation of ascorbic acid in creams has been found to be

inhibited by the addition of compounds such as citric acid tartaric acid and boric acid in

creams These compounds show a stabilizing effect on the photodegradation of ascorbic

acid in the order citric acid gt tartaric acid gt boric acid The lower effect of boric acid

may be due to its interaction with the emulsifying agents and humectants Boric acid

exerts this effect by complex formation with ascorbic acid Citric acid and tartaric acid

are antioxidant synergists and in combination with ascorbic acid may exert a stabilizing

effect on its degradation

184

Salient Features of the Work

In the present work an attempt has been made to study the effects of solvent

characteristics formulation factors particularly the emulsifying agents in terms of the

carbon chain length and humectants in terms of viscosity medium pH drug

concentration redox potential and interactions with other vitamins and stabilizers on the

kinetics of photodegradation of ascorbic acid in cream preparations The study may

provide useful information to improve the photostability and efficacy of ascorbic acid in

cream preparations

SUGGESTIONS

The present work may provide guidelines for a systematic study of the stability of

drug substances in cream ointment preparations and the evaluation of the influence of

formulation variables such as emulsifying agents and humectants concentration pH

polarity viscosity redox potential on the rate of degradation and stabilization of drug

substances This may enable the formulator in the judicious design of formulations that

have improved stability and efficacy for therapeutic use The kinetic parameters may

throw light on the comparative stability of the preparations and help in the choice of

appropriate formulation ingredients

REFERENCES

186

Abe Y Horii H Taniguchi S (1986) A theoretical study of the oxidation mechanism

of triose reductone in reference to L-ascorbic acid Can J Chem 64 360-365

Ahmad I (1968) A Study of the Degradation of Riboflavin and Related Compounds

PhD Thesis University of London London

Ahmad I Rapson HDC (1990) Multicomponent spectrophotometric assay of

riboflavin and photoproducts J Pharm Biomed Anal 8 217-223

Ahmad I Vaid FHM (2006) Photochemistry of flavins in aqueous and organic

solvents In Silva E Edwards AM Eds Flavins Photochemistry and

Photobiology Royal Society of Chemistry Cambridge Chap 2

Ahmad I Tollin G (1981) Solvent effect on flavin electron transfer reactions

Biochemistry 20 5925-5928

Ahmad I Jamil N Zoha SMS (1973) Statistical evaluation of antioxidants used in

the manufacture of ascorbic acid tablets J Sci Univ Kar 2 269-287

Ahmad I Fasihullah Q Vaid FHM (2004b) A study of simultaneous photolysis and

photoaddition reactions of riboflavin in aqueous solution J Photochem Photobiol B

Biol 75 13-20

Ahmad I Fasihullah Q Vaid FHM (2005) Effect of phosphate buffer on

photodegradation reactions of riboflavin in aqueous solution J Photochem

Photobiol B Biol 78 229-234

Ahmad I Fasihullah Q Vaid FHM (2006a) Effect of light intensity and wavelength

on photodegradation reactions of riboflavin in aqueous solution J Photochem

Photobiol B Biol 82 21-27

187

Ahmad I Fasihullah Q Vaid F H M (2006b) Photolysis of formylmethylflavin in

aqueous and organic solvents Photochem Photobiol Sci 5 680-685

Ahmad I Ahmed S Sheraz MA Vaid FHM (2008) Effect of borate buffer on the

photolysis of riboflavin in aqueous solution J Photochem Photobiol B Biol 93 82-

87

Ahmad I Ahmed S Sheraz MA Vaid FHM (2009c) Borate toxicity effect on

drug stability and analytical applications In Chung MP Ed Handbook on

Borates Chemistry Production and Applications Nova Science Publishers Inc

New York in press

Ahmad I Sheraz MA Ahmed S Vaid FHM (2009d) Analytical applications of

borates Mater Sci Res J in press

Ahmad I Fasihullah Q Noor A Ansari IA Ali QNM (2004a) Photolysis of

riboflavin in aqueous solution a kinetic study Int J Pharm 280 199-208

Ahmad I Ahmed S Sheraz MA Aminuddin M Vaid FHM (2009a) Effect of

caffeine complexation on the photolysis of riboflavin in aqueous solution a kinetic

study Chem Pharm Bull in press

Ahmad I Ahmed S Sheraz MA Vaid FHM Ansari IA (2009b) Effect of

divalent ions on photodegradation kinetics and pathways of riboflavin in aqueous

solution Photochem Photobiol Sci accepted

Ahmed S (2009) Effect of Complexing Agents on the Photodegradation of Riboflavin

in Aqueous Solution PhD Thesis Baqai Medical University Karachi

Ahmad T (1978) Studies on the Stability of Sulphacetamide and Degradation Products

PhD Thesis University of Karachi Karachi

188

Ahmad T Ahmad I (1983) The effect of antioxidants on the photolysis of aqueous

sulfacetamide and sulfanilamide solutions Pol J Pharmacol Pharm 35 69-75

Albini A Fasani E Eds (1998) Drugs Photochemistry and Photostability The Royal

Society of Chemistry Cambridge

Allwood MC (1984a) Factors influencing the stability of ascorbic acid in total

parenteral nutrition infusions J Clin Hosp Pharm 9 75-85

Allwood MC (1984b) Compatibility and stability of TPN mixtures in big bags J Clin

Pharm Ther 9 181-198

Allwood MC Kearney MCJ (1998) Compatibility and stability of additives in

parenteral nutrition admixtures Nutrition 14 697-706

Al-Meshal IA Hassan MMA (1982) Ascorbic Acid In Florey K Ed Analytical

Profiles of Drug Substances Vol 11 Academic Press New York pp 45-78

Anderson NH Johnston D McLelland MA Munden P (1991) Photostability

testing of drug substances and drug products in UK pharmaceutical laboratories J

Pharm Biomed Anal 9 443-449

Ansari IA (2002) Photolysis and Interactions of Cyanocobalamin with some B-

Vitamins and Ascorbic Acid in Parenteral Solutions PhD Thesis University of

Karachi Karachi

Ansari IA Vaid Vaid HM Ahmad I (2004) Spectral Study of Photolysis of

Aqueous Cyanocobalamin Solutions in Presence of Vitamins B and C Pak J Pharm

Sci 17 83-93

Arcus CL Zilva SS (1940) The photochemical decomposition of L-ascorbic acid II

Biochem J 34 61-66

189

Arger CB Rupp D Lo P (1996) Preservation of dispersed systems In Liberman

HA Rieger MM Banker GS Eds Pharmaceutical Dosage Forms Disperse

Systems 2nd ed vol1 Marcel Dekker New York Chap9

Asard H May J Smirnoff N Eds (2004) Vitamin C Function and Biochemistry in

Animals and Plants BIOS Scientific Publishers New York

Asker AF Canady D Cobb C (1985) Influence of DL-methionine on the

photostability of ascorbic acid solutions Drug Dev Ind Pharm 11 2109-2125

Ault RG Baird DK Carrington HC Haworth WN Herbert R Hirst EL

Percival EGV Smith F Stacey M (1933) Synthesis of d- and of l-ascorbic acid

and of analogous substances J Chem Soc 1419-1423

Austria R Semenzato A Bettero A (1997) Stability of vitamin C derivatives in

solution and topical formulations J Pharm Biomed Anal 15 795-801

Baertschi SW Alsante KM (2005) Stress testing The chemistry of drug degradation

In Baertschi SW Ed Pharmaceutical Stress testing Predicting Drug Degradation

Taylor amp Francis London Chap 3

Bailey CW Bright JR Jasper JJ (1945) A study of the binary system nicotinamide-

ascorbic acid J Am Chem Soc 67 1184-1186

Ball GFM (2006) Vitamins in Foods Analysis Bioavailability and Stability CRC

Press Boca Raton FL Chap 15

Banhegyi G Marcolongo P Puskas F Fulceri R Mandl J Benedetti A (1998)

Dehydroascorbate and ascorbate transport in rat liver microsomal vesicles J Biol

Chem 273 2758-2762

190

Bardolph EM Taylor RH (1997) Sailors scurvy science and authority J R Soc

Med 90 238

Barel AO Paye M Maibach HI Eds (2001) Handbook of Cosmetic Science and

Technology Marcel Dekker New York

Barry BW (1983) Dermatological Formulations Percutaneous Absorption Marcel

Dekker New York

Barry BW (2002) Transdermal drug delivery In Aulton ME Ed Pharmaceutics

The Science of Dosage Form Design Churchill Livingstone London Chap 33

Basu TK Temple NJ Garg ML (1999) Antioxidants in Human Health and

Disease CABI Publishing Oxford

Becher P (1965) Emulsions Theory and Practice Reinhold New York p 1

Beissenhirtz M Scheller F Lisdat F (2003) Immobilized cytochrome c sensor in

organicaqueous media for the characterization of hydrophilic and hydrophobic

antioxidants Electroanalysis 15 1425-1435

Berlett BS Stadtman ER (1997) Protein oxidation in aging disease and oxidative

stress J Biol Chem 272 20313-20316

Betageri G Prabhu S (2002) Semisolid preparations In Swarbrick J Boylan JC

Eds Encyclopedia of Pharmaceutical Technology 2nd ed Marcel Dekker New

York pp 2436-2457

Bielski BHJ (1982) Chemistry of ascorbic acid radicals In Seib PA Tolbert BM

Eds Ascorbic Acid Chemistry Metabolism and Uses Advances in Chemistry

Series American Chemical Society Washington DC pp 81-100

191

Bielski BHJ Allen AO Schwartz HA (1981) Mechanism of disproportionation of

ascorbate radicals J Am Chem Soc 103 3516-3518

Billany M (2002) Suspensions and emulsions In Aulton ME Ed Pharmaceutics

The Science of Dosage Form Design 2nd ed Churchill Livingstone London Chap

23

Bisby RH Morgan CG Hamblett I Gorman AA (1999) Quenching of singlet

oxygen by trolox C ascorbate and amino acids effect of pH and temperature J Phys

Chem 103 7454-7459

Bissett DL (2006) Anti-aging skin care formulations In Draelos ZD Thaman LA

Eds Cosmetic Formulation of Skin Care Products Taylor amp Francis Group New

York Chap 11

Blaugh SM Hajratwala B (1972) Kinetics of aerobic oxidation of ascorbic acid J

Pharm Sci 61 556-562

Block LH (1996) Pharmaceutical emulsions and microemulsions In Lieberman HA

Rieger MM Banker GS Eds Pharmaceutical Dosage Forms Disperse Systems

Vol 2 2nd ed Chap 2

Bolliger HR Konig A (1969) Water-soluble vitamins In Stahl E Ed Thin-layer

Chromatography Springer-Verlag Berlin pp 304-306

Bonwstra JA De Graaff A Gooris GS Nijsse J Wiechers JW Van Aelst AC

(2003) Water distribution and related morphology in human stratum corneum at

different hydration levels J Invest Dermatol 120 750-758

Bors SR (2005) Scurvy Thomas Dunne Books St Martinrsquos Griffen New York

192

Bors W Buettner GR (1997) The vitamin C radical and its reactions In Packer L

Fuchs J Eds Vitamin C in Health and Disease Marcel Dekker New York Chap

4

Braun L Puskas F Csala M Meszaros G Mandl J Banhegyi G (1997)

Ascorbate as a substrate for glycolysis or gluconeogenesis evidence for an interorgan

ascorbate cycle Free Radic Biol Med 23 804-808

British Pharmacopoeia (2009) Her Majestyrsquos Stationary Office London Electronic

version

Buettner G (1993) The pecking order of free radicals and antioxidants lipid

peroxidation α-tocopherol and ascorbate Arch Biochem Biphys 300 535-543

Buettner GR Schafer FQ (2004) Ascorbate as an antioxidant In Asard H May

JM Smirnoff N Eds Vitamin C Function and Biochemistry in Animals and

Plants BIOS Scientific Publishers Oxford Chap 10

Burke KE (2006) Topical nutritional antioxidants In Draelos ZD Thaman LA

Eds Cosmetic Formulation of Skin Care Products Taylor amp Francis Group New

York Chap 21

Burns JJ Ed (1961) Vitamin C Ann NY Acad Sci 92 1

Byrom P Turnbull JH (1967) Excited states of flavin coenzymes II Anaerobic

oxidation of amino acids by excited riboflavin derivatives Photochem Photobiol 6

125-131

Cadenas E Packer L (2002) Handbook of Antioxidants 2nd ed Marcel Dekker New

York Chap 7-9

193

Capellmann M Bolt HM (1992) Simultaneous determination of ascorbic acid and

dehydroascorbic acid by HPLC with postcolumn derivatisation and fluorometric

detection Fresenius J Anal Chem 342 462-466

Cairns WL Metzler DE (1971) Photochemical degradation of flavins VI A new

photoproduct and its use in studying photolytic mechanism J Am Chem Soc 93

2772-2777

Carlotti ME Ugazio E Sapino S Peira E Gallarate M (2006) Photodegradation

of retinol and anti-aging effectiveness of two commercial emulsions J Cosmet Sci

57 261-277

Carpenter K (1986) The History of Scurvy and Vitamin C Cambridge University Press

Cambridge

Carr AC Zhu BZ Frei B (2000) Potential antiatherogenic mechanisms of ascorbate

(vitamin C) and alpha-tocopherol (vitamin E) Circ Res 87 349-354

Carstensen JT (1972) Theory of Pharmaceutical Systems Academic Press New York

pp 211-214

Chaudhri SK Jain NK (2009) History of cosmetics Asian J Pharm 3 164-167

Chessex P Lovoic JC Roulean T Brochu P St Louis P Levy E Al Varez F

(2002) Photooxidation of parenteral multivitamins induced hepatic steatosis in a

neonatal guinea pig model of intravenous infusion Pediatr Res 52 958-963

Connors KA Amidon GL Stella VJ (1986) Chemical Stability of Pharmaceuticals

A Handbook for Pharmacists 2nd ed Wiley New York pp 105-106 142 208-220

194

Dabrowski K Huiterleitner S (1989) Application of a simultaneous assay of ascorbic

acid dehydroascorbic acid and ascorbic sulfate in biological matter Analyst

(London) 114 83-87

Dale JK Booth RE (1976) In Hoover JE Ed Dispensing of Medication 8th ed

Mack Easton Pennsylvania pp 479-481

Darr D Dunston S Faust H Pinnell S (1996) Effectiveness of antioxidants (vitamin

C and E) with and without sunscreens as topical photoprotectants Acta Derm

Venereol 76 264-268

Davey MW Montagu MV Inze D Sanmartin M Kanellis A Smirnoff N

Benzie IJJ Strain JJ Favell D Fletcher J (2000) Plant L-ascorbic acid

chemistry function metabolism bioavailability and effect of processing J Sci Food

Agr 80 825-860

Davies MB Austin J Partridge DA (1991) Vitamin C Its Chemistry and

Biochemistry The Royal Society of Chemistry Cambridge

De La Rochette A Birlouez-Aragon I Silva E Morliere P (2003) Advanced

glycation endproducts as UVA photosensitizers of tryptophan and ascorbic acid

consequences for the lens Biochem Biophys Acta 1621 235-241

De La Rochette A Silva E Birlouez-Aragon I Mancini M Edwards AM

Morliere P (2000) Riboflavin photodegradation and photosensitization effects are

highly dependent on oxygen and ascorbate concentration Photochem Photobiol 72

815-820

195

De Vries H Beijersbergen Van Henegouwen GMJ Huff FA (1984) Photochemical

decomposition of chloramphenicol in a 025 eye drop and in a therapeutic

intraocular concentration Int J Pharm 20 265-271

Dean RT Fu S Stocker R Davies MJ (1997) Biochemistry and pathology of

radicalmediated protein oxidation Biochem J 324 1-18

DeRitter E (1982) Vitamins in pharmaceutical formulations J Pharm Sci 71 1073-

1096

Deutsch JC (1998a) Ascorbic acid oxidation by hydrogen peroxide Anal Biochem

255 1-7

Deutsch JC (1998b)Spontaneous hydrolysis and dehydration of dehydroascorbic acid

in aqueous solution Anal Biochem 260 223-229

Deutsch JC (1998c)oxygen-accepting antioxidants which arise during ascorbate

oxidation Anal Biochem 265 238-245

Dodsun KY Young ER Soliman AGM (1992) Determination of total vitamin C

in various food matrices by liquid chromatography and fluorescence detection J

AOAC Int 75 887-891

Dollery C Ed (1999) Therapeutic Drugs 2nd ed Vol 1 Churchill Livingstone

London pp A213-A216

Doores S (2002) pH control agents and acidulants In Branen LA Davidson PM

Salminen S Thorngate JH Eds Food Additives Marcel Dekker New York p

637

Eberlein-Koumlnig B Ring J (2005) Relevance of vitamins C and E in cutaneous

photoprotection J Cosmet Dermatol 4 4-9

196

Ehrlich M Rao J Pabby A Goldman MP (2006) Improvement in the appearance

of wrinkles with topical transforming growth factor beta(1) and l-ascorbic acid

Dermatol Surg 32 618-625

EIRI Board (2007) Profitable Small Scale Manufacture of Cosmetics Engineers India

Research Institute Nai Sarak Delhi

Eitenmiller RR Ye L Landen WOJr (2008) Vitamin Analysis for the Health and

Food Sciences 2nd ed CRC Press Boca Raton FL Chap 5

Elia S (2009) Nutrition In Kumar P Clark M Eds Kumar and Clarkrsquos Clinical

Medicine 7th ed Elsevier Limited Chap 5

Elmore AR (2005) Final report of the safety assessment of L-ascorbic acid calcium

ascorbate magnesium ascorbate magnesium ascorbyl phosphate sodium ascorbate

and sodium ascorbyl phosphate as used in cosmetics Int J Toxicol 24 51-111

Erb C Nau-Staudt K Flammer J Nau W (2004) Ascorbic acid as a free radical

scavenger in porcine and bovine aqueous humour Ophthal Res 36 38-42

Espinal-Perez LE Moncada B Castanedo-Cazares JP (2004) A double-blind

randomized trial of 5 ascorbic acid vs 4 hydroquinone in melasma Int J

Dermatol 43 604-607

Fall HH Petering HG (1956) Metabolic inhibitors 1 67-Dimethyl-9-

formylmethylisoalloxazine67-dimethyl-9-(2-hydroxyethyl)-isoalloxazine and

derivatives J Am Chem Soc 78 377-381

Farahmand S Tajerzadeh H Farboud ES (2006) Formulation and evaluation of a

vitamin C multiple emulsion Pharm Dev Technol 11 255-261

197

Fasani E Albini A (2005) Photostability Stress Testing Predicting Drug Degradation

Taylor amp Francis London Chap 10

Fasihullah Q (1988) Photolysis of Flavins in Aqueous and Organic Solvents PhD

Thesis University of Karachi Karachi

Fasman GD Ed (1976) CRC Handbook of Biochemistry and Molecular Biology 3rd

ed Physical Chemical Data Vol 1 CRC Press Cleveland Ohio pp 122-130

Ferreira SLC Bandeira MLSF Lemos VA Dos Santos HC Spinola Costa

AC De Jesus DS (1997) Sensitive spectrophotometric determination of ascorbic

acid in fruit juices and pharmaceutical formulations using 2-(5-bromo-2-pyridylazo)-

5-diethylaminophenol (Br-PADAP) Anal Bioanal Chem 357 1174-1178

Fessenden RW Verma NC (1978) A time-resolved electron spin resonance study of

the oxidation of ascorbic acid by hydroxyl radical Biophys J 24 413-430

Finholt P Paulssen RB Higuchi T (1963) Rate of anaerobic degradation of ascorbic

acid in aqueous solution J Pharm Sci 52 948-954

Florence AT Attwood D (2006) Drug Stability Physicochemical Principles of

Pharmacy 4th ed Pharmaceutical Press London p 411

Flynn GL (2002) Cutaneous and transdermal delivery-processes and systems of

delivery In Banker GS and Rhodes CT Eds Modern Pharmaceutics Marcel

Dekker New York Chap 8

Foote CS (1991) Definition of Type I and Type II Photosensitized oxidation

Photochem Photobiol 54 659

Fornaro A Coicher N (1998) L-ascorbic acid complexation and redox reactions with

some transition metal ions Quni Nova 21 642-650

198

Fraga CG Motchink PA Wyrobek AJ Rempel DM Ames BN (1996)

Smoking and low antioxidant levels increase oxidative damage to sperm DNA

Mutat Res 351 199-203

Fraga CG Motchnik PA Shigenaga MK Helbock HJ Jacob RA Ames BN

(1991) Ascorbic acid protects against endogenous oxidative DNA damage in human

sperm Proc Natl Acad Sci USA 88 11003-11006

Frenich AG Torres ME Vega AB Vidal JL Bolanos PP (2005) Determination

of ascorbic acid and carotenoids in food commodities by liquid chromatography with

mass spectrometry detection J Agric Food Chem 53 7371-7376

Fung HL (1990) Drug stability In Banker GS Rhodes CT Eds Modern

Pharmaceutics 2nd ed Marcel Dekker New York Chap 6

Gallarate M Carlotti ME Trotta M Bovo S (1999) On the stability of ascorbic

acid in emulsified systems for topical and cosmetic use Int J Pharm 188 233-241

Ganshirt H Malzacher A (1960) Separation of several vitamins of the B group and C

by chromatography Naturwiss 47 279-280

Gardner WH (1972) Acidulants in food processing In Furia TE Ed Handbook of

Food Additives 2nd ed Vol 1 CRC Press Cleveland OH pp 225ndash270

Garrett ER (1967) Kinetics and mechanisms in stability of drugs In Bean HS

Beckett AH Carless JE Eds Advances in Pharmaceutical Sciences Vol 2

Academic Press London Chap 1

Garti N Aserin A (1996) Pharmaceutical emulsions double emulsions and

microemulsions In Benita S Ed Microencapsulation Methods and Industrial

Applications Marcel Dekker New York pp 411-534

199

Gensler M Rossmann A Schmidt HL (1995) Detection of added L-ascorbic acid in

fruit juice by isotope ratio mass spectrometry J Agric Food Chem 43 2662-2666

Gibbons E Allwood MC Neal T Hardy G (2001) Degradation of dehydroascorbic

acid in parenteral nutrition mixtures J Pharm Biomed Anal 25 605-611

Guitton C Gros P Comtat M (2007) Direct electrochemical measurements in dermo-

cosmetic creams In Alegret Merkoci Eds Comprehensive Analytical Chemistry

Elsevier Amsterdam 49 e59-e68

Guttman DE Brooke D (1963) Solution phase interaction of nicotinamide with

ascorbic acid J Pharm Sci 52 941-945

Gyorgy P Pearson WN Eds (1967) Vitamin Methods Vol VII Academic Press

New York pp 27-49

Hagg M Ylikoski S Kumpulainen J (1994) Vitamin C and α- and β-carotene in

vegetables consumed in Finland during 1988-1989 and 1992-1993 J Food Compos

Anal 7 252-259

Hagg M Ylikoski S Kumpulainen A (1995) Vitamin C content in fruits and berries

consumed in Finland J Food Compos Anal 8 12-20

Hall NK Chapman TM Kim HJ Min DB (2009) Antioxidant mechanisms of

Trolox and ascorbic acid on the oxidation of riboflavin in milk under light Food

Chem in press doi 101016jfoodchem200905017

Halliwell B (1996) Vitamin C antioxidant or pro-oxidant in vivo Free Radic Res 25

439-454

200

Halliwell B Whiteman M (1997) Antioxidant and prooxidant properties of vitamin C

In Packer L Fuchs J Eds Vitamin C in Health and Disease Marcel Dekker New

York pp 25-94

Halver JE Felton SP (2001) Analytical enigmas in assaying for vitamers C In

Dabrowski K Ed Ascorbic Acid in Aquatic Organisms CRC Press Boca Raton

Chap 3

Hanck A Ed (1982) Vitamin C New Clinical Applications in Immunology Lipid

Metabolism and Cancer Hans Huber Bern

Hashmi MH (1973) Assay of Vitamins in Pharmaceutical Preparations John Wiley

New York Chap 13

Hatchard CG Parker CA (1956) A new sensitive chemical actinometer II Potassium

ferrioxalate as a standard chemical actinometer Proc R Soc London Ser A A235

518-536

Heber GK Markovic B Hayes A (2006) An immunohistological study of anhydrous

topical ascorbic acid compositions on ex vivo human skin J Cosmet Dermatol 5

150-156

Heelis PF Parsons BJ Phillips GO McKellar JF (1981) The flavin sensitized

photooxidation of ascorbic acid a continuous and flash photolysis study Photochem

Photobiol 37 7-13

Henry JA Ed (1997) The New Guide to Medicines and Drugs The British Medical

Association 4th ed Dorling Kindersley London p 434

Hickey S Roberts H (2004) Ascorbate-The Science of Vitamin C Electronic Version

(ISBN 1-4116-0724-4)

201

Higdon JV Frei B (2002) The Antioxidant Vitamin C and E AOAC Press Arlington

VA pp 1-16

Hirst EL Zilva SS (1933) Ascorbic acid as the anti-scorbutic factor Biochem J 27

1271-1278

Ho AHL Puri A Sugden JK (1994) Effect of sweetening agents on the light

stability of aqueous solutions of L-ascorbic acid Int J Pharm 107 199-203

Holmstrom B Oster G (1961) Riboflavin as an electron donor in photochemical

reactions J Am Chem Soc 83 1867-1871

Homann P Gaffron H (1964) Photochemistry and metal catalyzes studies in a flavin

sensitized oxidation of ascorbic acid Photochem Photobiol 3 499-515

Hughes DE (1985) Irreversible reaction kinetics of the aerobic oxidation of ascorbic

acid Anal Chem 57 555-558

Humbert PG Haftek M Creidi P Lapiegravere C Nusgens B Richard A Schmitt D

Rougier A Zahouani H (2003) Topical ascorbic acid on photoaged skin Clinical

topographical and ultrastructural evaluation double-blind study vs placebo Exp

Dermatol 12 237-244

ICH Harmonized Tripartite Guideline (1997) QIB Photostability Testing of New Drug

Substances and Products Federal Register 62 27115-27122

Ichikawa M Ide N Shiraishi S Ono K (2005) Effect of various halide salts on the

incompatibility of cyanocobalamin and ascorbic acid in aqueous solution Chem

Pharm Bull 53 688-690

202

Ihara H Shino Y Aoki Y Hashizume N Minegishi N (2000) A simple and rapid

method for the routine assay of total ascorbic acid in serum and plasma using

ascorbate oxidase and o-phenylenediamine J Nutr Sci Vitaminol 46 321-324

Im-Emsap W Paeratakul O Siepmann J (2002) Disperse Systems In Banker GS

Rhodes CT Ed Modern Pharmaceutics Maecel Dekker New York Chap 9

Irache JM Ezpeleta I Vega FA (1993) HPLC determination of antioxidant

synergists and ascorbic acid in some fatty pharmaceuticals cosmetics and food

Chromatographia 35 232-23

Iwase H Ono I (1994) Determination of ascorbic acid in human plasma by high-

performance liquid chromatography with electrochemical detection using a

hydroxyapatite cartridge for precolumn deproteinization J Chromatogr B Biomed

Appl 655 195-200

Jain S Tiwari AK Jain NK (2006) Topical products In Jain NK Ed

Pharmaceutical Product Development CBS Publishers New Delhi Chap 7

Jamil N Ahmad I (1984) Moisture content of ascorbic acid tablets stored at various

temperatures and humidities Pak J Sci Ind Res 27 252

Jamil N Ahmad I Beg AE (1980a) The effect of UV and tungsten light on physical

characteristics of ascorbic acid tablets Laboratory prepared samples IV Kar Univ

J Sci 8 77-87

Jamil N Ahmad I Beg AE (1980b) The effect of UV and tungsten light on physical

characteristics of ascorbic acid tablets Commercial samples III Kar Univ J Sci 8

117-126

203

Jentzsch A Aikens P (2005) RetiSTARTM

for cosmetic formulations-stabilized retinol

In Rosen MR Ed Delivery System Handbook for Personal Care and Cosmetic

Products Technology Applications and Formulations William Andrew Publishing

Norwich NY pp 862-866

Johnston CS Steinberg FM Rucker RB (2007) Ascorbic Acid In Zempleni J

Rucker RB McCormick DB Suttie JW (Eds) Handbook of Vitamins 4th ed

CRC Press Boca Raton FL Chap 15

Jung MY Kim SM Kim SY (1995) Riboflavin-sensitized photooxidation of

ascorbic acid kinetics and amino acid effects Food Chem 53 397-403

Kall MA Anderson CJ (1999) Improved method for simultaneous determination of

ascorbic acid and dehydroascorbic acid isoascorbic acid and dehydroisoascorbic acid

in food and biological samples J Chromatogr B Biomed Sci Appl 730 101-111

Kampfenkel K Van Montagu M Inze D (1995) Extraction and determination of

ascorbate and dehydroascorbate from plant tissue Anal Biochem 225 165-167

Kaplan DL Moloney SJ Troy WR Dickens MS Pinnell SR (1989) A new

stabilized ascorbic acid solution percutaneous absorption and effect on relative

collagen synthesis J Cutan Aging Cosmet Derm I 115-121

Karatepe M (2004) Simultaneous determination of ascorbic acid and free

malondialdehyde in human serum by HPLC-UV LC GC (North America) 22 362-

365

Karlsen J (1996) Light-activated drugs and drug formulations in drug targeting In

Tonneson HH Ed The Photostability of Drugs and Formulations Taylor amp

Francis London Chap 8

204

Kassem MA Kassem AA Ammar HO (1969a) Studies on the stability of

injectable L-ascorbic acid solutions I Effect of pH solvent light and container

Pharm Acta Helv 44 611-623

Kassem MA Kassem AA Ammar HO (1969b) Studies on the stability of

injectable L-ascorbic acid solutions II Effect of metal ions and oxygen content of

solvent water Pharm Acta Helv 44 667-675

Kassem MA Kassem AA Ammar HO (1972) Studies on the stability of injectable

L-ascorbic acid solutions 3 Effect of metal-complexing agents Pharm Acta Helv

47 89-97

Kaushansky K Kipps TJ (2006) Hematopoietic agents growth factors minerals and

vitamins In Brunton L Ed Goodman amp Gilmans The Pharmacologic Basis of

Therapeutics 11th ed digital ed McGraw-Hill Companies USA Chap 53

Kearney MCJ Allwood MC Martin H Neal T Hardy G (1998) The influence

of amino acid source on the stability of ascorbic acid in TPN mixtures Nutrition 14

173-178

Kim H Kirshenbaum LJ Rosenithol I Riesz P (1993) Photosensitized formation

of ascorbate radicals by riboflavin an ESR study Photochem Photobiol 57 777-

784

Kimoto K Terada S Yamaguchi T (1997) Analysis of ascorbic acid

dehydroascorbic acid and transformation products by ion-pairing high-performance

liquid chromatography with multi-wavelength ultraviolet and electrochemical

detection Methods Enzymol 279 3-12

205

King CG Burns JJ Ed (1975) Second Conference on Vitamin C Ann N Y Acad

Sci 258

Kitagawa Y (1968) The effects of ultraviolet rays on vitamin C Eiyo To Shokuryo 20

416-421 through CA (1968) 68 111560f

Klick S Muijselaar PG Waterval J EichingerT Korn C Gerding TK Debets

AJ de Griend CS van den Beld C Somsen GW De Jong GJ (2005)Toward

a generic approach for stress testing of drug substances and drug products Pharm

Tech 29 48-66

Konings EJ (2006) Water-soluble vitamins J AOAC Int 89 285-288

Kwon BM Foote CS (1988) Chemistry of singlet oxygen 50 Hydroperoxide

intermediates in the photooxygenation of ascorbic acid J Am Chem Soc 110

6582-6583

Kwon BM Foote CS Khan SI (1989) Photooxygenation of ascorbic acid

derivatives and model compounds J Am Chem Soc 111 1854-1860

Laborie S Lavoie JC Chessex P (1998) Paradoxical role of ascorbic acid and

riboflavin in solutions of total parenteral nutrition implication in photoinduced

peroxide generation Pediatr Res 43 601-601

Laborie S Lavoie JC Pineault M Chessex P (1999) Protecting solutions of

parenteral nutrition from peroxidation JPEN 23 104-108

Laborie S Lavoie JC Pineault M Chessex P (2000) Contribution of multivitamin

air and light in the generation of peroxides in adult and neonatal parenteral nutrition

solutions Ann Pharmacother 34440-445

206

Laborie S Lavoie JC Rouleau T Chessex P (2002) Multivitamin solutions for

enteral supplementation a source of peroxides Nutrition 18 470-473s

Lachman L DeLuca P Akers MJ (1986) Kinetic principles and stability testing In

Lachman L Liberman HA Kanig JL Eds The Theory and Practice of

Industrial Pharmacy 3rd ed Lea amp Febiger Philadelphia Chap 26

Laidler KJ (1987) Chemical Kinetics 3rd ed Harper and Row New York pp 183-

185

Lambert WE de Leenheer A (1992) In Nollet LML Ed Food Analysis by HPLC

Marcel Dekker New York Chap 10

Lavoie JC Chessex P Rouleau T Migneault D Comte B (2004) Light-induced

byproducts of vitamin C in multivitamin solutions Clin Chem 50 135-140

Lee JS Kim JW Han SH Chang IS Kang HH Lee OS Oh SG Suh KD

(2004) The stabilization of L-ascorbic acid in aqueous solution and water-in-oil-in-

water double emulsion by controlling pH and electrolyte concentration J Cosmet

Sci 55 1-12

Lee KM Kwon JY Lee KW Lee HJ (2009) Ascorbic acid 6-palmitate

suppresses gap-junctional intercellular communication through phosphorylation of

connexin 43 via activation of the MEK-ERK pathway Mutat Res 660 51-56

Leacutevecircque N Mac-Mary S Muret P Makki S Aubin F Kantelip JP Heusegravele C

SS Humbert P (2005) Evaluation of a sunscreen photoprotective effect by ascorbic

acid assessment in human dermis using microdialysis and gas chromatography mass

spectrometry Exp Dermatol 14 176-181

207

Levine M (1986) New concepts in the biology and biochemistry of ascorbic acid N

Engl J Med 314 892-902

Lewin S (1976) Vitamin C Its Molecular Biology and Medical Potential Academic

Press New York

Liau LS Lee BL New AL Ong CN (1993) Determination of plasma ascorbic

acid by high-performance liquid chromatography with ultraviolet and electrochemical

detection J Chromatogr 612 63-70

Lin JY Selim MA Shea CR Grichnik JM Omar MM Monteiro-Riviere NA

Pinnell SR (2003) UV photoprotection by combination topical antioxidants vitamin

C and vitamin E J Am Acad Dermatol 48 866-874

Lin FH Lin JY Gupta RD Tournas JA Burch JA Selim MA Monteiro-

Riviere NA Grichnik JM Zielinski J Pinnell SR (2005) Ferulic acid

stabilizes a solution of vitamins C and E and doubles its photoprotection of skin J

Invest Dermatol 125 826-832

Lindahl T (1993) Instability and decay of the primary structure of DNA Nature 362

709-715

Litner CJ (1973) Drug Product Stability In Cooper MS Ed Quality Control in

Pharmaceutical Industry Vol 2 Academic Press New York p 185

Liu TZ Chin N Kiser MD Bigler WN (1982) Specific spectrophotometry of

ascorbic acid in serum or plasma by use of ascorbate oxidase Clin Chem 28 2225-

2228

Lu GW Flynn GL (2009) Cutaneous and transdermal delivery-processes and

systems of delivery In Florence AT Siepmann J Eds Modern Pharmaceutics-

208

Applications and Advances Vol 2 5th ed Informa Healthcare Inc New York

Chap 3

Lykkesfeldt J (2000) Determination of ascorbic acid and dehydroascorbic acid in

biological samples by high-performance liquid chromatography using subtraction

methods reliable reduction with tris [2-carboxyethyl] phosphine hydrochloride Anal

Biochem 282 89-93

Macek TJ (1960) Stability problems with some vitamins in pharmaceuticals Am J

Pharm 132 433-455

Mader WJ (1961) Vitamins In Higuchi T Brochmann-Hanssen E Eds

Pharmaceutical Analysis Interscience New York pp 689-693

Maggi N Pasqualucci CR Ballotta R Sensi P (1966) Rifampicin a new orally

active rifamycin Chemotherapy 11 285-292

Maia AM Baby AR Pinto CA Yasaka WJ Suenaga E Kaneko TM Velasco

MV (2006) Influence of sodium metabisulfite and glutathione on the stability of

vitamin C in OW emulsion and extemporaneous aqueous gel Int J Pharm 322

130-135

Mancini M Edwards AM Becker MI De Ioannes A Silva E (2000) Reactivity

of monoclonal antibodies against a tryptophan-riboflavin adduct toward irradiated

and non-irradiated bovine-eye-lens protein fractions an indicator of in vivo visible

light mediated phototransformations J Photochem Photobiol B Biol 55 9-15

Manoharan M Schwille PO (1994) Measurement of ascorbic acid in human plasma

and urine by high performance liquid chromatography Results in healthy subjects

209

and patients with idiopathic calcium urolithiasis J Chromatogr B Biomed Appl

654 134-139

Margolis SA Schmidt HL (1996) Measurement of ascorbic acid in human plasma

and serum stability intralaboratory repeatability and interlaboratory reproducibility

Clin Chem 42 1257-1262

Mason P (2007) Dietary Supplements 3rd ed Pharmaceutical Press pp 330-339

Massad WA Bertolotti S Garcia N (2004) Kinetics and mechanism of the vitamin

B2-sensitized photooxidation of isoproterenol Photochem Photobiol 79 428-433

Matsubayashi T Sakaeda T Kita T Kurimoto Y Nakamura T Nishiguchi K

Fujita T Kamiyama F Yamamoto A Okumura K (2003) Intradermal

concentration of hydroquinone after application of hydroquinone ointments is higher

than its cytotoxic concentration Biol Pharm Bull 26 1365-1367

McBride MM Moore WM (1967) The photochemistry of riboflavin II

Polarographic studies on the hydroxyethyl and formylmethyl analogues of riboflavin

Photochem Photobiol 6 103-113

McDowell LR (2000) Vitamins in Animal and Human Nutrition 2nd ed Iowa State

University Press Chap 15

Meister A (1994) Glutathione-ascorbic acid antioxidant system in animals J Biol

Chem 269 9397-9400

Meister A (1995) Mitochondrial changes associated with glutathione deficiency

Biochim Biophys Acta 1271 35-42

Min DB Boff JM (2002) Chemistry and reaction of singlet oxygen in foods

CRFSFS 1 58-72

210

Moeslinger T Brunner M Volf I Spieckermann PG (1995) Spectrophotometric

determination of ascorbic acid and dehydroascorbic acid Clin Chem 41 1177-1181

Moffat AC Osselton MD Widdop B (2004) Clarkersquos Analysis of Drugs and

Poisons 3rd ed Pharmaceutical Press London pp 649-650

Moore DE (2004) Standardization of kinetic studies of photodegradation reactions In

Tonnesen HH Ed Photostability of Drugs and Drug Formulations 2nd ed CRC

Press Boca Raton FL Chap 3

Moura T Gaudy D Jacob M Cassanas G (1994) pH influence on the stability of

ascorbic acid spray-drying solutions Pharm Acta Helv 69 77-80

Murray JC Burch JA Streilein RD Iannacchione MA Hall RP Pinnell SR

(2008) A topical antioxidant solution containing vitamins C and E stabilized by

ferulic acid provides protection for human skin against damage caused by ultraviolet

irradiation J Am Acad Dermatol 59 418-425

Mushran SP Agarwal MC (1977) Mechanistic studies on the oxidation of ascorbic

acid 5 J Sci Res (India) 36 274-283

Nerlo H Palak W Tomaszek J (1968) Stabilization of vitamin preparations II

Syrup of vitamin D3 and its stabilization Acta Polon Pharm 25 183-188

Niki E Noguchi N (1997) Protection of human low-density lipoprotein from oxidative

modification by vitamin C In Packer L Fuchs J Eds Vitamin C in Health and

Disease Marcel Dekker New York pp 183-192

Nishikimi M Fukuyama R Minoshima S Shimizu N Yagi K (1994) Cloning and

chromosomal mapping of the human nonfunctional gene for L-gulono-gamma-

211

lactone oxidase the enzyme for L-ascorbic acid biosynthesis missing in man J Biol

Chem 269 13685-13688

Njus D Wigle M Kelly PM Kipp BH Schlegel HB (2001) Mechanism of

ascorbic acid oxidation by Cytochrome B561 Biochemistry 40 11905-11911

Nusgens BV Humbert P Rougier A Colige AC Haftek M Lambert CA

Richard A Creidi P Lapiegravere CM (2001) Topically applied vitamin C enhances

the mRNA level of collagens I and III their processing enzymes and tissue inhibitor

of matrix metalloproteinase 1 in the human dermis J Invest Dermatol 116 853-859

Ogata Y Kosugi Y (1969) Solvent effect on the autooxidation of L-ascorbic acid

Tetrahedron 25 1055-1062

OrsquoKeefe T (2001) Ascorbic acid and stable ascorbate esters as sources of vitamin C in

aquaculture feeds ASA Technical Bulletin AQ48 wwwsoyaquaorg

OrsquoNeil MJ Ed (2001) The Merck Index 13th ed Merck amp Co Inc Rahway NJ

USA Electronic version

Ortega GR Delgado JN (1998) Vitamins and related compounds In Wilson and

Gisvoldrsquos Textbook of Organic Medicinal and Pharmaceutical Chemistry 10th ed

Lippincott-Raven Philadelphia Chap 27

Ortwerth BJ Monnier VM (1997) Protein glycation by the oxidation products of

ascorbic acid In Packer L Fuchs J Eds Vitamin C in Health and Disease Marcel

Dekker New York pp 123ndash142

Ozer O Muguet V Roy E Grossiord JL Seiller M (2000) Stability study of

WOW viscosified multiple emulsions Drug Dev Ind Pharm 26 1185-1189

Packer I Fuchs J (1999) Vitamin C in Health and Disease Marcel Dekker New York

212

Packer JE Slater TF Wilson RL (1979) Direct observation of a free radical

interaction between vitamin E and vitamin C Nature 278 737-738

Packer L Traber MG Kraemer K Frei B (2002) The Antioxidant Vitamins C and

E AOCS Press Illinois

Padayatty SJ Katz A Wang Y Eck P Kwon O Lee JH Chen S Corpe C

Dutta A Dutta SK Levine M (2003) Vitamin C as an antioxidant Evaluation of

its role in disease prevention J Am Coll Nutr 22 18-35

Parker CA (1953) A new sensitive chemical actinometer I Some trials with potassium

ferrioxalate Proc Roy Soc (Lond) A220 104-116

Parker JN Parker PM (2003) Antioxidants ICON Group International

Pastore P Rizzetto T Curcuruto O Cin MD Zaramella A Martin D (2001)

Characterization of dehydroascorbic acid solutions by liquid chromatography mass

spectrometry Rapid Commun Mass Spectrom 15 2051-2057

Pelletier O (1985) In Augustin J Klein BP Becker D Venugopal PB Eds

Methods of Vitamins Assay Wiley New York pp 303-347

Peyrat-Maillard MN Bonnely S Rondini L Berset C (2001) Effect of vitamin E

and vitamin C on the antioxidant activity of malt rootlets extracts Lebensmittel-

Wissenschaft Und-Technological 34 176-182

Pinnell SR (2003) Cutaneous photodamage oxidative stress and topical antioxidant

protection J Am Acad Dermatol 48 1-19

Pinnell SR Madey DL (1998) Topical vitamin C in skin care Aesthet Surg J 18

468-470

213

Pinnell SR Yang H Omar M Monteiro-Riviere N DeBuys HV Walker LC

Wang Y Levine M (2001) Topical L-ascorbic acid percutaneous absorption

studies Dermatol Surg 27 137-142

Placzek M Gaube S Kerkmann U Gilbertz KP Herzinger T Haen E Przybilla

B (2005) Ultraviolet B-induced DNA damage in human epidermis is modified by

the antioxidants ascorbic acid and D-alpha-tocopherol J Invest Dermatol 124 304-

307

Puskas F Gergely PJr Banki K Perl A (2000) Stimulation of the pentose

phosphate pathway and glutathione levels by dehydroascorbate the oxidized form of

vitamin C FASEB J 14 1352-1361

Puskas F Gergely P Niland B Banki K Perl A (2002) Differential regulation of

hydrogen peroxide and Fas-dependent apoptosis pathways by dehydroascorbate the

oxidized form of vitamin C Antioxid Redox Signal 4 357-369

Racz I (1989) Drug Formulation Wiley New York pp 121-122

Ramu A Mehta MM (2000) The riboflavin-mediated photooxidation of doxorubicin

Cancer Chemoth Pharm 46 449-458

Raschke T Koop U Duumlsing HJ Filbry A Sauermann K Jaspers S Wenck H

Wittern KP (2004) Topical activity of ascorbic acid from in vitro optimization to in

vivo efficacy Skin Pharmacol Physiol 17 200-206

Reed RA Harmon P Manas D Wasylaschuk W Galli C Biddell R Bergquist

PA Hunke W Templeton AC Ip D (2003) The role of excipients and package

components in the photostability of liquid formulations PDA J Pharm Sci Tech 57

351-368

214

Reichstein T Griissner A Oppcnuuer R (1933) Synthese der d- und l-Ascorbinsaumlure

(C-Vitamin) Helv Chim Acta 16 1019-1033

Riehl J Maupin C Layloff T (1995) On the choice of a photolysis source for the

photostability testing of pharmaceuticals Pharmacop Forum 21 1654-1663

Rivlin RS (2007) Riboflavin (vitamin B2) In Zempleni J Rucker RB McCormick

DB Suttie JW Eds Handbook of Vitamins 4th ed Taylor amp Francis CRC

Press Boca Raton FL p 241

Rogers AR Yacomeni JA (1971) The effect of pH on the aerobic degradation of

ascorbic acid solutions J Pharm Pharmacol 23 218S

Rosen MR Ed (2005) Delivery System Handbook for Personal Care and Cosmetic

Products-Technology Applications and Formulations William Andrew Publishing

Norwich NY

Rosenberg HR (1945) Chemistry and Physiology of the Vitamins Interscience New

York pp 289-338

Roth HJ Eger K Troschutz R (1991) Pharmaceutical Chemistry Vol 2 Drug

Analysis Ellis Horwood London pp 282-288

Rowe RC Sheskey PJ Quinn ME (2009) Handbook of Pharmaceutical Excipients

6th ed Pharmaceutical Press London pp 43-47 181-183 731-732

Roacutezanowska M Bober A Burke JM Sarna T (1997) The role of retinal pigment

epithelium melanin in photoinduced oxidation of ascorbate Photochem Photobiol

65 472-479

Rozman B Gasperlin M (2007) Stability of vitamins C and E in topical

microemulsions for combined antioxidant therapy Drug Deliv 14 235-245

215

Rozman B Gasperlin M Tinois-Tessoneaud E Pirot F Falson F (2009)

Simultaneous absorption of vitamins C and E from topical microemulsions using

reconstructed human epidermis as a skin model Eur J Pharm Biopharm 72 69-75

Ruumlmelin A Fauth U Halmagyi M (1999) Determination of ascorbic acid in plasma

and urine by high performance liquid chromatography with ultraviolet detection Clin

Chem Lab Med 37 533-536

Rumsey SC Levine M (2000) Vitamin C In Song WO Beecher GR

Eitenmiller RR Eds Modern Analytical Methodologies in Fat- and Water-Soluble

Vitamins Wiley New York Chap 13

Saari JC Baker EM Sauberlich HE (1967) Thin-layer chromatographic separation

of the oxidative degradation products of ascorbic acid Anal Biochem 18 173-177

Salkić M Keran H Jašić M (2007) Direct spectrophotometric determination of l-

ascorbic acid in the presence of potassium cyanide Agric Conspec Sci 72 371-375

Salvador A Chisvert A (2007) Analysis of Cosmetic Products Elsevier Amsterdam

Sattar A DeMan JM Alexander JC (1977) Light-induced degradation of vitamins

II Kinetic studies on ascorbic acid decomposition in solution Can Inst Food Sci

Technol J 10 65-68

Satoh K Sakagami H Terasaka H Ida Y Fujisawa S (2000) Interaction between

hydroquinone and ascorbic acid derivatives quenching effects of organic solvents

Antican Res 20 1577-1581

Sauberlich HE (1994a) Pharmacology of vitamin C Annu Rev Nutr 14 371-391

Sauberlich HE (1994b) Vitamin C and Cancer In Kritchevsky D Carroll K Eds

Nutrition and Disease Update Cancer AOCS Press Champaign IL pp 111-172

216

Sauermann K Jaspers S Koop U Wenck H (2004) Topically applied vitamin C

increases the density of dermal papillae in aged human skin BMC Dermatol 4 13

Sawicka J (1991) The influence of excipients and technological process on

cholecalciferol stability and its liberation from tablets Pharmazie 46 519-521

Scheuplein RJ Blank IH (1973) Mechanism of percutaneous absorption IV

Penetration of non-electrolytes (alcohols) from aqueous solutions and from pure

liquids J Invest Dermatol 60 286-296

Schick B (1943) Scurvy and pine needles Science 98 241-242

Schuman Jorns M Schollnhammer G Hemmerich P (1975) Intramolecular addition

of the riboflavin side chain Anion-catalysed neutral photochemistry Eur J

Biochem 57 35-48

Seib PA Tolbert BM (1982) Ascorbic Acid Chemistry Metabolism and Uses

Advances in Chemistry Series 200 American Chemical Society Washington DC

Sequeira F Vozone C (2000) Photostability studies of drug substances and products

Practical implications of the ICH guideline Pharm Tech 24 30-35

Shacter E (2000) Quantification and significance of protein oxidation in biological

samples Drug Metab Rev 32 307-326

Shah VP (1997) FDA Guidance for Industry Document Nonsterile Semisolid Dosage

Forms Centre for Drug Evaluation and Research Rockville MD pp 1-40

Sheraz MA Ahmed S Ahmad S (2007) Developments in the clinical and food

analysis of vitamin C J Baqai Med Univ 10 19-24

217

Shindo Y Witt E Han D Packer L (1994) Dose-response effect of acute ultraviolet

irradiation on antioxidants and molecular markers of oxidation in murine epidermis

and dermis J Invest Dermatol 102 470-475

Sibi M Batcu A Gheorghiu I (1953) Vitamin oxidation-reduction effect II Effect of

light on the oxidation-reduction reactions of lactoflavin Studii cercetari stiinf

(Romania) 4 291-305 through CA (1956) 50 14816g

Sidhu DS Sugden JK (1992) Effect of food dyes in the photostability of aqueous

solutions of L-ascorbic acid Int J Pharm 83 263-266

Silva E Quina FH (2006) Photoinduced processes in the eye lens do flavins really

play a role In Silva E Edwards AM Eds Flavins Photochemisry and

Photobiology The Royal Society of Chemistry Cambridge Chap 7

Silva E Ugarte P Andrade A Edwards AM (1994) Riboflavin-sensitized photo-

processes of tryptophan J Photochem Photobiol B Biol 23 43-48

Sim AK (1972) Ascorbic acid-A scurvy past and present Chem Indust 8 160-165

Singh S (2006) Stability testing during product development In Jain NK ED

Pharmaceutical Product Development CBS Publishers New Delhi Chap 9

Singh S Bakshi M (2000) Guidance on conduct of stress testing to determine inherent

stability of drugs Pharm Tech 24 1-14

Sinko PJ (2006) Chemical Kinetics and Stability Martinrsquos Physical Pharmacy and

Pharmaceutical Sciences 5th ed Lippincott Williams amp Wilkins Baltimore Chap

8 15 18

218

Smith JL Canham JE Kirkland WD Wells PA (1988) Effect of intralipid amino

acids container temperature and duration of storage on vitamin stability in total

parenteral nutrition admixtures J Parenter Enteral Nutr 12 478-483

Speek AJ Schrijver J Schreurs WHP (1984) Fluorimetric determination of total

vitamin C and total isovitamin C in food stuffs and beverages by high-performance

liquid chromatography with precolumn derivatization J Agric Food Chem 32 352-

355Spratley JE Hellstroumlm SO Mattsson CK Pais-Clemente M (2001)

Topical ascorbic acid reduces myringosclerosis in perforated tympanic membranes A

study in the rat Ann Otol Rhinol Laryngol 110 585-591

Spiclin P Homer M Zupancic A Gasperlin M (2003) Sodium ascorbyl phosphate

in topical microemulsions Int J Pharm 256 65-73

Stadtman ER (1991) Ascorbic acid and oxidative inactivation of proteins Am J Clin

Nutr 54 1125S-1128S

Stone I (1969) The Healing Factor Vitamin C Against Disease Grosset and Dunlap

New York

Sugden JK (1985) Photostability of cosmetic materials Int J Cosmet Sci 7 165-173

Swarbrick J Rubino JT Rubino OP (2006) Coarse Dispersions In Hendrickson

R Ed Remington The Science and Practice of Pharmacy 21st ed Lippincott

Williams amp Wilkins Baltimore Chap22

Sweetman SC Ed (2009) Martindale The Complete Drug Reference 36th ed

Pharmaceutical Press London Electronic version

219

Szent-Gyoumlrgi A (1928) Observation on function of peroxidase systems and chemistry

of adrenal cortex description of new carbohydrate derivative Biochem J 22 1387-

1389

Szymula M (2005) The influence of ascorbic acid on the rheological properties of the

microemulsion region of the SDSpentanolwater system J Cosmet Sci 56 267-

277

Taqui Khan MM Martell AE (1967) Metal ion and metal chelate catalyzed oxidation

of ascorbic acid by molecular oxygen 1 Cupric acid and ferric ion catalyzed

oxidation J Am Chem Soc 89 4176-4185

Tangney CC (1988) Analysis of vitamin C in biological samples with an emphasis on

recent chromatographic techniques Prog Clin Biol Res 259 331-362

Tatsumi K Ichikawa H Wada S (1992) Flavin-sensitized photooxidation of

substituted phenols in natural water J Contam Hydrol 9 207-219

Tessier F Birlouez-Aragon I Tjani C Guilland JC (1996) Validation of a

micromethod for determining oxidized and reduced vitamin C in plasma by HPLC-

fluorescence Int J Vitam Nutr Res 66 166-170

Thatcher SR Mansfield RK Miller RB Davis CW Baertschi SW (2001a)

Pharmaceutical photostability a technical and practical interpretation of the ICH

guideline and its application to pharmaceutical stability Part 1 Pharm Tech 25 98-

110

Thatcher SR Mansfield RK Miller RB Davis CW Baertschi SW (2001b)

Pharmaceutical photostability a technical and practical interpretation of the ICH

220

guideline and its application to pharmaceutical stability Part 2 Pharm Tech 25 50-

62

Thiele JJ Dreher F Packer L (2000) Antioxidant defense systems in skin In

Elsner P Maibach HI Eds Cosmeceuticals- Drugs vs Cosmetics Marcel

Dekker New York Chap 11

Thomas DP (1997) Sailors scurvy and science J R Soc Med 90 50-54

Thomson CO Trenerry VC (1995) A rapid method for the determination of total L-

ascorbic acid in fruits and vegetables by micellar electrokinetic capillary

chromatography Food Chem 53 43-50

Tonnesen HH (1991) Photochemical degradation of components in drug formulations

1 An approach to the standardization of degradation studies Pharmazie 46 263-265

Tonnesen HH (2002) Photodecomposition of Drugs In Swarbrick J Boylan JC

Eds Encyclopedia of Pharmaceutical Technology Vol 3 Marcel Dekker New

York pp 2197-2203

Tonnesen HH Ed (2004) The Photostability of Drugs and Drug Formulations

Culinary and Hospitality Industry Publications Services Weimex TX

Tonnesen HH Karlsen J (1997) A comment on photostability testing according to

ICH guideline calibration of light sources PharmEuropa 9 735-736

Tonnesen HH Moore DE (1993) Photochemical degradation of components in drug

formulations Pharm Technol Int 5 27-33

Tournas JA Lin FH Burch JA Selim MA Monteiro-Riviere NA Zielinski

JE Pinnell SR (2006) Ubiquinone idebenone and kinetin provide ineffective

221

photoprotection to skin when compared to a topical antioxidant combination of

vitamins C and E with ferulic acid J Invest Dermatol 126 1185-1187

Traber MG (2007) Vitamin E In Zempleni J Rucker RB McCormick DB

Suttie JW Eds Handbook of Vitamins 4th ed Taylor amp Francis CRC Press

Boca Raton FL Chap 4

Traikovich SS (1999) Use of topical ascorbic acid and its effect on photodamaged skin

topography Arch Otolaryngol Head Neck Surg 125 1091-1098

Treadwell GE Cairns WL Metzler DE (1968) Photochemical degradation of

flavins V Chromatographic studies of the products of photolysis of riboflavin J

Chromatogr 35 376-388

Truswell AS (2003) ABC of Nutrition 4th ed BMJ Publishing Group Chap 10

Tsao CS Young M (1985) Analysis of ascorbic acid derivatives by high-performance

liquid chromatography with electrochemical detection J Chromatogr 13 855-856

Underberg WJM (1978) Oxidative degradation of pharmaceutically important

phenothiazines III Kinetics and mechanism of promethazine oxidation J Pharm Sci

67 1133-1138

Urso ML Clarkson PM (2003) Oxidative stress exercise and antioxidant

supplementation Toxicology 189 41-54

Vaid FHM (1998) Photolysis and Interaction of Thiamine Hydrochloride with

Riboflavin in Aqueous Solution PhD Thesis University of Karachi Karachi

Vaid FHM Shaikh RH Ansari IA Ahmad I (2005) Spectral study of the

photolysis of aqueous thiamine hydrochloride and ascorbic acid solution in the

presence and absence of riboflavin J Chem Soc Pak 27 227-232

222

Vaid F H M Shaikh RH Ansari I A Ahmad I (2006) Chromatographic study of

the photolysis of aqueous thiamine hydrochloride and ascorbic acid solutions in the

presence and absence of riboflavin J Chem Soc Pak 28 481-464

Valvani SC (2000) Industrial stability testing in the United States and computerization

of stability data In Carstensen JT Rhodes CT Eds Drug Stability Principles

and Practices 3rd

ed Marcel Dekker New York pp 534-536

Vanderslice JT Higgs DJ (1988) Chromatographic separation of ascorbic acid

isoascorbic acid dehydroascorbic acid and dehydroisoascorbic acid and their

quantitation in food products J Micronutr Anal 4 109-118

Vanderslice JT Higgs DJ Hayes JM Block G (1990) Ascorbic acid and

dehydroascorbic acid content of foods-as-eaten J Food Comp Anal 3 105-118

Vanderslice JT Higgs DJ (1993) Quantitative determination of ascorbic acid

dehydroascorbic acid by HPLC in foods and other matrices J Nutr Biochem 4 184-

190

Varvaresou A Tsirivas E Iakovou K Gikas E Papathomas Z Vonaparti A

Panderi I (2006) Development and validation of a reversed-phase ion-pair liquid

chromatography method for the determination of magnesium ascorbyl phosphate and

melatonin in cosmetic creams Anal Chim Acta 573-574 284-290

Verma KK Jain A Verma A Chaurasia A (1991) Spectrophotometric

determination of ascorbic acid in pharmaceuticals by background correction and flow

injection Analyst (London) 116 641-645

Vimaladevi M (2005) Textbook of Cosmetics CBS Publishers New Dehli Chap 2

and 17

223

Wallwork SC Grant DJW (1977) Physical Chemistry for Students of Pharmacy and

Biology 3rd ed Longmann New York pp 502-505

Waugh WA King CG (1932) Isolation and identification of vitamin C J Biol

Chem 97 325-331

Wechtersbach L Cigic B (2007) Reduction of dehydroascorbic acid at low pH J

Biochem Biophys Methods 70 767-772

Weiner N (1996) Introduction In Liberman HA Rieger MM Banker GS Eds

Pharmaceutical Dosage Forms Disperse Systems 2nd ed vol1 Marcel Dekker

New York Chap1

Werner W (1949) The reaction of l-ascorbic and d-isoascorbic acid with nicotinic acid

and its amide J Org Chem 14 22-26

Wiechers JW (2003) Formulating for Efficacy Proceedings of the 2003 IFSCC

Conference Seoul Korea

Wiechers JW (2005) Optimizing skin delivery of active ingredients from emulsions

from theory to practice In Rosen MR Ed Delivery System Handbook for

Personal Care and Cosmetic Products-Technology Applications and Formulations

William Andrew Inc Norwich NY Chap 20

Wille JJ (2005) Thixogel-Novel topical delivery systems for hydrophobic plant actives

In Rosen MR Ed Delivery System Handbook for Personal Care and Cosmetic

Products-Technology Applications and Formulations William Andrew Inc

Norwich NY Chap 36

224

Wright JL (1986) Vitamin A monograph In Connors KA Amidon GL Stella

VJ Eds Chemical Stability of Pharmaceuticals A Handbook for Pharmacists 2nd

ed Wiley New York pp 791-803

Yao AA Wathelet B Thonart P (2009) Effect of protective compounds on the

survival electrolyte leakage and lipid degradation of freeze-dried Weissella

paramesenteroides LC11 during storage J Microbiol Biotechnol 19 810-817

Yeh SY Lach JL (1961) Stability of morphine in aqueous solutions III Kinetics of

morphine degradation in aqueous solution J Pharm Sci 50 35-42

Yoshimura A Ohno T (1988) Lumiflavin-sensitized photooxygenation of indole

Photochem Photobiol 48 561-565

Yoshioka S Stella VJ (2000) Stability of Drugs and Dosage Forms Kluwer

Academic Plenum Publishers New York Chap 2

Zaeslein C (1982) Vitamins in the Field of Medicine Hoffman La Roche Basle pp

89-96

Zeng W Martinuzzi F MacGregor A (2005) Development and application of a novel

UV method for the analysis of ascorbic acid J Pharm Biomed Anal 36 1107-1111

Zhang GF Chen HY (2000) Chemiluminescence studies of the oxidation of ascorbic

acid with copper (II) catalyzed by halide anions and its applications to the

determination of halide anions and ascorbic acid Anal Sci 16 1317-1321

Zhang L Lerner S Rustrum WV Hofmann GA (1999) Electroporation-mediated

topical delivery of vitamin C for cosmetic applications Bioelectrochem Bioenerg

48 453-461

225

Zilva SS (1932) The non-specificity of the phenolindophenol reducing capacity of

lemon juice and its fractions as a measure of their antiscorbutic activity Biochem J

26 1624-1627

226

AUTHORrsquoS PUBLICATIONS

The author obtained his B Pharm degree in 2003 and joined the post graduate

program securing an M Phil degree in Pharmaceutics in 2006 from Baqai Medical

University He is a co-author of following publications

CHAPTER IN BOOK

1 Chapter on ldquoBorate Toxicity Effect on Drug Stability and Analytical

Applicationsrdquo by Iqbal Ahmad Sofia Ahmed MA Sheraz and Faiyaz H M

Vaid In Handbook on Borates Chemistry Production and Applications (MP

Chung Ed) Nova Science Publishers Inc NY USA (in press)

PAPERS PUBLISHED

INTERNATIONAL

2 Iqbal Ahmad Sofia Ahmed MA Sheraz and Faiyaz HM Vaid ldquoEffect of Borate

Buffer on the Photolysis of Riboflavin in Aqueous Solutionrdquo Journal of

Photochemistry and Photobiology B Biology 93 82-87 (2008)

3 Iqbal Ahmad Sofia Ahmed MA Sheraz M Aminuddin and Faiyaz HM Vaid

ldquoEffect of Caffeine Complexation on the Photolysis of Riboflavin in Aqueous

Solution A Kinetic Studyrdquo Chemical and Pharmaceutical Bulletin 57 (2009)

published online September 14 2009

4 Iqbal Ahmad MA Sheraz Sofia Ahmed and Faiyaz HM Vaid ldquoAnalytical

Applications of Boratesrdquo Materials Science Research Journal (in press)

5 Iqbal Ahmad Sofia Ahmed MA Sheraz Kefi Iqbal and Faiyaz HM Vaid

ldquoPharmacological Aspects of Boratesrdquo International Journal of Medical and

Biological Frontiers (in press)

6 Iqbal Ahmad Sofia Ahmed MA Sheraz Faiyaz HM Vaid and Izhar A Ansari

ldquoEffect of Divalent Ions on Photodegradation Kinetics and Pathways of

Riboflavin in Aqueous Solutionrdquo Photochemical and Photobiological Sciences

accepted

227

NATIONAL

7 Sofia Ahmed MA Sheraz and Iqbal Ahmad ldquoAdvances in Antioxidant Activity of

Vitamin Erdquo Journal of Baqai Medical University 10 13-18 (2007)

8 MA Sheraz Sofia Ahmed and Iqbal Ahmad ldquoDevelopments in the Clinical and

Food Analysis of Vitamin Crdquo Journal of Baqai Medical University 10 19-24

(2007)

9 A Azmi SNH Naqvi M Usman MA Sheraz and Sofia Ahmed ldquoPancreatic

Glucagon in Certain Ungulates Comparative Study of Extraction and

Bioassayrdquo Pakistan Journal of Entomology 20 23-28 (2005)

10 Iqbal Ahmad Sofia Ahmed MA Sheraz Faiyaz HM Vaid and S Hasan

ldquoAdvances in Biochemical Functions and the Photochemistry of Flavins and

Flavoproteinsrdquo Pakistan Journal of Pharmaceutical Sciences in press

11 MA Sheraz Sofia Ahmed and Iqbal Ahmad ldquoEffect of Borates on the Stability of

Chemical and Pharmaceutical Compoundsrdquo Journal of Baqai Medical University

accepted

PAPERS SUBMITTED

12 Iqbal Ahmad MA Sheraz Sofia Ahmed Riaz H Shaikh and Faiyaz HM Vaid

ldquoPhotostability of Ascorbic Acid in Organic Solvents and Cream Formulationsrdquo

Chemical and Pharmaceutical Bulletin

13 Iqbal Ahmad MA Sheraz Sofia Ahmed Riaz H Shaikh and Faiyaz HM Vaid

ldquoPhotochemical Interaction of Ascorbic Acid with Riboflavin Nicotinamide and

Alpha-Tocopherol in Cream Formulationsrdquo Journal of Cosmetic Science

14 Iqbal Ahmad Kefi Iqbal Sofia Ahmed MA Sheraz ldquoApplications of Laser Flash

Photolysis Spectroscopy and Electron Microscopy in Photopolymerization and

Development of Glass Ionomer Dental Cementsrdquo Materials Science Research

Journal

15 Sofia Ahmed MA Sheraz M Aminuddin I Ahmad and Faiyaz HM Vaid ldquoA

Rapid Titrimetric Assay for Quantitation of Vitamin B1 in Neat and

Pharmaceutical Preparationsrdquo Pakistan Journal of Pharmaceutical Sciences

• 01 SZ-786
• 02 SZ-title
• 03 SZ-Certificate
• 04 SZ-Abstract
• 05 SZ-Acknowledgement
• 06 SZ-Dedication
• 07 SZ-Contents
• 08 SZ-Chapter 1
• 09 SZ-Chapter 2
• 10 SZ-Chapter 3
• 11 SZ-Object of Present Investigation
• 12 SZ-Chapter 4
• 13 SZ-Chapter 5
• 14 SZ-Chapter 6
• 15 SZ-Chapter 7
• 16 SZ-Conclusion
• 17 SZ-References
• 18 SZ-Authors Publications

iv

ABSTRACT

The present investigation is based on a study of the photodegradation of ascorbic

acid (vitamin C) in organic solvents and in oil-in-water cream preparations containing a

combination of emulsifying agents and humectants It also involves the study of the effect

of other vitamins (riboflavin nicotinamide and alpha-tocopherol) and certain compounds

acting as stabilizing agents (citric acid tartaric acid and boric acid) on the rate of

photodegradation of ascorbic acid in cream preparations The photodegradation of

ascorbic acid in organic solvents and cream preparations (pH 40ndash70) leads to the

formation of dehydroascorbic acid which is also biologically active The kinetics of

photodegradation of ascorbic acid alone and in combination with other vitamins in

creams has been studied using a UV spectrophotometric method and the official

iodimetric method respectively These methods were validated in the presence and

absence of other vitamins stabilizing agents under the experimental conditions

employed The recoveries of ascorbic acid in creams are in the range of 90ndash96 and the

reproducibility of the analytical methods is within plusmn 5

The apparent first-order rate constants (kobs) for the photodegradation of ascorbic

acid in aqueous organic solvents (029ndash040 times 10ndash3

minndash1

) and in creams (044ndash142 times

10ndash3

minndash1

) have been determined A linear relationship has been observed between kobs

and solvent dielectric constant reciprocal of solvent viscosity indicating the dependence

of the rate of photodegradation on solvent characteristics

In the creams the photodegradation of ascorbic acid appears to be affected by the

concentration of other vitamins pH of the medium carbon chain length of the

emulsifying agents (myristic acid palmitic acid and stearic acid) viscosity of the

v

humectant (ethylene glycol propylene glycol and glycerin) and redox potentials of

ascorbic acid The study indicates that the relative polar character of the emulsifying

agent and the ionized state and redox potential of ascorbic acid at a particular pH are

important factors in the photodegradation of ascorbic acid in creams

The second-order rate constants (kprime) (320 times 10ndash2

ndash 189 Mndash1

minndash1

) for the

photochemical interaction of ascorbic acid and the individual vitamins (riboflavin

nicotinamide alpha-tocopherol) along with the values of k0 obtained from the intercepts

of the plots of kobs versus vitamin concentration are also reported The values of k0

indicate that riboflavin and nicotinamide act as photosensitizing agents and alpha-

tocopherol acts as a stabilizing agent in the photodegradation of ascorbic acid in the

creams The kobs verses pH profiles for the photodegradation of ascorbic acid in creams

represents sigmoid type curves indicating the oxidation of the ionized form (AHndash) of

ascorbic acid (pKa1 41) with pH The AHndash species appears to be more susceptible to

photooxidation than the non-ionized form of ascorbic acid The effect of stabilizing

agents on the photodegradation of ascorbic acid has been found to be in the order of citric

acid gt tartaric acid gt boric acid The low activity of boric acid may be to some extent due

to its interaction with the emulsifying agents and humectants The polarity of the

emulsifying acids also plays a part in the rate of degradation of ascorbic acid Reaction

schemes for the photodegradation of ascorbic acid and its photochemical interaction with

riboflavin nicotinamide and alpha-tocopherol have been presented

vi

ACKNOWLEDGMENTS

I am highly grateful to All Mighty Allah who guided me in all difficulties and

provided me strength to overcome the problems during this work

Words are confined and inefficacious to express my immense gratitude to my

respectable supervisor Prof Dr Iqbal Ahmad Department of Pharmaceutical

Chemistry for his guidance encouragement keen interest and above all giving his

valuable time suggestions and attention His personality has been a source of constant

inspiration through out my research work

I would like to extend my sincere thanks to Prof Lt Gen (R) Dr Syed Azhar

Ahmed Vice Chancellor Baqai Medical University for his personal interest and

constant encouragement through out the study

It is my great desire to express my gratitude to Prof Dr Syed Fazal Hussain

CEO Baqai Institute of Pharmaceutical Sciences for his cooperation and attention and

providing all the facilities of the Institute at my disposal during the research work

I am also thankful to Mrs Shaukat Khalid Dean Faculty of Pharmaceutical

Sciences for her support during the study

I feel honored to express my sincere thanks and indebtedness to Prof Dr

Khursheed Ali Khan Department of Pharmaceutics Prof Dr Aminuddin Department

of Pharmaceutical Chemistry and Dr Faiyaz H M Vaid Chairman Department of

Pharmaceutical Chemistry Faculty of Pharmacy University of Karachi who helped me

selflessly with their invaluable suggestions through out the research work

vii

I feel immense pleasure to pay my sincere and special thanks to Ms Sofia

Ahmed Assistant Professor and In charge Department of Pharmaceutics who lent all

sort of cooperation and spared no effort in helping me during this work

Special thanks are due to Mr Saif-ur-Rehman Khatak Deputy Drug Controller

for his cooperation and help during this study

I acknowledge with sincere thanks the contribution of Tabros Pharmaceutical

Industry Karachi for providing me the opportunity to use their facilities for certain

measurements without which the completion of this work would not have been possible

I highly appreciate the technical services rendered by Mr Anees Mr Wajahat

and Mr Sajjad in pursuance of this study

I am very grateful to Mrs Prof Dr Iqbal Ahmad for her kindness and generous

hospitality during my innumerable visits to their residence

Last but not the least I would like to express my immense indebtedness to My

Gracious Parents Beloved Brothers and Sisters for their moral support kindness and

encouragement throughout my career

I am also thankful to all my students for their affectionate feelings

M A S

viii

To

My Beloved Parents amp

Late Prof Dr S Sabir Ali for their interest and endless support

ix

CONTENTS

Chapter Page

ABSTRACT iv

ACKNOWLEDGEMENTS vi

I INTRODUCTION 1

11 HISTORICAL BACKGROUND 2

12 PHYSICOCHEMICAL CHARACTERISTICS OF

ASCORBIC ACID

2

13 CHEMISTRY OF ASCORBIC ACID 3

131 Nomenclature and Structure 3

132 Chemical Stability 3

14 BIOCHEMICAL FUNCTIONS 7

15 ANTIOXIDANT ACTIVITY 8

16 PHOTOSTABILITY OF DRUGS 9

17 KINETIC TREATMENTS OF PHOTOCHEMICAL

REACTIONS

12

18 LITERATURE ON ASCORBIC ACID 15

II PHOTODEGRADATION REACTIONS AND ASSAY OF

ASCORBIC ACID

17

21 PHOTODEGRADATION REACTIONS 18

211 Photodegradation of Ascorbic Acid 18

212 Effect of Various Substances on Photodegradation of Ascorbic

Acid

20

213 Photosensitized Oxidation of Ascorbic Acid 22

214 Ascorbic Acid Interaction with Other Water-Soluble Vitamins 25

22 ASSAY OF ASCORBIC ACID 26

221 Spectrophotometric Methods 26

222 Fluorimetric Methods 28

x

223 Mass spectrometric Methods 28

224 Chromatographic Methods 28

225 Enzymatic Methods 29

226 Commercial Kits for Clinical Analysis 30

227 Analysis in Creams 30

III FORMULATION AND STABILITY OF CREAM

PREPARATIONS

31

31 FORMULATION OF CREAM PREPARATIONS 32

311 Choice of Emulsion Type 32

312 Choice of Oil Phase 33

313 Emulsion Consistency 33

314 Choice of Emulsifying Agent 34

315 Formulation by the HLB Method 34

316 Concept of Relative Polarity Index 35

32 FORMULATION OF ASCORBIC ACID CREAMS 37

33 STABILITY OF CREAMS 39

331 Physical Stability 39

332 Chemical Stability 39

333 Microbial Stability 40

334 Stability of Ascorbic Acid in Liquid Formulations 41

335 Stability of Ascorbic Acid in Emulsions and Creams 41

336 Stability Testing of Emulsions 45

3361 Macroscopic examination 46

3362 Globule size analysis 46

3363 Change in viscosity 46

3364 Accelerated stability tests 46

337 FDA Guidelines for Semisolid Preparations 46

xi

OBJECT OF PRESENT INVESTIGATION 48

IV MATERIALS AND METHODS 51

41 MATERIALS 52

42 METHODS 55

421 Cream Formulations 55

422 Preparation of Creams 56

423 Thin-Layer Chromatography 57

424 pH Measurements 57

425 Ultraviolet and Visible Spectrometry 58

426 Photolysis of Ascorbic Acid 59

4261 Creams 59

4262 Aqueous and organic solvents 59

4263 Storage of creams in dark 59

427 Measurement of Light Intensity 59

428 Procedure 60

4281 Calculation 62

429 Viscosity Measurements 63

4210 Assay method 65

42101 UV spectrophotometric method for the assay of creams

containing ascorbic acid alone

65

42102 Iodimetric method for the assay of ascorbic acid in creams

containing riboflavin nicotinamide and alpha-tocopherol 65

42103 Spectrophotometric method for the assay of ascorbic acid in

aqueous and organic solvents

67

V PHOTODEGRADATION OF ASCORBIC ACID IN

ORGANIC SOLVENTS AND CREAM FORMULATIONS

68

51 INTRODUCTION 69

52 PHOTOPRODUCTS OF ASCORBIC ACID 71

xii

53 SPECTRAL CHARACTERISTICS OF PHOTOLYSED

SOLUTIONS

71

54 ASSAY OF ASCORBIC ACID IN CREAMS AND

SOLUTIONS

73

55 EFFECT OF SOLVENT 74

56 EFFECT OF CONCENTRATION 80

57 EFFECT OF CARBON CHAIN LENGTH OF

EMULSIFYING AGENT

88

58 EFFECT OF VISCOSITY 94

59 EFFECT OF pH 94

510 EFFECT OF REDOX POTENTIAL 96

511 PRIMARY PHOTOCHEMICAL REACTIONS IN THE

OXIDATION OF ASCORBIC ACID

97

512 DEGRADATION OF ASCORBIC ACID IN THE DARK 98

VI PHOTOCHEMICAL INTERACTION OF ASCORBIC

ACID WITH RIBOFLAVIN NICOTINAMIDE AND

ALPHA-TOCOPHEROL IN CREAM FORMULATIONS

109

61 INTRODUCTION 110

62 ABSORPTION CHARACTERISTICS OF PHOTOLYSED

CREAMS

114

63 PHOTOPRODUCTS OF ASCORBIC ACID AND OTHER

VITAMINS

114

64 ASSAY METHOD 116

65 KINETICS OF PHOTODEGRADATION OF ASCORBIC

ACID

117

66 INTERACTION OF RIBOFLAVIN WITH ASCORBIC ACID 128

67 INTERACTION OF NICOTINAMIDE WITH ASCORBIC

ACID

129

68 INTERACTION OF ΑLPHA-TOCOPHEROL WITH

ASCORBIC ACID

130

69 EFFECT OF CARBON CHAIN LENGTH OF

EMULSIFYING AGENT

130

xiii

610 EFFECT OF VISCOSITY OF CREAMS 132

611 DEGRADATION OF ASCORBIC ACID IN THE PRESENCE

OF OTHER VITAMINS IN THE DARK

135

VII STABILIZATION OF ASCORBIC ACID WITH CITRIC

ACID TARTARIC ACID AND BORIC ACID IN CREAM

FORMULATIONS

141

71 INTRODUCTION 142

72 CREAM FORMULATIONS 142

73 PRODUCTS OF ASCORBIC ACID

PHOTODEGRADATION

145

74 SPECTRAL CHANGES IN PHOTODEGRADED CREAMS 145

75 ASSAY OF ASCORBIC ACID IN CREAMS 145

76 KINETICS OF PHOTODEGRADATION 146

77 EFFECT OF STABILIZING AGENTS 146

78 DEGRADATION OF ASCORBIC ACID IN PRESENCE OF

STABILIZING AGENTS IN THE DARK

162

79 EFFECT OF ADDITIVES ON TRANSMISSION OF

ASCORBIC ACID

163

CONCLUSIONS AND SUGGESTIONS 179

CONCLUSIONS 180

SUGGESTIONS 184

REFERENCES 185

AUTHORrsquoS PUBLICATIONS 226

CHAPTER I

INTRODUCTION

2

11 HISTORICAL BACKGROUND

The disease scurvy which now is known as a condition due to a deficiency of

ascorbic acid in the diet has considerable historical significance (Schick 1943

Carpenter 1986 Bardolph and Taylor 1997 Thomas 1997 Bors 2005) Zilva (1932)

isolated the antiscorbutic activity factor from a crude fraction of lemon and showed that

the activity was destroyed by oxidation and protected by reducing agents Waugh and

King (1932) isolated crystalline vitamin C from lemon juice and showed it to be the

antiscorbutic factor Szent-Gyorgyi (1928) had isolated the same factor from pepper in

connection with his biological oxidation-reduction studies Hirst and Zilva (1933)

identified the antiscorbutic factor as ascorbic acid Early work on the chemical

identification and elucidation of the structure of ascorbic acid has been well documented

(Carpenter 1986) The first synthesis of L-ascorbic acid was achieved almost

simultaneously by Ault et al (1933) and Reichstein et al (1933)

Plants and most animals synthesize their own vitamin C but humans lack this

ability due to the deficiency in an enzyme L-gulono-gamma-lactone oxidase that

catalyzes the terminal step in ascorbic acid biosynthesis (Nishikimi et al 1994)

Therefore humans obtain this vitamin from diet and or vitamin supplements to not only

avoid the development of scurvy but also for overall well being (Stone 1969 Lewin

1976 Davies et al 1991) The minimal daily requirement for ascorbic acid in healthy

adults is 40ndash60 mg (Truswell 2003 Mason 2007 Eitenmiller et al 2008 Elia 2009)

12 PHYSICOCHEMICAL CHARACTERISTICS OF ASCORBIC ACID

The important physicochemical characteristics of ascorbic acid (Table 1) involved

in its identification and degradation are described by many authors (Connors et al 1986

3

OrsquoNeil 2001 Moffat et al 2004 Sinko 2006 Johnston et al 2007) The most

important chemical property of ascorbic acid is the reversible oxidation to semidehydro-

L-ascorbic acid and further oxidation to dehydro-L-ascorbic acid This property is the

basis for its physiological activity In addition the proton on oxygenndash3 is acidic (pKa1 =

417) which contributes to the acidic nature of ascorbic acid (1)

13 CHEMISTRY OF ASCORBIC ACID

131 Nomenclature and Structure

The IUPAC-IUB Commission on Biochemical Nomenclature changed the name

vitamin C (2-oxo-L-theo-hexono-4-lactone-23-enediol) to ascorbic acid or L-ascorbic

acid in 1965 (Johnston et al 2007) The chemical structure of ascorbic acid (1) is

HO OH

O

OHHO

H

(1)

O

The molecule has a near planar five-membered ring with two chiral centers

which contain four stereoisomers

132 Chemical Stability

Ascorbic acid is sensitive to air and light and is kept in a well-closed container

protected from light (British Pharmacopoeia 2009) The degradation reactions of

ascorbic acid in aqueous solution depend on a number of factors such as pH temperature

presence of oxygen or metal It is not very stable in aqueous media at room

temperature and undergoes oxidative degradation to dehydroascorbic acid and

4

Table 1 Physicochemical characteristics of ascorbic acid

Empirical formula C6H8O6

Molar mass 17613

Crystalline form Monoclinic mix of platelets and needles

Melting point 190 to 192 degC

[α]25

+205deg to +215deg

pH

5 mg ml

50 mg ml

~3

~2

pKa 417 1157 (20deg)

Redox potential

(dehydroascorbic acid ascorbate)

(H+ ascorbate

ndash)

ndash174 mV

+282 mV

Solubility g ml

Water

Ethanol absolute

Ether chloroform benzene

033

002

Insoluble

UV spectrum

Absorption maximum [A(1 1 cm)]

pH 20

pH 70

245 nm [695]

265 nm [940]

Infrared spectrum

Principal peaks (Nujol mull)

1026 (CminusOH str) 1111(CminusOminusC str) 1312

(minusCminusOminus str) 1653 (C=O str) 990 (C=C str)

cmndash1

Mass spectrum

Principal ions at mz

29 41 39 42 69 116 167 168

D

5

23-diketogulonic acid The stability of ascorbic acid and dehydroascorbic acid can be

improved by lowering the pH below 2 (Wechtersbach and Cigic 2007) Above pH 7

alkali-catalyzed degradation by cleavage at Cndash1 or Cndash2 results in a number of

compounds mainly monondash dindash and tricarboxylic acids (Connors et al 1986 Bors and

Buettner 1997 Halliwell and Whiteman 1997) The oxidative degradation of ascorbic

acid and dehydroascorbic acid in parenteral nutrition mixtures is catalyzed by trace

elements particularly copper (Allwood 1984ab Allwood et al 1992 Allwood and

Kearney 1998 Kearney et al 1998 Gibbons et al 2001) Stabilized ascorbic acid

preparations in hydroalcoholic vehicle (Kaplan et al 1989) and aquaculture feeds

(OrsquoKeefe 2001) have been reported The various oxidation products of ascorbic acid are

shown in Fig 1

It is interesting to note that in addition to redox and acid-base properties ascorbic

acid can exist as a free radical (Bielski et al 1981 Bielski 1982 Halliwell 1996 Bors

and Buettner 1997) The ascorbate radical anion is an important intermediate in the

reactions involving oxidants and ascorbic acidrsquos antioxidant activity Rate constants for

the generation of ascorbate radicals are in the range of 104ndash10

8 s

ndash1 When ascorbate

radicals are generated by oxyanions the rate constants are of the order of 104ndash10

7 s

ndash1

when generated by halide radicals 106ndash10

8 s

ndash1 and when generated by tocopherols and

flavonoids radicals 106ndash10

8 s

ndash1 (Bielski 1982 Halliwell and Whiteman 1997) The

ascorbate radicals decay usually by disproportionation However a change in ionic

strength or pH can influence the rate of dismutation of ascorbic acid Certain oxyanions

such as phosphates accelerate dismutation (Bielski et al 1981) The acceleration is

attributed to the activity of various protonated forms of phosphate to donate a proton

6

Fig 1 Oxidation products of ascorbic acid

O

OHOH

H

OO

OHOH

H

OO

OHOH

H

O

Ascorbyl radical anion

(interm ediate)

Ascorbic acid

(1)

-e- -2H

+

+e- +2H

+

-e-

+e-

Dehydroascorbic acid

(2)

23-diketo-L-gulonic acid

O xalic acid

+

L-Threonic acid

L-Xylose

+

C O 2

CO 2

L-Xylonic acid

+

L-Lyxonic acid CO 2

HO OH O O-

O O

7

efficiently to the ascorbate radical particularly the dimer form of ascorbate

The unusual stability of the ascorbate radical in biological systems dictates that

accessory enzymatic systems be made available to reduce the potential transient

accumulation of ascorbate radical The excess ascorbate radicals may initiate a chain of

free-radicals reactions In plants NADHmonodehydroascorbate reductase maintains

ascorbic acid in its reduced form NADHmonodehydroascorbate reductase plays a major

role in stress related responses in plants Glutathione dehydroascorbate reductase serves

this purpose in animal tissues Such enzymes keep ascorbic acid operating at maximum

efficiency so that other enzyme systems may take advantage of the univalent redox

cycling capacity of ascorbate (Asard et al 2004 Johnston et al 2007)

The anaerobic degradation of ascorbic acid has been studied by Finholt et al

(1963) Under these conditions the molecule is dehydrated and hydrolyzed in aqueous

solution to give furfural and carbon dioxide The rate of degradation is maximum at pH

41 corresponding to the pKa of ascorbic acid This has been suggested due to the

formation of a saltndashacid complex in solution The reaction is dependent on buffer

concentration but has relatively small effect of ionic strength

14 BIOCHEMICAL FUNCTIONS

Ascorbic acid plays an essential role in the activities of several enzymes It is vital

for the growth and maintenance of healthy bones teeth gums ligaments and blood

vessels It is important for the manufacture of certain neurotransmitters and adrenal

hormones Ascorbic acid is required for the utilization of folic acid and the absorption of

iron It is also necessary for normal immune responses to infection and for wound healing

(Henry 1997)

8

Ascorbic acid deprivation and scurvy include a range of signs and symptoms that

involves defects in specific enzymatic processes (Johnston et al 2007) The

administration of ascorbic acid improves most of the signs of chemically induced

glutathione (L-γ-glutamyl-L-cysteine-glycine GSH) deficiency (Meister 1994) The

effect is very pronounced in newborn rats which do not efficiently synthesize ascorbic

acid in contrast to adult rats and guinea pigs When L-buthionine-(SR)-sulphoxime is

administered in addition to the loss in GSH there is a marked increase in

dehydroascorbic acid This has led to the hypothesis that GSH is very important to

dehydroascorbic acid reduction and as a sequence to ascorbic acid recycling (Meister

1995)

Ascorbic acid also possesses pro-oxidant properties and may cause apoptosis

lymphoid and myeloid cells It has been shown that dehydroascorbic acid also stimulates

the antioxidant defenses in some cells by preferentially importing dehydroascorbate over

ascorbate (Braun et al 1997 Banhegyi et al 1998 Puskas et al 2000 2002)

15 ANTIOXIDANT ACTIVITY

Ascorbic acid is known to readily scavenge reactive oxygen and nitrogen species

such as superoxide and hydroperoxyl radicals aqueous peroxyl radicals singlet oxygen

ozone peroxynitrite nitrogen dioxide nitroxide radicals and hypochlorous acid Excess

of such products has been associated with lipids (Niki and Noguchi 1997 Carr et al

2000 Urso and Clarkson 2003) DNA (Fraga et al 1991 1996 Lindahl 1993) and

protein oxidation (Stadtman 1991 Berlett and Stadtman 1997 Dean et al 1997

Ortwerth and Monnier 1997 Padayatty et al 2003)

9

The electron donor character of ascorbate may be responsible for many of its

known biological functions Inspite of the availability of ascorbic acid to influence the

production of hydroxyl and alkoxyl radicals it remains uncertain whether this is the

principal effect or mechanism that occurs in vivo There is a good evidence that ascorbic

acid protects lipids in biological fluids as an antioxidant (Johnston et al 2007) A

detailed account of the function of ascorbate as an antioxidant and its reactions with

reactive nitrogen species and singlet oxygen has been reported by Packer et al (2002)

and Buettner and Schafer (2004)

Ascorbic acid (Eordm ndash0115 V pH 52 Sinko 2006) has been used as an antioxidant

for the stabilization of drugs with a higher oxidation potential These drugs include

morphine (Yeh and Lach 1961) vitamin A (Wright 1986) rifampin (Maggi et al

1966) cholecalciferol (Nerlo et al 1968 Sawicka 1991) promethazine (Underberg

1978) and sulphacetamide and sulphanilamide (Ahmad and Ahmad 1983)

16 PHOTOSTABILITY OF DRUGS

Many drug substances are sensitive to light (British Pharmacopoeia 2009) and

may degrade in pharmaceutical formulations to inactive or toxic compounds This could

make a product therapeutically inactive while in use by the patients The

photodegradation (photolysis) of drug substances may occur not only during storage but

also during the use of the product It may involve several mechanisms including

oxidation reduction hydrolysis decarboxylation isomerization rearrangement and other

reactions Normal sunlight or room light may cause substantial degradation of drug

molecules The study of degradation of drug substances under the action of UVvisible

light is relevant to the process of drug development for several reasons such as

10

Exposure to light can influence the stability of a drug formulation resulting in the

loss of potency

Inappropriate exposure to light of the raw material or the final product can lead to

the formation of toxic photoproducts that are dangerous to health

Information about the stability of drug substances and formulations is needed to

predict the shelf-life of the final product (Tonnesen and Moore 1993)

The development of light-activated drugs involves activation of the compound

through photochemical reactions (Tonnesen 1991)

Adverse effects due to the formation of minor degradation products during

storage and administration have been reported (de Vries et al 1984) The drugs

substances may also cause light-induced side effects after administration to the patient by

interaction with endogenous substances The study of the photochemical properties of

drug substances and formulated products is an integral part of formulation development

to ensure the safety and efficacy of the product

The photodegradation of drug substances occurs as a result of the absorption of

radiation energy by a molecule (A) to produce an excited state species (A) (11) The

absorbed energy can be lost either by a radiative process involving fluorescence or

phosphorescence (12) or by a physical or chemical radiationless process The physical

process results in the loss of energy as heat (13) or by collisional quenching (14) The

chemical decay leads to the formation of a new species (15) The whole process is

represented as

11

A A (11)

A A + hυprime (12)

A A + heat (13)

A + A 2A (14)

A product (s) (15)

According to the Stark-Einstein law the absorption of one quantum of radiation

results in the formation of one excited molecule which may take part in several

photochemical processes [Eqs (11)ndash(15)] The quantum yield φ for any one of these

processes is defined by

Number of molecules undergoing the photochemical process φ =

Number of quanta absorbed

Considering a pure photochemical reaction the quantum yield has a value of 0ndash1

however if A is a radical that can take part in a free-radical chain reaction so that the

absorption of energy simply initiates the reaction then each quantum of energy may

result in the decomposition of molecules and φ may appear to be greater than 1 (Connors

et al 1986)

Detailed information on the photostability and photodegradation of drug

substances including vitamins alone or in solid or liquid formulations is available in the

reviews published by DeRitter (1982) Albini and Fasani (1998) Sequeira and Vozone

(2000) Tonnesen (2002 2004) Yoshioka and Stella (2002) Min and Boff (2002) Reed

et al (2003) Fasani and Albini (2005) and Sinko (2006) The photostability of cosmetic

materials has been reviewed by Sugden (1985) Important aspects dealing with the

photostability testing of drug substances have been dealt by Anderson et al (1991)

k1

k2

k3

k4

hυ

12

Tonnesen and Moore (1993) Tonnesen and Karlsen (1997) Riehl et al (1995) ICH

(1997) Singh and Bakshi (2000) Valvani (2000) Thatcher et al (2001ab) Fasani and

Albini (2005) Klick et al (2005) Singh (2006) and Ahmad and Vaid (2006)

17 KINETIC TREATMENT OF PHOTOCHEMICAL REACTIONS

The kinetic treatment of photochemical reactions with reference to the

photostability of drug substances has been considered by Moore (2004) and is presented

in this section

The photostability testing of a drug substance at the preformulation stage involves

a study of the drugrsquos rate of degradation in solution on exposure to light for a period of

time The value of the degradation rate constant depends very much on the design of the

experimental conditions (eg concentration solvent pH irradiation source oxygen

content) The factors that determine the rate of a photochemical reaction are simply the

rate at which the radiation is absorbed by the test sample (ie the number N of photons

absorbed per second) and the efficiency of the photochemical process (ie the quantum

yield of the reaction φ) For a monochromatic photon source the number of photons

absorbed depends upon the intensity of the photon source and the absorbance at that

wavelength of the absorbing species The rate of a photochemical reaction is defined as

Rate = number of molecules transformed per second = N φ (16)

In the first instance the rate can be determined for a homogeneous liquid sample

in which the only photon absorption is due to the drug molecule undergoing

transformation with the restriction that the concentration is low so that the drug does not

absorb all of the available radiation in the wavelength range corresponding to its

13

absorption spectrum The value of N can be derived at a particular wavelength λ and is

given by

Nλ = Iλ ndash It = Iλ (1 ndash 10ndashA

) (17)

where Iλ and It are the incident and transmitted radiation intensities respectively and A is

the absorbance of the sample at the wavelength of irradiation This expression can be

expanded as a power series

Nλ = 2303 Iλ (A + A22 + A

36 + hellip) (18)

When the absorbance is low (Alt 002) the second- and higher-order terms are negligible

and the expression simplifies to the first term in Eq 18 Given the Beerrsquos law relation

between absorbance and concentration N can be seen to be directly proportional to

concentration

Nλ = 2303 Iλ A = 2303 Iλ ελ b C (19)

where ελ is the molar absorptivity at wavelength λ C the molar concentration of the

absorbing species and b the optical path length of the reaction vessel Now Iλ and ελ vary

with wavelength so the expression must be integrated over the relevant wavelength range

where each has a non-zero value

N = 2303 b C int (Iλ ελ) dλ integrated from λ1 to λ2 (110)

Thus

Rate = 2303 b C φ int (Iλ ελ) dλ (111)

Now the overlap integral (int Iλ ελ dλ) is a constant for a particular combination of photon

source and absorbing substance b is determined by the reaction vessel chosen and φ is a

characteristic of the reaction Thus by grouping the constant terms into an overall

constant k1 the expression is simplified to a first-order kinetic equation

14

Rate = ndashd [Drug] dt = k1C (112)

The integrated form of Eq 112 can be expressed in exponential form (Eq 113) or

logarithmic form (Eq 114)

[Drug]t = [Drug]0 endashk1t

(113)

ln [Drug]t = ln [Drug]0 ndash k1t (114)

Verification of first-order kinetics is obtained when a plot of the logarithm of the

concentration of drug remaining is linear with slope equal to (ndashk1)

Eq 112 predicts that a photodegradation reaction studied at low concentrations in

solution will follow first-order kinetics however the rate constant derived from a study

performed in one laboratory will not be the same as that found in another The reason for

this is the inherent difficulty in reproducing exactly the experimental arrangement of

photon source and sample irradiation geometry Therefore the relative values of the rate

constants are useful in a given experimental arrangement for making comparisons of

degradation of the absorbing substance in different formulations eg those containing

ingredients designed to inhibit the photoreaction The use of rate constants is helpful for

comparative purposes when studying a number of different reaction mixtures under the

same irradiation conditions such as the effect of pH on the degradation of a drug

However the reaction order and numerical values of the rate constants are relative to the

specific conditions used

15

18 LITERATURE ON ASCORBIC ACID

A large number of reviews have been published on various aspects of ascorbic

acid A list of important reviews is given below

Chemistry biochemical functions and related aspects

Rosenberg (1945) Burns (1961) King and Burns (1975) Sim (1972) Hanck

(1982) Zaeslein (1982) Seib and Tolbert (1982) Carpenter (1986) Levine

(1986) Davies et al (1991) Halliwell and Whiteman (1997) Ortega and Delgado

(1998) Asard et al (2004) Hickey and Roberts (2004) Johnston et al (2007)

Eitenmiller (2008)

Chemical and pharmaceutical stability

Macek (1960) Garrett (1967) Carstensen (1972) Dale and Booth (1976) Hashmi

(1973) Litner (1973) DeRitter (1982) Allwood (1984ab) Allwood and Kearney

(1998) Connors et al (1986) Smith et al (1988) Racz (1989) Roth et al 1991

Ball (2006) Eitenmiller et al (2008) Sweetman (2009)

Methods of assay and chromatography

Mader (1961) Gyorgy and Pearson (1967) Bolliger and Konig (1969) Hashmi

(1973) Al-Meshal and Hassan (1982) Pelletier (1985) Lambert and deLeenheer

(1992) Halver and Felton (2001) Moffat et al (2004) Ball (2006) Eitenmiller et

al (2008)

Pharmacology and related aspects

Levine (1986) Dollery (1999) Sauberlich (1994ab) McDowell (2000)

Kaushansky and Kipps (2006) Sweetman (2009)

16

Antioxidant activity

Basu et al (1999) Shacter (2000) Thiele et al (2000) Cadenas and Packer

(2002) Packer et al (2002) Padayathy et al (2003) Parker and Parker (2003)

Burke (2006) Johnston et al (2007)

Cosmetic Preparations

Barel et al (2001) Salvador and Chisvert (2007) Rosen (2005) Bissett (2006)

Chaudhri and Jain (2009)

CHAPTER II

PHOTODEGRADATION

REACTIONS AND ASSAY

OF ASCORBIC ACID

18

21 PHOTODEGRADATION REACTIONS

211 Photodegradation of Ascorbic Acid

Aqueous ascorbic acid (1) solutions are degraded by UV light to give

dehydroascorbic acid (2) (Arcus and Zilva 1940) Ascorbic acid degradation at a

concentration of 52 and 50 mg on UV irradiation for 2 hours gave a loss of 43 and 8

respectively Dehydroascorbic acid solutions are more stable to UV light than the

ascorbic acid (Kitagawa 1968) In many natural products the vitamin is oxidized on

exposure to air and light (OrsquoNeil 2001) When solutions of multivitamin preparations are

exposed to light H2O2 as well as organic peroxides are generated and specific

byproducts that differ from dehydroascorbic acid and 23-diketogulonic acid (3) are

produced (Lavoie et al 2004)

In aqueous neutral or alkaline solution ascorbic acid (1) undergoes chemical or

photochemical oxidation to dehydroascorbic acid (2) which upon saponification of the

lactone ring under the influence of the base water produces 23-diketo-L-gulonic acid (an

α szlig- diketogulonic acid) (3) This acid undergoes further oxidation to oxalic acid (4) and

L-threonic acid (5) (Racz 1989) (Fig 2a) At room temperature oxalic acid (4) is also

formed along with threonolactone (6) by photochemical degradation of ascorbic acid (1)

in the presence of singlet oxygen (1O2) (Silva and Quina 2006) (Fig 2a) The low-

temperature photooxygenation of ascorbic acid (1) gives a mixture of unstable

hydroperoxide ketones (7) and (8) which on standing interconvert and cyclize to

hydroperoxyhemiketal (9) The hydroperoxyhemiketal breaks down on warming to

produce the oxalate esters of threonic acid (10) (Fig 2b) (Kwon and Foote 1988)

19

COOH

COOH

O

OHHO

O

HOH2C

HO2

O

O

HO

OO

O O2H

OHHO

O

HOH2C

OH

O

O

OH

O2H

OO

HO O2CCO2H

(1)hv

room temperature

(4)(6)

(1)hv

85 oC

(7)

(a)

(8)

+

cyclization

(9)

ring cleavage

(b)

(10)

(2)

OH O

OHHO

OH O O

(3)

OH OH

OH

OH O

O

OH

1O2 [O]

+

(5)

COOH

COOH

(4)

+

OH

Fig 2 Photooxidation of ascorbic acid at room and low temperature

20

An important consideration in the stability of ascorbic acid in total parenteral

nutrition (TPN) solutions is the generation of hydrogen peroxide in the presence of light

(Laborie et al 1998 1999 2000 2002 Chessex et al 2002) This may result from the

oxidation of ascorbate anion by molecular oxygen (Homann and Gaffron 1964 Taqui

Khan and Martell 1967 Mushran and Agarwal 1977 Hughes 1985 De La Rochette et

al 2000) leading to further degradation of ascorbic acid (Deutsch 1998a 1998b

1998c) The kinetics and mechanism of oxidation reactions of ascorbic acid have been

studied by several workers (Taqui Khan and Martell 1967 Ogata and Kosugi 1969

Blaugh and Hajratwala 1972 Fessenden and Verma 1978 Abe et al 1986 Kwon et al

1989 Fornaro and Coicher 1998 Njus et al 2001)

The photostability of various ascorbic acid tablets on exposure to UV light has

been studied and the influence of antioxidants and moisture on the potency loss of

ascorbic acid has been evaluated The physical characteristics of ascorbic acid tablets are

also affected on UV irradiation (Ahmad et al 1973 Jamil et al 1980ab Jamil and

Ahmad 1984)

212 Effect of Various Substances on Photodegradation of Ascorbic Acid

The oxidation-reduction reactions of ascorbic acid in the presence of riboflavin at

pH 8ndash9 under the influence of light have been studied Under these conditions ascorbic

acid is a more active H donor to riboflavin than phenolphthalein (Sibi et al 1953)

Riboflavin has been found to catalyze the photodegradation of ascorbic acid solutions

during exposure to light and air The losses of ascorbic acid are markedly increased by

the presence of Cu2+

and Fe3+

ions under light exposed and unexposed conditions (Sattar

et al 1977) A spectral study of the UV photolysis of ascorbic acid solutions in the

21

presence of riboflavin has shown that the degradation of ascorbic acid is enhanced to the

extent of about 15 (Vaid et al 2005) The influence of DL- methionine on the

photostability of ascorbic acid solutions has also been studied DL- methionine (10 mg

) enhances the photostability of ascorbic acid (40 mg ) in acetate and phosphate

buffers but not in citrate buffer at pH 45 The photoprotective action of DL-methionine

on ascorbic acid appears to be influenced by its concentration pH of the medium and the

buffer species (Asker et al 1985)

The degradation of ascorbic acid solutions on irradiation with simulated sunlight

in the presence of the food dye quinolone yellow (E 104) is enhanced However this

effect is reversed by the addition of mannitol indicating that this dye facilitates the

photogeneration of hydroxyl radicals which may cause degradation of the vitamin The

incorporation of triplet quenchers enhances the stability of substrate solutions suggesting

that the dye acts as a triplet sensitizer to facilitate the reaction (Sidhu and Sugden 1992)

The photostability of ascorbic acid solutions is enhanced by sweetening agents (mannitol

sorbitol sucrose dextrose and Canderal) at 5 wv concentration However the addition

of stoichiometric amounts of hydrogen peroxide as a source of hydroxyl radicals and 2

2rsquo-azobis (2-amidopropane) as a source of hydroperoxyl radicals results in diminished

stability of ascorbic acid solutions The diminished activity may be due to the action of

hydroperoxyl radicals in the presence of hydroxyl radical scavengers (Ho et al 1994)

Metal-complexing agents (eg disodium ethylenediaminetetraacetic acid N-

hydroxylethyl ethylenediaminetetraacetic acid 8-hydroxyquinoline) have a stabilizing

effect on the photolysis of ascorbic acid injectable solutions (Kassem et al 1969ab

22

1972) This may be due to the interaction of these agents with metal ions and other

impurities

213 Photosensitized Oxidation of Ascorbic Acid

In the presence of visible light a photosensitizer such as riboflavin can exhibit

photosensitizing properties through a mixed Type IndashType II mechanism (Yoshimura and

Ohno 1988 Foote 1991 Silva et al 1994 Silva and Quina 2006) as presented below

Type I mechanism (low oxygen concentration)

RF + hυ rarr 1RF rarr

3RF (21)

3RF + SH rarr RF

middot ndash + SH

middot + rarr RFH

middot + S

middot (22)

RFmiddot ndash

+ O2 rarr RF + O2middot ndash

(23)

2RFHmiddot rarr RF + RFH2 (24)

RFH2 + O2 rarr RF + H2O2 (25)

H2O2 + O2middot ndashrarr

ndashOH +

middotOH + O2 (26)

Smiddot and or SH

middot +

+ H2O2 O2middot ndash

rarr Soxid (27)

Type II mechanism (high oxygen concentration)

RF + hυ rarr 1RF rarr

3RF (28)

3RF + O2 rarr RF +

1O2 (29)

SH + 1O2 rarr Soxid (210)

In these reactions RF 1RF and

3RF represent RF in the ground state and in the excited

singlet and triplet states respectively RFmiddot ndash

RFHmiddot and RFH2 are the radical anion the

radical and the reduced form of RF SH is the reduced substrate and SHmiddot

+ S

middot and Soxid

23

represent the intermediate radical cation the radical and the oxidized form of the

substrate respectively

An early study of the riboflavin-sensitized photooxidation of ascorbic acid has

been carried out by flash photolysis (Heelis et al 1981) ESR spectrometry has been

used to investigate the photosensitized formation of ascorbate radicals by riboflavin (Kim

et al 1993) The photochemical behavior of a system consisting of ascorbate ion (AHndash)

and riboflavin has been studied by Mancini et al (2000) and De La Rochette et al (2000

2003) The photosensitized processes were examined as a function of oxygen pressure

and the efficiency of RF induced degradation of AHndash

at various oxygen concentrations

was compared on the basis of the respective initial photosensitization quantum yields

(Table 2)

In this reaction a Type I photosensitization mechanism (Karlsen 1996) implies a

direct electron transfer between AHndash and the RF triplet-excited state followed by the

oxidation of semioxidized ascorbyl radical (AHmiddot) by molecular oxygen or some other

reactive species On the contrary in a Type II photosensitization mechanism singlet

oxygen is produced directly by energy transfer from the RF triplet-excited state to

molecular oxygen and the singlet oxygen then oxidizes the AHndash Thus by irradiating

under increasing oxygen pressure it is possible to control the relative prevalence and

efficiency of Type I or Type II mechanisms The absence of a linear relationship between

the quantum yields of ascorbate degradation and oxygen concentration indicates that the

photosensitization mechanism involved in not exclusively Type II

24

Table 2 Initial quantum yield (φ) for ascorbate (AHndash) degradation during

photosensitization by RF (35 microM) in solutions irradiated at 365 nm and

37ordmC

O2 103 times φ (AH

ndash)a

0

5

20

14 plusmn 06

1670 plusmn 220

1940 plusmn 200

a Data are the mean plusmn SD of three independent experiments

25

In the presence of RF and O2 the quantum yields for degradation of ascorbate ion

have been found to be greater than one suggesting the participation of chain reactions

initiated by the ascorbyl radical as given by the following reactions

3RF + AH

ndash rarr RFmiddot

ndash + AHmiddot (211)

AHmiddot + O2 rarr A + HO2middot (212)

HO2middot + AHndash rarr H2O2 + AHmiddot (213)

The generation of the ascorbyl radical by the reaction between the RF excited-

triplet state and the ascorbate ion (Eq 211) is the only step that requires the absorption of

photons (to form the excited-triplet state of RF) The subsequent reactions (Eqs 212 and

213) are independent of light and lead to further degradation of the ascorbate ion In the

presence of transition metal ions such as Fe3+

in trace amounts in the buffer solution

containing RF and ascorbate ions further oxidation of ascorbate ion could also occur As

a result the reduced form of the metal ion (ie Fe2+

) can be generated by the metal

catalyzed oxidation of ascorbate ion This has been confirmed by the significant decrease

in the AHndash photooxidation quantum yield in the presence of the metal chelator EDTA

which inactivates the trace amounts of iron present in the buffer solution The quantum

yields for the photosensitized oxidation of ascorbate ion are decreased twofold at 20 O2

and fourfold at 5 O2 concentration in the presence of EDTA (Silva and Quina 2006)

Amino acids have been found to affect the photosensitized oxidation of ascorbic acid

(Jung et al 1995)

214 Ascorbic Acid Interaction with Other Water-Soluble Vitamins

The stability of ascorbic acid is reported to be enhanced in syrups containing B-

complex vitamins (Connors et al 1986) This may be due to the increased viscosity of

the syrups inhibiting the oxidation of ascorbic acid The rate of photolysis in solution

26

containing cyanocobalamin and ascorbic acid is reported to decrease with an increase in

pH (Ansari et al 2004) where as use of certain halide salts has been reported to be

beneficial in stabilizing pharmaceutical products and dietary supplements when vitamin

B12 and vitamin C are combined in solution (Ichikawa et al 2005) When a solution of

multivitamins is exposed to light it is reported that organic peroxidases are generated and

the concentration of ascorbic acid decreases (Lavoie et al 2004)

22 ASSAY OF ASCORBIC ACID

Recent accounts of the development and application of analytical methods to the

determination of ascorbic acid in pharmaceuticals biological samples and food materials

are reported in the literature (Rumsey and Levine 2000 Halver and Felton 2001 Moffat

et al 2004 Ball 2006 Sheraz et al 2007 Eitenmiller et al 2008 Salkic and Kubicek

2008) Most of these methods are based on the application of spectrophotometric

fluorimetric and chromatographic techniques to suit the requirements of a particular assay

and are summarized below

221 Spectrophotometric Methods

Spectrophotometric methods are the most widely used methods for the assay of

ascorbic acid in aqueous solution Ascorbic acid exhibits strong absorption in the

ultraviolet region (absorption maxima 243 nm at pH 2 and 265 nm at pH 4ndash10 OrsquoNeil

2001 Moffat et al 2004 British Pharmacopoeia 2009) This is the basis of

spectrophotometric methods for the determination of the vitamins in pure solutions and in

sample preparations where no interference is observed from UV absorbing impurities

The value of A (1 1 cm) at the analytical wavelength of 245 nm (pH 20) is high (695)

which makes the method very sensitive for the determination of mg quantities of the

27

vitamin Treatment of the material to be analyzed with ascorbic acid oxidase is often used

as a blank to correct for the presence of interfering substances in biological samples (Liu

et al 1982) A spectrophotometric method for the determination of ascorbic acid in

pharmaceuticals by background correction (245 nm) has been reported (Verma et al

1991) The direct determination of ascorbic acid in mixtures involves the use of 22prime-

dipyridyl as a colorimetric reagent The method is based on the reduction of Fe (III) by

ascorbic acid to Fe (II) which reacts with 2 2prime-dipyridyl to form a colored complex

(absorption maximum 510 nm) that can be used for quantitative determination (Margolis

and Schmidt 1996) A spectrophotometric method has been developed for the

determination of ascorbic acid and its oxidation product dehydroascorbic acid in

biological samples (Moeslinger et al 1995) A sensitive method has been reported for

the determination of ascorbic acid in pharmaceutical formulations and fruit juices by

interaction with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (Br-PADAP) (Ferreira

et al 1997) A novel UV method has been developed for the analysis of ascorbic acid in

methanol at 245 nm in various formulations (Zeng et al 2005)

Ascorbic acid in aqueous solutions has been assayed at 244 nm (pH ~2) (Ogata

and Kosugi 1969) 245 nm (pH 35) (Blaugh and Hajratwala 1972) 264 nm (pH 7)

(Salkic et al 2007) 265 nm (pH 7) (Hashmi 1973) 275 nm (pH 41 and 70) (Heelis et

al 1981) 265 nm (pH 7) (Al-Meshal and Hassan 1982) 245 nm (pH ~2) (Verma et al

1991) and 265 nm (pH ~7) (Erb et al 2004) Dehydroascorbic acid and 23-

diketogulonic acid do not significantly absorb in this region (Pelletier 1985 Davies et

al 1991 Rumsey and Levine 2000) and therefore do not interfere with the assay of

ascorbic acid in degraded solutions

28

222 Fluorimetric Methods

Fluorimetry is a highly sensitive technique for the determination of fluorescent

compounds or fluorescent derivatives of non-fluorescent compounds The technique has

been used for the detection of microg quantities of ascorbic acid Methods based on

fluorimetric (Kampfenkel et al 1995) and chemiluminescence detection (Zhang and

Chen 2000) provide highly sensitive methods for the determination of ascorbic acid in

plant and other materials

223 Mass Spectrometric Methods

Conventional and isotope mass spectrometric techniques have also been used for

the analysis of ascorbic acid Isotope ratio mass spectrometry is particularly useful and

sensitive when 13

C ascorbic acid is used as a standard in the analysis of complex matrices

(Gensler et al 1995)

224 Chromatographic Methods

High-performance liquid chromatographic (HPLC) methods have extensively

been employed for the determination of ascorbic acid in biological samples These

methods include ion exchange reversed phase and ion-pairing HPLC protocols

Spectrophotometric fluorimetric and electrochemical detection has been used in the

HPLC analysis of ascorbic acid The electrochemical detection is used for the

simultaneous determination of ascorbic acid dehydroascorbic acid and their isomers and

derivatives A number of HPLC methods have been developed for the detection and

determination of ascorbic acid and its oxidation products and derivatives in biological

samples and plant materials (Tsao and Young 1985 Tangney 1988 Dabrowski and

Huiterleitner 1989 Thomson and Trenerry 1995 Kimoto et al 1997 Kall and

29

Anderson 1999 Rumelin et al 1999 Lykkesfeldt 2000 Zhang et al 2000 Pastore et

al 2001 Frenich et al 2005) The limit of detection of ascorbic acid in plasma or urine

with UV detection lies in the range of 100-120 microg (Liau et al 1993 Manoharan and

Schwille 1994) Fluorescence detection of ascorbic acid and dehydroascorbic acid in

plasma and its comparison with coulometric detection has been reported (Tessier et al

1996) A liquid chromatography-diode-array detection (LCndashDAD) method has been

reported for the determination of 10 water-soluble and 10 fat-soluble vitamins including

ascorbic acid in pharmaceutical preparations with a coefficient of variation lt 65

(Konings 2006)

Liquid chromatography methods based on precolumn and o-phenylenediamine

(OPD) derivatization have been used for the determination of total vitamin C and total

isovitamin C in foods and dehydro forms of the vitamin Isoascorbic acid has been used

as an internal standard in the analysis (Speek et al 1985 Vanderslice et al 1990

Dodsun et al 1992 Vanderslice and Higgs 1988 1993 Hagg et al 1994 1995) The

limits of detection of ascorbic acid by HPLC using different detectors are in the range of

16ndash400 microgl (Capellmann and Bolt 1992 Iwase and Ono 1994 Karatepe 2004)

225 Enzymatic Methods

Enzymatic methods using ascorbate oxidase are specific and have the advantage

of selectively measuring the biological activity of ascorbic acid in serum or plasma (Liu

et al 1982) Ascorbate oxidase and OPD derivatization has been used to develop a rapid

automated method for the routine assay of ascorbic acid in serum and plasma The

method has a sample throughput of 100h (Ihara et al 2000)

30

226 Commercial Kits for Clinical Analysis

Commercial kits (eg Immunodiagnostic Germany Biovision USA) are also

used for the determination of ascorbic acid in biological samples (serum or plasma) in

clinical laboratories

227 Analysis in Creams

The general methods for the analysis of active ingredients and excipients in

cosmetic products including creams are described by Salvador and Chisvert (2007)

Ascorbic acid and derivatives in creams have been determined by liquid chromatography

(Irache et al 1993 Varvaresou et al 2006) gas chromatography-mass spectrometry

(Leveque et al 2005) and electrochemical methods (Beissenhirtz et al 2003 Guitton et

al 2007)

CHAPTER III

FORMULATION AND

STABILITY OF CREAM

PREPARATIONS

32

31 FORMULATION OF CREAM PREPARATIONS

Traditionally emulsions have been defined as dispersions of macroscopic droplets

of one liquid in another liquid with a droplet diameter approximately in the range of 05-

100 microm (Becher 1965) According to the definition of International Union of Pure and

Applied Chemistry (IUPAC) (1971) ldquoIn an emulsion liquid droplets and or liquid

crystals are dispersed in a liquidrdquo

Creams are semisolid emulsions intended for external applications They are often

composed of two phases Oil-in-water (ow) emulsions are most useful as water-washable

bases whereas water-in-oil (wo) emulsions are emollient and cleansing agents The

active ingredient is often dissolved in one or both phases thus creating a three-phase

system Patients often prefer a wo cream to an ointment because the cream spreads more

readily is less greasy and the evaporating water soothes the inflamed tissue OW creams

(vanishing creams) rub into the skin the continuous phase evaporates and increases the

concentration of a water-soluble drug in the adhering film The concentration gradient for

drug across the stratum corneum therefore increases promoting percutaneous absorption

(Barry 2002 Betageri and Prabhu 2002)

The various factors involved in the formulation of emulsions and topical products

have been discussed by Block (1996) Barry (2002) and Jain et al (2006) and are briefly

presented in the following sections

311 Choice of Emulsion Type

Oil-in-water emulsions are used for the topical application of water-soluble drugs

mainly for local effect They do not have the greasy texture associated with oily bases

and are therefore pleasant to use and easily washed from skin surfaces Moisturizing

33

creams designed to prevent moisture loss from the skin and thus inhibit drying of the

stratum corneum are more efficient if formulated as ow emulsions which produce a

coherent water-repellent film

312 Choice of Oil Phase

Many emulsions for external use contain oils that are present as carriers for the

active ingredient It must be realized that the type of oil used may also have an effect both

on the viscosity of the product and on the transport of the drug into the skin (Barry

2002) One of the most widely used oils for this type of preparation is liquid paraffin

This is one of a series of hydrocarbons which also includes hard paraffin soft paraffin

and light liquid paraffin They can be used individually or in combination with each other

to control emulsion consistency This will ensure that the product can be spread easily but

will be sufficiently viscous to form a coherent film over the skin The film-forming

capabilities of the emulsion can be further modified by the inclusion of various waxes

such as bees wax carnauba wax or higher fatty alcohols

313 Emulsion Consistency

A consideration of the texture or feel of a product intended for external use is

important A wo preparation will have a greasy texture and often exhibits a higher

apparent viscosity than ow emulsions This fact imparts a feeling of richness to many

cosmetic formulations Oil-in-water emulsions will however feel less greasy or sticky on

application to the skin will be absorbed more readily because of their lower oil content

and can be more easily washed from skin surface Ideally emulsions should exhibit the

rheological properties of plasticity pseudoplasticity and thixotropy Emulsions of high

apparent viscosity for external use (cream) are of a semisolid consistency There are

34

several methods by which the rheological properties of an emulsion can be controlled

(Billany 2002)

314 Choice of Emulsifying Agent

The choice of emulgent to be used would depend on factors such as its

emulsifying ability route of administration and toxicity Most of the non-ionic emulgents

are less irritant and less toxic than their anionic and cationic counter parts Some

emulgents such as the ionic alkali soaps often have a high pH and are thus unsuitable for

application to broken skin Even in normal intact skin with a pH of 55 the application of

such alkaline materials can cause irritation Some emulsifiers in particular wool fat can

cause sensitizing reactions in susceptible people The details of various types of

emulsifying agents are available in the literature (Betageri and Prabhu 2002 Billany

2002 Swarbrick et al 2006)

315 Formulation by the HLB Method

The physically stable emulsions are best achieved by the presence of a condensed

layer of emulgent at the oil water interface and the complex interfacial films formed by a

blend of an oil-soluble emulsifying agent with a water-soluble one produces the most

satisfactory emulsions

It is possible to calculate the relative quantities of the emulgents necessary to

produce the most physically stable emulsions for a particular formulation with water

combination This approach is called the hydrophilic-lipophilic balance (HLB) method

Each surfactant is allocated an HLB number representing the relative properties of the

lipophilic and hydrophilic parts of the molecule High numbers (up to a theoretical

number of 20) therefore indicates a surfactant exhibiting mainly hydrophilic or polar

35

properties whereas low numbers represent lipophilic or non-polar characteristics Each

type of oil requires an emulgent of a particular HLB number in order to ensure a stable

product For an ow emulsion the more polar the oil phase the more polar must be the

emulgent system (Billany 2002 Im-Emsap et al 2002 Swarbrick et al 2006)

316 Concept of Relative Polarity Index

In the ingredient selection in cosmetic formulations a new concept of relative

polarity index (RPI) has been presented (Wiechers 2005) The physicochemical

characteristics of the ingredients determine their skin delivery to a greater extent than the

formulation type The cosmetic formulation cannot change the chemistry of the active

molecule that needs to penetrate to a specific site within the skin However the

formulation type can be selected based on the polarity of the active ingredient and the

desired site of action for the active ingredient For optimum skin delivery the solubility of

the active ingredient needs to be as high as possible (to create a large concentration

gradient) and as small as possible (to create a large partition coefficient) To achieve this

it is necessary to determine the following parameters

The total amount dissolved in the formulation that is available for skin penetration

the higher this amount the more will penetrate until a solution concentration is

reached in the skin therefore a high absolute solubility in the formulation is required

The polarity of the formulation relative to that of the stratum corneum if an active

ingredient dissolves better in the stratum corneum than in the formulation then the

partition of the active ingredient will favour the stratum corneum therefore a low

(relative to that in the stratum corneum) solubility in the formulation is required

(Wiechers 2005)

36

These requirements can be met by considering the concept of RPI (Wiechers

2003 2005) In this systematic approach it is essential to consider the stratum corneum

as another solvent with its own polarity The stratum corneum appears to behave very

similarly to and in a more polar fashion than butanol with respect to its solubilizing

ability for active ingredients (Scheuplein and Blank 1973) The polarity of stratum

corneum as expressed by its octanol water partition coefficient is 63

The relative polarity index may be used to compare the polarity of an active

ingredient with both that of the skin and that of the oil phase of a cosmetic formulation

predominantly consisting of emollients It may be visualized as a vertical line with a high

polarity at the top and a high lipophilicity at the bottom The polarity is expressed as the

log10 of the octanol water coefficient For example the relative polarity index values of

glycerin and isostearyl isostearate are -176 and 2698 respectively (Wiechers 2005) In

order to use the concept of the relative polarity index three numbers (on log10 scale) are

required

The polarity of the stratum corneum is set at 08 However in reality this value will

change with the hydration state of the stratum corneum that is determined in part by

the external relative humidity (Bonwstra et al 2003)

The polarity of the active molecule

The polarity of the formulation

For multiphase or multipolarity systems like emulsions the active ingredient is dissolved

in the phase For example in an ow emulsion where a lipophilic active ingredient is

dissolved in the oil phase it is the polarity of the homogenous mixture of the lipophilic

active ingredient and internal oil For the same lipophilic active in a wo emulsion it is

37

the polarity of the homogenous mixture of the lipophilic active ingredients and external

oil For water-soluble active ingredients it is the polarity of the homogenous mixture of

the hydrophilic active ingredient and the aqueous phase regardless whether it is internal

(wo emulsions) or external (ow emulsions)

Once the active ingredient and the formulation type have been chosen it is

necessary to create the delivery system that will effectively deliver the molecule The

concept of relative polarity index allows the formulator to select the polarity of the phase

in which the active ingredient is incorporated on the basis of its own properties and those

of the stratum corneum In order to achieve maximum delivery the polarity of the active

ingredient and the stratum corneum need to be considered In order to improve the skin

delivery of active ingredients the first step involves selecting a primary emollient with a

polarity close to that of the active ingredient in which it will have a high solubility The

second step is to reduce the solubility of the active ingredient in the primary emollient via

the addition of a secondary emollient with a different polarity and therefore lower

solubility for the active ingredient This approach has shown a 3-4 fold increase in skin

penetration with out increasing the amount of active ingredients in the formulation

(Wiechers 2005)

32 FORMULATION OF ASCORBIC ACID CREAMS

Ascorbic acid is a water-soluble material and is included frequently in skin care

formulations to restore skin health It is very unstable and is easily oxidized in aqueous

solution This vitamin is known to be a reducing agent in biological systems and causes

the reduction of both oxygen- and nitrogen- based free radicals (Higdon and Frei 2002)

It can also act as a co-antioxidant with the tocopheroxyl radical to regenerate alpha-

38

tocopherol (Packer et al 1979 Buettner 1993 Peyrat-Maillard et al 2001) In this

reaction the two vitamins act synergistically Alpha-tocopherol first functions as the

primary antioxidant that reacts with an organic free radical Thereafter ascorbic acid

reacts with the free radical tocopheroxyl to general alpha-tocopherol In physiological

conditions the ascorbyl radical formed by regenerating tocopherol is then converted back

to ascorbate by the redox cycle (Davies et al 1991) The interaction of ascorbic acid

with a redox partner such as alpha-tocopherol has been found useful to slow its oxidation

and prolong its action

The instability of ascorbic acid makes this antioxidant active ingredient a

formulation challenge to deliver to the skin and retain its effective form In addition to its

use in combination with alpha-tocopherol in cream formulations the stability of ascorbic

acid may be improved by its use in the form of a fatty acid ester such as ascorbyl

palmitate Ascorbyl palmitate has been used in thixogel formulations and is typically

incorporated into the mineral oil phase Preliminary experiments have shown that it could

be slowly released from the starch-oil emulsion matrix and act as an antioxidant (Wille

2005)

Various physical and chemical factors involved in the formulation of cream

preparations have been discussed in the above sections For polar and air light sensitive

compounds such as ascorbic acid it is important to consider factors such as the choice of

formulation ingredients polar character of formulation HLB value pH viscosity etc to

achieve stability

39

33 STABILITY OF CREAMS

331 Physical Stability

The most important consideration with respect to pharmaceutical and cosmetic

emulsions (creams) is the stability of the finished product The stability of a

pharmaceutical emulsion is characterized by the absence of coalescence of the internal

phase absence of creaming and maintenance of elegance with respect to appearance

odor color and other physical properties An emulsion is a dynamic system however

any flocculation and resultant creaming represent potential steps towards complete

coalescence of the internal phase In pharmaceutical emulsions creaming results as a lack

of uniformity of drug distribution and poses a problem to the pharmaceutical

compounder Another important factor in the stabilization of emulsions is phase inversion

which involves the change of emulsion type from ow to wo or vice versa and is

considered as a case of instability The four major phenomena associated with the

physical instability of emulsions are flocculation creaming coalescence and breaking

These have been discussed by Garti and Aserin (1996) Im-Emsap et al (2002) and Sinko

(2006)

332 Chemical Stability

The instability of a drug may lead to the loss of its concentration through a

chemical reaction under normal or stress conditions This results in a reduction of the

potency and is a well-recognized cause of poor product quality The degradation of the

drug may make the product esthetically unacceptable if significant changes in color or

odor have occurred The degradation product may also be a toxic substance The various

pathways of chemical degradation of a drug depend on the structural characteristics of the

40

drug and may involve hydrolysis dehydration isomerization and racemization

decarboxylation and elimination oxidation photodegradation drug-excipients and drug-

drug interactions Factors determining the chemical stability of drug substances include

intrinsic factors such as molecular structure of the drug itself and environmental factors

such as temperature light pH buffer species ionic strength oxygen moisture additives

and excipients The application of well-established kinetic principles may throw light on

the role of each variable in altering the kinetics of degradation and to provide valuable

insight into the mechanism of degradation (Baertschi and Alsante 2005 Yoshioka and

Stella 2002 Lachman et al 1986) The chemical stability of individual components

within an emulsion system may be very different from their stability after incorporation

into other formulation types For example many unsaturated oils are prone to oxidation

and their degree of exposure to oxygen may be influenced by factors that affect the extent

of molecular dispersion (eg droplet size) This could be particularly troublesome in

emulsions because emulsification may introduce air into the product and because of the

high interfacial contact area between the phases (Barry 2002) The use of antioxidants

retards oxidation of unsaturated oils minimizes changes in color and texture and prevents

rancidity in the formulation Moreover they can retard the degradation of certain active

ingredients such as vitamin C (Vimaladevi 2005) The stability problems of dispersed

systems and the factors leading to these stability problems have been discussed by

Weiner (1996) and Lu and Flynn (2009)

333 Microbial Stability

Topical bases often contain aqueous and oily phases together with carbohydrates

and proteins and are susceptible to bacterial and fungal attack Microbial growth spoils

41

the formulation and is a potential toxic hazard Therefore topical formulations need

appropriate preservatives to prevent microbial growth and to maintain their quality and

shelf-life (Barry 2002 Arger et al 1996) Cream formulations may contain fats and oils

with high percentage of unsaturated linkages that are susceptible to oxidation degradation

and development of rancidity The addition of antioxidants retards oxidation of fats and

oils minimizes changes in color and texture and prevents rancidity in the formulation

Moreover they can retard the degradation of certain active ingredients such as vitamin C

These aspects in relation to dermatological formulations have been discussed by Barry

(1983 2002) and Vimaladevi 2005)

334 Stability of Ascorbic Acid in Liquid Formulations

Ascorbic acid is very unstable in aqueous solution Different workers have studied

the stability of ascorbic acid in liquid formulations (Connors et al 1986 Austria et al

1997) Its shelf-life can be prolonged by appropriate choice of vehicle and control of

other variables such as pH stabilizers temperature light and oxygen (Table 3)

Similarly the stability of various concentrations of ascorbic acid in solution form may

vary depending upon the type of solvent used (Table 4) (Connors et al 1986 Satoh et

al 2000 Lee et al 2004 Zeng et al 2005)

335 Stability of Ascorbic Acid in Emulsions and Creams

Ascorbic acid exerts several functions on skin such as collagen synthesis

depigmentation and antioxidant activity Ultraviolet radiation generates reactive oxygen

species (ROS) which produce some harmful effects on the skin including photocarcinoma

and photoaging In order to combat these problems ascorbic acid as an antioxidant has

42

Table 3 Effect of vehicles on the stability of ascorbic acid ( ascorbic acid remaining in

solutions after storage at room temperature) (Connors et al 1986)

Storage Time (days) No Vehicle

30 60 90 120 180 240 360

1 Corn Syrup 965 925 920 920 900 860 760

2 Sorbitol 990 990 990 970 960 925 890

3 4 Carboxymethyl

Cellulose

840 680 565 380 ndash ndash ndash

4 Glycerin 100 100 990 990 970 935 920

5 Propylene glycol 995 990 980 945 920 900 900

6 Syrup USP 100 100 980 980 930 900 880

7 Syrup 212 gL 880 810 775 745 645 590 440

8 25 Tragacanth 785 620 510 320 ndash ndash ndash

9 Saturated solution of

Dextrose

990 935 875 800 640 580 510

10 Distilled Water 900 815 745 675 405 185 ndash

11 50 Propylene glycol +

50 Glycerin

980 ndash 960 ndash 933 ndash ndash

12 25 Distilled Water +

75 Sorbo (70 solution

of Sorbitol)

955 954 ndash 942 930 ndash ndash

13 50 Glycerin + 50

Sorbo

982 984 978 ndash ndash 914 ndash

43

Table 4 Stability of various concentrations of ascorbic acid in water propylene glycol

and USP syrup at room temperature ( of ascorbic acid remaining in solution)

(Connors et al 1986)

Storage Time (days) Concentration

(mg ml)

Solvent

30 60 90 120 180 240 360

10 Water 930 840 820 670 515 410 ndash

50 Water 940 920 880 795 605 590 300

100 Water 970 930 910 835 705 680 590

10 Propylene glycol 100 985 980 975 960 920 860

50 Propylene glycol 100 970 980 980 980 965 935

100 Propylene glycol 100 100 100 100 990 100 925

10 Syrup 100 100 980 990 970 960 840

50 Syrup 100 100 100 100 990 100 960

100 Syrup 100 100 100 100 100 100 995

44

been used in various dosage forms and in different concentrations (Darr et al 1996

Gallarate et al 1999 Zhang et al 1999 Pinnell et al 2001 Lee et al 2004 Raschke

et al 2004 Elmore 2005 Farahmand et al 2006 Maia et al 2006) Ascorbic acid has

good photoprotective ability against UVA-mediated phototoxicity (Darr et al 1996) A

variety of formulations containing ascorbic acid or its derivatives have been studied in

order to evaluate their stability and delivery through the skin (Gallarate et al 1999

Zhang et al 1999 Ozer et al 2000 Pinnell et al 2001 Lee et al 2004 Raschke et al

2004 Farahmand et al 2006) Formulations containing derivatives of ascorbic acid are

found to be more stable than ascorbic acid but they do not produce the same effect as that

of the parent compound probably due to the lack of redox properties (Heber et al 2006)

Effective delivery of ascorbic acid through topical preparations is a major factor that

should be critically evaluated as it may be dependent upon the nature or type of the

formulation (Gallarate et al 1999 Pinnell et al 2001) The pH of the formulation

should be on the acidic side (~ pH 35) for effective penetration of the vitamin in the skin

(Pinnell et al 2001) and for its stabilization in the formulation (Gallarate et al 1999)

Some other antioxidants such as alpha-tocopherol ferulic acid and sodium metabisulphite

have also been used in combination with ascorbic acid for the purpose of its stabilization

in topical formulations and to produce some synergistic effects (Darr et al 1996 Lin et

al 2005 Maia et al 2006 Tournas et al 2006) Effect of some rheological properties

such as viscosity and dielectric constant on the stability of ascorbic acid in emulsions has

also been investigated (Connors et al 1986) Viscosity of the medium is an important

factor that should be considered for the purpose of ascorbic acid stability as higher

viscosity formulations have shown some degree of protection (Ozer et al 2000

45

Szymula 2005) Along with other factors formulation type also plays an important role in

the stability of ascorbic acid It is reported that ascorbic acid is more stable in emulsified

system as compared to aqueous solutions (Gallarate et al 1999 Lee et al 2004) In

multiemulsions ascorbic acid is reported to be more stable as compared to simple

emulsions (Gallarate et al 1999 Ozer et al 2000 Lee et al 2004 Farahmand et al

2006)

Ascorbic acid and its derivatives have been used in a variety of cosmetic

formulations as an antioxidant pH adjuster anti-aging and photoprotectant (Elmore

2005) The control of instability of ascorbic acid poses a significant challenge in the

development of cosmetic formulations It is also reported that certain metal ions or

enzyme systems effectively convert ascorbic acidrsquos antioxidant action to pro-oxidant

activity (Elmore 2005) Therefore utilization of an effective antioxidant system is

required to maintain the stability of vitamin C in various preparations (Zhang et al 1999

Pinnell et al 2001 Maia et al 2006) The chemical stability of ascorbic acid has been

studied in emulsions and creams by several workers (Darr et al 1996 Gallarate et al

1999 Lee et al 2004 Raschke et al 2004 Elmore 2005 Farahmand et al 2006)

however there is a lack of information on the photostability of ascorbic acid in cream

formulations

336 Stability Testing of Emulsions

The stability testing of emulsions (creams) may be carried out by performing the

following tests (Billany 2002)

46

3361 Macroscopic examination

The assessment of the physical stability of an emulsion is made by an

examination of the degree of creaming or coalescence occurring over a period of time

This involves the calculation of the ratio of the volume of the creamed or separated part

of the emulsion and the total volume A comparison of these values can be made for

different products

3362 Globule size analysis

An increase in mean globule size with time (coupled with a decrease in globule

numbers) indicates that coalescence is the cause of this behavior This can be used to

compare the rates of coalescence for a variety of emulsion formulations For this purpose

microscopic examination or electronic particle counting devices (coulter counter) or

laser diffraction sizing are widely used

3363 Change in viscosity

Many factors may influence the viscosity of emulsions A change in apparent

viscosity may result from any variation in globule size or number or in the orientation or

migration of emulsifier over a period of time

3264 Accelerated stability tests

In order to compare the relative stabilities of a range of similar products it is

necessary to speed up the processes of creaming and coalescence by storage at elevated

temperatures and then carrying out the tests described in the above sections

337 FDA guidelines for semisolid preparations

According to FDA draft guidelines to the industry (Shah 1997) semisolid

preparations (eg creams) should be evaluated for appearance clarity color

47

homogencity odour pH consistency viscosity particle size distribution (when feasible)

assay degradation products preservative and antioxidant content (if present) microbial

limits sterility and weight loss when appropriate Additionally samples from

production lot or approved products are retained for stability testing in case of product

failure in the field Retained samples can be tested along with returned samples to

ascertain if the problem was manufacturing or storage related Appropriate stability data

should be provided for products supplied in closed-end tubes to support the maximum

anticipated use period during patient use and after the seal is punctured allowing product

contact with the cap cap lever Creams in large containers including tubes should be

assayed by sampling at the surface top middle and bottom of the container In addition

tubes should be sampled near the crimp The objective of stability testing is to determine

whether the product has adequate shelf-life under market and use conditions

48

OBJECT OF PRESENT INVESTIGATION

Ascorbic acid (vitamin C) is extensively used as a single ingredient or in

combination with vitamin B complex and other vitamins in the form of drops injectables

lotions and syrups It is an ingredient of anti-aging cosmetic products alone or along with

alpha-tocopherol (vitamin E) Ascorbic acid exerts several functions on the skin as

collagen synthesis depigmentation and antioxidant activity It protects the signs of

degenerative skin conditions caused by oxy-radical damage In solutions and creams

ascorbic acid is susceptible to air and light and undergoes oxidative degradation to

dehydroascorbic acid and inactive products The degradation is influenced by

temperature viscosity and polarity of the medium and is catalysed by metal ions

particularly Cu+2

Fe+2

and Zn+2

One of the major problems faced in cream preparations is the instability of

ascorbic acid as it may be exposed to light during formulation manufacturing and

storage and the possibility of photochemical degradation can not be neglected The

behaviour of ascorbic acid in light is of particular interest since no systematic kinetic

studies have been conducted on its photodegradation in these preparations under various

conditions The study of the formulation variables such as emulsifier humectants and pH

may throw light on the photostabilization of ascorbic acid in creams

The main object of this investigation is to study the behaviour of ascorbic acid in

cream preparations on exposure to UV light in the pharmaceutically useful pH range An

important aspect of the work is to study the interaction of ascorbic acid with other

vitamins such as riboflavin nicotinamide and alpha-tocopherol and the effect of certain

stabilizers such as citric acid tartaric acid and boric acid on its photodegradation In

49

addition it is intended to study the photolysis of ascorbic acid in organic solvents to

evaluate the effect of solvent characteristics (eg dielectric constant and viscosity) on the

stability of the vitamin The study of all these aspects may provide useful information to

improve the photostability and efficacy of ascorbic acid in cream preparations

An outline of the proposed plan of work is presented as follows

1 To prepare a number of oil-in-water cream formulations based on different

emulsifying agents and humectants containing ascorbic acid alone and in

combination with other vitamins and stabilizing agents

2 To perform photodegradation studies on ascorbic acid in creams using a UV

irradiation source with emission corresponding to the absorption maximum of

ascorbic acid

3 To identify the photoproducts of ascorbic acid in creams using chromatographic

and spectrophotometric methods

4 To apply appropriate and validated analytical methods for the assay of ascorbic

acid alone and in combination with other vitamins and stabilizing agents

5 To study the effect of solvent characteristics such as dielectric constant and

viscosity on the photolysis of ascorbic acid in aqueous and organic solvents

6 To evaluate the kinetics of photodegradation of ascorbic acid and its interactions

with other vitamins (riboflavin nicotinamide and alpha-tocopherol) in creams

7 To evaluate the effect of carbon chain length of the emulsifying agent and the

viscosity of the humectant on the photodegradation of ascorbic acid

50

8 To develop relationships between the rate of photodegradation of ascorbic acid

and the concentration pH carbon chain length of emulsifier viscosity of the

creams

9 To determine the effect of compounds such as citric acid tartaric acid and boric

acid used as stabilizing agents on the rate of photodegradation and stabilization

of ascorbic acid in creams

10 To present reaction schemes for the photodegradation of ascorbic acid and its

interactions with other vitamins

CHAPTER IV

MATERIALS

AND

METHODS

52

41 MATERIALS

Vitamins and Related Compounds

L-Ascorbic Acid vitamin C (5R)-5-[(1S)-12-dihydroxyethyl]-34-dihydroxyfuran-2(5H)-

one Merck

C6H8O6 Mr 1761

Dehydroascorbic Acid L-threo-23-hexodiulosonic acid γ-lactone Sigma

C6H6O6 Mr 1741

23-Diketogulonic Acid

C6H8O7 Mr 192

It was prepared according to the method of Homann and Gaffron (1964) by the

hydrolysis of dehydroascorbic acid

Riboflavin vitamin B2 (310-dihydro-78-dimethyl-10-[(2S3S4R)-2345-

tetrahydroxypentyl] benzopteridine-24-dione) Merck

C17H20N4O6 Mr 3764

Nicotinamide vitamin B3 (pyridine-3-carboxamide) Merck

C6H6N2O Mr 1221

Alpha-Tocopherol vitamin E ((2R)-2578-tetramethyl-2-[(4R8R)-4812-

trimethyltridecyl]-34-dihydro-2H-1-benzopyran-6-ol) Merck

C29H50O2 Mr 4307

Formylmethylflavin (78-dimethyl-10-formylmethylisoalloxazine)

C14H12N4O3 Mr 2843

53

Formylmethylflavin was synthesized according to the method of Fall and Petering

(1956) by the periodic acid oxidation of riboflavin It was recrystallized from absolute

methanol dried in vacuo and stored in the dark in a refrigerator

Lumichrome (78-dimethylalloxazine) Sigma

C12H10N4O2 Mr 2423

It was stored in the dark in a desiccator

Stabilizers

Boric Acid orthoboric acid Merck

H3BO3 Mr 618

Citric Acid 2-hydroxypropane-123-tricarboxylic acid Merck

C6H8O7H2O Mr 2101

L-Tartaric acid [(2R3R)-23-dihydroxybutanedioic acid] Merck

C4H6O6 Mr 1501

Emulsifying Agents

Stearic Acid (95) octadecanoic acid Merck

C18H36O2 Mr 2845

Palmitic Acid hexadecanoic acid Merck

C16H32O2 Mr 2564

Myristic Acid tetradecanoic acid Merck

C14H28O2 Mr 2284

Cetyl alcohol hexadecan-1-ol Merck

C16H34O Mr 2424

54

Humectants

Glycerin glycerol (propane-123-triol) Merck

C3H8O3 Mr 921

Propylene glycol (RS)-propane-12-diol Merck

C3H8O2 Mr 7610

Ethylene glycol ethane-12-diol Merck

C2H6O2 Mr 6207

Potassium Ferrioxalate Actinometry

Potassium Ferrioxalate

K3Fe(C2O4)3 3H2O Mr 4912

Potassium Ferrioxalate was prepared according to the method of Hatchard and

Parker (1956) Three volumes of 15 M potassium oxalate was mixed with one volume of

15 M ferric chloride with vigorous stirring The yellow green precipitate of potassium

ferrioxalate was recrystallized twice from water dried at 45 ordmC and stored in the dark in a

desiccator

Reagents

All the reagents and solvents used were of analytical grade obtained from BDH

Merck

Water

Freshly boiled distilled water was used throughout the work

55

42 METHODS

421 Cream Formulations

On the basis of the various cream formulations reported in the literature (Block

1996 Flynn 2002 Betageri and Prabhu 2002 Vimaladevi 2005 EIRI Board Lu and

Flynn 2009) the following basic formula was used for the preparation of oil-in-water

creams containing ascorbic acid

Oil phase Percentage (ww)

Emulsifier

Myristic palmitic stearic acid

Cetyl alcohol

120

30

Aqueous phase

Humectant

Ethylene glycol propylene glycol glycerin

50

Active ingredient

Ascorbic acid

20 (0114 M)

Neutralizer

Potassium hydroxide

10

Continuous phase

Distilled water

QS

Additional ingredientsa

Vitamins

Riboflavin (Vitamin B2)

Nicotinamide (Vitamin B3)

Alpha-Tocopherol (Vitamin E)

0002ndash001 (053ndash266times10ndash4

M)

028ndash140 (0023ndash0115 M)

017ndash086 (0395ndash200times10ndash2

M)

Stabilizers

Citric acid

Tartaric acid

Boric acid

010ndash040 (0476ndash190times10ndash2

M)

010ndash040 (067ndash266times10ndash2

M)

010ndash040 (0016ndash0065 M)

a The vitamin stabilizer concentrations used were found to be effective in promotion

inhibition of photodegradation of ascorbic acid in cream formulations

56

422 Preparation of Creams

The emulsifiers were melted at 70ndash80 ordmC in a glass jar immersed in a water bath

Ascorbic acid was separately dissolved in a small portion of distilled water Potassium

hydroxide and humectant were dissolved in the remaining portion of water and mixed

with the oily phase with constant stirring until the formation of a thick white mass It was

cooled to ~40 ordmC and the ascorbic acid solution was added The thick mass was mixed

using a mechanical mixer with a glass stirrer at 1000 rpm for 5 minutes The pH of the

cream was adjusted to the desired value and the contents again mixed for 10 minutes at

500 rpm All the creams were prepared under uniform conditions to maintain their

individual physical characteristics and stored at room temperature in airtight glass

containers protected from light

In the case of other vitamins nicotinamide was dissolved along with ascorbic acid

in water and added to the cream Riboflavin or alpha-tocopherol were directly added to

the cream and mixed thoroughly according to the procedure described above

In the case of stabilizing agents (citric tartaric and boric acids) the individual

compounds were dissolved in the ascorbic acid solution and added to the cream followed

by the procedure described above

The details of the various cream formulations used in this study are given in

chapters 5ndash7

57

423 Thin-Layer Chromatography (TLC)

The following TLC systems were used for the separation and identification of

ascorbic acid and photodegradation products

Adsorbent a) Silica gel GF 254 (250-microm) precoated plates

(Merck)

Solvent systems S1 acetic acid-acetone-methanol-benzene

(552070 vv) (Ganshirt and Malzacher 1960)

S2 ethanol-10 acetic acid-water (9010 vv)

(Bolliger and Konig 1969)

S3 acetonitrile-butylnitrile-water (66332 vv)

(Saari et al 1967)

Temperature 25ndash27 ordmC

Location of spots Ascorbic acid UV light 254 nm (Uvitec lamp

UK)

Dehydroascorbic acid Spray with a 3 aqueous

phenylhydrazine hydrochloride solution

424 pH Measurements

The measurements of pH of aqueous solutions and cream formulations were

carried out using an Elmetron LCD display pH meter (modelndashCP501 sensitivity plusmn 001

pH units) (Poland) with a combination electrode The electrode was calibrated

automatically in the desired pH range (25 ordmC) using the following buffer solutions

58

Phthalate pH 4008

Phosphate pH 6865

Disodium tetraborate pH 9180

The electrode was immersed directly into the cream (British Pharmacopoeia

2009) kept for few seconds to equilibrate and the pH adjusted in the range of 40ndash70

with phosphoric acid sodium hydroxide solution

425 Ultraviolet and Visible Spectrometry

The absorbance measurements and spectral determinations were performed on

Shimadzu UVndashVisible recording spectrophotometer (model UVndash1601) using matched

silica cells of 10 mm path length The cells were employed always in the same orientation

using appropriate control solutions in the reference beam The baseline was automatically

corrected by the built-in baseline memory at the initializing period Auto-zero adjustment

was made by a one-touch operation The instrument checked the wavelength calibration

(6561 nm) using the deuterium lamp at the initializing period The absorbance scale was

periodically checked using the following calibration standard (British Pharmacopoeia

2009)

0057ndash0063 gl of potassium dichromate in 0005 M sulphuric acid

The specific absorbance [A(1 1 cm)] of the solution should match the

following values with the stated limit of tolerance

Wavelength

(nm)

Specific absorbance

A (1 1 cm)

Maximum

tolerance

235 1245 1229ndash1262

257 1445 1428ndash1462

313 486 470ndash503

350 1073 1056ndash109

430 159 157ndash161

59

426 Photolysis of Ascorbic Acid

4261 Creams

A 2 g quantity of the cream was uniformly spread on several rectangular glass

plates (5 times 15 cm) covered with a 1 cm tape on each side to give a 1 mm thick layer The

plates were irradiated in a dark chamber using a Philips 30 watt TUV tube (100

emission at 254 nm the wavelength absorbed by ascorbic acid at pH 4ndash7) fixed

horizontally at a distance of 30 cm from the centre of the plates Each plate was removed

at appropriate interval and the cream was subjected to spectrophotometric assay and

chromatographic examination

4262 Aqueous and organic solvents

A 10ndash3

M solution of ascorbic acid (50 ml) prepared in water (pH 70 005 M

phosphate buffer) or in an organic solvent in a 100 ml beaker (Pyrex) was placed in a

water bath maintained at 20 plusmn 1 ordmC The solution was irradiated with the Philips 30 watt

TUV tube in a dark chamber as stated above Samples were withdrawn at appropriate

intervals for assay and chromatography

4263 Storage of creams in dark

In order to determine the stability of various cream formulations in the dark

samples were stored at room temperature in a cupboard protected from light for a period

of three months The samples were analyzed periodically for the content of ascorbic acid

and the presence of any degradation product

427 Measurement of Light Intensity

The potassium ferrioxalate actinometry was used for the measurement of light

intensity of the radiation source employed in this work This actinometer has been

60

developed by Parker (1953) and Hatchard and Parker (1956) and is considered as the

most useful actinometer over a wide range of wavelengths (254ndash577 nm) It has been

used by Holmstrom and Oster (1961) Byrom and Turnbull (1967) McBride and Moore

(1967) Ahmad (1968) Ahmad (1978) Ahmad et al (2004a 2004b 2005 2006a

2006b 2008 2009ab) Fasihullah (1988) Vaid (1998) Ansari (2002) and Ahmad (2009)

for the measurement of light intensity

The irradiation of potassium ferrioxalate solutions in sulphuric acid results in the

reduction of ferric ion to ferrous ion according to the following reaction

2Fe [(C2O4)3]3ndash

rarr 2 Fe (C2O4) + 3 (C2O4)2ndash

+ 2CO2 (31)

The amount of Fe2+

ions formed in the reaction may be determined by

complexation with 110-phenanthroline to give a red colored complex The absorbance of

the complex is measured at 510 nm

428 Procedure

An oxygen free 0006 M solution of potassium ferrioxalate (2947 gl) in 01 N

H2SO4 was placed in the reaction vessel and irradiated with the lamp used for the

photolysis of riboflavin The irradiation was carried out under nitrogen (90ndash120

bubblesminute) which also caused stirring of the solution The temperature of the

reaction vessel was maintained at 25 plusmn 1 ordmC during the reaction

An aliquot of the photolysed solution (1ndash2 ml) was pipetted out at suitable

intervals (up to 30 minutes) into a 10 ml volumetric flask to which was then added 09

ml of N H2SO4 + 1 ml (01) 110-phenanthroline + 05 ml buffer (60 ml N CH3COONa

+ 36 ml N H2SO4 made up to 100 ml with distilled water) The flask was made up

to the mark with distilled water (final pH 35) thoroughly shaken to mix the contents and

61

Fig 3 Spectral power distribution of TUV 30 W tube (Philips)

62

allowed to stand for one hour in the dark to develop the colorndashcomplex The absorbance

of the phenanthrolinendashferrous complex was measured in a 1 cm cell at 510 nm using the

appropriate solution as blank The amount of Fe2+

ions formed was determined from the

calibration graph The calibration graph was constructed in a similar manner using

several dilutions of 1 times 10ndash6

mole ml Fe2+

in 01 N H2SO4 (freshly prepared by dilution

from standardized 01 M FeSO4 in 01 N H2SO4) (Fig 8) The experimental value of the

molar absorptivity of Fe2+

complex as determined from the slope of the calibration graph

is equal to 111 times 104 M

ndash1 cm

ndash1 and is in agreement with the value reported by Parker

(1953) Using the values of the known quantum yield for ferrioxalate actinometer at

different wavelengths (Hatchard and Parker 1956) the number of Fe2+

ions formed

during photolysis the time of exposure and the fraction of the light absorbed by the

length of the actinometer solution employed the light intensity incident just inside the

front window of the photolysis cell can be calculated In the present case total absorption

of the light has been assumed

4281 Calculation

The number of Fe2+

ions formed during photolysis (nFe

2+) is given by the

equation

6023 times 1020

V1 V3 A Σ

n Fe

2+ =

V2 1 ε (32)

where V1 is the volume of the actinometer solution irradiated (ml)

V2 is the volume of the aliquot taken for analysis (ml)

V3 is the final volume to which the aliquot V2 is diluted (ml)

1 is the path length of the spectrophotometer cell used (1 cm)

A is the measured absorbance of the final solution at 510 nm

63

ε is the molar absorptivity of the Fe2+

complex (111 times 104 M

ndash1 cm

ndash1)

The number of quanta absorbed by the actinometer nabs can then be obtained as follows

n Fe

2+

Σ nabs = ф

(33)

where ф is the quantum yield for the Fe2+

formation at the desired wavelength

The number of quanta per second per cell nabs is therefore given by

Σ nabs 6023 times 1020

V1 V3 A nabs =

t =

ф V2 1 ε t (34)

where t is the irradiation time of the actinometer in seconds

The relative spectral energy distribution of the radiation source (Fig 3) shows

100 emission at 254 nm the wavelength used for the photolysis of ascorbic acid (λmax

265 nm at pH 4ndash7) The energy emitted by the radiation source at various wavelengths

can be calculated using the equation

1197 times 105

E (KJ molndash1

) = λ nm

(35)

The quantum efficiency of ferrioxalate actinometer at the wavelength absorbed by

ascorbic acid (ie 254 nm) is high although the sensitivity drops over 450 nm The

average intensity of the TUV tube used in this study was determined as 556 plusmn 012 times

1018

quanta sndash1

429 Viscosity Measurements

The viscosity of the cream formulations was measured with a Brookfield RV

viscometer (Model DV-II + Pro USA) The instrument was calibrated using the

manufacturerrsquos viscosity standard A 200 g quantity of the cream was placed in a beaker

and the spindle (TE) was dipped into the cream It was rotated at a speed of 06 rpm for

64

00

02

04

06

08

10

12

0 2 4 6 8 10 12

Concentration of Fe++

times 105 M

Ab

sorb

an

ce a

t 51

0 n

m

Fig 4 Calibration graph for the determination of K3Fe(C2O4)3

65

one minute and the viscosity was recorded at 25plusmn1 ordmC The test was repeated three times

to account for the experimental variability and the average viscosity was noted

4210 Assay Methods

42101 UV spectrophotometric method for the assay of creams containing ascorbic

acid alone

The creams were thoroughly mixed a quantity of 2 g was accurately weighed and

the assay of ascorbic acid was carried out by the UV method of Zeng et al (2005) In the

case of photodegraded creams (2 g) the material was completely removed from the glass

plate and transferred to a volumetric flask The method involved extraction of ascorbic

acid with methanol (3 times 10 ml) adjustment of the pH of combined methanolic solutions

to 20 (with H3PO4) dilution of the final solution with acidified methanol (pH 20) to 100

ml and measurement of the absorbance at 245 nm using appropriate blank The

concentration of ascorbic acid was calculated using 560 as the value of specific

absorbance [A (1 1 cm)] at the analytical wavelength (Table 5)

The same method was used for the assay of ascorbic acid in creams stored in the

dark and in the presence of individual stabilizing agents (citric tartaric and boric acids)

42102 Iodimetric method for the assay of ascorbic acid in creams containing

riboflavin nicotinamide and alpha-tocopherol

The assay of ascorbic acid in creams in the presence of riboflavin nicotinamide

and alpha-tocopherol was carried out according to the procedure of British

Pharmacopoeia (2009) as follows

The photolysed cream (2 g) was completely scrapped from the glass plate and

transferred to a flask containing 40 ml of distilled water and 10 ml of 1 M sulphuric acid

66

Table 5 Calibration data for ascorbic acid showing linear regression analysisa

λ max 245 nm

Concentration range 01ndash10 times 10ndash4

M (0176ndash1761 mg )

Slope 9920

SE (plusmn) of slope 00114

Intercept 00012

Correlation coefficient 09996

Molar absorptivity (ε) 9920 Mndash1

cmndash1

Specific absorbance [A (1 1 cm)] 560

a Values represent a mean of five determinations

67

was added The solution was titrated with 002 M iodine solution using 1 ml of starch

solution as indicator until a persistent violet-blue color was obtained Each ml of 002 M

iodine solution is equivalent to 352 mg of C6H8O6 The same method was used for the

assay of ascorbic acid in creams stored in the dark

42103 Spectrophotometric method for the assay of ascorbic acid in aqueous and

organic solvents

A 1 ml aliquot of the photolysed solutions of ascorbic acid in water or in an

organic solvent was evaporated to dryness under nitrogen at room temperature and the

residue redissolved in a small volume of methanol The solution was transferred to a 10

ml volumetric flask made up to volume with acidified methanol (pH 20) and the

absorbance measured at 245 nm using an appropriate blank The content of ascorbic acid

in the solutions was determined using 9920 Mndash1

cmndash1

as the value of molar absorptivity at

the analytical wavelength (Table 5)

CHAPTER V

PHOTODEGRADATION OF

ASCORBIC ACID IN

ORGANIC SOLVENTS AND

CREAM FORMULATIONS

69

51 INTRODUCTION

Ascorbic acid (vitamin C) is an essential micronutrient that performs important

metabolic functions (Packer and Fuchs 1999 Davey et al 2000 Johnston et al 2007)

It is an ingredient of anti-aging cosmetic products (Darr et al 1996 Gallarate et al

1999 Traikovich 1999 Zhang et al 1999 Ozer et al 2000 Nusgens et al 2001

Pinnell et al 2001 2003 Lee et al 2004 Raschke et al 2004 Sauermann et al 2004

Elmore 2005 Jentzsch et al 2005 Lin et al 2005 Placzek et al 2005 Carlotti et al

2006 Farahmand et al 2006 Heber et al 2006 Maia et al 2006 Tournas et al 2006)

and exerts several functions on the skin as collagen synthesis depigmentation and

antioxidant activity (Nusgens et al 2001 Spiclin et al 2003) As an antioxidant it

protects skin by neutralizing reactive oxygen species generated on exposure to sunlight

(Shindo et al 1994) In biological systems it reduces both oxygenndash and nitrogenndash based

free radicals (Higdon and Frei 2002) and thus delays the aging process In view of the

instability of ascorbic acid in skin care formulations (Bissett 2006) it is often used in

combination with another redox partner such as alpha-tocopherol (vitamin E) to retard its

oxidation (Wille 2005)

The details of the cream formulations used in this study are given in Table 6 The

results obtained on the photodegradation of ascorbic acid in aqueous organic solvents

and cream formulations are discussed in the following sections

70

Table 6 Composition of cream formulations containing ascorbic acid

Ingredients Cream

No pH

SA PA MA CA AH2 GL PG EG PH DW

1 a 4 + minus minus + + + minus minus + +

b 5 + minus minus + + + minus minus + +

c 6 + minus minus + + + minus minus + +

d 7 + minus minus + + + minus minus + +

2 a 4 minus + minus + + + minus minus + +

b 5 minus + minus + + + minus minus + +

c 6 minus + minus + + + minus minus + +

d 7 minus + minus + + + minus minus + +

3 a 4 minus minus + + + + minus minus + +

b 5 minus minus + + + + minus minus + +

c 6 minus minus + + + + minus minus + +

d 7 minus minus + + + + minus minus + +

4 a 4 + minus minus + + minus + minus + +

b 5 + minus minus + + minus + minus + +

c 6 + minus minus + + minus + minus + +

d 7 + minus minus + + minus + minus + +

5 a 4 minus + minus + + minus + minus + +

b 5 minus + minus + + minus + minus + +

c 6 minus + minus + + minus + minus + +

d 7 minus + minus + + minus + minus + +

6 a 4 minus minus + + + minus + minus + +

b 5 minus minus + + + minus + minus + +

c 6 minus minus + + + minus + minus + +

d 7 minus minus + + + minus + minus + +

7 a 4 + minus minus + + minus minus + + +

b 5 + minus minus + + minus minus + + +

c 6 + minus minus + + minus minus + + +

d 7 + minus minus + + minus minus + + +

8 a 4 minus + minus + + minus minus + + +

b 5 minus + minus + + minus minus + + +

c 6 minus + minus + + minus minus + + +

d 7 minus + minus + + minus minus + + +

9 a 4 minus minus + + + minus minus + + +

b 5 minus minus + + + minus minus + + +

c 6 minus minus + + + minus minus + + +

d 7 minus minus + + + minus minus + + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PG = propylene glycol EG = ethylene glycol

PH = potassium hydroxide DW = distilled water

71

52 PHOTOPRODUCTS OF ASCORBIC ACID

The photolysis of ascorbic acid (AH2) in aqueous and organic solvents and in

cream formulations on UV irradiation leads to the formation of dehydroascorbic acid

(DHA) as detected by TLC along with the undegraded AH2 using the solvent systems A

B and C The identification of DHA was carried out by comparison of the Rf value and

spot color with those of the authentic compound The formation of DHA on

photooxidation of ascorbic acid solutions has previously been reported (Homan and

Gaffron 1964 Sattar et al 1977 Heelis et al 1981 Rozanowska et al 1997 Lavoie et

al 2004) DGA the hydrolysis product of DHA (Homan and Gaffron 1964) could not

be detected under the present experimental conditions

53 SPECTRAL CHARACTERISTICS OF PHOTOLYSED SOLUTIONS

A typical set of the UV absorption spectra of photolysed solutions of AH2 in

methanol is shown in Fig 5 There is a gradual loss of absorbance around 245 nm with

time as a result of the oxidation of the molecule to DHA (Pelletier 1985 Davies et al

1991 Rumsey and Levine 2000) which does not absorb in this region due to the loss of

conjugation Similar absorption changes are observed on the photolysis of AH2 in other

organic solvents and in the methanolic extracts of cream formulations However the

magnitude of these changes varies with the rate of photolysis in a particular solvent or

cream and appears to be a function of the polar character pH and viscosity of the

medium

72

Fig 5 UV absorption spectra of photolysed solutions of ascorbic acid in methanol at

0 40 80 120 160 220 and 300 min

73

54 ASSAY OF ASCORBIC ACID IN CREAMS AND SOLUTIONS

The assay of AH2 in creams and solutions has been carried out in acidified

methanol (pH 20) according to the UV spectrophotometric method of Zeng et al (2005)

Aqueous solutions of AH2 (~pH 2) exhibit absorption maxima at 243 nm (OrsquoNeil 2001

Moffat et al 2004 Sweetman 2009) 244 nm (Ogata and Kosugi 1969) and 245 nm

(Verma et al 1991 Johnston et al 2007) The absorption maxima of AH2 in methanol

and phosphate buffer (pH 25) occur at 245 nm (Zeng et al 2005) Since dilute solutions

of AH2 are highly susceptible to oxidation the pH was adjusted to 20 with phosphoric

acid to convert the molecule to the non-ionized form (99) to minimize degradation

during the assay AH2 in acidified methanol (pH 20) was found to exhibit the absorption

maximum at 245 nm as reported by Zeng et al (2005) The method was also used for the

assay of AH2 in aqueous and organic solvents

The validity of Beerrsquos law relation in the concentration range used was confirmed

prior to the assay The calibration data for AH2 at the analytical wavelength are presented

in Table 5 (Chapter 4) The correlation coefficient (r = 09996) indicates a good linear

relationship over the concentration range employed The values of specific absorbance

and molar absorptivity at 245 nm determined from the slope of the curve are in good

agreement with those reported by previous workers (Davies et al 1991 Johnston et al

2007) The method of Zeng et al (2005) has been found to be satisfactory for the assay of

AH2 in cream formulations and solutions and has been used to study the kinetics of

photolysis reactions The method was validated before its application to the assay of AH2

in photolysed creams The reproducibility of the method was confirmed by the analysis of

known amounts of AH2 in the concentration range likely to be found in photodegraded

74

creams The values of the recoveries of AH2 in creams by the UV spectrophotometric

method are in the range of 90ndash96 The values of RSD for the assays indicate the

precision of the method within plusmn5 (Table 7)

In order to compare the UV spectrophotometric method with the British

Pharmacopoeia iodimetric method (2009) using a dilute iodine solution (002 M) the

creams were simultaneously assayed for AH2 content by the two methods and the results

are reported in Table 8 The statistical evaluation of the accuracy and precision of the two

methods was carried out by the application of the F test and the t test respectively The F

test showed that there is no significant difference between the precision of the two

methods and the calculated value of F is lower than the critical value (F = 639 P = 005)

in each case The t test indicated that the calculated t values are lower than the tabulated t

values (t = 2776 P = 005) suggesting that at 95 confidence level the differences

between the results of the two methods are statistically non-significant Thus the accuracy

and precision of the UV spectrophotometric method is comparable to that of the official

iodimetric method for the assay of AH2 in cream formulations The results of the assays

of AH2 in aqueous organic solvents and cream formulations are reported in Table 9

55 EFFECT OF SOLVENT

The influence of solvent on the rate of degradation of drugs is of considerable

importance to the formulator since the stability of drug species in solution media may be

predicted on the basis of their chemical reactivity The reactivity of drugs in a particular

medium depends to a large extent on solvent characteristics such as the dielectric

constant and viscosity (Connors et al 1986 Yoshioka and Stella 2000 Sinko 2006)

75

Table 7 Recovery of ascorbic acid added to cream formulationsa

Cream

Formulationb

Added

(mg)

Found

(mg)

Recovery

()

RSD

()

1a 400

200

380

183

950

915

21

25

2b 400

200

371

185

928

925

15

25

3c 400

200

375

181

938

905

11

31

4d 400

200

384

189

960

945

13

21

5b 400

200

370

189

925

945

14

26

6c 400

200

369

190

923

950

10

22

7d 400

200

374

182

935

910

17

39

8c 400

200

380

188

950

940

15

33

9d 400

200

367

189

918

945

20

42

a Values expressed as a mean of three to five determinations

b The cream formulations represent combinations of each emulsifier (stearic acid

palmitic acid myristic acid) with each humectant (glycerin propylene glycol ethylene

glycol) to observe the efficiency of methanol to extract AH2 from different creams

(Table 6)

76

Table 8 Assay of ascorbic acid in creams using UV spectrophotometric and iodimetric

methods

Ascorbic acid (mg) Cream

Formulationb Added UV method

a

Iodimetric

methoda

Fcalc tcalc

1a 40

20

380 plusmn 081

183 plusmn 046

375 plusmn 095

185 plusmn 071

138

238

245

104

2b 40

20

371 plusmn 056

185 plusmn 047

373 plusmn 064

193 plusmn 038

130

065

181

200

3c 40

20

375 plusmn 040

181 plusmn 056

374 plusmn 046

183 plusmn 071

132

160

101

223

4d 40

20

384 plusmn 051

189 plusmn 039

381 plusmn 066

190 plusmn 052

167

178

176

231

5b 40

20

370 plusmn 052

189 plusmn 050

372 plusmn 042

185 plusmn 067

065

179

162

125

6c 40

20

369 plusmn 037

190 plusmn 042

371 plusmn 058

188 plusmn 056

245

177

122

197

7d 40

20

374 plusmn 062

182 plusmn 072

370 plusmn 070

184 plusmn 082

127

129

144

168

8c 40

20

380 plusmn 058

188 plusmn 062

375 plusmn 075

192 plusmn 060

167

094

123

162

9d 40

20

367 plusmn 072

189 plusmn 080

365 plusmn 082

187 plusmn 075

149

092

130

203

Theoretical values (P = 005) for F is 639 and for t is 2776

a Mean plusmn SD (n = 5)

b Table 6

77

Table 9 Photodegradation of ascorbic acid in cream formulations

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) pHa 40 50 60 70

0 383 382 384 383

60 374 369 366 361

120 361 354 346 325

180 351 345 325 305

240 345 327 301 284

1

300 336 316 287 264

0 380 383 382 379

60 371 376 362 346

120 359 357 342 320

180 352 345 322 301

240 341 335 299 283

2

300 336 321 291 261

0 384 376 381 385

60 377 367 360 358

120 366 348 334 324

180 356 337 317 305

240 343 320 301 282

3

300 335 307 273 253

78

Table 9 continued

0 377 378 386 372

60 365 361 371 355

120 353 345 347 322

180 344 327 325 298

240 332 320 306 279

4

300 317 303 284 252

0 381 367 372 373

60 372 358 358 353

120 360 337 336 321

180 352 325 320 302

240 341 313 300 284

5

300 327 302 278 256

0 376 386 380 377

60 366 372 350 350

120 353 347 323 316

180 337 334 308 298

240 329 320 291 274

6

300 313 306 267 245

79

Table 9 continued

0 380 372 378 380

60 373 362 350 354

120 358 340 329 321

180 344 328 304 300

240 332 315 292 283

7

300 319 302 272 252

0 380 381 378 361

60 368 364 361 335

120 355 354 340 313

180 342 340 315 281

240 337 331 303 269

8

300 323 314 281 248

0 378 382 370 375

60 370 369 349 342

120 356 347 326 321

180 339 333 298 291

240 326 314 277 271

9

300 313 302 265 242

a The values at pH 40ndash70 represent the formulations a to d of each cream respectively

80

In order to observe the effect of solvent dielectric constant the apparent first-

order rate constants (kobs) for the photolysis of AH2 in alcoholic solvents (Table 10) were

plotted against the dielectric constants of the solvents A linear relationship indicated the

dependence of the rates of photolysis on solvent dielectric constant (Fig 6) This implies

the involvement of a polar intermediate in the reaction to facilitate the formation of the

degradation products as suggested by Ahmad and Tollin (1981) in the case of flavin

electron transfer reactions The effect of solvent polarity has been observed on the

autooxidation of AH2 in organic solvents (Ogata and Kosugi 1969)

Another solvent parameter affecting the rate of a chemical reaction is viscosity

which can greatly influence the stability of oxidisable substances (Wallwork and Grant

1977 Laidler 1987 Fung 1990) A plot of kobs for the photolysis of AH2 against the

reciprocal of solvent viscosity (Table 10) is linear showing that an increase in solvent

viscosity results in a decrease in the rate of photolysis (Fig 7) The viscosity of the liquid

affects the rate at which molecules can diffuse through the solution This in turn may

affect the rate at which a compound can suffer oxidation at the liquid surface This

applies to AH2 and an increase in the viscosity of the medium makes access to air at the

surface more difficult to prevent oxidation (Wallwork and Grant 1977)

56 EFFECT OF CONCENTRATION

In order to observe the effect of concentration (Table 11) on the photostability of

AH2 in a cream using stearic palmitic and myristic acids as emulsifying agents and

glycerin as humectant plots of log concentration versus time were constructed (Fig 8)

and the apparent first-order rate constants were determined (Table 12) A graph of kobs

against concentration of AH2 (Fig 9) exhibited an apparent linear relationship between

81

Table 10 Apparent first-order rate constants (kobs) for the photolysis of ascorbic acid in

water and organic solvents

Solvent Dielectric

Constant (25 ordmC)

Viscosity

(mPas) ndash1

kobs times104

(minndash1

)

Water 785 1000 404

Methanol 326 1838 324

Ethanol 243 0931 316

1-Propanol 201 0514 302

1-Butanol 178 0393 295

82

00

20

40

60

80

0 10 20 30 40 50 60 70 80

Dielectric constant

k (

min

ndash1)

Fig 6 A plot of kobs for photolysis of ascorbic acid against solvent dielectric constant

(times) Water () methanol () ethanol (diams) 1-propanol () 1-butanol

83

00

10

20

30

40

50

00 05 10 15 20

Viscosity (mPas)ndash1

k times

10

4 (m

inndash1)

Fig 7 A plot of kobs for photolysis of ascorbic acid against reciprocal of solvent

viscosity Symbols are as in Fig 6

84

Table 11 Effect of concentration on the photodegradation of ascorbic acid in cream

formulations at pH 60

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) 05 10 15 20 25

0 95 191 290 379 471

60 90 182 277 358 453

120 82 167 260 339 431

180 77 158 239 311 401

240 70 144 225 298 382

1

300 64 134 210 282 363

0 92 186 287 380 472

60 88 175 272 369 453

120 82 160 251 342 429

180 75 152 238 326 405

240 71 144 225 309 392

2

300 65 134 215 289 366

0 94 182 286 376 470

60 87 171 265 352 454

120 78 152 251 337 426

180 69 143 227 315 404

240 62 129 215 290 378

3

300 58 119 195 271 353

85

05

10

15

20

25

06

08

10

12

14

16

18

log

co

nce

ntr

ati

on

(m

g)

a

05

10

15

20

25

06

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

b

05

10

15

20

25

06

08

10

12

14

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

c

Fig 8 Log concentration versus time plots for the photodegradation of ascorbic acid at

various concentrations in creams at pH 60 a) stearic acid b) palmitic acid

c) myristic acid

86

Table 12 Apparent first-order rate constants (kobs) for the photodegradation of various

ascorbic acid concentrations in cream formulations at pH 60

kobs times 103 (min

ndash1)a Cream

Formulationb 05 10 15 20 25

1 133

(0994)

120

(0993)

111

(0995)

101

(0994)

090

(0994)

2 118

(0992)

108

(0994)

098

(0993)

093

(0992)

084

(0994)

3 169

(0994)

144

(0995)

126

(0994)

109

(0993)

097

(0992)

a The values in parenthesis are correlation coefficients

b Table 6

87

Stearic acid

Palmitic acid

Myristic acid

00

05

10

15

20

25

00 05 10 15 20 25

Ascorbic acid concentration ()

kob

s (min

ndash1)

Fig 9 A plot of kobs for photodegradation against ascorbic acid concentrations in cream

formulations

88

the two values Thus the rate of degradation of AH2 is faster at a lower concentration on

exposure to the same intensity of light This may be due to a relatively greater number of

photons available for excitation of the molecule at lower concentration compared to that

at a higher concentration The AH2 concentrations of creams used in this study are within

the range (1ndash15) reported by previous workers for topical applications to skin (Kaplan

et al 1989 Traikovich et al 1999 Nusgens et al 2001 Matsubayashi et al 2003

Espinal-Perez et al 2004 Sauermann et al 2004 Lin et al 2005 Heber et al 2006)

57 EFFECT OF CARBON CHAIN LENGTH OF EMULSIFYING AGENT

The values of kobs for the photodegradation of AH2 (2) in various cream

formulations are reported in Table 13 The first-order plots for the photodegradation of

AH2 at pH 4ndash7 in various cream formulations are shown in Fig 10ndash12 The plots of kobs

against carbon chain length of the emulsifying agents are shown in Fig 13 They indicate

that the photodegradation of AH2 is affected by the emulsifying agent in the order

myristic acid gt stearic acid gt palmitic acid

These acids possess a polar character (Yao et al 2009) and the carbon chain of the acid

may play a part in the photostability of AH2 However the results indicate that in the

presence of palmitic acid AH2 exhibits greater stability as indicated by the plots of kobs

versus the carbon chain length of the emulsifying agents (Fig 13) This could be

predominantly due to the interaction of AH2 with palmitic acid in the cream to impart it

greater stability Ascorbic acid-6-palmitate is known to be an antioxidant in cosmetic

preparations (Lee et al 2009) and food products (Doores 2002)

In view of the above observations it may be suggested that the photodegradation

of AH2 could involve a polar semiquinone intermediate (Johnston et al 2007) which

89

Table 13 First-order rate constants (kobs) for the photodegradation of ascorbic acid in

cream formulations

kobs times 103 (min

ndash1)abc

Cream

Formulation pH 40 50 60 70

1 044

(0992)

064

(0994)

100

(0995)

126

(0995)

2 042

(0992)

060

(0991)

095

(0992)

120

(0995)

3 047

(0993)

069

(0993)

107

(0991)

137

(0995)

4 056

(0993)

072

(0992)

104

(0994)

131

(0993)

5 050

(0991)

067

(0992)

097

(0991)

124

(0992)

6 061

(0992)

079

(0993)

113

(0992)

140

(0994)

7 060

(0992)

071

(0993)

108

(0994)

133

(0992)

8 053

(0991)

062

(0992)

099

(0994)

126

(0993)

9 065

(0991)

081

(0996)

117

(0993)

142

(0995)

a The rate constants at pH 40ndash70 represent the values for formulations a to d of each

cream respectively

b The values in parenthesis are correlation coefficients

c The values of rate constants are relative and depend on specific experimental conditions

including light intensity

The estimated error is plusmn5

90

12

13

14

15

16

17

log

co

nce

ntr

ati

on

(m

g)

1

12

13

14

15

16

17

log

co

nce

ntr

ati

on

(m

g)

2

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

3

Fig 10 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (1ndash3) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

91

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

4

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

5

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

6

Fig 11 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (4ndash6) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

92

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

7

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

8

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

9

Fig 12 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (7ndash9) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

93

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

ko

bs times

10

3 (m

inndash

1)

1-3

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

ko

bs times

10

3 (

min

ndash1)

4-6

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

Carbon chain length

ko

bs times

10

3 (

min

ndash1)

7-9

Fig 13 Plots of kobs for photodegradation of ascorbic acid in creams (1ndash9) against carbon

chain length of emulsifier () Stearic acid () palmitic acid () myristic acid

Humectant used glycerin (1ndash3) propylene glycol (4ndash6) ethylene glycol (7ndash9)

94

depending on the polar character of the medium undergoes oxidation with varying rates

This is similar to the behavior of the photolysis of riboflavin analogs which is dependent

on the polar character of the medium (Ahmad and Tollin 1981) The effect of carbon

chain length on the transdermal delivery of an active ingredient has been discussed (Lu

and Flynn 2009)

58 EFFECT OF VISCOSITY

The plots of rates of AH2 degradation in cream formulations (Table 13) as a

function of carbon chain length (Fig 13) indicate that the rates vary with the humectant

and hence the viscosity of the medium in the order

ethylene glycol gt propylene glycol gt glycerin

This is in agreement with the rate of photolysis of AH2 in organic solvents that

higher the viscosity of the medium lower the rate of photolysis Thus apart from the

carbon chain length of the emulsifier viscosity of the humectant added to the cream

formulation appears to play an important part in the stability of AH2 The stabilizing

effect of viscosity imparting substances on AH2 solutions has been reported (Stone 1969

Kassem et al 1969ab)

59 EFFECT OF pH

The kobsndashpH profiles for the photodegradation of AH2 in various creams (1ndash9) at

pH 4ndash7 (Fig 14) represent a sigmoid type curve indicating the oxidation of the ionized

form (AHndash) of AH2 (pKa 41) (OrsquoNeil 2001) with pH The AH

ndash species appears to be

more susceptible to photooxidation than the non-ionized form (AH2) The behavior of

AH2 on photooxidation in the pH range 4ndash7 is similar to that observed for the chemical

oxidation of AH2 by molecular oxygen (DeRitter 1982) and involves the interaction of

95

Stearic Acid (1)

Palmitic Acid (2)

Myristic Acid (3)

04

06

08

10

12

14

kob

s times

10

3 (m

inndash

1)

Stearic Acid (4)

Palmitic Acid (5)

Myristic Acid (6)

04

06

08

10

12

14

ko

bs times

10

3 (

min

ndash1)

Stearic Acid (7)

Palmitic Acid (8)

Myristic Acid (9)

04

06

08

10

12

14

30 40 50 60 70

pH

ko

bs

times 1

03

(min

ndash1)

Fig 14 The kobsndashpH profiles for the photodegradation of ascorbic acid in creams (1ndash9)

Glycerin

Propylene glycol

Ethylene glycol

96

AH2 with singlet oxygen on UV irradiation (Silva and Quina 2006) The AHndash species

(predominant in the pH range 42ndash70 557ndash999) is more reactive towards singlet

oxygen than its protonated form the AH2 molecule as suggested by Bisby et al (1999)

and therefore the rate of photooxidation is higher in the pH range above 41

corresponding to the pKa1 of AH2 The major goal of a ratendashpH profile is to determine

the optimum pH range for a particular formulation Several workers have studied the

ratendashpH profiles of the chemical oxidation of AH2 in the pH range 2ndash7 (Garrett 1967

Taqui Khan and Martell 1967 Rogers and Yacomeni 1971 Blaugh and Hajratwala

1972 DeRitter 1982 Moura et al 1994) however the kinetics of photooxidation of

AH2 in cream formulations has so far not been reported

510 EFFECT OF REDOX POTENTIAL

The photooxidation of AH2 is also influenced by its redox potential which varies

with pH The greater photostability of AH2 at pH 5ndash6 compared to that at pH 7 and above

is due to its lower rate of oxidationndashreduction in this range (Eordm pH 50 = +0127 V)

(OrsquoNeil 2001) The increase in the rate of photooxidation with pH is due to a

corresponding increase in the redox potential (Eordm pH 70 = +0058 V) (Fasman 1976) of

AH2 and is similar to the photolysis behavior of riboflavin at pH 5ndash6 (Eordm pH 50 = ndash0117

V) (Sinko 2006) compared to that at pH 70 (Eordm pH 70 = ndash 0207 V) (Ahmad et al

2004a Sinko 2006) Since the ionization as well as the redox potentials of AH2 are a

function of pH the rate of photooxidation depends upon the specific species present and

its redox behavior at a particular pH

97

511 PRIMARY PHOTOCHEMICAL REACTIONS IN THE OXIDATION OF

ASCORBIC ACID

A reaction scheme based on general photochemical principles for the important

reactions involved in the photooxidation of ascorbic acid is presented below

0AH2 hv k1

1AH2 (51)

1AH2 k2 Products (52)

1AH2 isc k3

3AH2 (53)

3AH2 k4 Products (54)

0AH

ndash hv k5

1AH

ndash (55)

1AH

ndash k6 Products (56)

1AH

ndash k7

3AH

ndash (57)

3AH

ndash k8 Products (58)

3AH

ndash +

0AH2 k9 AH٠

ndash + AH٠ (59)

2 AH٠ k10 A + AH2 (510)

3AH2 +

3O2 k11

0AH2 +

1O2 (511)

AHndash +

1O2 k12

3AH

ndash +

3O2 (512)

AH٠ + 1O2 k13 AHOO٠ (513)

AHOO٠ k14 A + HO2٠ (514)

AHOO٠ + 0AH2 k15 AH٠ + AHOOH (515)

AHOOH k16 secondary reaction

A + H2O2 (516)

According to this reaction scheme the ground state ascorbic acid species (0AH2

0AH

ndash) each is excited to the lowest singlet state (

1AH2

1AH

ndash) by the absorption of a

quantum of UV light (51 55) These excited states may directly be converted to

98

photoproducts (52 56) or may undergo intersystem crossing (isc) to form the excited

triplet states (53 57) The excited triplet states may then degrade to the photoproducts

(54 58) The monoascorbate triplet (3AH

ndash) may react with the ground state ascorbic

acid to form a monoascorbate radical anion (AH٠ndash) and a monoascorbate radical (AH٠)

(59) Two AH٠ radical species may lead to the formation of an oxidized (A) and a

reduced ascorbic acid molecule (AH2) (510) Ascorbic acid triplet (3AH2) may react with

molecular oxygen (3O2) to yield singlet oxygen (

1O2) (511) which may then react with

monoascorbate anion (AHndash) to form the excited triplet state (

3AH

ndash) (512) or with

monoascorbate radical to form a peroxyl radical (AHOO٠) (513) The peroxyl radical

may yield dehydroascorbic acid (A) (514) or react with ground state ascorbic acid to

give monoascorbate radical and a reduced species AHOOH (515) The reduced species

may give rise to dehydroascorbic acid and hydrogen peroxide (516)

512 DEGRADATION OF ASCORBIC ACID IN THE DARK

In view of the instability of AH2 and to observe its degradation in the dark the

creams were stored in airtight containers at room temperature in a cupboard for a period

of about 3 months and assayed for AH2 content at appropriate intervals The analytical

data (Table 14) were subjected to kinetic treatment (Fig 15ndash17) and the apparent first-

order rate constants for the degradation of AH2 were determined (Table 15) The values

of the rate constants indicate that the degradation of AH2 in the dark is about 70 times

slower than those of the creams exposed to UV irradiation (Table 13) The degradation of

AH2 in creams in the dark is due to chemical oxidation (Section 132) and occurs in the

order of emulsifying agents (Fig 18)

myristic acid gt stearic acid gt palmitic acid

99

Table 14 Degradation of ascorbic acid in the dark in cream formulations

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(days) pHa 40 50 60 70

0 383 382 384 383

10 354 340 313 278

20 309 306 279 245

40 244 209 183 161

60 172 166 131 105

1

80 145 114 81 61

0 380 383 382 379

10 360 343 350 335

20 322 310 301 294

40 266 250 211 186

60 233 211 168 142

2

80 182 153 114 89

0 384 376 381 385

10 368 350 340 318

20 318 273 273 266

40 223 199 172 155

60 174 132 117 84

3

80 122 97 66 54

100

Table 14 continued

0 377 378 386 372

10 350 334 334 318

20 314 268 256 244

40 238 208 182 136

60 179 155 107 94

4

80 128 101 79 59

0 381 367 372 373

10 350 293 300 320

20 299 266 270 263

40 220 191 192 184

60 183 153 139 129

5

80 149 115 87 76

0 376 386 380 377

10 312 320 314 251

20 255 282 226 199

40 175 194 159 131

60 139 128 99 74

6

80 102 81 55 41

101

Table 14 continued

0 380 372 378 380

10 323 330 333 323

20 288 273 276 224

40 212 174 182 146

60 152 133 108 83

7

80 100 82 66 56

0 380 381 378 361

10 333 320 310 310

20 281 266 260 257

40 230 189 171 177

60 156 148 128 111

8

80 123 96 78 66

0 378 382 370 375

10 313 295 281 300

20 256 247 257 203

40 194 178 151 133

60 119 114 88 74

9

80 88 68 49 39

a The values at pH 40ndash70 represent the formulations a to d of each cream respectively

102

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

1

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

2

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

3

Fig 15 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (1ndash3) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

103

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

4

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

5

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

6

Fig 16 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (4ndash6) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

104

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

7

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

8

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

9

Fig 17 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (7ndash9) at pH 40 (times) 50 () 60 () and 70 ()

Palmitic acid

Myristic acid

Stearic acid

105

Table 15 First-order rate constants (kobs) for the degradation of ascorbic acid in cream

formulations in the dark

kobs times 102 (day

ndash1)abc

Cream

Formulation pH 40 50 60 70

1 128

(0991)

152

(0994)

191

(0995)

220

(0994)

2 091

(0992)

110

(0991)

152

(0993)

182

(0992)

3 148

(0991)

176

(0995)

220

(0993)

254

(0995)

4 137

(0992)

161

(0993)

205

(0994)

236

(0995)

5 121

(0991)

141

(0994)

175

(0993)

195

(0993)

6 162

(0992)

194

(0995)

237

(0994)

265

(0994)

7 164

(0994)

189

(0994)

222

(0993)

246

(0996)

8 143

(0994)

167

(0995)

193

(0996)

212

(0993)

9 184

(0995)

208

(0994)

251

(0992)

280

(0996)

a The rate constants at pH 40ndash70 represent the values for formulations a to d of each

cream respectively

b The values in parenthesis are correlation coefficients

c The values of rate constants are relative and depend on specific experimental

conditions

The estimated error is plusmn5

106

pH 4

pH 5

pH 6

pH 7

00

10

20

30

k times

10

2 (d

ayndash

1)

1-3

pH 4

pH 5

pH 6

pH 7

00

10

20

30

k times

10

2 (

da

yndash1)

4-6

pH 4

pH 5

pH 6

pH 7

00

10

20

30

12 14 16 18

Carbon chain length

k times

10

2 (

da

yndash1)

7-9

Fig 18 Plots of kobs for degradation of ascorbic acid in the dark in creams (1ndash9) against

carbon chain length of emulsifier () Stearic acid () palmitic acid

() myristic acid Humectant used glycerin (1ndash3) propylene glycol (4ndash6)

ethylene glycol (7ndash9)

107

Although it is logical to expect a linear relationship between the rate of degradation and

the carbon chain length of the emulsifier due to its polar character (Yao et al 2009) it

has not been observed in the present case The reason for the slowest rate of degradation

of AH2 in the presence of palmitic acid appears to be due to the interaction of AH2 with

palmitic acid (Lee et al 2009) as explained in Section 57

The degradation of AH2 also appears to be affected by the viscosity of the cream

in the order of humectant (Fig 19)

ethylene glycol gt propylene glycol gt glycerin

Thus the presence of glycerin imparts the most stabilizing effect on the degradation of

AH2 This is the same order as observed in the case of photodegradation of AH2 in the

creams The airtight containers used for the storage of creams make the access of air to

the creams difficult to cause chemical oxidation of AH2 However it has been observed

that the degradation of AH2 is highest in the upper layer of the creams compared to that

of the middle and the bottom layers Therefore the creams were thoroughly mixed before

sampling for the assay of AH2 However the scattering in kinetic plots (Fig 15ndash17) is

due to non-uniform distribution of AH2 in degraded creams

The effect of pH on the degradation of AH2 in the creams (Fig 19) shows that the

degradation increases with an increase in pH as observed in the case of photodegradation

of AH2 in the creams This is due to an increase in the ionization and redox potential of

AH2 with pH causing greater oxidation of the molecule and has been discussed in

Sections 59 and 510

108

Stearic Acid (1)

Palmitic Acid (2)

Myristic Acid (3)

00

10

20

30

k times

10

2 (d

ayndash

1)

Glycerin

Stearic Acid (4)

Palmitic Acid (5)

Myristic Acid (6)

00

10

20

30

k times

10

2 (

da

yndash1)

Propylene glycol

Stearic Acid (7)

Palmitic Acid (8)

Myristic Acid (9)

00

10

20

30

30 40 50 60 70

pH

k times

10

2 (d

ayndash

1)

Ethylene glycol

Fig 19 The kobsndashpH profiles for the degradation of ascorbic acid in the dark in creams

(1ndash9)

CHAPTER VI

PHOTOCHEMICAL INTERACTION

OF ASCORBIC ACID WITH

RIBOFLAVIN NICOTINAMIDE

AND ALPHA-TOCOPHEROL IN

CREAM FORMULATIONS

110

61 INTRODUCTION

It is now medically recognized that sagging skin and other signs of degenerative

skin conditions such as wrinkles and age spots are caused primarily by oxy-radical

damage Ascorbic acid can accelerate wound healing protect fatty tissues from oxidative

damage and play an integral role collagen synthesis (Zhang et al 1999) It is used in

cosmetic preparations for its anti-aging depigmentation and antioxidant properties

(Spiclin 2003 Ehrlich et al 2006) It is also used in combination with other vitamins

such as alpha-tocopherol as a co-antioxidant to stabilize cosmetic preparations (Eberlein-

Koumlnig and Ring 2005 Bissett 2006 Murray 2008) Ascorbic acid in the presence of air

or light is known to interact with alpha-tocopherol (Packer et al 2002 Johnston et al

2007) riboflavin (Homann and Gaffron 1964 Sattar et al 1977 Heelis et al 1981

Kim et al 1993 Jung et al 1995 De La Rochette et al 2000 2003 Lavoie et al

2004 Vaid et al 2005 Ahmad and Vaid 2006 Silva and Quina 2006) and

nicotinamide (Bailey et al 1945 Werner et al 1949 Guttman and Brooke 1963

DeRitter 1982) The present work involves a study of the effect of alpha-tocopherol

riboflavin and nicotinamide on the photostability of ascorbic acid in cream formulations

to observe whether the interaction in these formulations leads to the stabilization of

ascorbic acid The chemical structures of nicotinamide (NA) alpha-tocopherol (TP)

riboflavin (RF) formylmethylflavin (FMF) and lumichrome (LC) are shown in Fig 20

The details of the cream formulations used in this study are given in Table 16

The results obtained on the photodegradation of ascorbic acid in cream formulations are

discussed in the following sections

111

Riboflavin

N

N

NH

N

CH2

CH

C OHH

CH OH

CH2OH

N

N

NH

N

CH2

CHO

Formylmethylflavin

N

N

NH

HN

Lumichrome

OH

N

NH2

O

Nicotinamide

O CH3

CH3

CH3

HO

H3C

CH3 CH3 CH3

CH3

Alpha-Tocopherol

O

O

H3C

H3C

H3C

H3C

O

O

H3C

H3C

O

O

Fig 20 Chemical structures of alpha-tocopherol nicotinamide riboflavin

formylmethylflavin and lumichrome

112

Table 16 Composition of cream formulations containing ascorbic acid (2) and other

vitamins

Ingredients Cream

No SA PA MA CA GL AH2 RFa NA

b TP

c PH DW

10 a + minus minus + + + a minus minus + +

b + minus minus + + + b minus minus + +

c + minus minus + + + c minus minus + +

d + minus minus + + + d minus minus + +

e + minus minus + + + e minus minus + +

11 a minus + minus + + + a minus minus + +

b minus + minus + + + b minus minus + +

c minus + minus + + + c minus minus + +

d minus + minus + + + d minus minus + +

e minus + minus + + + e minus minus + +

12 a minus minus + + + + a minus minus + +

b minus minus + + + + b minus minus + +

c minus minus + + + + c minus minus + +

d minus minus + + + + d minus minus + +

e minus minus + + + + e minus minus + +

13 a + minus minus + + + minus a minus + +

b + minus minus + + + minus b minus + +

c + minus minus + + + minus c minus + +

d + minus minus + + + minus d minus + +

e + minus minus + + + minus e minus + +

14 a minus + minus + + + minus a minus + +

b minus + minus + + + minus b minus + +

c minus + minus + + + minus c minus + +

d minus + minus + + + minus d minus + +

e minus + minus + + + minus e minus + +

113

Table 16 continued

15 a minus minus + + + + minus a minus + +

b minus minus + + + + minus b minus + +

c minus minus + + + + minus c minus + +

d minus minus + + + + minus d minus + +

e minus minus + + + + minus e minus + +

16 a + minus minus + + + minus minus a + +

b + minus minus + + + minus minus b + +

c + minus minus + + + minus minus c + +

d + minus minus + + + minus minus d + +

e + minus minus + + + minus minus e + +

17 a minus + minus + + + minus minus a + +

b minus + minus + + + minus minus b + +

c minus + minus + + + minus minus c + +

d minus + minus + + + minus minus d + +

e minus + minus + + + minus minus e + +

18 a minus minus + + + + minus minus a + +

b minus minus + + + + minus minus b + +

c minus minus + + + + minus minus c + +

d minus minus + + + + minus minus d + +

e minus minus + + + + minus minus e + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PH = potassium hydroxide DW = distilled water

RF = riboflavin NA = nicotinamide TP = alpha-tocopherol

a RF(g ) a = 0002 b = 0004 c = 0006 d = 0008 e = 0010

b NA (g ) a = 028 b = 056 c = 084 d = 112 e = 140

c TP (g ) a = 017 b = 034 c = 052 d = 069 e = 086

The molar concentrations of these vitamins are given in Section 421

114

62 ABSORPTION CHARACTERISTICS OF PHOTOLYSED CREAMS

A typical set of the absorption spectra of the methanolic extracts (pH 20) of the

freshly prepared and photolysed creams containing AH2 and TP is shown in Fig 21 AH2

in acidified methanol exhibits absorption maximum at 245 nm (Zeng et al 2005) as

observed in Fig 21 The absorption due to TP at 284 nm (Moffat et al 2004) was

cancelled by using an appropriate blank containing an equivalent concentration of TP

The gradual decrease in absorption at around 245 nm during UV irradiation indicates the

transformation of AH2 to DHA which does not absorb in this region (Davies et al 1991)

as a result of the loss of C3=C2 chromophore Similar spectral changes around 245 nm are

observed in the presence of RF and NA which also strongly absorb in this region A

decrease in the absorption of AH2 around 266 nm in aqueous solution (pH 60) in the

presence of RF has been reported (Vaid et al 2005) The spectral changes and loss of

absorbance in methanolic extracts of creams depends on the rate of photolysis of AH2 in

the presence of these vitamins

63 PHOTOPRODUCTS OF ASCORBIC ACID AND OTHER VITAMINS

The UV irradiation of AH2 in cream formulations (pH 60) in the presence of RF

NA and TP results in the degradation of AH2 and RF and the following photoproducts

have been identified on comparison of their RF values and spot color fluorescence with

those of the authentic compounds

AH2 DHA

RF FMF LC CMF

In the TLC systems used NA and TP did not show the formation of any

degradation product in creams

115

Fig 21 UV absorption spectra of methanolic extracts of photodegraded ascorbic acid in

cream at 0 60 120 180 300 and 480 min

116

The formation of DHA in the photooxidation of AH2 has previously been reported by

many workers (Homann and Gaffron 1964 Sattar et al 1977 Heelis et al 1981

Rozanowska et al 1997 Lavoie et al 2004 Vaid et al 2006) RF is sensitive to light in

aqueous solutions (DeRitter 1982 British Pharmacopoeia 2009 Sweetman 2009) and is

known to form a number of products under aerobic conditions (Treadwell et al 1968

Cairns and Metzler 1971 Schuman Jorns et al 1975 Ahmad and Rapson 1990 Ahmad

and Vaid 2006 Ahmad et al 2004ab 2005 2008 Vaid et al 2006) It has been found

to degrade on UV irradiation in cream formulations to give FMF LC and CMF and these

products have been reported in the photolysis of RF by the workers cited above The

formation of these products may be affected by the interaction of AH2 and RF in creams

(Section 66) NA and TP individually did not appear to form any photoproduct of their

own directly or on interaction with AH2 in creams and may influence the degradation of

AH2 on UV irradiation

64 ASSAY METHOD

In view of the presence of RF (absorption maxima 223 267 373 and 444 nm)

(British Pharmacopoeia 2009) NA (absorption maximum 261 nm) (Moffat et al 2004)

and TP (absorption maximum 284 nm) (Moffat et al 2004) in the cream formulations

and the interference of these vitamins with the absorption of AH2 (absorption maximum

265 nm) (Davies et al 1991) the direct spectrophotometric method cannot be applied for

the determination of AH2 Therefore the iodimetric method (British Pharmacopoeia

2009) was used to determine AH2 in cream formulations The method was validated in

the presence of RF NA and TP before its application to the determination of AH2 in

photodegraded creams The reproducibility of the method has been confirmed by the

117

assay of known concentrations of AH2 in the range present in photodegraded creams The

recovery of AH2 in the creams has been found to be in the range 90ndash96 The values of

RSD indicate that the precision of the method is within plusmn5 (Table 17) and it can be

applied to study the kinetics of AH2 photolysis in cream formulations The assay data on

AH2 in various cream formulations are given in Table 18

65 KINETICS OF PHOTODEGRADATION OF ASCORBIC ACID

Several chemical and physical factors play a role in the photodegradation of AH2

in the presence of other vitamins (RF NA TP) and affect the rate of its degradation in

cream formulations The present work involves the study of photodegradation of AH2 in

cream formulations containing glycerin as humectant as AH2 has been found to be most

stable in these creams (Chapter 5) The apparent first-order rate constants (kobs) for the

photodegradation of AH2 in the presence of other vitamins in cream formulations

derived from the kinetic plots (Fig 22ndash24) are reported in Table 19 The second-order

rate constants (correlation coefficients 0991ndash0996) determined from the slopes of the

graphs of kobs versus vitamin concentration for the individual vitamins (Fig 25) and the

values of k0 determined from the intercept on the vertical axis at zero concentration are

reported in Table 20 The values of k0 give an indication of the effect of other vitamins on

the rate of degradation of AH2 These values are about 13 times lower than the values of

kobs obtained at the highest concentrations of RF and NA indicating that RF and NA both

accelerate the photodegradation of AH2 in creams RF is known to act as a

photosensitizer for the degradation of AH2 (Section 66) and therefore its presence in

creams would accelerate the degradation of AH2 The increase in the rate of

photodegradation of AH2 in the presence of NA has not previously been reported NA

118

Table 17 Recovery of ascorbic acid in cream formulations in the presence of other

vitamins by iodimetric methoda

Cream

Formulationb

Added

(mg )

Found

(mg )

Recovery

()

RSD

()

10e (RF) 400

200

373

187

933

935

29

22

11e (RF) 400

200

379

187

948

935

25

31

12e (RF) 400

200

375

188

938

940

29

28

13e (NA) 400

200

382

191

955

955

23

27

14e (NA) 400

200

380

185

950

925

19

26

15e (NA) 400

200

379

191

948

955

21

17

16e (TP) 400

200

368

183

920

915

29

44

17e (TP) 400

200

391

195

978

975

11

13

18e (TP) 400

200

377

182

943

910

32

37

a Values expressed as a mean of three to five determinations

b The cream formulations represent all the emulsifiers (stearic acid palmitic acid

myristic acid) to observe the efficiency of iodimetric method for the recovery of

ascorbic acid in presence of the highest concentration of vitamins (Table 16)

119

Table 18 Photodegradation of ascorbic acid in cream formulations in the presence of

other vitamins

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) a b C d e

0 373 372 374 372 375

60 362 354 354 360 359

150 342 336 336 332 334

240 315 314 308 310 302

10 (RF)

330 301 291 288 281 282

0 380 379 376 374 374

60 370 366 362 362 361

150 343 337 340 332 328

240 329 323 320 313 310

11 (RF)

330 307 301 294 288 282

0 379 380 375 372 376

60 362 366 361 351 342

150 341 335 319 307 312

240 310 306 295 284 282

12 (RF)

330 285 278 263 254 243

120

Table 18 continued

0 372 370 371 368 365

60 361 358 348 350 349

120 342 343 329 326 330

180 327 325 319 312 308

240 317 309 299 289 285

13 (NA)

300 299 291 283 278 273

0 386 380 375 378 370

60 371 362 365 362 355

120 359 351 343 339 336

200 341 332 325 316 311

14 (NA)

300 313 303 296 294 280

0 375 371 374 370 366

60 362 356 352 352 345

120 343 332 336 326 314

200 323 315 311 295 293

15 (NA)

300 293 283 275 270 259

121

Table 18 continued

0 380 378 380 377 377

60 362 365 369 369 371

120 351 352 360 360 364

180 340 346 349 353 355

240 331 334 343 343 346

16 (TP)

300 320 323 330 332 337

0 383 380 378 380 377

60 372 371 372 373 370

120 363 360 361 366 365

180 348 348 350 356 355

240 341 343 343 348 348

17 (TP)

300 330 332 336 339 341

0 380 383 377 375 373

60 364 370 366 367 366

120 352 356 351 352 351

180 334 338 339 343 342

240 324 328 324 332 330

18 (TP)

300 307 315 317 318 322

122

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

10

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

11

ab

c

de

13

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

12

Fig 22 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing riboflavin (a) 0002 (b) 0004 (c) 0006 (d) 0008

(e) 0010

Stearic acid

Palmitic acid

Myristic acid

123

abcde

13

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

13

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

14

abcde

13

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

15

Fig 23 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing nicotinamide (a) 028 (b) 056 (c) 084 (d) 112 (e) 140

Stearic acid

Palmitic acid

Myristic acid

124

abcde

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

16

abcde

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

17

abcde

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

18

Fig 24 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing alpha-tocopherol (a) 017 (b) 034 (c) 052 (d) 069

(e) 086

Stearic acid

Myristic acid

Palmitic acid

125

Table 19 First-order rate constants (kobs) for the photodegradation of ascorbic acid in the

presence of other vitamins in cream formulations

kobs times 103 (min

ndash1)ab

Cream

formulationd

Other

vitaminc

a b C d e

10 RF 068

(0991)

073

(0996)

079

(0995)

085

(0992)

089

(0995)

11 RF 065

(0992)

070

(0992)

073

(0994)

080

(0995)

086

(0993)

12 RF 087

(0993)

096

(0995)

109

(0993)

116

(0994)

127

(0992)

13 NA 073

(0993)

081

(0992)

088

(0994)

096

(0994)

101

(0993)

14 NA 069

(0992)

074

(0992)

080

(0991)

086

(0995)

094

(0995)

15 NA 083

(0994)

090

(0993)

101

(0993)

109

(0994)

115

(0995)

16 TP 055

(0991)

051

(0994)

046

(0994)

042

(0993)

038

(0991)

17 TP 050

(0995)

045

(0993)

041

(0992)

038

(0995)

034

(0994)

18 TP 070

(0996)

066

(0996)

060

(0994)

055

(0993)

051

(0993)

a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental conditions

including the light intensity

c Vitamin concentrations (andashe) are as given in Table 16

d All the creams contain glycerin as humectant

The estimated error is plusmn5

126

00

05

10

15

00 10 20 30

Riboflavin concentration (M times 104)

kob

s times

10

3 (

min

ndash1)

10-12

00

05

10

15

00 20 40 60 80 100 120

Nicotinamide concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

13-15

00

02

04

06

08

00 04 08 12 16 20

Alpha-Tocopherol concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

16-18

Fig 25 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against individual vitamin concentration in cream formulations (10ndash18)

127

Table 20 First-order rate constants (k0)a for the photodegradation of ascorbic acid in the

absence of other vitamins and second-order rate constants (k) for the

photochemical interaction of ascorbic acid with RF NA and TP

Cream

formulation

Other

vitamin

k0 times 103

(minndash1

)

k

(Mndash1

minndash1

)

Correlation

coefficient

10 RF 062 102 0994

11 RF 059 097 0992

12 RF 077 189 0995

13 NA 066 032 times 10ndash2

0995

14 NA 062 027 times 10ndash2

0993

15 NA 074 037 times 10ndash2

0994

16 TP 059 110 times 10ndash2b

0996

17 TP 053 096 times 10ndash2b

0992

18 TP 075 123 times 10ndash2b

0994

a

The variations in the values of k0 are due to the degradation of AH2 in the presence of

different emulsifying agents in cream formulations

b Values for the inhibition of photodegradation of AH2

128

forms a complex with AH2 (Section 67) and may also act as a photosensitizer for AH2 by

energy transfer in the excited state on UV irradiation The absorption maximum of NA

(261 nm) (Moffat et al 2004) is very close to that of AH2 (265 nm) (Davies et al 1991)

and the possibility of energy transfer in the excited state (Moore 2004) is greater leading

to the photodegradation of AH2

The value of k0 is about 13 times greater than the values of kobs obtained for the

degradation of AH2 in the presence of the highest concentrations of TP in the creams

This indicates that TP has a stabilising effect on the photodegradation of AH2 in the

cream formulations This is in agreement with the view that the TP acts as a redox partner

with AH2 to retard its oxidation (Wille 2005) Thus among the three vitamins studied

only TP appears to have a stabilising effect on photodegradation of AH2 The

photochemical interaction of individual vitamins with AH2 is discussed below

66 INTERACTION OF RIBOFLAVIN WITH ASCORBIC ACID

The interaction of RF with the ascorbate ion (AHndash) may be represented by the

following reactions proposed by Silva and Quina (2006)

RF rarr 1RF (61)

1RF rarr

3RF (62)

3RF + AH

ndash rarr RF

ndashmiddot + AHmiddot (63)

AHmiddot + O2 rarr A + HO2middot (64)

HO2middot + AHndash rarr H2O2 + AHmiddot (65)

RF on the absorption of a quantum of light is promoted to the excited singlet state (1RF)

(61) 1RF may undergo intersystem crossing (isc) to form the excited triplet state (

3RF)

(62) The excited triplet state may react with the ascorbate ion to generate the ascorbyl

hv

isc

129

radical (AH) (63) (Kim et al 1993) The ascorbyl radical reacts with oxygen to give

dehydroascorbic acid (A) and peroxyl radical (HO2) (64) This radical may interact with

ascorbate ion to generate further ascorbyl radicals (65) These radicals may again take

part in the sequence of reactions to form A The role of RF in this reaction is to act as a

photosensitiser in the oxidation of ascorbic acid to A Ascorbic acid is reported to protect

riboflavin in milk under the influence of light by reacting with singlet oxygen (Hall et al

2009) (Section 511)

67 INTERACTION OF NICOTINAMIDE WITH ASCORBIC ACID

NA is known to form a 11 complex with ascorbic acid (Guttman and Brooke

1963 OrsquoNeil 2001 Doores 2002) The complexation of NA and AH2 may result from

the donor-acceptor interaction between AH2 (donor) and NA (acceptor) as observed in

the case of tryptophan and NA (Florence and Attwood 2006) In the presence of light the

interaction may cause reduction of NA (NAH) to form the ascorbyl radical (AH) ((66)-

(68)) which is oxidized to dehydroascorbic acid (A) (69) The NAH may be oxidized to

NA and H2O2 (610)

NA rarr 1NA (66)

1NA rarr

3NA (67)

3NA + AH2 rarr NAH + AHmiddot (68)

2 AH٠ rarr A + AH2 (69)

NAH + O2 rarr NA + H2O2 (610)

The proposed reactions suggest that on photochemical interaction AH2 undergoes

photosensitised oxidation in the presence of NA indicating that the photostability of

ascorbic acid is affected by NA

isc

130

68 INTERACTION OF ΑLPHA-TOCOPHEROL WITH ASCORBIC ACID

TP is an unstable compound and its oxidation by air results in the formation of an

epoxide which than produces a quinone that is inactive (Connors et al 1986) TP is

destroyed by sun light and artificial light containing the wavelengths in the UV region

(Ball 2006) It interacts with other antioxidants such as ascorbic acid (AH2) according to

the following reactions

TPndashO + AH2 rarr TP + AHmiddot (611)

2 AHmiddot rarr A + AH2 (612)

TP + AHmiddot rarr TPndashO + AH2 (613)

The tocopheroxyl radical (TPndashO) reacts with AH2 to regenerate TP and form the

ascorbyl radical (AHmiddot) (611) This radical undergoes further reactions as described in

equations (64) and (65) (Traber 2007) It may also disproportionate back to A and AH2

(612) TP reacts with AHmiddot to produce again the TPndashO radical and AH2 Thus in the

presence of light TP leads to the oxidation of AH2 which is ultimately regenerated in the

reaction (Davies et al 1991) Ascorbic acid has the ability to act as a co-antioxidant with

the TPndashO to regenerate TP (Packer et al 1979 2002) In this manner AH2 and TP act

synergistically to function in a redox cycle and AH2 is stabilized in the cream

formulations and microemulsions (Rozman and Gasperlin 2007 Rozman et al 2009)

69 EFFECT OF CARBON CHAIN LENGTH OF EMULSIFYING AGENT

The graphs of kobs for the photodegradation of AH2 in the presence of RF NA and

TP versus the carbon chain length of emulsifying agents are shown in Fig 26 It appears

that the photodegradation of AH2 in the presence of all the three vitamins in the creams

lies in the order

131

Fig 26 Plots of k for photodegradation of ascorbic acid in creams (10ndash18) against

carbon chain length of emulsifier () Stearic acid () palmitic acid

() myristic acid

00

05

10

15

20

25

k

(Mndash

1 m

inndash

1)

00

05

10

15

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash

1 m

inndash

1)

132

myristic acid gt stearic acid gt palmitic acid

The same order of emulsifying agents has been observed in the absence of the

added vitamins (Section 57) The polar character of these acids (Yao et al 2009) on the

basis of their carbon chain length may play a part in the photostability of AH2 The

greater stability of AH2 in creams in the presence of palmitic acid (Fig 26) may be due to

the interaction of AH2 with palmitic acid as discussed in Section 57 Ascorbic acid-6-

palmitate is known to be an antioxidant in cosmetic preparations (Lee et al 2009) and

food products (Doores 2002)

610 EFFECT OF VISCOSITY OF CREAMS

The plots of kobs for the degradation of AH2 in the presence of the highest

concentration of vitamins versus reciprocal of the viscosity of creams (Table 21) are

linear (Fig 27) and indicate that the increase in cream viscosity leads to a decrease in the

rate of degradation of AH2 The slopes of the plots indicate the effect of viscosity on the

interaction of AH2 with other vitamins in the order

riboflavin gt nicotinamide gt alpha-tocopherol

The relatively slow rate of degradation of AH2 in creams containing palmitic acid may be

due to the interaction of AH2 with the vitamins as well as palmitic acid (Lee et al 2009)

Thus viscosity is an important factor in the stability of AH2 in cream formulations and

may affect its rate of interaction with other vitamins It has been suggested that an

increase in the viscosity of the medium makes access to air at the surface more difficult to

prevent the oxidation of a drug (Wallwork and Grant 1977) This is in agreement with

the photolysis of AH2 in aqueous and organic solvents cream formulations (Chapter 5)

and aerobic oxidation of Ah2 in syrups (Blaug and Hajratwala 1972)

133

Table 21 Average viscosity of cream formulations containing different emulsifying

agents and glycerin as humectant (25 plusmn 1 ordmC) and the photodegradation rate

constants of AH2

Cream No Emulsifying

agent

Viscosityab

(mPa s)

kobs times 103c

10 (RF)

13 (NA)

16 (TP)

Stearic acid 9000 089

101

038

11 (RF)

14 (NA)

17 (TP)

Palmitic acid 8600 086

094

034

12 (RF)

15 (NA)

18 (TP)

Myristic acid 7200 127

115

051

a plusmn10

b Average viscosity of creams containing the individual vitamins (RF NA TP)

c The values have been obtained in the presence of highest concentration of the

vitamins

134

00

05

10

15

20

25

30

100 110 120 130 140

Viscosity (mPa s)ndash1

times 103

kob

s (m

inndash1)

Fig 27 Plots of kobs in the presence of highest concentration of vitamins versus

reciprocal of the viscosity of creams () riboflavin

( ) nicotinamide (- - -- - -) alpha-tocopherol

135

611 DEGRADATION OF ASCORBIC ACID IN THE PRESENCE OF OTHER

VITAMINS IN THE DARK

In order to observe the effect of riboflavin nicotinamide and alpha-tocopherol on

the degradation of AH2 in the creams stored in the dark the AH2 contents of the creams

were assayed at appropriate intervals (Table 22) The apparent first-order rate constants

determined from the kinetic plots (Fig 28) for the degradation of AH2 in the presence of

the highest concentrations of the individual vitamins in cream formulations (10ndash18) are

reported in Table 23 These rate constants indicate that the overall degradation of AH2 in

the presence of the highest concentration of the individual vitamins (RF NA and TP) is

about 70 times slower than that obtained on the exposure of creams to UV irradiation

This decrease in the rate of degradation of AH2 in the creams is the same as observed in

the case of AH2 alone In the absence of light the degradation of AH2 occurs due to

chemical oxidation (Section 132) and does not appear to be affected by the presence of

riboflavin and nicotinamide as indicated by the comparisons of the values of kobs in the

presence and absence of these vitamins (Table 15 and 23) In the presence of alpha-

tocopherol the degradation is slower than that in the presence of riboflavin and

nicotinamide This may be due to some interaction of AH2 and alpha-tocopherol causing

stabilisation of AH2 in the creams

As observed in the case of AH2 degradation alone in creams in the dark the AH2

degradation in the presence of the highest concentrations of other vitamins also occurs in

the same order of emulsifying agents (Fig 29)

myristic acid gt stearic acid gt palmitic acid

136

Table 22 Degradation of ascorbic acid in cream formulations in the dark in presence of

highest concentration of other vitamins

Concentration of ascorbic acid (mg 2g) Cream

No Time (days) 0 10 20 40 60 80

10e (RF) 375 285 233 171 110 69

11e (RF) 374 341 281 221 148 113

12e (RF) 372 259 203 130 89 59

13e (NA) 365 330 255 187 126 81

14e (NA) 370 321 289 219 159 109

15e (NA) 366 289 249 159 110 63

16e (TP) 377 359 321 261 211 159

17e (TP) 377 366 333 275 228 191

18e (TP) 373 361 304 252 200 167

137

02

07

12

17lo

g c

on

cen

tra

tio

n (

mg

)

10-12Riboflavin

02

07

12

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

13-15Nicotinamide

10

12

14

16

18

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

16-18Alpha-Tocopherol

Fig 28 First-order plots for the degradation of ascorbic acid in the dark in presence of

other vitamins using the emulsifying agents (minusminusminusminus) Stearic acid

(minus minusminus minus) palmitic acid (----) myristic acid

138

Table 23 First-order rate constants (kobs) for the degradation of ascorbic acid in presence

of other vitamins in cream formulations in the dark

Cream

formulation

Other

vitaminc

kobs times 102

(dayndash1

)ab

10e RF 204

(0995)

11e RF 156

(0992)

12e RF 222

(0992)

13e NA 189

(0995)

14e NA 151

(0993)

15e NA 214

(0995)

16e TP 100

(0994)

17e TP 088

(0995)

18e TP 105

(0993)

a The values in parenthesis are correlation coefficients and range from 0991ndash0996 due to

some variations in AH2 distribution in the creams

b The values of rate constants are relative and depend on specific experimental

conditions

c Vitamin concentrations (andashe) are as given in Table 16

The estimated error is plusmn5

139

Riboflavin

Nicotinamide

Alpha-Tocopherol

00

10

20

30

12 14 16 18Carbon chain length

ko

bs times

10

2 (

da

yndash1)

Fig 29 Plots of kobs for degradation of ascorbic acid in the dark in creams (10ndash18)

against carbon chain length of the emulsifier () Stearic acid () palmitic acid

() myristic acid

140

This indicates that the rate of degradation of AH2 is slowest in the creams containing

palmitic acid as the emulsifying agent The reason for AH2 degradation in the dark in this

order has already been explained in section 512

CHAPTER VII

STABILIZATION OF

ASCORBIC ACID WITH

CITRIC ACID TARTARIC

ACID AND BORIC ACID IN

CREAM FORMULATIONS

142

71 INTRODUCTION

Ascorbic acid is an ingredient of cosmetic preparations (Section 51) and is

sensitive to light (Rowe et al 2009 Sweetman 2009 British Pharmacopoeia 2009)

degrading to dehydroascorbic acid on UV irradiation by photooxidation (Kitagawa

1968) The photosensitivity of ascorbic acid makes it unstable in pharmaceutical and

cosmetic preparations (DeRitter 1982) The present work is an attempt to study the

photodegradation of ascorbic acid in cream formulations in the presence of certain

compounds (eg citric acid tartaric acid and boric acid) to investigate their role in the

stabilization of the vitamin on exposure to light and in the dark Citric acid and tartaric

acid are used as an antioxidant synergist (Rowe et al 2009) and boric acid is a

complexing agent for hydroxy compounds (Ahmad et al 2009cd)

72 CREAM FORMULATIONS

The details of the various cream formulations used in this study are given in Table

24 and the results obtained on the photodegradation of ascorbic acid in the presence of

stabilizing agents in these formulations are discussed in the following sections

143

Table 24 Composition of cream formulations containing ascorbic acid (2) and

stabilizers

Ingredients Cream

No SA PA MA CA GL PG EG AH2 CTa TA

b BA

c PH DW

19 a + minus minus + + minus minus + a minus minus + +

b + minus minus + + minus minus + b minus minus + +

c + minus minus + + minus minus + c minus minus + +

20 a minus + minus + + minus minus + a minus minus + +

b minus + minus + + minus minus + b minus minus + +

c minus + minus + + minus minus + c minus minus + +

21 a minus minus + + + minus minus + a minus minus + +

b minus minus + + + minus minus + b minus minus + +

c minus minus + + + minus minus + c minus minus + +

22 a + minus minus + + minus minus + minus a minus + +

b + minus minus + + minus minus + minus b minus + +

c + minus minus + + minus minus + minus c minus + +

23 a minus + minus + + minus minus + minus a minus + +

b minus + minus + + minus minus + minus b minus + +

c minus + minus + + minus minus + minus c minus + +

24 a minus minus + + + minus minus + minus a minus + +

b minus minus + + + minus minus + minus b minus + +

c minus minus + + + minus minus + minus c minus + +

25 a + minus minus + + minus minus + minus minus a + +

b + minus minus + + minus minus + minus minus b + +

c + minus minus + + minus minus + minus minus c + +

26 a minus + minus + + minus minus + minus minus a + +

b minus + minus + + minus minus + minus minus b + +

c minus + minus + + minus minus + minus minus c + +

27 a minus minus + + + minus minus + minus minus a + +

b minus minus + + + minus minus + minus minus b + +

c minus minus + + + minus minus + minus minus c + +

144

Table 24 continued

28 a + minus minus + minus + minus + minus minus a + +

b + minus minus + minus + minus + minus minus b + +

c + minus minus + minus + minus + minus minus c + +

29 a minus + minus + minus + minus + minus minus a + +

b minus + minus + minus + minus + minus minus b + +

c minus + minus + minus + minus + minus minus c + +

30 a minus minus + + minus + minus + minus minus a + +

b minus minus + + minus + minus + minus minus b + +

c minus minus + + minus + minus + minus minus c + +

31 a + minus minus + minus minus + + minus minus a + +

b + minus minus + minus minus + + minus minus b + +

c + minus minus + minus minus + + minus minus c + +

32 a minus + minus + minus minus + + minus minus a + +

b minus + minus + minus minus + + minus minus b + +

c minus + minus + minus minus + + minus minus c + +

33 a minus minus + + minus minus + + minus minus a + +

b minus minus + + minus minus + + minus minus b + +

c minus minus + + minus minus + + minus minus c + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PG = propylene glycol EG = ethylene glycol

PH = potassium hydroxide DW = distilled water CT = citric acid TA = tartaric acid

BA = boric acid

a CT (g ) a = 01 b = 02 c = 04

b TA (g ) a = 01 b = 02 c = 04

c BA (g ) a = 01 b = 02 c = 04

145

73 PRODUCTS OF ASCORBIC ACID PHOTODEGRADATION

The photodegradation of AH2 in cream formulations leads to the formation of

DHA as detected by TLC and reported earlier in the photolysis of AH2 in aqueous

solutions (Vaid et al 2006) and cream formulations (Sections 52 and 63) AH2 and

DHA in the methanolic extracts of the degraded creams were identified by comparison of

their Rf and color of the spots with those of the reference standards DHA is also

biologically active (Gardner 1972 Doores 2002) but its further degradation to 23-

diketo-gulonic acid (DGA) results in the loss of vitamin activity (Section 132)

However this product has not been detected in the present cream formulations

Therefore the creams may still possess their biological efficacy

74 SPECTRAL CHANGES IN PHOTODEGRADED CREAMS

In order to observe the spectral changes in photodegraded creams in the presence

of stabilizing agents the absorption spectra of the methanolic extracts of a degraded

cream were determined The spectra show a gradual loss of absorbance around 245 nm

due to the oxidation of AH2 to DHA on UV irradiation and similar to that shown for the

photodegradation of AH2 alone in Fig 5 DHA has negligible absorbance around 245 nm

(Davies et al 1991) and therefore it does not interfere with the absorbance of AH2 in

methanolic solutions The spectral changes and loss of absorbance around 245 nm in

methanolic solution depend on the extent of photooxidation of AH2 in a particular cream

75 ASSAY OF ASCORBIC ACID IN CREAMS

The UV spectrophotometric method (Zeng et al 2005) has previously been

applied to the determination of AH2 in cream formulations (Section 54) The absorbance

of the methanolic extracts of creams containing AH2 during photodegradation was used

146

to determine the concentration of AH2 The method was validated in the presence of citric

acid (CT) tartaric acid (TA) and boric acid (BA) before its application to the evaluation

of the kinetics of AH2 degradation in cream formulations The recovery of AH2 in creams

has been found to be in the range of 90ndash96 and is similar to that reported in Table 7

The reproducibility of the method lies within plusmn5 The assay data on the degradation of

AH2 in various creams in the presence of the stabilizing agents are reported in Table 25

76 KINETICS OF PHOTODEGRADATION

The effect of CT TA and BA as stabilizing agents on the photodegradation of

AH2 was studied by adding 01ndash04 of each compound to the cream formulations (19ndash

33) at pH 60 This concentration range is normally used for the stabilization of drugs in

pharmaceutical preparations (Im-Emsap et al 2002) The apparent first-order rate

constants (kobs) determined from the plots of log concentration versus time (Fig 30ndash34)

are reported in Table 26 The second-order rate constants (k) determined from the plots

of kobs versus concentration of the individual compounds (Fig 35ndash36) are given in Table

27 The values of k indicate the rate of inhibition of photodegradation of AH2 by each

compound

77 EFFECT OF STABILIZING AGENTS

In order to compare the effectiveness of CT TA and BA as stabilizing agents for

AH2 plots of k versus carbon chain length of the emulsifying agents were constructed

(Fig 37) The k values for the interaction of these compounds with AH2 are in the order

citric acid gt tartaric acid gt boric acid

The curves indicate that the highest interaction of these compounds with AH2 is in the

order

147

Table 25 Photodegradation of ascorbic acid in cream formulations in the presence of

stabilizers

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) a b c

0 374 378 379

60 362 362 372

120 349 355 367

210 333 335 349

19 (CT)

300 319 322 336

400 296 309 324

0 381 378 380

60 368 370 369

120 355 363 364

210 344 345 355

20 (CT)

300 328 335 341

400 312 319 331

21 (CT) 0 368 370 374

60 355 356 360

120 340 344 343

210 321 322 333

300 296 299 315

400 272 285 299

148

Table 25 continued

0 375 374 378

60 363 363 368

120 352 354 362

210 329 335 345

22 (TA)

300 307 314 333

400 292 299 313

0 370 377 374

60 364 365 368

120 352 357 357

210 332 344 349

23 (TA)

300 317 330 335

400 301 310 322

24 (TA) 0 376 379 377

60 367 369 368

120 351 348 352

210 325 330 344

300 306 317 326

400 284 294 310

149

Table 25 continued

0 370 375 380

60 356 362 359

120 331 339 344

210 311 318 330

25 (BA)

300 279 288 305

400 260 269 283

0 377 375 370

60 364 363 361

120 351 353 351

210 331 332 337

26 (BA)

300 323 324 325

400 301 307 313

27 (BA) 0 380 377 375

60 369 368 366

120 333 338 341

210 305 313 318

300 292 294 304

400 262 266 281

150

Table 25 continued

0 373 376 378

60 348 349 360

120 329 336 339

210 315 312 323

28 (BA)

300 282 283 299

400 249 264 280

0 370 373 380

60 358 355 367

120 343 346 356

210 325 329 347

29 (BA)

300 307 312 325

400 287 295 315

30 (BA) 0 369 375 372

60 353 358 362

120 321 330 335

210 283 294 303

300 265 281 293

400 242 254 270

151

Table 25 continued

0 374 376 379

60 348 366 352

120 324 340 337

210 303 319 322

31 (BA)

300 275 289 293

400 243 260 275

0 370 374 375

60 355 354 366

120 339 344 345

210 313 319 330

32 (BA)

300 288 297 308

400 261 271 290

33 (BA) 0 377 380 377

60 357 361 367

120 324 335 339

210 288 294 307

300 270 280 293

400 233 248 265

Creams 19ndash27 contain glycerin 28ndash30 contain propylene glycol and 31ndash33 contain

ethylene glycol as humectants

152

a

b

c

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

19

ab

c

14

15

16

log

co

nce

ntr

ati

on

(m

g)

20

a

b

c

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

21

Fig 30 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and citric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

153

a

b

c

14

15

16lo

g c

on

cen

tra

tio

n (

mg

)

22

a

b

c

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

23

a

b

c

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

24

Fig 31 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and tartaric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

154

ab

c

13

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

25

abc

13

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

26

ab

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

27

Fig 32 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and boric acid (a) 01 (b) 02 (c) 04

Palmitic acid

Stearic acid

Myristic acid

155

a

b

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

28

ab

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

29

a

b

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

30

Fig 33 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing propylene glycol and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

156

a

b

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

31

ab

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

32

a

b

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

33

Fig 34 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing ethylene glycol and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

157

Table 26 Apparent first-order rate constants (kobs) for the photodegradation of ascorbic

acid in presence of different stabilizers in cream formulations

kobs times 103 (min

ndash1)ab

Cream

formulation Stabilizer

c

a b c

19 CT 057

(0995)

050

(0992)

041

(0991)

20 CT 049

(0996)

043

(0995)

034

(0993)

21 CT 076

(0995)

067

(0995)

055

(0992)

22 TA 065

(0995)

058

(0995)

046

(0991)

23 TA 054

(0994)

047

(0993)

038

(0994)

24 TA 072

(0996)

063

(0992)

049

(0991)

25 BA 091

(0994)

086

(0995)

071

(0993)

26 BA 055

(0994)

050

(0993)

042

(0993)

27 BA 095

(0995)

089

(0992)

074

(0996)

28 BA 097

(0995)

088

(0992)

075

(0993)

29 BA 064

(0994)

057

(0991)

047

(0993)

30 BA 110

(0994)

100

(0996)

084

(0992)

31 BA 105

(0995)

094

(0994)

078

(0992)

32 BA 088

(0994)

079

(0993)

066

(0993)

33 BA 120

(0995)

108

(0993)

091

(0993) a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental conditions

including the light intensity

c Stabilizer concentrations (andashc) are as given in Table 24

The estimated error is plusmn5

158

00

04

08

12

00 04 08 12 16 20

Citric acid concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

19-21

00

04

08

12

00 05 10 15 20 25

Tartaric acid concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

22-24

Fig 35 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against citric acid (19ndash21) and tartaric acid concentrations (22ndash24) in cream

formulations

159

00

04

08

12k

ob

s times

10

3 (

min

ndash1)

25-27

00

04

08

12

00 20 40 60

ko

bs times

10

3 (

min

ndash1)

28-30

00

04

08

12

00 20 40 60

Boric acid concentration (M times 102)

kob

s times

10

3 (

min

ndash1)

30-33

Fig 36 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against boric acid concentrations in cream formulations (25ndash33)

Propylene glycol

Glycerin

Ethylene glycol

160

Table 27 First-order rate constants (k0)a for the photodegradation of ascorbic acid in the

absence of stabilizers and second-order rate constants (k) for the interaction of

ascorbic acid with CT TA and BA

Cream

formulation Stabilizers

k0 times 103

(minndash1

)

k times 102

(Mndash1

minndash1

)

Correlation

coefficient

19 CT 062 111 0991

20 CT 053 103 0994

21 CT 082 145 0995

22 TA 071 092 0995

23 TA 059 080 0993

24 TA 080 118 0996

25 BA 098 041 0994

26 BA 059 026 0994

27 BA 102 044 0995

28 BA 104 046 0992

29 BA 069 033 0995

30 BA 118 054 0994

31 BA 113 053 0995

32 BA 095 045 0995

33 BA 129 060 0993

a The variations in the values of k0 are due to the presence of different emulsifying agents

in cream formulations

161

00

04

08

12

16

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash1

min

ndash1)

18-33

a

b

e

cd

Fig 37 Plots of k for photodegradation of ascorbic acid in creams (19ndash33) against

carbon chain length of emulsifier () Stearic acid () palmitic acid ()

myristic acid (a) CT (b) TA (c) BA with glycerin (d) BA with propylene

glycol (e) BA with ethylene glycol

162

myristic acid gt stearic acid gt palmitic acid

In the case of myristic acid and stearic acid it may be explained on the basis of the

decreasing polarity (Yao et al 2009) It is interesting to observe the lowest rates of

interaction of these compounds in the creams containing palmitic acid This could be due

to the interaction of AH2 with palmitic acid to form a palmitate derivative in addition to

its interaction with the individual stabilizing agents CT and TA are known to act as

antioxidant synergists (Rowe et al 2009 Sweetman 2009) and in this capacity may

inhibit the photooxidation of AH2 as indicated by the values of the degradation rate

constants in the presence of these compounds The addition of CT to nutritional

supplements is known to inhibit the oxidation of AH2 (Doores 2002) Boric acid forms a

complex with AH2 (Rivlin 2007) and there by may inhibit its degradation Boric acid

may also interact with glycerin added to the creams as a humectant and form a complex

(Rowe et al 2009) This may influence its interaction and stabilizing effect on AH2 in

creams as indicated by the lower k values compared to those in the presence of CT and

TA It has further been observed that the k values for BA are greater in propylene glycol

and ethylene glycol compared to those in glycerin (Table 27) Again this may be due to

greater interaction of BA with glycerin compared to other humectants in the creams

78 DEGRADATION OF ASCORBIC ACID IN PRESENCE OF STABILIZING

AGENTS IN THE DARK

An important factor in the formulation of cosmetic preparations is to ensure the

chemical and photostability of the active ingredient by the use of appropriate stabilizing

agents The choice of these agents would largely depend on the nature and

physicochemical characteristics of the active ingredient AH2 possesses a redox system

163

and can be easily oxidized by air or light In order to observe the effect of CT TA and

BA on the stability of AH2 the cream formulations containing the individual compounds

were stored in the dark for a period of about three months and the rate of degradation of

AH2 was determined The assay data are reported in Table 28 and the kinetic plots are

shown in Fig 38ndash42 The values of apparent first-order rate constants for the degradation

of AH2 in the presence of the stabilizing agents are reported in Table 29 The second

order-rate constants for the interaction of CT TA and BA with AH2 are reported in Table

30 (Fig 43ndash44) The plots of k against the carbon chain length of the emulsifiers are

shown in Fig 45 The kinetic data indicate the same pattern of rates of degradation and

interaction of AH2 with these compounds as observed in the presence of light except that

the rates are much slower in the dark Thus the stabilizing agents are equally effective in

inhibiting the rate of degradation of AH2 in the dark The effect of emulsifying agents and

the humectants on the rate of degradation of AH2 in the presence of the stabilizers has

been discussed in the above Section 77

79 EFFECT OF ADDITIVES ON TRANSMISSION OF ASCORBIC ACID

In order to observe the effect of additives (citric tartaric and boric acids) on the

transmission characteristics of ascorbic acid (0002 mg100 ml) in methanol containing

the highest concentration of the additives (004) used in this study the transmission

spectra were measured It has been found that these additives produce a hypsochromic

shift in the absorption maximum of ascorbic acid This may result in the reduction of the

fraction of light absorbed by ascorbic acid to the extent of about 10 and thus influence

the rate of photodegradation reactions However since all the additives produce similar

effects the rate constants can be considered on a comparative basis

164

Table 28 Degradation of ascorbic acid in cream formulations in the presence of

stabilizers in the dark

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(days) a b c

0 374 378 379

10 355 346 362

20 326 328 342

40 293 297 322

19 (CT)

60 264 269 295

80 241 245 262

0 381 378 380

10 361 364 372

20 339 350 348

40 309 312 330

20 (CT)

60 279 286 301

80 260 266 282

21 (CT) 0 368 370 374

10 342 346 364

20 310 321 348

40 278 282 313

60 249 251 278

80 217 228 249

165

Table 28 continued

0 375 374 378

10 339 344 351

20 317 326 336

40 282 288 306

22 (TA)

60 251 258 280

80 222 235 252

0 370 377 374

10 340 354 355

20 332 336 343

40 297 303 310

23 (TA)

60 266 282 294

80 238 248 267

24 (TA) 0 376 379 377

10 341 339 350

20 306 319 323

40 263 284 279

60 223 241 249

80 196 202 223

166

Table 28 continued

0 370 375 380

10 331 341 334

20 287 289 301

40 225 247 245

25 (BA)

60 189 185 214

80 141 154 170

0 377 375 370

10 355 357 349

20 326 314 324

40 264 267 286

26 (BA)

60 232 238 254

80 189 199 211

27 (BA) 0 380 377 375

10 346 339 337

20 309 288 301

40 233 241 260

60 192 196 211

80 140 147 163

167

Table 28 continued

0 373 376 378

10 314 322 333

20 267 281 305

40 217 233 253

28 (BA)

60 167 177 204

80 122 135 151

0 370 373 380

10 336 329 343

20 283 277 306

40 233 243 267

29 (BA)

60 189 190 217

80 144 154 173

30 (BA) 0 369 375 372

10 308 319 329

20 255 275 310

40 210 226 244

60 158 163 191

80 113 131 147

168

Table 28 continued

0 374 376 379

10 303 311 329

20 266 260 289

40 211 219 239

31 (BA)

60 155 158 178

80 112 121 149

0 370 374 375

10 314 323 339

20 276 280 305

40 222 233 258

32 (BA)

60 172 187 193

80 126 136 162

33 (BA) 0 377 380 377

10 308 306 320

20 254 265 280

40 205 214 237

60 144 155 175

80 107 118 138

169

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

19

abc

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

20

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

21

Fig 38 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing citric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

170

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

22

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

23

ab

c

12

13

14

15

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

24

Fig 39 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing tartaric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

171

a

b

c

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

25

abc

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

26

ab

c

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

27

Fig 40 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing glycerin and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

172

a

b

c

10

12

14

16

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

28

ab

c

10

12

14

16

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

29

a

b

c

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

30

Fig 41 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing propylene glycol and boric acid (a) 01 (b) 02 (c)

04

Palmitic acid

Stearic acid

Myristic acid

173

ab

c

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

31

ab

c

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

32

a

b

c

08

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

33

Fig 42 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing ethylene glycol and boric acid (a) 01 (b) 02 (c)

04

Myristic acid

Palmitic acid

Stearic acid

174

Table 29 Apparent first-order rate constants (kobs) for the degradation of ascorbic acid in

presence of different stabilizers in cream formulations in the dark

kobs times 102 (day

ndash1)ab

Cream

formulation Stabilizer

c

a b c

19 CT 055

(0994)

052

(0992)

044

(0991)

20 CT 048

(0995)

046

(0995)

038

(0992)

21 CT 064

(0994)

061

(0995)

052

(0994)

22 TA 063

(0994)

058

(0995)

049

(0996)

23 TA 054

(0995)

050

(0995)

041

(0994)

24 TA 081

(0995)

075

(0993)

066

(0995)

25 BA 118

(0996)

113

(0994)

097

(0994)

26 BA 087

(0995)

079

(0993)

068

(0994)

27 BA 124

(0995)

114

(0994)

101

(0993)

28 BA 134

(0995)

124

(0996)

110

(0992)

29 BA 116

(0996)

108

(0992)

096

(0995)

30 BA 142

(0993)

131

(0995)

115

(0995)

31 BA 145

(0995)

137

(0992)

117

(0995)

32 BA 130

(0996)

120

(0993)

107

(0994)

33 BA 153

(0995)

141

(0994)

122

(0994) a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental

conditions

c Stabilizer concentrations (andashc) are as given in Table 24

The estimated error is plusmn5

175

176

Table 30 First-order rate constants (k0)a for the degradation of ascorbic acid in the

absence of stabilizers and second-order rate constants (k) for the chemical

interaction of ascorbic acid with CT TA and BA in the dark

Cream

formulation Stabilizers

k0 times 102

(dayndash1

)

k times 102

(Mndash1

dayndash1

)

Correlation

coefficient

19 CT 060 797 0996

20 CT 052 723 0995

21 CT 069 850 0994

22 TA 068 710 0996

23 TA 058 636 0994

24 TA 086 758 0994

25 BA 126 444 0993

26 BA 092 375 0992

27 BA 131 480 0991

28 BA 141 488 0993

29 BA 122 418 0994

30 BA 149 531 0991

31 BA 155 578 0996

32 BA 137 472 0994

33 BA 163 627 0996

a The variations in the values of k0 are due to the presence of different emulsifying agents

in cream formulations

177

00

04

08

12

00 04 08 12 16 20

Citric acid concentration (M times 102)

kob

s times

10

2 (

da

yndash1)

19-21

00

04

08

12

00 05 10 15 20 25

Tartaric acid concentration (M times 102)

kob

s times

10

2 (

da

yndash1)

22-24

Fig 35 Plots of first-order rate constants (kobs) for the degradation of ascorbic acid in the

dark against citric acid (19ndash21) and tartaric acid (22ndash24) concentrations in

cream formulations

178

00

10

20

00 20 40 60

kob

s times

10

3 (

min

ndash1)

25-27

00

10

20

00 20 40 60

kob

s times

10

3 (

min

ndash1)

28-30

00

10

20

00 20 40 60

Boric acid concentration (M times 102)

kob

s times

10

3 (

min

ndash1)

30-33

Fig 44 Plots of first-order rate constants (kobs) for the degradation of ascorbic acid in the

dark against boric acid concentrations in cream formulations (25ndash33)

Glycerin

Propylene glycol

Ethylene glycol

179

00

04

08

12

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash

1 d

ayndash

1)

18-33

b

a

e

dc

Fig 45 Plots of k for degradation of ascorbic acid in the dark in creams (19ndash33) against

carbon chain length of emulsifier () Stearic acid () palmitic acid ()

myristic acid (a) CT (b) TA (c) BA with glycerin (d) BA with propylene

glycol (e) BA with ethylene glycol

CONCLUSIONS

AND

SUGGESTIONS

180

CONCLUSIONS

The main conclusions of the present study on the photodegradation of the ascorbic

acid in organic solvents and cream formulations are as follows

1 Identification of Photodegradation Products

The photodegradation of ascorbic acid in aqueous organic solvents and

laboratory prepared oil-in-water cream preparations on UV irradiation leads to the

formation of dehydroascorbic acid No further degradation products of dehydroascorbic

acid have been detected under the present experimental conditions The product was

identified by comparison of its Rf value and color of the spot with those of the authentic

compound by thin-layer chromatography and spectral changes

2 Assay of Ascorbic Acid

Ascorbic acid in aqueous organic solvents and cream preparations was assayed

in acidified methanolic solutions (pH 20) at 245 nm using a UV spectrophotometric

method Ascorbic acid in combination with other vitamins (riboflavin nicotinamide and

alpha-tocopherol) was assayed by the official iodimetric method due to interference by

these vitamins at the analytical wavelength Both analytical methods were validated

under the experimental conditions employed before their application to the assay of

ascorbic acid The recoveries of ascorbic acid in cream preparations are in the range of

90ndash96 and the reproducibility of both methods are within plusmn5 The F test and the t test

show that there is no significant difference between the precision of the two methods and

therefore these methods can be applied to the assay of ascorbic acid in cream

preparations with comparable results

181

3 Kinetics of Photodegradation

a) Photodegradation of ascorbic acid in organic solvents

Ascorbic acid degradation follows apparent first-order kinetics in aqueous

organic solvents A plot of the first-order rate constants (log kobs) versus solvent dielectric

constant is linear with positive slope indicating an increase in the rate with dielectric

constant On the contrary a plot of kobs verses reciprocal of solvent viscosity is linear with

a positive slope showing a decrease in the rate with solvent viscosity Thus the rate of

photodegradation of ascorbic acid (an oxidizable drug) depends on the solvent

characteristics

b) Photodegradation of ascorbic acid in cream preparations

Ascorbic acid has been found to follow apparent first-order kinetics in cream

preparations and the rate of degradation is affected by the following factors

i Effect of concentration

An apparent linear relationship has been observed between log kobs and

concentration (05ndash25) of ascorbic acid in a cream preparation Thus the rate of

degradation of ascorbic acid appears to be faster at a lower concentration

compared to that of a higher concentration on exposure to the same intensity of

light

ii Effect of carbon chain length of the emulsifying agent

The plots of kobs verses carbon chain length of the emulsifying agent show that the

photodegradation of ascorbic acid is affected in the order myristic acid gt stearic

acid gt palmitic acid This is predominantly due to the interaction of ascorbic acid

with palmitic acid and the carbon chain length (measure of relative polar

182

character) of the emulsifying acid probably does not play a part in the

photodegradation kinetics of ascorbic acid in creams This is evident from the

non-linear relationship between the rate constants for ascorbic acid degradation

and the carbon chain length of the emulsifying acids

iii Effect of viscosity

The values of kobs for the photodegradation of ascorbic acid in cream preparations

are in the order of humectant ethylene glycol gt propylene glycol gt glycerin

showing that the rates of degradation are influenced by the viscosity of the

humectant and decrease with an increase in the viscosity as observed in the case

of organic solvents

iv Effect of pH

The log kndashpH profiles for the photodegradation of ascorbic acid in creams

represent sigmoid type curves indicating an increase in the rate of oxidation of the

molecule with ionization (pH 42ndash70 557ndash999) The AHndash species appears to

be more susceptible to oxidation than the non-ionized molecule in the pH range

studied

v Effect of redox potential

The values of kobs show that the rate of photooxidation of ascorbic acid is

influenced by its redox potential which varies with pH The greater photostability

of ascorbic acid at pH 5ndash6 compared to that at pH 7 and above is due to its lower

rate of oxidation-reduction in the lower range The increase in the rate of

photooxidation with pH is due to a corresponding increase in the redox potential

of ascorbic acid

183

c) Photodegradation of ascorbic acid in the presence of other vitamins (riboflavin

nicotinamide alpha-tocopherol) in cream preparations

The photodegradation of ascorbic acid is affected by the presence of other

vitamins in creams The kinetic data on the photochemical interactions indicate that

riboflavin and nicotinamide act as photosensitizers in the degradation of ascorbic acid

and have an adverse effect on the photostability of the vitamin in creams Whereas

alpha-tocopherol exerts an inhibitory effect on the degradation of ascorbic acid by acting

as a redox partner in the creams Thus a combination of ascorbic acid and alpha-

tocopherol has a synergistic effect on the stabilization of ascorbic acid in creams These

vitamins do not appear to influence the rate of degradation of ascorbic acid in the dark

d) Photodegradation of ascorbic acid in the presence of citric acid tartaric acid and

boric acid in cream preparations

The rate of photodegradation of ascorbic acid in creams has been found to be

inhibited by the addition of compounds such as citric acid tartaric acid and boric acid in

creams These compounds show a stabilizing effect on the photodegradation of ascorbic

acid in the order citric acid gt tartaric acid gt boric acid The lower effect of boric acid

may be due to its interaction with the emulsifying agents and humectants Boric acid

exerts this effect by complex formation with ascorbic acid Citric acid and tartaric acid

are antioxidant synergists and in combination with ascorbic acid may exert a stabilizing

effect on its degradation

184

Salient Features of the Work

In the present work an attempt has been made to study the effects of solvent

characteristics formulation factors particularly the emulsifying agents in terms of the

carbon chain length and humectants in terms of viscosity medium pH drug

concentration redox potential and interactions with other vitamins and stabilizers on the

kinetics of photodegradation of ascorbic acid in cream preparations The study may

provide useful information to improve the photostability and efficacy of ascorbic acid in

cream preparations

SUGGESTIONS

The present work may provide guidelines for a systematic study of the stability of

drug substances in cream ointment preparations and the evaluation of the influence of

formulation variables such as emulsifying agents and humectants concentration pH

polarity viscosity redox potential on the rate of degradation and stabilization of drug

substances This may enable the formulator in the judicious design of formulations that

have improved stability and efficacy for therapeutic use The kinetic parameters may

throw light on the comparative stability of the preparations and help in the choice of

appropriate formulation ingredients

REFERENCES

186

Abe Y Horii H Taniguchi S (1986) A theoretical study of the oxidation mechanism

of triose reductone in reference to L-ascorbic acid Can J Chem 64 360-365

Ahmad I (1968) A Study of the Degradation of Riboflavin and Related Compounds

PhD Thesis University of London London

Ahmad I Rapson HDC (1990) Multicomponent spectrophotometric assay of

riboflavin and photoproducts J Pharm Biomed Anal 8 217-223

Ahmad I Vaid FHM (2006) Photochemistry of flavins in aqueous and organic

solvents In Silva E Edwards AM Eds Flavins Photochemistry and

Photobiology Royal Society of Chemistry Cambridge Chap 2

Ahmad I Tollin G (1981) Solvent effect on flavin electron transfer reactions

Biochemistry 20 5925-5928

Ahmad I Jamil N Zoha SMS (1973) Statistical evaluation of antioxidants used in

the manufacture of ascorbic acid tablets J Sci Univ Kar 2 269-287

Ahmad I Fasihullah Q Vaid FHM (2004b) A study of simultaneous photolysis and

photoaddition reactions of riboflavin in aqueous solution J Photochem Photobiol B

Biol 75 13-20

Ahmad I Fasihullah Q Vaid FHM (2005) Effect of phosphate buffer on

photodegradation reactions of riboflavin in aqueous solution J Photochem

Photobiol B Biol 78 229-234

Ahmad I Fasihullah Q Vaid FHM (2006a) Effect of light intensity and wavelength

on photodegradation reactions of riboflavin in aqueous solution J Photochem

Photobiol B Biol 82 21-27

187

Ahmad I Fasihullah Q Vaid F H M (2006b) Photolysis of formylmethylflavin in

aqueous and organic solvents Photochem Photobiol Sci 5 680-685

Ahmad I Ahmed S Sheraz MA Vaid FHM (2008) Effect of borate buffer on the

photolysis of riboflavin in aqueous solution J Photochem Photobiol B Biol 93 82-

87

Ahmad I Ahmed S Sheraz MA Vaid FHM (2009c) Borate toxicity effect on

drug stability and analytical applications In Chung MP Ed Handbook on

Borates Chemistry Production and Applications Nova Science Publishers Inc

New York in press

Ahmad I Sheraz MA Ahmed S Vaid FHM (2009d) Analytical applications of

borates Mater Sci Res J in press

Ahmad I Fasihullah Q Noor A Ansari IA Ali QNM (2004a) Photolysis of

riboflavin in aqueous solution a kinetic study Int J Pharm 280 199-208

Ahmad I Ahmed S Sheraz MA Aminuddin M Vaid FHM (2009a) Effect of

caffeine complexation on the photolysis of riboflavin in aqueous solution a kinetic

study Chem Pharm Bull in press

Ahmad I Ahmed S Sheraz MA Vaid FHM Ansari IA (2009b) Effect of

divalent ions on photodegradation kinetics and pathways of riboflavin in aqueous

solution Photochem Photobiol Sci accepted

Ahmed S (2009) Effect of Complexing Agents on the Photodegradation of Riboflavin

in Aqueous Solution PhD Thesis Baqai Medical University Karachi

Ahmad T (1978) Studies on the Stability of Sulphacetamide and Degradation Products

PhD Thesis University of Karachi Karachi

188

Ahmad T Ahmad I (1983) The effect of antioxidants on the photolysis of aqueous

sulfacetamide and sulfanilamide solutions Pol J Pharmacol Pharm 35 69-75

Albini A Fasani E Eds (1998) Drugs Photochemistry and Photostability The Royal

Society of Chemistry Cambridge

Allwood MC (1984a) Factors influencing the stability of ascorbic acid in total

parenteral nutrition infusions J Clin Hosp Pharm 9 75-85

Allwood MC (1984b) Compatibility and stability of TPN mixtures in big bags J Clin

Pharm Ther 9 181-198

Allwood MC Kearney MCJ (1998) Compatibility and stability of additives in

parenteral nutrition admixtures Nutrition 14 697-706

Al-Meshal IA Hassan MMA (1982) Ascorbic Acid In Florey K Ed Analytical

Profiles of Drug Substances Vol 11 Academic Press New York pp 45-78

Anderson NH Johnston D McLelland MA Munden P (1991) Photostability

testing of drug substances and drug products in UK pharmaceutical laboratories J

Pharm Biomed Anal 9 443-449

Ansari IA (2002) Photolysis and Interactions of Cyanocobalamin with some B-

Vitamins and Ascorbic Acid in Parenteral Solutions PhD Thesis University of

Karachi Karachi

Ansari IA Vaid Vaid HM Ahmad I (2004) Spectral Study of Photolysis of

Aqueous Cyanocobalamin Solutions in Presence of Vitamins B and C Pak J Pharm

Sci 17 83-93

Arcus CL Zilva SS (1940) The photochemical decomposition of L-ascorbic acid II

Biochem J 34 61-66

189

Arger CB Rupp D Lo P (1996) Preservation of dispersed systems In Liberman

HA Rieger MM Banker GS Eds Pharmaceutical Dosage Forms Disperse

Systems 2nd ed vol1 Marcel Dekker New York Chap9

Asard H May J Smirnoff N Eds (2004) Vitamin C Function and Biochemistry in

Animals and Plants BIOS Scientific Publishers New York

Asker AF Canady D Cobb C (1985) Influence of DL-methionine on the

photostability of ascorbic acid solutions Drug Dev Ind Pharm 11 2109-2125

Ault RG Baird DK Carrington HC Haworth WN Herbert R Hirst EL

Percival EGV Smith F Stacey M (1933) Synthesis of d- and of l-ascorbic acid

and of analogous substances J Chem Soc 1419-1423

Austria R Semenzato A Bettero A (1997) Stability of vitamin C derivatives in

solution and topical formulations J Pharm Biomed Anal 15 795-801

Baertschi SW Alsante KM (2005) Stress testing The chemistry of drug degradation

In Baertschi SW Ed Pharmaceutical Stress testing Predicting Drug Degradation

Taylor amp Francis London Chap 3

Bailey CW Bright JR Jasper JJ (1945) A study of the binary system nicotinamide-

ascorbic acid J Am Chem Soc 67 1184-1186

Ball GFM (2006) Vitamins in Foods Analysis Bioavailability and Stability CRC

Press Boca Raton FL Chap 15

Banhegyi G Marcolongo P Puskas F Fulceri R Mandl J Benedetti A (1998)

Dehydroascorbate and ascorbate transport in rat liver microsomal vesicles J Biol

Chem 273 2758-2762

190

Bardolph EM Taylor RH (1997) Sailors scurvy science and authority J R Soc

Med 90 238

Barel AO Paye M Maibach HI Eds (2001) Handbook of Cosmetic Science and

Technology Marcel Dekker New York

Barry BW (1983) Dermatological Formulations Percutaneous Absorption Marcel

Dekker New York

Barry BW (2002) Transdermal drug delivery In Aulton ME Ed Pharmaceutics

The Science of Dosage Form Design Churchill Livingstone London Chap 33

Basu TK Temple NJ Garg ML (1999) Antioxidants in Human Health and

Disease CABI Publishing Oxford

Becher P (1965) Emulsions Theory and Practice Reinhold New York p 1

Beissenhirtz M Scheller F Lisdat F (2003) Immobilized cytochrome c sensor in

organicaqueous media for the characterization of hydrophilic and hydrophobic

antioxidants Electroanalysis 15 1425-1435

Berlett BS Stadtman ER (1997) Protein oxidation in aging disease and oxidative

stress J Biol Chem 272 20313-20316

Betageri G Prabhu S (2002) Semisolid preparations In Swarbrick J Boylan JC

Eds Encyclopedia of Pharmaceutical Technology 2nd ed Marcel Dekker New

York pp 2436-2457

Bielski BHJ (1982) Chemistry of ascorbic acid radicals In Seib PA Tolbert BM

Eds Ascorbic Acid Chemistry Metabolism and Uses Advances in Chemistry

Series American Chemical Society Washington DC pp 81-100

191

Bielski BHJ Allen AO Schwartz HA (1981) Mechanism of disproportionation of

ascorbate radicals J Am Chem Soc 103 3516-3518

Billany M (2002) Suspensions and emulsions In Aulton ME Ed Pharmaceutics

The Science of Dosage Form Design 2nd ed Churchill Livingstone London Chap

23

Bisby RH Morgan CG Hamblett I Gorman AA (1999) Quenching of singlet

oxygen by trolox C ascorbate and amino acids effect of pH and temperature J Phys

Chem 103 7454-7459

Bissett DL (2006) Anti-aging skin care formulations In Draelos ZD Thaman LA

Eds Cosmetic Formulation of Skin Care Products Taylor amp Francis Group New

York Chap 11

Blaugh SM Hajratwala B (1972) Kinetics of aerobic oxidation of ascorbic acid J

Pharm Sci 61 556-562

Block LH (1996) Pharmaceutical emulsions and microemulsions In Lieberman HA

Rieger MM Banker GS Eds Pharmaceutical Dosage Forms Disperse Systems

Vol 2 2nd ed Chap 2

Bolliger HR Konig A (1969) Water-soluble vitamins In Stahl E Ed Thin-layer

Chromatography Springer-Verlag Berlin pp 304-306

Bonwstra JA De Graaff A Gooris GS Nijsse J Wiechers JW Van Aelst AC

(2003) Water distribution and related morphology in human stratum corneum at

different hydration levels J Invest Dermatol 120 750-758

Bors SR (2005) Scurvy Thomas Dunne Books St Martinrsquos Griffen New York

192

Bors W Buettner GR (1997) The vitamin C radical and its reactions In Packer L

Fuchs J Eds Vitamin C in Health and Disease Marcel Dekker New York Chap

4

Braun L Puskas F Csala M Meszaros G Mandl J Banhegyi G (1997)

Ascorbate as a substrate for glycolysis or gluconeogenesis evidence for an interorgan

ascorbate cycle Free Radic Biol Med 23 804-808

British Pharmacopoeia (2009) Her Majestyrsquos Stationary Office London Electronic

version

Buettner G (1993) The pecking order of free radicals and antioxidants lipid

peroxidation α-tocopherol and ascorbate Arch Biochem Biphys 300 535-543

Buettner GR Schafer FQ (2004) Ascorbate as an antioxidant In Asard H May

JM Smirnoff N Eds Vitamin C Function and Biochemistry in Animals and

Plants BIOS Scientific Publishers Oxford Chap 10

Burke KE (2006) Topical nutritional antioxidants In Draelos ZD Thaman LA

Eds Cosmetic Formulation of Skin Care Products Taylor amp Francis Group New

York Chap 21

Burns JJ Ed (1961) Vitamin C Ann NY Acad Sci 92 1

Byrom P Turnbull JH (1967) Excited states of flavin coenzymes II Anaerobic

oxidation of amino acids by excited riboflavin derivatives Photochem Photobiol 6

125-131

Cadenas E Packer L (2002) Handbook of Antioxidants 2nd ed Marcel Dekker New

York Chap 7-9

193

Capellmann M Bolt HM (1992) Simultaneous determination of ascorbic acid and

dehydroascorbic acid by HPLC with postcolumn derivatisation and fluorometric

detection Fresenius J Anal Chem 342 462-466

Cairns WL Metzler DE (1971) Photochemical degradation of flavins VI A new

photoproduct and its use in studying photolytic mechanism J Am Chem Soc 93

2772-2777

Carlotti ME Ugazio E Sapino S Peira E Gallarate M (2006) Photodegradation

of retinol and anti-aging effectiveness of two commercial emulsions J Cosmet Sci

57 261-277

Carpenter K (1986) The History of Scurvy and Vitamin C Cambridge University Press

Cambridge

Carr AC Zhu BZ Frei B (2000) Potential antiatherogenic mechanisms of ascorbate

(vitamin C) and alpha-tocopherol (vitamin E) Circ Res 87 349-354

Carstensen JT (1972) Theory of Pharmaceutical Systems Academic Press New York

pp 211-214

Chaudhri SK Jain NK (2009) History of cosmetics Asian J Pharm 3 164-167

Chessex P Lovoic JC Roulean T Brochu P St Louis P Levy E Al Varez F

(2002) Photooxidation of parenteral multivitamins induced hepatic steatosis in a

neonatal guinea pig model of intravenous infusion Pediatr Res 52 958-963

Connors KA Amidon GL Stella VJ (1986) Chemical Stability of Pharmaceuticals

A Handbook for Pharmacists 2nd ed Wiley New York pp 105-106 142 208-220

194

Dabrowski K Huiterleitner S (1989) Application of a simultaneous assay of ascorbic

acid dehydroascorbic acid and ascorbic sulfate in biological matter Analyst

(London) 114 83-87

Dale JK Booth RE (1976) In Hoover JE Ed Dispensing of Medication 8th ed

Mack Easton Pennsylvania pp 479-481

Darr D Dunston S Faust H Pinnell S (1996) Effectiveness of antioxidants (vitamin

C and E) with and without sunscreens as topical photoprotectants Acta Derm

Venereol 76 264-268

Davey MW Montagu MV Inze D Sanmartin M Kanellis A Smirnoff N

Benzie IJJ Strain JJ Favell D Fletcher J (2000) Plant L-ascorbic acid

chemistry function metabolism bioavailability and effect of processing J Sci Food

Agr 80 825-860

Davies MB Austin J Partridge DA (1991) Vitamin C Its Chemistry and

Biochemistry The Royal Society of Chemistry Cambridge

De La Rochette A Birlouez-Aragon I Silva E Morliere P (2003) Advanced

glycation endproducts as UVA photosensitizers of tryptophan and ascorbic acid

consequences for the lens Biochem Biophys Acta 1621 235-241

De La Rochette A Silva E Birlouez-Aragon I Mancini M Edwards AM

Morliere P (2000) Riboflavin photodegradation and photosensitization effects are

highly dependent on oxygen and ascorbate concentration Photochem Photobiol 72

815-820

195

De Vries H Beijersbergen Van Henegouwen GMJ Huff FA (1984) Photochemical

decomposition of chloramphenicol in a 025 eye drop and in a therapeutic

intraocular concentration Int J Pharm 20 265-271

Dean RT Fu S Stocker R Davies MJ (1997) Biochemistry and pathology of

radicalmediated protein oxidation Biochem J 324 1-18

DeRitter E (1982) Vitamins in pharmaceutical formulations J Pharm Sci 71 1073-

1096

Deutsch JC (1998a) Ascorbic acid oxidation by hydrogen peroxide Anal Biochem

255 1-7

Deutsch JC (1998b)Spontaneous hydrolysis and dehydration of dehydroascorbic acid

in aqueous solution Anal Biochem 260 223-229

Deutsch JC (1998c)oxygen-accepting antioxidants which arise during ascorbate

oxidation Anal Biochem 265 238-245

Dodsun KY Young ER Soliman AGM (1992) Determination of total vitamin C

in various food matrices by liquid chromatography and fluorescence detection J

AOAC Int 75 887-891

Dollery C Ed (1999) Therapeutic Drugs 2nd ed Vol 1 Churchill Livingstone

London pp A213-A216

Doores S (2002) pH control agents and acidulants In Branen LA Davidson PM

Salminen S Thorngate JH Eds Food Additives Marcel Dekker New York p

637

Eberlein-Koumlnig B Ring J (2005) Relevance of vitamins C and E in cutaneous

photoprotection J Cosmet Dermatol 4 4-9

196

Ehrlich M Rao J Pabby A Goldman MP (2006) Improvement in the appearance

of wrinkles with topical transforming growth factor beta(1) and l-ascorbic acid

Dermatol Surg 32 618-625

EIRI Board (2007) Profitable Small Scale Manufacture of Cosmetics Engineers India

Research Institute Nai Sarak Delhi

Eitenmiller RR Ye L Landen WOJr (2008) Vitamin Analysis for the Health and

Food Sciences 2nd ed CRC Press Boca Raton FL Chap 5

Elia S (2009) Nutrition In Kumar P Clark M Eds Kumar and Clarkrsquos Clinical

Medicine 7th ed Elsevier Limited Chap 5

Elmore AR (2005) Final report of the safety assessment of L-ascorbic acid calcium

ascorbate magnesium ascorbate magnesium ascorbyl phosphate sodium ascorbate

and sodium ascorbyl phosphate as used in cosmetics Int J Toxicol 24 51-111

Erb C Nau-Staudt K Flammer J Nau W (2004) Ascorbic acid as a free radical

scavenger in porcine and bovine aqueous humour Ophthal Res 36 38-42

Espinal-Perez LE Moncada B Castanedo-Cazares JP (2004) A double-blind

randomized trial of 5 ascorbic acid vs 4 hydroquinone in melasma Int J

Dermatol 43 604-607

Fall HH Petering HG (1956) Metabolic inhibitors 1 67-Dimethyl-9-

formylmethylisoalloxazine67-dimethyl-9-(2-hydroxyethyl)-isoalloxazine and

derivatives J Am Chem Soc 78 377-381

Farahmand S Tajerzadeh H Farboud ES (2006) Formulation and evaluation of a

vitamin C multiple emulsion Pharm Dev Technol 11 255-261

197

Fasani E Albini A (2005) Photostability Stress Testing Predicting Drug Degradation

Taylor amp Francis London Chap 10

Fasihullah Q (1988) Photolysis of Flavins in Aqueous and Organic Solvents PhD

Thesis University of Karachi Karachi

Fasman GD Ed (1976) CRC Handbook of Biochemistry and Molecular Biology 3rd

ed Physical Chemical Data Vol 1 CRC Press Cleveland Ohio pp 122-130

Ferreira SLC Bandeira MLSF Lemos VA Dos Santos HC Spinola Costa

AC De Jesus DS (1997) Sensitive spectrophotometric determination of ascorbic

acid in fruit juices and pharmaceutical formulations using 2-(5-bromo-2-pyridylazo)-

5-diethylaminophenol (Br-PADAP) Anal Bioanal Chem 357 1174-1178

Fessenden RW Verma NC (1978) A time-resolved electron spin resonance study of

the oxidation of ascorbic acid by hydroxyl radical Biophys J 24 413-430

Finholt P Paulssen RB Higuchi T (1963) Rate of anaerobic degradation of ascorbic

acid in aqueous solution J Pharm Sci 52 948-954

Florence AT Attwood D (2006) Drug Stability Physicochemical Principles of

Pharmacy 4th ed Pharmaceutical Press London p 411

Flynn GL (2002) Cutaneous and transdermal delivery-processes and systems of

delivery In Banker GS and Rhodes CT Eds Modern Pharmaceutics Marcel

Dekker New York Chap 8

Foote CS (1991) Definition of Type I and Type II Photosensitized oxidation

Photochem Photobiol 54 659

Fornaro A Coicher N (1998) L-ascorbic acid complexation and redox reactions with

some transition metal ions Quni Nova 21 642-650

198

Fraga CG Motchink PA Wyrobek AJ Rempel DM Ames BN (1996)

Smoking and low antioxidant levels increase oxidative damage to sperm DNA

Mutat Res 351 199-203

Fraga CG Motchnik PA Shigenaga MK Helbock HJ Jacob RA Ames BN

(1991) Ascorbic acid protects against endogenous oxidative DNA damage in human

sperm Proc Natl Acad Sci USA 88 11003-11006

Frenich AG Torres ME Vega AB Vidal JL Bolanos PP (2005) Determination

of ascorbic acid and carotenoids in food commodities by liquid chromatography with

mass spectrometry detection J Agric Food Chem 53 7371-7376

Fung HL (1990) Drug stability In Banker GS Rhodes CT Eds Modern

Pharmaceutics 2nd ed Marcel Dekker New York Chap 6

Gallarate M Carlotti ME Trotta M Bovo S (1999) On the stability of ascorbic

acid in emulsified systems for topical and cosmetic use Int J Pharm 188 233-241

Ganshirt H Malzacher A (1960) Separation of several vitamins of the B group and C

by chromatography Naturwiss 47 279-280

Gardner WH (1972) Acidulants in food processing In Furia TE Ed Handbook of

Food Additives 2nd ed Vol 1 CRC Press Cleveland OH pp 225ndash270

Garrett ER (1967) Kinetics and mechanisms in stability of drugs In Bean HS

Beckett AH Carless JE Eds Advances in Pharmaceutical Sciences Vol 2

Academic Press London Chap 1

Garti N Aserin A (1996) Pharmaceutical emulsions double emulsions and

microemulsions In Benita S Ed Microencapsulation Methods and Industrial

Applications Marcel Dekker New York pp 411-534

199

Gensler M Rossmann A Schmidt HL (1995) Detection of added L-ascorbic acid in

fruit juice by isotope ratio mass spectrometry J Agric Food Chem 43 2662-2666

Gibbons E Allwood MC Neal T Hardy G (2001) Degradation of dehydroascorbic

acid in parenteral nutrition mixtures J Pharm Biomed Anal 25 605-611

Guitton C Gros P Comtat M (2007) Direct electrochemical measurements in dermo-

cosmetic creams In Alegret Merkoci Eds Comprehensive Analytical Chemistry

Elsevier Amsterdam 49 e59-e68

Guttman DE Brooke D (1963) Solution phase interaction of nicotinamide with

ascorbic acid J Pharm Sci 52 941-945

Gyorgy P Pearson WN Eds (1967) Vitamin Methods Vol VII Academic Press

New York pp 27-49

Hagg M Ylikoski S Kumpulainen J (1994) Vitamin C and α- and β-carotene in

vegetables consumed in Finland during 1988-1989 and 1992-1993 J Food Compos

Anal 7 252-259

Hagg M Ylikoski S Kumpulainen A (1995) Vitamin C content in fruits and berries

consumed in Finland J Food Compos Anal 8 12-20

Hall NK Chapman TM Kim HJ Min DB (2009) Antioxidant mechanisms of

Trolox and ascorbic acid on the oxidation of riboflavin in milk under light Food

Chem in press doi 101016jfoodchem200905017

Halliwell B (1996) Vitamin C antioxidant or pro-oxidant in vivo Free Radic Res 25

439-454

200

Halliwell B Whiteman M (1997) Antioxidant and prooxidant properties of vitamin C

In Packer L Fuchs J Eds Vitamin C in Health and Disease Marcel Dekker New

York pp 25-94

Halver JE Felton SP (2001) Analytical enigmas in assaying for vitamers C In

Dabrowski K Ed Ascorbic Acid in Aquatic Organisms CRC Press Boca Raton

Chap 3

Hanck A Ed (1982) Vitamin C New Clinical Applications in Immunology Lipid

Metabolism and Cancer Hans Huber Bern

Hashmi MH (1973) Assay of Vitamins in Pharmaceutical Preparations John Wiley

New York Chap 13

Hatchard CG Parker CA (1956) A new sensitive chemical actinometer II Potassium

ferrioxalate as a standard chemical actinometer Proc R Soc London Ser A A235

518-536

Heber GK Markovic B Hayes A (2006) An immunohistological study of anhydrous

topical ascorbic acid compositions on ex vivo human skin J Cosmet Dermatol 5

150-156

Heelis PF Parsons BJ Phillips GO McKellar JF (1981) The flavin sensitized

photooxidation of ascorbic acid a continuous and flash photolysis study Photochem

Photobiol 37 7-13

Henry JA Ed (1997) The New Guide to Medicines and Drugs The British Medical

Association 4th ed Dorling Kindersley London p 434

Hickey S Roberts H (2004) Ascorbate-The Science of Vitamin C Electronic Version

(ISBN 1-4116-0724-4)

201

Higdon JV Frei B (2002) The Antioxidant Vitamin C and E AOAC Press Arlington

VA pp 1-16

Hirst EL Zilva SS (1933) Ascorbic acid as the anti-scorbutic factor Biochem J 27

1271-1278

Ho AHL Puri A Sugden JK (1994) Effect of sweetening agents on the light

stability of aqueous solutions of L-ascorbic acid Int J Pharm 107 199-203

Holmstrom B Oster G (1961) Riboflavin as an electron donor in photochemical

reactions J Am Chem Soc 83 1867-1871

Homann P Gaffron H (1964) Photochemistry and metal catalyzes studies in a flavin

sensitized oxidation of ascorbic acid Photochem Photobiol 3 499-515

Hughes DE (1985) Irreversible reaction kinetics of the aerobic oxidation of ascorbic

acid Anal Chem 57 555-558

Humbert PG Haftek M Creidi P Lapiegravere C Nusgens B Richard A Schmitt D

Rougier A Zahouani H (2003) Topical ascorbic acid on photoaged skin Clinical

topographical and ultrastructural evaluation double-blind study vs placebo Exp

Dermatol 12 237-244

ICH Harmonized Tripartite Guideline (1997) QIB Photostability Testing of New Drug

Substances and Products Federal Register 62 27115-27122

Ichikawa M Ide N Shiraishi S Ono K (2005) Effect of various halide salts on the

incompatibility of cyanocobalamin and ascorbic acid in aqueous solution Chem

Pharm Bull 53 688-690

202

Ihara H Shino Y Aoki Y Hashizume N Minegishi N (2000) A simple and rapid

method for the routine assay of total ascorbic acid in serum and plasma using

ascorbate oxidase and o-phenylenediamine J Nutr Sci Vitaminol 46 321-324

Im-Emsap W Paeratakul O Siepmann J (2002) Disperse Systems In Banker GS

Rhodes CT Ed Modern Pharmaceutics Maecel Dekker New York Chap 9

Irache JM Ezpeleta I Vega FA (1993) HPLC determination of antioxidant

synergists and ascorbic acid in some fatty pharmaceuticals cosmetics and food

Chromatographia 35 232-23

Iwase H Ono I (1994) Determination of ascorbic acid in human plasma by high-

performance liquid chromatography with electrochemical detection using a

hydroxyapatite cartridge for precolumn deproteinization J Chromatogr B Biomed

Appl 655 195-200

Jain S Tiwari AK Jain NK (2006) Topical products In Jain NK Ed

Pharmaceutical Product Development CBS Publishers New Delhi Chap 7

Jamil N Ahmad I (1984) Moisture content of ascorbic acid tablets stored at various

temperatures and humidities Pak J Sci Ind Res 27 252

Jamil N Ahmad I Beg AE (1980a) The effect of UV and tungsten light on physical

characteristics of ascorbic acid tablets Laboratory prepared samples IV Kar Univ

J Sci 8 77-87

Jamil N Ahmad I Beg AE (1980b) The effect of UV and tungsten light on physical

characteristics of ascorbic acid tablets Commercial samples III Kar Univ J Sci 8

117-126

203

Jentzsch A Aikens P (2005) RetiSTARTM

for cosmetic formulations-stabilized retinol

In Rosen MR Ed Delivery System Handbook for Personal Care and Cosmetic

Products Technology Applications and Formulations William Andrew Publishing

Norwich NY pp 862-866

Johnston CS Steinberg FM Rucker RB (2007) Ascorbic Acid In Zempleni J

Rucker RB McCormick DB Suttie JW (Eds) Handbook of Vitamins 4th ed

CRC Press Boca Raton FL Chap 15

Jung MY Kim SM Kim SY (1995) Riboflavin-sensitized photooxidation of

ascorbic acid kinetics and amino acid effects Food Chem 53 397-403

Kall MA Anderson CJ (1999) Improved method for simultaneous determination of

ascorbic acid and dehydroascorbic acid isoascorbic acid and dehydroisoascorbic acid

in food and biological samples J Chromatogr B Biomed Sci Appl 730 101-111

Kampfenkel K Van Montagu M Inze D (1995) Extraction and determination of

ascorbate and dehydroascorbate from plant tissue Anal Biochem 225 165-167

Kaplan DL Moloney SJ Troy WR Dickens MS Pinnell SR (1989) A new

stabilized ascorbic acid solution percutaneous absorption and effect on relative

collagen synthesis J Cutan Aging Cosmet Derm I 115-121

Karatepe M (2004) Simultaneous determination of ascorbic acid and free

malondialdehyde in human serum by HPLC-UV LC GC (North America) 22 362-

365

Karlsen J (1996) Light-activated drugs and drug formulations in drug targeting In

Tonneson HH Ed The Photostability of Drugs and Formulations Taylor amp

Francis London Chap 8

204

Kassem MA Kassem AA Ammar HO (1969a) Studies on the stability of

injectable L-ascorbic acid solutions I Effect of pH solvent light and container

Pharm Acta Helv 44 611-623

Kassem MA Kassem AA Ammar HO (1969b) Studies on the stability of

injectable L-ascorbic acid solutions II Effect of metal ions and oxygen content of

solvent water Pharm Acta Helv 44 667-675

Kassem MA Kassem AA Ammar HO (1972) Studies on the stability of injectable

L-ascorbic acid solutions 3 Effect of metal-complexing agents Pharm Acta Helv

47 89-97

Kaushansky K Kipps TJ (2006) Hematopoietic agents growth factors minerals and

vitamins In Brunton L Ed Goodman amp Gilmans The Pharmacologic Basis of

Therapeutics 11th ed digital ed McGraw-Hill Companies USA Chap 53

Kearney MCJ Allwood MC Martin H Neal T Hardy G (1998) The influence

of amino acid source on the stability of ascorbic acid in TPN mixtures Nutrition 14

173-178

Kim H Kirshenbaum LJ Rosenithol I Riesz P (1993) Photosensitized formation

of ascorbate radicals by riboflavin an ESR study Photochem Photobiol 57 777-

784

Kimoto K Terada S Yamaguchi T (1997) Analysis of ascorbic acid

dehydroascorbic acid and transformation products by ion-pairing high-performance

liquid chromatography with multi-wavelength ultraviolet and electrochemical

detection Methods Enzymol 279 3-12

205

King CG Burns JJ Ed (1975) Second Conference on Vitamin C Ann N Y Acad

Sci 258

Kitagawa Y (1968) The effects of ultraviolet rays on vitamin C Eiyo To Shokuryo 20

416-421 through CA (1968) 68 111560f

Klick S Muijselaar PG Waterval J EichingerT Korn C Gerding TK Debets

AJ de Griend CS van den Beld C Somsen GW De Jong GJ (2005)Toward

a generic approach for stress testing of drug substances and drug products Pharm

Tech 29 48-66

Konings EJ (2006) Water-soluble vitamins J AOAC Int 89 285-288

Kwon BM Foote CS (1988) Chemistry of singlet oxygen 50 Hydroperoxide

intermediates in the photooxygenation of ascorbic acid J Am Chem Soc 110

6582-6583

Kwon BM Foote CS Khan SI (1989) Photooxygenation of ascorbic acid

derivatives and model compounds J Am Chem Soc 111 1854-1860

Laborie S Lavoie JC Chessex P (1998) Paradoxical role of ascorbic acid and

riboflavin in solutions of total parenteral nutrition implication in photoinduced

peroxide generation Pediatr Res 43 601-601

Laborie S Lavoie JC Pineault M Chessex P (1999) Protecting solutions of

parenteral nutrition from peroxidation JPEN 23 104-108

Laborie S Lavoie JC Pineault M Chessex P (2000) Contribution of multivitamin

air and light in the generation of peroxides in adult and neonatal parenteral nutrition

solutions Ann Pharmacother 34440-445

206

Laborie S Lavoie JC Rouleau T Chessex P (2002) Multivitamin solutions for

enteral supplementation a source of peroxides Nutrition 18 470-473s

Lachman L DeLuca P Akers MJ (1986) Kinetic principles and stability testing In

Lachman L Liberman HA Kanig JL Eds The Theory and Practice of

Industrial Pharmacy 3rd ed Lea amp Febiger Philadelphia Chap 26

Laidler KJ (1987) Chemical Kinetics 3rd ed Harper and Row New York pp 183-

185

Lambert WE de Leenheer A (1992) In Nollet LML Ed Food Analysis by HPLC

Marcel Dekker New York Chap 10

Lavoie JC Chessex P Rouleau T Migneault D Comte B (2004) Light-induced

byproducts of vitamin C in multivitamin solutions Clin Chem 50 135-140

Lee JS Kim JW Han SH Chang IS Kang HH Lee OS Oh SG Suh KD

(2004) The stabilization of L-ascorbic acid in aqueous solution and water-in-oil-in-

water double emulsion by controlling pH and electrolyte concentration J Cosmet

Sci 55 1-12

Lee KM Kwon JY Lee KW Lee HJ (2009) Ascorbic acid 6-palmitate

suppresses gap-junctional intercellular communication through phosphorylation of

connexin 43 via activation of the MEK-ERK pathway Mutat Res 660 51-56

Leacutevecircque N Mac-Mary S Muret P Makki S Aubin F Kantelip JP Heusegravele C

SS Humbert P (2005) Evaluation of a sunscreen photoprotective effect by ascorbic

acid assessment in human dermis using microdialysis and gas chromatography mass

spectrometry Exp Dermatol 14 176-181

207

Levine M (1986) New concepts in the biology and biochemistry of ascorbic acid N

Engl J Med 314 892-902

Lewin S (1976) Vitamin C Its Molecular Biology and Medical Potential Academic

Press New York

Liau LS Lee BL New AL Ong CN (1993) Determination of plasma ascorbic

acid by high-performance liquid chromatography with ultraviolet and electrochemical

detection J Chromatogr 612 63-70

Lin JY Selim MA Shea CR Grichnik JM Omar MM Monteiro-Riviere NA

Pinnell SR (2003) UV photoprotection by combination topical antioxidants vitamin

C and vitamin E J Am Acad Dermatol 48 866-874

Lin FH Lin JY Gupta RD Tournas JA Burch JA Selim MA Monteiro-

Riviere NA Grichnik JM Zielinski J Pinnell SR (2005) Ferulic acid

stabilizes a solution of vitamins C and E and doubles its photoprotection of skin J

Invest Dermatol 125 826-832

Lindahl T (1993) Instability and decay of the primary structure of DNA Nature 362

709-715

Litner CJ (1973) Drug Product Stability In Cooper MS Ed Quality Control in

Pharmaceutical Industry Vol 2 Academic Press New York p 185

Liu TZ Chin N Kiser MD Bigler WN (1982) Specific spectrophotometry of

ascorbic acid in serum or plasma by use of ascorbate oxidase Clin Chem 28 2225-

2228

Lu GW Flynn GL (2009) Cutaneous and transdermal delivery-processes and

systems of delivery In Florence AT Siepmann J Eds Modern Pharmaceutics-

208

Applications and Advances Vol 2 5th ed Informa Healthcare Inc New York

Chap 3

Lykkesfeldt J (2000) Determination of ascorbic acid and dehydroascorbic acid in

biological samples by high-performance liquid chromatography using subtraction

methods reliable reduction with tris [2-carboxyethyl] phosphine hydrochloride Anal

Biochem 282 89-93

Macek TJ (1960) Stability problems with some vitamins in pharmaceuticals Am J

Pharm 132 433-455

Mader WJ (1961) Vitamins In Higuchi T Brochmann-Hanssen E Eds

Pharmaceutical Analysis Interscience New York pp 689-693

Maggi N Pasqualucci CR Ballotta R Sensi P (1966) Rifampicin a new orally

active rifamycin Chemotherapy 11 285-292

Maia AM Baby AR Pinto CA Yasaka WJ Suenaga E Kaneko TM Velasco

MV (2006) Influence of sodium metabisulfite and glutathione on the stability of

vitamin C in OW emulsion and extemporaneous aqueous gel Int J Pharm 322

130-135

Mancini M Edwards AM Becker MI De Ioannes A Silva E (2000) Reactivity

of monoclonal antibodies against a tryptophan-riboflavin adduct toward irradiated

and non-irradiated bovine-eye-lens protein fractions an indicator of in vivo visible

light mediated phototransformations J Photochem Photobiol B Biol 55 9-15

Manoharan M Schwille PO (1994) Measurement of ascorbic acid in human plasma

and urine by high performance liquid chromatography Results in healthy subjects

209

and patients with idiopathic calcium urolithiasis J Chromatogr B Biomed Appl

654 134-139

Margolis SA Schmidt HL (1996) Measurement of ascorbic acid in human plasma

and serum stability intralaboratory repeatability and interlaboratory reproducibility

Clin Chem 42 1257-1262

Mason P (2007) Dietary Supplements 3rd ed Pharmaceutical Press pp 330-339

Massad WA Bertolotti S Garcia N (2004) Kinetics and mechanism of the vitamin

B2-sensitized photooxidation of isoproterenol Photochem Photobiol 79 428-433

Matsubayashi T Sakaeda T Kita T Kurimoto Y Nakamura T Nishiguchi K

Fujita T Kamiyama F Yamamoto A Okumura K (2003) Intradermal

concentration of hydroquinone after application of hydroquinone ointments is higher

than its cytotoxic concentration Biol Pharm Bull 26 1365-1367

McBride MM Moore WM (1967) The photochemistry of riboflavin II

Polarographic studies on the hydroxyethyl and formylmethyl analogues of riboflavin

Photochem Photobiol 6 103-113

McDowell LR (2000) Vitamins in Animal and Human Nutrition 2nd ed Iowa State

University Press Chap 15

Meister A (1994) Glutathione-ascorbic acid antioxidant system in animals J Biol

Chem 269 9397-9400

Meister A (1995) Mitochondrial changes associated with glutathione deficiency

Biochim Biophys Acta 1271 35-42

Min DB Boff JM (2002) Chemistry and reaction of singlet oxygen in foods

CRFSFS 1 58-72

210

Moeslinger T Brunner M Volf I Spieckermann PG (1995) Spectrophotometric

determination of ascorbic acid and dehydroascorbic acid Clin Chem 41 1177-1181

Moffat AC Osselton MD Widdop B (2004) Clarkersquos Analysis of Drugs and

Poisons 3rd ed Pharmaceutical Press London pp 649-650

Moore DE (2004) Standardization of kinetic studies of photodegradation reactions In

Tonnesen HH Ed Photostability of Drugs and Drug Formulations 2nd ed CRC

Press Boca Raton FL Chap 3

Moura T Gaudy D Jacob M Cassanas G (1994) pH influence on the stability of

ascorbic acid spray-drying solutions Pharm Acta Helv 69 77-80

Murray JC Burch JA Streilein RD Iannacchione MA Hall RP Pinnell SR

(2008) A topical antioxidant solution containing vitamins C and E stabilized by

ferulic acid provides protection for human skin against damage caused by ultraviolet

irradiation J Am Acad Dermatol 59 418-425

Mushran SP Agarwal MC (1977) Mechanistic studies on the oxidation of ascorbic

acid 5 J Sci Res (India) 36 274-283

Nerlo H Palak W Tomaszek J (1968) Stabilization of vitamin preparations II

Syrup of vitamin D3 and its stabilization Acta Polon Pharm 25 183-188

Niki E Noguchi N (1997) Protection of human low-density lipoprotein from oxidative

modification by vitamin C In Packer L Fuchs J Eds Vitamin C in Health and

Disease Marcel Dekker New York pp 183-192

Nishikimi M Fukuyama R Minoshima S Shimizu N Yagi K (1994) Cloning and

chromosomal mapping of the human nonfunctional gene for L-gulono-gamma-

211

lactone oxidase the enzyme for L-ascorbic acid biosynthesis missing in man J Biol

Chem 269 13685-13688

Njus D Wigle M Kelly PM Kipp BH Schlegel HB (2001) Mechanism of

ascorbic acid oxidation by Cytochrome B561 Biochemistry 40 11905-11911

Nusgens BV Humbert P Rougier A Colige AC Haftek M Lambert CA

Richard A Creidi P Lapiegravere CM (2001) Topically applied vitamin C enhances

the mRNA level of collagens I and III their processing enzymes and tissue inhibitor

of matrix metalloproteinase 1 in the human dermis J Invest Dermatol 116 853-859

Ogata Y Kosugi Y (1969) Solvent effect on the autooxidation of L-ascorbic acid

Tetrahedron 25 1055-1062

OrsquoKeefe T (2001) Ascorbic acid and stable ascorbate esters as sources of vitamin C in

aquaculture feeds ASA Technical Bulletin AQ48 wwwsoyaquaorg

OrsquoNeil MJ Ed (2001) The Merck Index 13th ed Merck amp Co Inc Rahway NJ

USA Electronic version

Ortega GR Delgado JN (1998) Vitamins and related compounds In Wilson and

Gisvoldrsquos Textbook of Organic Medicinal and Pharmaceutical Chemistry 10th ed

Lippincott-Raven Philadelphia Chap 27

Ortwerth BJ Monnier VM (1997) Protein glycation by the oxidation products of

ascorbic acid In Packer L Fuchs J Eds Vitamin C in Health and Disease Marcel

Dekker New York pp 123ndash142

Ozer O Muguet V Roy E Grossiord JL Seiller M (2000) Stability study of

WOW viscosified multiple emulsions Drug Dev Ind Pharm 26 1185-1189

Packer I Fuchs J (1999) Vitamin C in Health and Disease Marcel Dekker New York

212

Packer JE Slater TF Wilson RL (1979) Direct observation of a free radical

interaction between vitamin E and vitamin C Nature 278 737-738

Packer L Traber MG Kraemer K Frei B (2002) The Antioxidant Vitamins C and

E AOCS Press Illinois

Padayatty SJ Katz A Wang Y Eck P Kwon O Lee JH Chen S Corpe C

Dutta A Dutta SK Levine M (2003) Vitamin C as an antioxidant Evaluation of

its role in disease prevention J Am Coll Nutr 22 18-35

Parker CA (1953) A new sensitive chemical actinometer I Some trials with potassium

ferrioxalate Proc Roy Soc (Lond) A220 104-116

Parker JN Parker PM (2003) Antioxidants ICON Group International

Pastore P Rizzetto T Curcuruto O Cin MD Zaramella A Martin D (2001)

Characterization of dehydroascorbic acid solutions by liquid chromatography mass

spectrometry Rapid Commun Mass Spectrom 15 2051-2057

Pelletier O (1985) In Augustin J Klein BP Becker D Venugopal PB Eds

Methods of Vitamins Assay Wiley New York pp 303-347

Peyrat-Maillard MN Bonnely S Rondini L Berset C (2001) Effect of vitamin E

and vitamin C on the antioxidant activity of malt rootlets extracts Lebensmittel-

Wissenschaft Und-Technological 34 176-182

Pinnell SR (2003) Cutaneous photodamage oxidative stress and topical antioxidant

protection J Am Acad Dermatol 48 1-19

Pinnell SR Madey DL (1998) Topical vitamin C in skin care Aesthet Surg J 18

468-470

213

Pinnell SR Yang H Omar M Monteiro-Riviere N DeBuys HV Walker LC

Wang Y Levine M (2001) Topical L-ascorbic acid percutaneous absorption

studies Dermatol Surg 27 137-142

Placzek M Gaube S Kerkmann U Gilbertz KP Herzinger T Haen E Przybilla

B (2005) Ultraviolet B-induced DNA damage in human epidermis is modified by

the antioxidants ascorbic acid and D-alpha-tocopherol J Invest Dermatol 124 304-

307

Puskas F Gergely PJr Banki K Perl A (2000) Stimulation of the pentose

phosphate pathway and glutathione levels by dehydroascorbate the oxidized form of

vitamin C FASEB J 14 1352-1361

Puskas F Gergely P Niland B Banki K Perl A (2002) Differential regulation of

hydrogen peroxide and Fas-dependent apoptosis pathways by dehydroascorbate the

oxidized form of vitamin C Antioxid Redox Signal 4 357-369

Racz I (1989) Drug Formulation Wiley New York pp 121-122

Ramu A Mehta MM (2000) The riboflavin-mediated photooxidation of doxorubicin

Cancer Chemoth Pharm 46 449-458

Raschke T Koop U Duumlsing HJ Filbry A Sauermann K Jaspers S Wenck H

Wittern KP (2004) Topical activity of ascorbic acid from in vitro optimization to in

vivo efficacy Skin Pharmacol Physiol 17 200-206

Reed RA Harmon P Manas D Wasylaschuk W Galli C Biddell R Bergquist

PA Hunke W Templeton AC Ip D (2003) The role of excipients and package

components in the photostability of liquid formulations PDA J Pharm Sci Tech 57

351-368

214

Reichstein T Griissner A Oppcnuuer R (1933) Synthese der d- und l-Ascorbinsaumlure

(C-Vitamin) Helv Chim Acta 16 1019-1033

Riehl J Maupin C Layloff T (1995) On the choice of a photolysis source for the

photostability testing of pharmaceuticals Pharmacop Forum 21 1654-1663

Rivlin RS (2007) Riboflavin (vitamin B2) In Zempleni J Rucker RB McCormick

DB Suttie JW Eds Handbook of Vitamins 4th ed Taylor amp Francis CRC

Press Boca Raton FL p 241

Rogers AR Yacomeni JA (1971) The effect of pH on the aerobic degradation of

ascorbic acid solutions J Pharm Pharmacol 23 218S

Rosen MR Ed (2005) Delivery System Handbook for Personal Care and Cosmetic

Products-Technology Applications and Formulations William Andrew Publishing

Norwich NY

Rosenberg HR (1945) Chemistry and Physiology of the Vitamins Interscience New

York pp 289-338

Roth HJ Eger K Troschutz R (1991) Pharmaceutical Chemistry Vol 2 Drug

Analysis Ellis Horwood London pp 282-288

Rowe RC Sheskey PJ Quinn ME (2009) Handbook of Pharmaceutical Excipients

6th ed Pharmaceutical Press London pp 43-47 181-183 731-732

Roacutezanowska M Bober A Burke JM Sarna T (1997) The role of retinal pigment

epithelium melanin in photoinduced oxidation of ascorbate Photochem Photobiol

65 472-479

Rozman B Gasperlin M (2007) Stability of vitamins C and E in topical

microemulsions for combined antioxidant therapy Drug Deliv 14 235-245

215

Rozman B Gasperlin M Tinois-Tessoneaud E Pirot F Falson F (2009)

Simultaneous absorption of vitamins C and E from topical microemulsions using

reconstructed human epidermis as a skin model Eur J Pharm Biopharm 72 69-75

Ruumlmelin A Fauth U Halmagyi M (1999) Determination of ascorbic acid in plasma

and urine by high performance liquid chromatography with ultraviolet detection Clin

Chem Lab Med 37 533-536

Rumsey SC Levine M (2000) Vitamin C In Song WO Beecher GR

Eitenmiller RR Eds Modern Analytical Methodologies in Fat- and Water-Soluble

Vitamins Wiley New York Chap 13

Saari JC Baker EM Sauberlich HE (1967) Thin-layer chromatographic separation

of the oxidative degradation products of ascorbic acid Anal Biochem 18 173-177

Salkić M Keran H Jašić M (2007) Direct spectrophotometric determination of l-

ascorbic acid in the presence of potassium cyanide Agric Conspec Sci 72 371-375

Salvador A Chisvert A (2007) Analysis of Cosmetic Products Elsevier Amsterdam

Sattar A DeMan JM Alexander JC (1977) Light-induced degradation of vitamins

II Kinetic studies on ascorbic acid decomposition in solution Can Inst Food Sci

Technol J 10 65-68

Satoh K Sakagami H Terasaka H Ida Y Fujisawa S (2000) Interaction between

hydroquinone and ascorbic acid derivatives quenching effects of organic solvents

Antican Res 20 1577-1581

Sauberlich HE (1994a) Pharmacology of vitamin C Annu Rev Nutr 14 371-391

Sauberlich HE (1994b) Vitamin C and Cancer In Kritchevsky D Carroll K Eds

Nutrition and Disease Update Cancer AOCS Press Champaign IL pp 111-172

216

Sauermann K Jaspers S Koop U Wenck H (2004) Topically applied vitamin C

increases the density of dermal papillae in aged human skin BMC Dermatol 4 13

Sawicka J (1991) The influence of excipients and technological process on

cholecalciferol stability and its liberation from tablets Pharmazie 46 519-521

Scheuplein RJ Blank IH (1973) Mechanism of percutaneous absorption IV

Penetration of non-electrolytes (alcohols) from aqueous solutions and from pure

liquids J Invest Dermatol 60 286-296

Schick B (1943) Scurvy and pine needles Science 98 241-242

Schuman Jorns M Schollnhammer G Hemmerich P (1975) Intramolecular addition

of the riboflavin side chain Anion-catalysed neutral photochemistry Eur J

Biochem 57 35-48

Seib PA Tolbert BM (1982) Ascorbic Acid Chemistry Metabolism and Uses

Advances in Chemistry Series 200 American Chemical Society Washington DC

Sequeira F Vozone C (2000) Photostability studies of drug substances and products

Practical implications of the ICH guideline Pharm Tech 24 30-35

Shacter E (2000) Quantification and significance of protein oxidation in biological

samples Drug Metab Rev 32 307-326

Shah VP (1997) FDA Guidance for Industry Document Nonsterile Semisolid Dosage

Forms Centre for Drug Evaluation and Research Rockville MD pp 1-40

Sheraz MA Ahmed S Ahmad S (2007) Developments in the clinical and food

analysis of vitamin C J Baqai Med Univ 10 19-24

217

Shindo Y Witt E Han D Packer L (1994) Dose-response effect of acute ultraviolet

irradiation on antioxidants and molecular markers of oxidation in murine epidermis

and dermis J Invest Dermatol 102 470-475

Sibi M Batcu A Gheorghiu I (1953) Vitamin oxidation-reduction effect II Effect of

light on the oxidation-reduction reactions of lactoflavin Studii cercetari stiinf

(Romania) 4 291-305 through CA (1956) 50 14816g

Sidhu DS Sugden JK (1992) Effect of food dyes in the photostability of aqueous

solutions of L-ascorbic acid Int J Pharm 83 263-266

Silva E Quina FH (2006) Photoinduced processes in the eye lens do flavins really

play a role In Silva E Edwards AM Eds Flavins Photochemisry and

Photobiology The Royal Society of Chemistry Cambridge Chap 7

Silva E Ugarte P Andrade A Edwards AM (1994) Riboflavin-sensitized photo-

processes of tryptophan J Photochem Photobiol B Biol 23 43-48

Sim AK (1972) Ascorbic acid-A scurvy past and present Chem Indust 8 160-165

Singh S (2006) Stability testing during product development In Jain NK ED

Pharmaceutical Product Development CBS Publishers New Delhi Chap 9

Singh S Bakshi M (2000) Guidance on conduct of stress testing to determine inherent

stability of drugs Pharm Tech 24 1-14

Sinko PJ (2006) Chemical Kinetics and Stability Martinrsquos Physical Pharmacy and

Pharmaceutical Sciences 5th ed Lippincott Williams amp Wilkins Baltimore Chap

8 15 18

218

Smith JL Canham JE Kirkland WD Wells PA (1988) Effect of intralipid amino

acids container temperature and duration of storage on vitamin stability in total

parenteral nutrition admixtures J Parenter Enteral Nutr 12 478-483

Speek AJ Schrijver J Schreurs WHP (1984) Fluorimetric determination of total

vitamin C and total isovitamin C in food stuffs and beverages by high-performance

liquid chromatography with precolumn derivatization J Agric Food Chem 32 352-

355Spratley JE Hellstroumlm SO Mattsson CK Pais-Clemente M (2001)

Topical ascorbic acid reduces myringosclerosis in perforated tympanic membranes A

study in the rat Ann Otol Rhinol Laryngol 110 585-591

Spiclin P Homer M Zupancic A Gasperlin M (2003) Sodium ascorbyl phosphate

in topical microemulsions Int J Pharm 256 65-73

Stadtman ER (1991) Ascorbic acid and oxidative inactivation of proteins Am J Clin

Nutr 54 1125S-1128S

Stone I (1969) The Healing Factor Vitamin C Against Disease Grosset and Dunlap

New York

Sugden JK (1985) Photostability of cosmetic materials Int J Cosmet Sci 7 165-173

Swarbrick J Rubino JT Rubino OP (2006) Coarse Dispersions In Hendrickson

R Ed Remington The Science and Practice of Pharmacy 21st ed Lippincott

Williams amp Wilkins Baltimore Chap22

Sweetman SC Ed (2009) Martindale The Complete Drug Reference 36th ed

Pharmaceutical Press London Electronic version

219

Szent-Gyoumlrgi A (1928) Observation on function of peroxidase systems and chemistry

of adrenal cortex description of new carbohydrate derivative Biochem J 22 1387-

1389

Szymula M (2005) The influence of ascorbic acid on the rheological properties of the

microemulsion region of the SDSpentanolwater system J Cosmet Sci 56 267-

277

Taqui Khan MM Martell AE (1967) Metal ion and metal chelate catalyzed oxidation

of ascorbic acid by molecular oxygen 1 Cupric acid and ferric ion catalyzed

oxidation J Am Chem Soc 89 4176-4185

Tangney CC (1988) Analysis of vitamin C in biological samples with an emphasis on

recent chromatographic techniques Prog Clin Biol Res 259 331-362

Tatsumi K Ichikawa H Wada S (1992) Flavin-sensitized photooxidation of

substituted phenols in natural water J Contam Hydrol 9 207-219

Tessier F Birlouez-Aragon I Tjani C Guilland JC (1996) Validation of a

micromethod for determining oxidized and reduced vitamin C in plasma by HPLC-

fluorescence Int J Vitam Nutr Res 66 166-170

Thatcher SR Mansfield RK Miller RB Davis CW Baertschi SW (2001a)

Pharmaceutical photostability a technical and practical interpretation of the ICH

guideline and its application to pharmaceutical stability Part 1 Pharm Tech 25 98-

110

Thatcher SR Mansfield RK Miller RB Davis CW Baertschi SW (2001b)

Pharmaceutical photostability a technical and practical interpretation of the ICH

220

guideline and its application to pharmaceutical stability Part 2 Pharm Tech 25 50-

62

Thiele JJ Dreher F Packer L (2000) Antioxidant defense systems in skin In

Elsner P Maibach HI Eds Cosmeceuticals- Drugs vs Cosmetics Marcel

Dekker New York Chap 11

Thomas DP (1997) Sailors scurvy and science J R Soc Med 90 50-54

Thomson CO Trenerry VC (1995) A rapid method for the determination of total L-

ascorbic acid in fruits and vegetables by micellar electrokinetic capillary

chromatography Food Chem 53 43-50

Tonnesen HH (1991) Photochemical degradation of components in drug formulations

1 An approach to the standardization of degradation studies Pharmazie 46 263-265

Tonnesen HH (2002) Photodecomposition of Drugs In Swarbrick J Boylan JC

Eds Encyclopedia of Pharmaceutical Technology Vol 3 Marcel Dekker New

York pp 2197-2203

Tonnesen HH Ed (2004) The Photostability of Drugs and Drug Formulations

Culinary and Hospitality Industry Publications Services Weimex TX

Tonnesen HH Karlsen J (1997) A comment on photostability testing according to

ICH guideline calibration of light sources PharmEuropa 9 735-736

Tonnesen HH Moore DE (1993) Photochemical degradation of components in drug

formulations Pharm Technol Int 5 27-33

Tournas JA Lin FH Burch JA Selim MA Monteiro-Riviere NA Zielinski

JE Pinnell SR (2006) Ubiquinone idebenone and kinetin provide ineffective

221

photoprotection to skin when compared to a topical antioxidant combination of

vitamins C and E with ferulic acid J Invest Dermatol 126 1185-1187

Traber MG (2007) Vitamin E In Zempleni J Rucker RB McCormick DB

Suttie JW Eds Handbook of Vitamins 4th ed Taylor amp Francis CRC Press

Boca Raton FL Chap 4

Traikovich SS (1999) Use of topical ascorbic acid and its effect on photodamaged skin

topography Arch Otolaryngol Head Neck Surg 125 1091-1098

Treadwell GE Cairns WL Metzler DE (1968) Photochemical degradation of

flavins V Chromatographic studies of the products of photolysis of riboflavin J

Chromatogr 35 376-388

Truswell AS (2003) ABC of Nutrition 4th ed BMJ Publishing Group Chap 10

Tsao CS Young M (1985) Analysis of ascorbic acid derivatives by high-performance

liquid chromatography with electrochemical detection J Chromatogr 13 855-856

Underberg WJM (1978) Oxidative degradation of pharmaceutically important

phenothiazines III Kinetics and mechanism of promethazine oxidation J Pharm Sci

67 1133-1138

Urso ML Clarkson PM (2003) Oxidative stress exercise and antioxidant

supplementation Toxicology 189 41-54

Vaid FHM (1998) Photolysis and Interaction of Thiamine Hydrochloride with

Riboflavin in Aqueous Solution PhD Thesis University of Karachi Karachi

Vaid FHM Shaikh RH Ansari IA Ahmad I (2005) Spectral study of the

photolysis of aqueous thiamine hydrochloride and ascorbic acid solution in the

presence and absence of riboflavin J Chem Soc Pak 27 227-232

222

Vaid F H M Shaikh RH Ansari I A Ahmad I (2006) Chromatographic study of

the photolysis of aqueous thiamine hydrochloride and ascorbic acid solutions in the

presence and absence of riboflavin J Chem Soc Pak 28 481-464

Valvani SC (2000) Industrial stability testing in the United States and computerization

of stability data In Carstensen JT Rhodes CT Eds Drug Stability Principles

and Practices 3rd

ed Marcel Dekker New York pp 534-536

Vanderslice JT Higgs DJ (1988) Chromatographic separation of ascorbic acid

isoascorbic acid dehydroascorbic acid and dehydroisoascorbic acid and their

quantitation in food products J Micronutr Anal 4 109-118

Vanderslice JT Higgs DJ Hayes JM Block G (1990) Ascorbic acid and

dehydroascorbic acid content of foods-as-eaten J Food Comp Anal 3 105-118

Vanderslice JT Higgs DJ (1993) Quantitative determination of ascorbic acid

dehydroascorbic acid by HPLC in foods and other matrices J Nutr Biochem 4 184-

190

Varvaresou A Tsirivas E Iakovou K Gikas E Papathomas Z Vonaparti A

Panderi I (2006) Development and validation of a reversed-phase ion-pair liquid

chromatography method for the determination of magnesium ascorbyl phosphate and

melatonin in cosmetic creams Anal Chim Acta 573-574 284-290

Verma KK Jain A Verma A Chaurasia A (1991) Spectrophotometric

determination of ascorbic acid in pharmaceuticals by background correction and flow

injection Analyst (London) 116 641-645

Vimaladevi M (2005) Textbook of Cosmetics CBS Publishers New Dehli Chap 2

and 17

223

Wallwork SC Grant DJW (1977) Physical Chemistry for Students of Pharmacy and

Biology 3rd ed Longmann New York pp 502-505

Waugh WA King CG (1932) Isolation and identification of vitamin C J Biol

Chem 97 325-331

Wechtersbach L Cigic B (2007) Reduction of dehydroascorbic acid at low pH J

Biochem Biophys Methods 70 767-772

Weiner N (1996) Introduction In Liberman HA Rieger MM Banker GS Eds

Pharmaceutical Dosage Forms Disperse Systems 2nd ed vol1 Marcel Dekker

New York Chap1

Werner W (1949) The reaction of l-ascorbic and d-isoascorbic acid with nicotinic acid

and its amide J Org Chem 14 22-26

Wiechers JW (2003) Formulating for Efficacy Proceedings of the 2003 IFSCC

Conference Seoul Korea

Wiechers JW (2005) Optimizing skin delivery of active ingredients from emulsions

from theory to practice In Rosen MR Ed Delivery System Handbook for

Personal Care and Cosmetic Products-Technology Applications and Formulations

William Andrew Inc Norwich NY Chap 20

Wille JJ (2005) Thixogel-Novel topical delivery systems for hydrophobic plant actives

In Rosen MR Ed Delivery System Handbook for Personal Care and Cosmetic

Products-Technology Applications and Formulations William Andrew Inc

Norwich NY Chap 36

224

Wright JL (1986) Vitamin A monograph In Connors KA Amidon GL Stella

VJ Eds Chemical Stability of Pharmaceuticals A Handbook for Pharmacists 2nd

ed Wiley New York pp 791-803

Yao AA Wathelet B Thonart P (2009) Effect of protective compounds on the

survival electrolyte leakage and lipid degradation of freeze-dried Weissella

paramesenteroides LC11 during storage J Microbiol Biotechnol 19 810-817

Yeh SY Lach JL (1961) Stability of morphine in aqueous solutions III Kinetics of

morphine degradation in aqueous solution J Pharm Sci 50 35-42

Yoshimura A Ohno T (1988) Lumiflavin-sensitized photooxygenation of indole

Photochem Photobiol 48 561-565

Yoshioka S Stella VJ (2000) Stability of Drugs and Dosage Forms Kluwer

Academic Plenum Publishers New York Chap 2

Zaeslein C (1982) Vitamins in the Field of Medicine Hoffman La Roche Basle pp

89-96

Zeng W Martinuzzi F MacGregor A (2005) Development and application of a novel

UV method for the analysis of ascorbic acid J Pharm Biomed Anal 36 1107-1111

Zhang GF Chen HY (2000) Chemiluminescence studies of the oxidation of ascorbic

acid with copper (II) catalyzed by halide anions and its applications to the

determination of halide anions and ascorbic acid Anal Sci 16 1317-1321

Zhang L Lerner S Rustrum WV Hofmann GA (1999) Electroporation-mediated

topical delivery of vitamin C for cosmetic applications Bioelectrochem Bioenerg

48 453-461

225

Zilva SS (1932) The non-specificity of the phenolindophenol reducing capacity of

lemon juice and its fractions as a measure of their antiscorbutic activity Biochem J

26 1624-1627

226

AUTHORrsquoS PUBLICATIONS

The author obtained his B Pharm degree in 2003 and joined the post graduate

program securing an M Phil degree in Pharmaceutics in 2006 from Baqai Medical

University He is a co-author of following publications

CHAPTER IN BOOK

1 Chapter on ldquoBorate Toxicity Effect on Drug Stability and Analytical

Applicationsrdquo by Iqbal Ahmad Sofia Ahmed MA Sheraz and Faiyaz H M

Vaid In Handbook on Borates Chemistry Production and Applications (MP

Chung Ed) Nova Science Publishers Inc NY USA (in press)

PAPERS PUBLISHED

INTERNATIONAL

2 Iqbal Ahmad Sofia Ahmed MA Sheraz and Faiyaz HM Vaid ldquoEffect of Borate

Buffer on the Photolysis of Riboflavin in Aqueous Solutionrdquo Journal of

Photochemistry and Photobiology B Biology 93 82-87 (2008)

3 Iqbal Ahmad Sofia Ahmed MA Sheraz M Aminuddin and Faiyaz HM Vaid

ldquoEffect of Caffeine Complexation on the Photolysis of Riboflavin in Aqueous

Solution A Kinetic Studyrdquo Chemical and Pharmaceutical Bulletin 57 (2009)

published online September 14 2009

4 Iqbal Ahmad MA Sheraz Sofia Ahmed and Faiyaz HM Vaid ldquoAnalytical

Applications of Boratesrdquo Materials Science Research Journal (in press)

5 Iqbal Ahmad Sofia Ahmed MA Sheraz Kefi Iqbal and Faiyaz HM Vaid

ldquoPharmacological Aspects of Boratesrdquo International Journal of Medical and

Biological Frontiers (in press)

6 Iqbal Ahmad Sofia Ahmed MA Sheraz Faiyaz HM Vaid and Izhar A Ansari

ldquoEffect of Divalent Ions on Photodegradation Kinetics and Pathways of

Riboflavin in Aqueous Solutionrdquo Photochemical and Photobiological Sciences

accepted

227

NATIONAL

7 Sofia Ahmed MA Sheraz and Iqbal Ahmad ldquoAdvances in Antioxidant Activity of

Vitamin Erdquo Journal of Baqai Medical University 10 13-18 (2007)

8 MA Sheraz Sofia Ahmed and Iqbal Ahmad ldquoDevelopments in the Clinical and

Food Analysis of Vitamin Crdquo Journal of Baqai Medical University 10 19-24

(2007)

9 A Azmi SNH Naqvi M Usman MA Sheraz and Sofia Ahmed ldquoPancreatic

Glucagon in Certain Ungulates Comparative Study of Extraction and

Bioassayrdquo Pakistan Journal of Entomology 20 23-28 (2005)

10 Iqbal Ahmad Sofia Ahmed MA Sheraz Faiyaz HM Vaid and S Hasan

ldquoAdvances in Biochemical Functions and the Photochemistry of Flavins and

Flavoproteinsrdquo Pakistan Journal of Pharmaceutical Sciences in press

11 MA Sheraz Sofia Ahmed and Iqbal Ahmad ldquoEffect of Borates on the Stability of

Chemical and Pharmaceutical Compoundsrdquo Journal of Baqai Medical University

accepted

PAPERS SUBMITTED

12 Iqbal Ahmad MA Sheraz Sofia Ahmed Riaz H Shaikh and Faiyaz HM Vaid

ldquoPhotostability of Ascorbic Acid in Organic Solvents and Cream Formulationsrdquo

Chemical and Pharmaceutical Bulletin

13 Iqbal Ahmad MA Sheraz Sofia Ahmed Riaz H Shaikh and Faiyaz HM Vaid

ldquoPhotochemical Interaction of Ascorbic Acid with Riboflavin Nicotinamide and

Alpha-Tocopherol in Cream Formulationsrdquo Journal of Cosmetic Science

14 Iqbal Ahmad Kefi Iqbal Sofia Ahmed MA Sheraz ldquoApplications of Laser Flash

Photolysis Spectroscopy and Electron Microscopy in Photopolymerization and

Development of Glass Ionomer Dental Cementsrdquo Materials Science Research

Journal

15 Sofia Ahmed MA Sheraz M Aminuddin I Ahmad and Faiyaz HM Vaid ldquoA

Rapid Titrimetric Assay for Quantitation of Vitamin B1 in Neat and

Pharmaceutical Preparationsrdquo Pakistan Journal of Pharmaceutical Sciences

• 01 SZ-786
• 02 SZ-title
• 03 SZ-Certificate
• 04 SZ-Abstract
• 05 SZ-Acknowledgement
• 06 SZ-Dedication
• 07 SZ-Contents
• 08 SZ-Chapter 1
• 09 SZ-Chapter 2
• 10 SZ-Chapter 3
• 11 SZ-Object of Present Investigation
• 12 SZ-Chapter 4
• 13 SZ-Chapter 5
• 14 SZ-Chapter 6
• 15 SZ-Chapter 7
• 16 SZ-Conclusion
• 17 SZ-References
• 18 SZ-Authors Publications

v

humectant (ethylene glycol propylene glycol and glycerin) and redox potentials of

ascorbic acid The study indicates that the relative polar character of the emulsifying

agent and the ionized state and redox potential of ascorbic acid at a particular pH are

important factors in the photodegradation of ascorbic acid in creams

The second-order rate constants (kprime) (320 times 10ndash2

ndash 189 Mndash1

minndash1

) for the

photochemical interaction of ascorbic acid and the individual vitamins (riboflavin

nicotinamide alpha-tocopherol) along with the values of k0 obtained from the intercepts

of the plots of kobs versus vitamin concentration are also reported The values of k0

indicate that riboflavin and nicotinamide act as photosensitizing agents and alpha-

tocopherol acts as a stabilizing agent in the photodegradation of ascorbic acid in the

creams The kobs verses pH profiles for the photodegradation of ascorbic acid in creams

represents sigmoid type curves indicating the oxidation of the ionized form (AHndash) of

ascorbic acid (pKa1 41) with pH The AHndash species appears to be more susceptible to

photooxidation than the non-ionized form of ascorbic acid The effect of stabilizing

agents on the photodegradation of ascorbic acid has been found to be in the order of citric

acid gt tartaric acid gt boric acid The low activity of boric acid may be to some extent due

to its interaction with the emulsifying agents and humectants The polarity of the

emulsifying acids also plays a part in the rate of degradation of ascorbic acid Reaction

schemes for the photodegradation of ascorbic acid and its photochemical interaction with

riboflavin nicotinamide and alpha-tocopherol have been presented

vi

ACKNOWLEDGMENTS

I am highly grateful to All Mighty Allah who guided me in all difficulties and

provided me strength to overcome the problems during this work

Words are confined and inefficacious to express my immense gratitude to my

respectable supervisor Prof Dr Iqbal Ahmad Department of Pharmaceutical

Chemistry for his guidance encouragement keen interest and above all giving his

valuable time suggestions and attention His personality has been a source of constant

inspiration through out my research work

I would like to extend my sincere thanks to Prof Lt Gen (R) Dr Syed Azhar

Ahmed Vice Chancellor Baqai Medical University for his personal interest and

constant encouragement through out the study

It is my great desire to express my gratitude to Prof Dr Syed Fazal Hussain

CEO Baqai Institute of Pharmaceutical Sciences for his cooperation and attention and

providing all the facilities of the Institute at my disposal during the research work

I am also thankful to Mrs Shaukat Khalid Dean Faculty of Pharmaceutical

Sciences for her support during the study

I feel honored to express my sincere thanks and indebtedness to Prof Dr

Khursheed Ali Khan Department of Pharmaceutics Prof Dr Aminuddin Department

of Pharmaceutical Chemistry and Dr Faiyaz H M Vaid Chairman Department of

Pharmaceutical Chemistry Faculty of Pharmacy University of Karachi who helped me

selflessly with their invaluable suggestions through out the research work

vii

I feel immense pleasure to pay my sincere and special thanks to Ms Sofia

Ahmed Assistant Professor and In charge Department of Pharmaceutics who lent all

sort of cooperation and spared no effort in helping me during this work

Special thanks are due to Mr Saif-ur-Rehman Khatak Deputy Drug Controller

for his cooperation and help during this study

I acknowledge with sincere thanks the contribution of Tabros Pharmaceutical

Industry Karachi for providing me the opportunity to use their facilities for certain

measurements without which the completion of this work would not have been possible

I highly appreciate the technical services rendered by Mr Anees Mr Wajahat

and Mr Sajjad in pursuance of this study

I am very grateful to Mrs Prof Dr Iqbal Ahmad for her kindness and generous

hospitality during my innumerable visits to their residence

Last but not the least I would like to express my immense indebtedness to My

Gracious Parents Beloved Brothers and Sisters for their moral support kindness and

encouragement throughout my career

I am also thankful to all my students for their affectionate feelings

M A S

viii

To

My Beloved Parents amp

Late Prof Dr S Sabir Ali for their interest and endless support

ix

CONTENTS

Chapter Page

ABSTRACT iv

ACKNOWLEDGEMENTS vi

I INTRODUCTION 1

11 HISTORICAL BACKGROUND 2

12 PHYSICOCHEMICAL CHARACTERISTICS OF

ASCORBIC ACID

2

13 CHEMISTRY OF ASCORBIC ACID 3

131 Nomenclature and Structure 3

132 Chemical Stability 3

14 BIOCHEMICAL FUNCTIONS 7

15 ANTIOXIDANT ACTIVITY 8

16 PHOTOSTABILITY OF DRUGS 9

17 KINETIC TREATMENTS OF PHOTOCHEMICAL

REACTIONS

12

18 LITERATURE ON ASCORBIC ACID 15

II PHOTODEGRADATION REACTIONS AND ASSAY OF

ASCORBIC ACID

17

21 PHOTODEGRADATION REACTIONS 18

211 Photodegradation of Ascorbic Acid 18

212 Effect of Various Substances on Photodegradation of Ascorbic

Acid

20

213 Photosensitized Oxidation of Ascorbic Acid 22

214 Ascorbic Acid Interaction with Other Water-Soluble Vitamins 25

22 ASSAY OF ASCORBIC ACID 26

221 Spectrophotometric Methods 26

222 Fluorimetric Methods 28

x

223 Mass spectrometric Methods 28

224 Chromatographic Methods 28

225 Enzymatic Methods 29

226 Commercial Kits for Clinical Analysis 30

227 Analysis in Creams 30

III FORMULATION AND STABILITY OF CREAM

PREPARATIONS

31

31 FORMULATION OF CREAM PREPARATIONS 32

311 Choice of Emulsion Type 32

312 Choice of Oil Phase 33

313 Emulsion Consistency 33

314 Choice of Emulsifying Agent 34

315 Formulation by the HLB Method 34

316 Concept of Relative Polarity Index 35

32 FORMULATION OF ASCORBIC ACID CREAMS 37

33 STABILITY OF CREAMS 39

331 Physical Stability 39

332 Chemical Stability 39

333 Microbial Stability 40

334 Stability of Ascorbic Acid in Liquid Formulations 41

335 Stability of Ascorbic Acid in Emulsions and Creams 41

336 Stability Testing of Emulsions 45

3361 Macroscopic examination 46

3362 Globule size analysis 46

3363 Change in viscosity 46

3364 Accelerated stability tests 46

337 FDA Guidelines for Semisolid Preparations 46

xi

OBJECT OF PRESENT INVESTIGATION 48

IV MATERIALS AND METHODS 51

41 MATERIALS 52

42 METHODS 55

421 Cream Formulations 55

422 Preparation of Creams 56

423 Thin-Layer Chromatography 57

424 pH Measurements 57

425 Ultraviolet and Visible Spectrometry 58

426 Photolysis of Ascorbic Acid 59

4261 Creams 59

4262 Aqueous and organic solvents 59

4263 Storage of creams in dark 59

427 Measurement of Light Intensity 59

428 Procedure 60

4281 Calculation 62

429 Viscosity Measurements 63

4210 Assay method 65

42101 UV spectrophotometric method for the assay of creams

containing ascorbic acid alone

65

42102 Iodimetric method for the assay of ascorbic acid in creams

containing riboflavin nicotinamide and alpha-tocopherol 65

42103 Spectrophotometric method for the assay of ascorbic acid in

aqueous and organic solvents

67

V PHOTODEGRADATION OF ASCORBIC ACID IN

ORGANIC SOLVENTS AND CREAM FORMULATIONS

68

51 INTRODUCTION 69

52 PHOTOPRODUCTS OF ASCORBIC ACID 71

xii

53 SPECTRAL CHARACTERISTICS OF PHOTOLYSED

SOLUTIONS

71

54 ASSAY OF ASCORBIC ACID IN CREAMS AND

SOLUTIONS

73

55 EFFECT OF SOLVENT 74

56 EFFECT OF CONCENTRATION 80

57 EFFECT OF CARBON CHAIN LENGTH OF

EMULSIFYING AGENT

88

58 EFFECT OF VISCOSITY 94

59 EFFECT OF pH 94

510 EFFECT OF REDOX POTENTIAL 96

511 PRIMARY PHOTOCHEMICAL REACTIONS IN THE

OXIDATION OF ASCORBIC ACID

97

512 DEGRADATION OF ASCORBIC ACID IN THE DARK 98

VI PHOTOCHEMICAL INTERACTION OF ASCORBIC

ACID WITH RIBOFLAVIN NICOTINAMIDE AND

ALPHA-TOCOPHEROL IN CREAM FORMULATIONS

109

61 INTRODUCTION 110

62 ABSORPTION CHARACTERISTICS OF PHOTOLYSED

CREAMS

114

63 PHOTOPRODUCTS OF ASCORBIC ACID AND OTHER

VITAMINS

114

64 ASSAY METHOD 116

65 KINETICS OF PHOTODEGRADATION OF ASCORBIC

ACID

117

66 INTERACTION OF RIBOFLAVIN WITH ASCORBIC ACID 128

67 INTERACTION OF NICOTINAMIDE WITH ASCORBIC

ACID

129

68 INTERACTION OF ΑLPHA-TOCOPHEROL WITH

ASCORBIC ACID

130

69 EFFECT OF CARBON CHAIN LENGTH OF

EMULSIFYING AGENT

130

xiii

610 EFFECT OF VISCOSITY OF CREAMS 132

611 DEGRADATION OF ASCORBIC ACID IN THE PRESENCE

OF OTHER VITAMINS IN THE DARK

135

VII STABILIZATION OF ASCORBIC ACID WITH CITRIC

ACID TARTARIC ACID AND BORIC ACID IN CREAM

FORMULATIONS

141

71 INTRODUCTION 142

72 CREAM FORMULATIONS 142

73 PRODUCTS OF ASCORBIC ACID

PHOTODEGRADATION

145

74 SPECTRAL CHANGES IN PHOTODEGRADED CREAMS 145

75 ASSAY OF ASCORBIC ACID IN CREAMS 145

76 KINETICS OF PHOTODEGRADATION 146

77 EFFECT OF STABILIZING AGENTS 146

78 DEGRADATION OF ASCORBIC ACID IN PRESENCE OF

STABILIZING AGENTS IN THE DARK

162

79 EFFECT OF ADDITIVES ON TRANSMISSION OF

ASCORBIC ACID

163

CONCLUSIONS AND SUGGESTIONS 179

CONCLUSIONS 180

SUGGESTIONS 184

REFERENCES 185

AUTHORrsquoS PUBLICATIONS 226

CHAPTER I

INTRODUCTION

2

11 HISTORICAL BACKGROUND

The disease scurvy which now is known as a condition due to a deficiency of

ascorbic acid in the diet has considerable historical significance (Schick 1943

Carpenter 1986 Bardolph and Taylor 1997 Thomas 1997 Bors 2005) Zilva (1932)

isolated the antiscorbutic activity factor from a crude fraction of lemon and showed that

the activity was destroyed by oxidation and protected by reducing agents Waugh and

King (1932) isolated crystalline vitamin C from lemon juice and showed it to be the

antiscorbutic factor Szent-Gyorgyi (1928) had isolated the same factor from pepper in

connection with his biological oxidation-reduction studies Hirst and Zilva (1933)

identified the antiscorbutic factor as ascorbic acid Early work on the chemical

identification and elucidation of the structure of ascorbic acid has been well documented

(Carpenter 1986) The first synthesis of L-ascorbic acid was achieved almost

simultaneously by Ault et al (1933) and Reichstein et al (1933)

Plants and most animals synthesize their own vitamin C but humans lack this

ability due to the deficiency in an enzyme L-gulono-gamma-lactone oxidase that

catalyzes the terminal step in ascorbic acid biosynthesis (Nishikimi et al 1994)

Therefore humans obtain this vitamin from diet and or vitamin supplements to not only

avoid the development of scurvy but also for overall well being (Stone 1969 Lewin

1976 Davies et al 1991) The minimal daily requirement for ascorbic acid in healthy

adults is 40ndash60 mg (Truswell 2003 Mason 2007 Eitenmiller et al 2008 Elia 2009)

12 PHYSICOCHEMICAL CHARACTERISTICS OF ASCORBIC ACID

The important physicochemical characteristics of ascorbic acid (Table 1) involved

in its identification and degradation are described by many authors (Connors et al 1986

3

OrsquoNeil 2001 Moffat et al 2004 Sinko 2006 Johnston et al 2007) The most

important chemical property of ascorbic acid is the reversible oxidation to semidehydro-

L-ascorbic acid and further oxidation to dehydro-L-ascorbic acid This property is the

basis for its physiological activity In addition the proton on oxygenndash3 is acidic (pKa1 =

417) which contributes to the acidic nature of ascorbic acid (1)

13 CHEMISTRY OF ASCORBIC ACID

131 Nomenclature and Structure

The IUPAC-IUB Commission on Biochemical Nomenclature changed the name

vitamin C (2-oxo-L-theo-hexono-4-lactone-23-enediol) to ascorbic acid or L-ascorbic

acid in 1965 (Johnston et al 2007) The chemical structure of ascorbic acid (1) is

HO OH

O

OHHO

H

(1)

O

The molecule has a near planar five-membered ring with two chiral centers

which contain four stereoisomers

132 Chemical Stability

Ascorbic acid is sensitive to air and light and is kept in a well-closed container

protected from light (British Pharmacopoeia 2009) The degradation reactions of

ascorbic acid in aqueous solution depend on a number of factors such as pH temperature

presence of oxygen or metal It is not very stable in aqueous media at room

temperature and undergoes oxidative degradation to dehydroascorbic acid and

4

Table 1 Physicochemical characteristics of ascorbic acid

Empirical formula C6H8O6

Molar mass 17613

Crystalline form Monoclinic mix of platelets and needles

Melting point 190 to 192 degC

[α]25

+205deg to +215deg

pH

5 mg ml

50 mg ml

~3

~2

pKa 417 1157 (20deg)

Redox potential

(dehydroascorbic acid ascorbate)

(H+ ascorbate

ndash)

ndash174 mV

+282 mV

Solubility g ml

Water

Ethanol absolute

Ether chloroform benzene

033

002

Insoluble

UV spectrum

Absorption maximum [A(1 1 cm)]

pH 20

pH 70

245 nm [695]

265 nm [940]

Infrared spectrum

Principal peaks (Nujol mull)

1026 (CminusOH str) 1111(CminusOminusC str) 1312

(minusCminusOminus str) 1653 (C=O str) 990 (C=C str)

cmndash1

Mass spectrum

Principal ions at mz

29 41 39 42 69 116 167 168

D

5

23-diketogulonic acid The stability of ascorbic acid and dehydroascorbic acid can be

improved by lowering the pH below 2 (Wechtersbach and Cigic 2007) Above pH 7

alkali-catalyzed degradation by cleavage at Cndash1 or Cndash2 results in a number of

compounds mainly monondash dindash and tricarboxylic acids (Connors et al 1986 Bors and

Buettner 1997 Halliwell and Whiteman 1997) The oxidative degradation of ascorbic

acid and dehydroascorbic acid in parenteral nutrition mixtures is catalyzed by trace

elements particularly copper (Allwood 1984ab Allwood et al 1992 Allwood and

Kearney 1998 Kearney et al 1998 Gibbons et al 2001) Stabilized ascorbic acid

preparations in hydroalcoholic vehicle (Kaplan et al 1989) and aquaculture feeds

(OrsquoKeefe 2001) have been reported The various oxidation products of ascorbic acid are

shown in Fig 1

It is interesting to note that in addition to redox and acid-base properties ascorbic

acid can exist as a free radical (Bielski et al 1981 Bielski 1982 Halliwell 1996 Bors

and Buettner 1997) The ascorbate radical anion is an important intermediate in the

reactions involving oxidants and ascorbic acidrsquos antioxidant activity Rate constants for

the generation of ascorbate radicals are in the range of 104ndash10

8 s

ndash1 When ascorbate

radicals are generated by oxyanions the rate constants are of the order of 104ndash10

7 s

ndash1

when generated by halide radicals 106ndash10

8 s

ndash1 and when generated by tocopherols and

flavonoids radicals 106ndash10

8 s

ndash1 (Bielski 1982 Halliwell and Whiteman 1997) The

ascorbate radicals decay usually by disproportionation However a change in ionic

strength or pH can influence the rate of dismutation of ascorbic acid Certain oxyanions

such as phosphates accelerate dismutation (Bielski et al 1981) The acceleration is

attributed to the activity of various protonated forms of phosphate to donate a proton

6

Fig 1 Oxidation products of ascorbic acid

O

OHOH

H

OO

OHOH

H

OO

OHOH

H

O

Ascorbyl radical anion

(interm ediate)

Ascorbic acid

(1)

-e- -2H

+

+e- +2H

+

-e-

+e-

Dehydroascorbic acid

(2)

23-diketo-L-gulonic acid

O xalic acid

+

L-Threonic acid

L-Xylose

+

C O 2

CO 2

L-Xylonic acid

+

L-Lyxonic acid CO 2

HO OH O O-

O O

7

efficiently to the ascorbate radical particularly the dimer form of ascorbate

The unusual stability of the ascorbate radical in biological systems dictates that

accessory enzymatic systems be made available to reduce the potential transient

accumulation of ascorbate radical The excess ascorbate radicals may initiate a chain of

free-radicals reactions In plants NADHmonodehydroascorbate reductase maintains

ascorbic acid in its reduced form NADHmonodehydroascorbate reductase plays a major

role in stress related responses in plants Glutathione dehydroascorbate reductase serves

this purpose in animal tissues Such enzymes keep ascorbic acid operating at maximum

efficiency so that other enzyme systems may take advantage of the univalent redox

cycling capacity of ascorbate (Asard et al 2004 Johnston et al 2007)

The anaerobic degradation of ascorbic acid has been studied by Finholt et al

(1963) Under these conditions the molecule is dehydrated and hydrolyzed in aqueous

solution to give furfural and carbon dioxide The rate of degradation is maximum at pH

41 corresponding to the pKa of ascorbic acid This has been suggested due to the

formation of a saltndashacid complex in solution The reaction is dependent on buffer

concentration but has relatively small effect of ionic strength

14 BIOCHEMICAL FUNCTIONS

Ascorbic acid plays an essential role in the activities of several enzymes It is vital

for the growth and maintenance of healthy bones teeth gums ligaments and blood

vessels It is important for the manufacture of certain neurotransmitters and adrenal

hormones Ascorbic acid is required for the utilization of folic acid and the absorption of

iron It is also necessary for normal immune responses to infection and for wound healing

(Henry 1997)

8

Ascorbic acid deprivation and scurvy include a range of signs and symptoms that

involves defects in specific enzymatic processes (Johnston et al 2007) The

administration of ascorbic acid improves most of the signs of chemically induced

glutathione (L-γ-glutamyl-L-cysteine-glycine GSH) deficiency (Meister 1994) The

effect is very pronounced in newborn rats which do not efficiently synthesize ascorbic

acid in contrast to adult rats and guinea pigs When L-buthionine-(SR)-sulphoxime is

administered in addition to the loss in GSH there is a marked increase in

dehydroascorbic acid This has led to the hypothesis that GSH is very important to

dehydroascorbic acid reduction and as a sequence to ascorbic acid recycling (Meister

1995)

Ascorbic acid also possesses pro-oxidant properties and may cause apoptosis

lymphoid and myeloid cells It has been shown that dehydroascorbic acid also stimulates

the antioxidant defenses in some cells by preferentially importing dehydroascorbate over

ascorbate (Braun et al 1997 Banhegyi et al 1998 Puskas et al 2000 2002)

15 ANTIOXIDANT ACTIVITY

Ascorbic acid is known to readily scavenge reactive oxygen and nitrogen species

such as superoxide and hydroperoxyl radicals aqueous peroxyl radicals singlet oxygen

ozone peroxynitrite nitrogen dioxide nitroxide radicals and hypochlorous acid Excess

of such products has been associated with lipids (Niki and Noguchi 1997 Carr et al

2000 Urso and Clarkson 2003) DNA (Fraga et al 1991 1996 Lindahl 1993) and

protein oxidation (Stadtman 1991 Berlett and Stadtman 1997 Dean et al 1997

Ortwerth and Monnier 1997 Padayatty et al 2003)

9

The electron donor character of ascorbate may be responsible for many of its

known biological functions Inspite of the availability of ascorbic acid to influence the

production of hydroxyl and alkoxyl radicals it remains uncertain whether this is the

principal effect or mechanism that occurs in vivo There is a good evidence that ascorbic

acid protects lipids in biological fluids as an antioxidant (Johnston et al 2007) A

detailed account of the function of ascorbate as an antioxidant and its reactions with

reactive nitrogen species and singlet oxygen has been reported by Packer et al (2002)

and Buettner and Schafer (2004)

Ascorbic acid (Eordm ndash0115 V pH 52 Sinko 2006) has been used as an antioxidant

for the stabilization of drugs with a higher oxidation potential These drugs include

morphine (Yeh and Lach 1961) vitamin A (Wright 1986) rifampin (Maggi et al

1966) cholecalciferol (Nerlo et al 1968 Sawicka 1991) promethazine (Underberg

1978) and sulphacetamide and sulphanilamide (Ahmad and Ahmad 1983)

16 PHOTOSTABILITY OF DRUGS

Many drug substances are sensitive to light (British Pharmacopoeia 2009) and

may degrade in pharmaceutical formulations to inactive or toxic compounds This could

make a product therapeutically inactive while in use by the patients The

photodegradation (photolysis) of drug substances may occur not only during storage but

also during the use of the product It may involve several mechanisms including

oxidation reduction hydrolysis decarboxylation isomerization rearrangement and other

reactions Normal sunlight or room light may cause substantial degradation of drug

molecules The study of degradation of drug substances under the action of UVvisible

light is relevant to the process of drug development for several reasons such as

10

Exposure to light can influence the stability of a drug formulation resulting in the

loss of potency

Inappropriate exposure to light of the raw material or the final product can lead to

the formation of toxic photoproducts that are dangerous to health

Information about the stability of drug substances and formulations is needed to

predict the shelf-life of the final product (Tonnesen and Moore 1993)

The development of light-activated drugs involves activation of the compound

through photochemical reactions (Tonnesen 1991)

Adverse effects due to the formation of minor degradation products during

storage and administration have been reported (de Vries et al 1984) The drugs

substances may also cause light-induced side effects after administration to the patient by

interaction with endogenous substances The study of the photochemical properties of

drug substances and formulated products is an integral part of formulation development

to ensure the safety and efficacy of the product

The photodegradation of drug substances occurs as a result of the absorption of

radiation energy by a molecule (A) to produce an excited state species (A) (11) The

absorbed energy can be lost either by a radiative process involving fluorescence or

phosphorescence (12) or by a physical or chemical radiationless process The physical

process results in the loss of energy as heat (13) or by collisional quenching (14) The

chemical decay leads to the formation of a new species (15) The whole process is

represented as

11

A A (11)

A A + hυprime (12)

A A + heat (13)

A + A 2A (14)

A product (s) (15)

According to the Stark-Einstein law the absorption of one quantum of radiation

results in the formation of one excited molecule which may take part in several

photochemical processes [Eqs (11)ndash(15)] The quantum yield φ for any one of these

processes is defined by

Number of molecules undergoing the photochemical process φ =

Number of quanta absorbed

Considering a pure photochemical reaction the quantum yield has a value of 0ndash1

however if A is a radical that can take part in a free-radical chain reaction so that the

absorption of energy simply initiates the reaction then each quantum of energy may

result in the decomposition of molecules and φ may appear to be greater than 1 (Connors

et al 1986)

Detailed information on the photostability and photodegradation of drug

substances including vitamins alone or in solid or liquid formulations is available in the

reviews published by DeRitter (1982) Albini and Fasani (1998) Sequeira and Vozone

(2000) Tonnesen (2002 2004) Yoshioka and Stella (2002) Min and Boff (2002) Reed

et al (2003) Fasani and Albini (2005) and Sinko (2006) The photostability of cosmetic

materials has been reviewed by Sugden (1985) Important aspects dealing with the

photostability testing of drug substances have been dealt by Anderson et al (1991)

k1

k2

k3

k4

hυ

12

Tonnesen and Moore (1993) Tonnesen and Karlsen (1997) Riehl et al (1995) ICH

(1997) Singh and Bakshi (2000) Valvani (2000) Thatcher et al (2001ab) Fasani and

Albini (2005) Klick et al (2005) Singh (2006) and Ahmad and Vaid (2006)

17 KINETIC TREATMENT OF PHOTOCHEMICAL REACTIONS

The kinetic treatment of photochemical reactions with reference to the

photostability of drug substances has been considered by Moore (2004) and is presented

in this section

The photostability testing of a drug substance at the preformulation stage involves

a study of the drugrsquos rate of degradation in solution on exposure to light for a period of

time The value of the degradation rate constant depends very much on the design of the

experimental conditions (eg concentration solvent pH irradiation source oxygen

content) The factors that determine the rate of a photochemical reaction are simply the

rate at which the radiation is absorbed by the test sample (ie the number N of photons

absorbed per second) and the efficiency of the photochemical process (ie the quantum

yield of the reaction φ) For a monochromatic photon source the number of photons

absorbed depends upon the intensity of the photon source and the absorbance at that

wavelength of the absorbing species The rate of a photochemical reaction is defined as

Rate = number of molecules transformed per second = N φ (16)

In the first instance the rate can be determined for a homogeneous liquid sample

in which the only photon absorption is due to the drug molecule undergoing

transformation with the restriction that the concentration is low so that the drug does not

absorb all of the available radiation in the wavelength range corresponding to its

13

absorption spectrum The value of N can be derived at a particular wavelength λ and is

given by

Nλ = Iλ ndash It = Iλ (1 ndash 10ndashA

) (17)

where Iλ and It are the incident and transmitted radiation intensities respectively and A is

the absorbance of the sample at the wavelength of irradiation This expression can be

expanded as a power series

Nλ = 2303 Iλ (A + A22 + A

36 + hellip) (18)

When the absorbance is low (Alt 002) the second- and higher-order terms are negligible

and the expression simplifies to the first term in Eq 18 Given the Beerrsquos law relation

between absorbance and concentration N can be seen to be directly proportional to

concentration

Nλ = 2303 Iλ A = 2303 Iλ ελ b C (19)

where ελ is the molar absorptivity at wavelength λ C the molar concentration of the

absorbing species and b the optical path length of the reaction vessel Now Iλ and ελ vary

with wavelength so the expression must be integrated over the relevant wavelength range

where each has a non-zero value

N = 2303 b C int (Iλ ελ) dλ integrated from λ1 to λ2 (110)

Thus

Rate = 2303 b C φ int (Iλ ελ) dλ (111)

Now the overlap integral (int Iλ ελ dλ) is a constant for a particular combination of photon

source and absorbing substance b is determined by the reaction vessel chosen and φ is a

characteristic of the reaction Thus by grouping the constant terms into an overall

constant k1 the expression is simplified to a first-order kinetic equation

14

Rate = ndashd [Drug] dt = k1C (112)

The integrated form of Eq 112 can be expressed in exponential form (Eq 113) or

logarithmic form (Eq 114)

[Drug]t = [Drug]0 endashk1t

(113)

ln [Drug]t = ln [Drug]0 ndash k1t (114)

Verification of first-order kinetics is obtained when a plot of the logarithm of the

concentration of drug remaining is linear with slope equal to (ndashk1)

Eq 112 predicts that a photodegradation reaction studied at low concentrations in

solution will follow first-order kinetics however the rate constant derived from a study

performed in one laboratory will not be the same as that found in another The reason for

this is the inherent difficulty in reproducing exactly the experimental arrangement of

photon source and sample irradiation geometry Therefore the relative values of the rate

constants are useful in a given experimental arrangement for making comparisons of

degradation of the absorbing substance in different formulations eg those containing

ingredients designed to inhibit the photoreaction The use of rate constants is helpful for

comparative purposes when studying a number of different reaction mixtures under the

same irradiation conditions such as the effect of pH on the degradation of a drug

However the reaction order and numerical values of the rate constants are relative to the

specific conditions used

15

18 LITERATURE ON ASCORBIC ACID

A large number of reviews have been published on various aspects of ascorbic

acid A list of important reviews is given below

Chemistry biochemical functions and related aspects

Rosenberg (1945) Burns (1961) King and Burns (1975) Sim (1972) Hanck

(1982) Zaeslein (1982) Seib and Tolbert (1982) Carpenter (1986) Levine

(1986) Davies et al (1991) Halliwell and Whiteman (1997) Ortega and Delgado

(1998) Asard et al (2004) Hickey and Roberts (2004) Johnston et al (2007)

Eitenmiller (2008)

Chemical and pharmaceutical stability

Macek (1960) Garrett (1967) Carstensen (1972) Dale and Booth (1976) Hashmi

(1973) Litner (1973) DeRitter (1982) Allwood (1984ab) Allwood and Kearney

(1998) Connors et al (1986) Smith et al (1988) Racz (1989) Roth et al 1991

Ball (2006) Eitenmiller et al (2008) Sweetman (2009)

Methods of assay and chromatography

Mader (1961) Gyorgy and Pearson (1967) Bolliger and Konig (1969) Hashmi

(1973) Al-Meshal and Hassan (1982) Pelletier (1985) Lambert and deLeenheer

(1992) Halver and Felton (2001) Moffat et al (2004) Ball (2006) Eitenmiller et

al (2008)

Pharmacology and related aspects

Levine (1986) Dollery (1999) Sauberlich (1994ab) McDowell (2000)

Kaushansky and Kipps (2006) Sweetman (2009)

16

Antioxidant activity

Basu et al (1999) Shacter (2000) Thiele et al (2000) Cadenas and Packer

(2002) Packer et al (2002) Padayathy et al (2003) Parker and Parker (2003)

Burke (2006) Johnston et al (2007)

Cosmetic Preparations

Barel et al (2001) Salvador and Chisvert (2007) Rosen (2005) Bissett (2006)

Chaudhri and Jain (2009)

CHAPTER II

PHOTODEGRADATION

REACTIONS AND ASSAY

OF ASCORBIC ACID

18

21 PHOTODEGRADATION REACTIONS

211 Photodegradation of Ascorbic Acid

Aqueous ascorbic acid (1) solutions are degraded by UV light to give

dehydroascorbic acid (2) (Arcus and Zilva 1940) Ascorbic acid degradation at a

concentration of 52 and 50 mg on UV irradiation for 2 hours gave a loss of 43 and 8

respectively Dehydroascorbic acid solutions are more stable to UV light than the

ascorbic acid (Kitagawa 1968) In many natural products the vitamin is oxidized on

exposure to air and light (OrsquoNeil 2001) When solutions of multivitamin preparations are

exposed to light H2O2 as well as organic peroxides are generated and specific

byproducts that differ from dehydroascorbic acid and 23-diketogulonic acid (3) are

produced (Lavoie et al 2004)

In aqueous neutral or alkaline solution ascorbic acid (1) undergoes chemical or

photochemical oxidation to dehydroascorbic acid (2) which upon saponification of the

lactone ring under the influence of the base water produces 23-diketo-L-gulonic acid (an

α szlig- diketogulonic acid) (3) This acid undergoes further oxidation to oxalic acid (4) and

L-threonic acid (5) (Racz 1989) (Fig 2a) At room temperature oxalic acid (4) is also

formed along with threonolactone (6) by photochemical degradation of ascorbic acid (1)

in the presence of singlet oxygen (1O2) (Silva and Quina 2006) (Fig 2a) The low-

temperature photooxygenation of ascorbic acid (1) gives a mixture of unstable

hydroperoxide ketones (7) and (8) which on standing interconvert and cyclize to

hydroperoxyhemiketal (9) The hydroperoxyhemiketal breaks down on warming to

produce the oxalate esters of threonic acid (10) (Fig 2b) (Kwon and Foote 1988)

19

COOH

COOH

O

OHHO

O

HOH2C

HO2

O

O

HO

OO

O O2H

OHHO

O

HOH2C

OH

O

O

OH

O2H

OO

HO O2CCO2H

(1)hv

room temperature

(4)(6)

(1)hv

85 oC

(7)

(a)

(8)

+

cyclization

(9)

ring cleavage

(b)

(10)

(2)

OH O

OHHO

OH O O

(3)

OH OH

OH

OH O

O

OH

1O2 [O]

+

(5)

COOH

COOH

(4)

+

OH

Fig 2 Photooxidation of ascorbic acid at room and low temperature

20

An important consideration in the stability of ascorbic acid in total parenteral

nutrition (TPN) solutions is the generation of hydrogen peroxide in the presence of light

(Laborie et al 1998 1999 2000 2002 Chessex et al 2002) This may result from the

oxidation of ascorbate anion by molecular oxygen (Homann and Gaffron 1964 Taqui

Khan and Martell 1967 Mushran and Agarwal 1977 Hughes 1985 De La Rochette et

al 2000) leading to further degradation of ascorbic acid (Deutsch 1998a 1998b

1998c) The kinetics and mechanism of oxidation reactions of ascorbic acid have been

studied by several workers (Taqui Khan and Martell 1967 Ogata and Kosugi 1969

Blaugh and Hajratwala 1972 Fessenden and Verma 1978 Abe et al 1986 Kwon et al

1989 Fornaro and Coicher 1998 Njus et al 2001)

The photostability of various ascorbic acid tablets on exposure to UV light has

been studied and the influence of antioxidants and moisture on the potency loss of

ascorbic acid has been evaluated The physical characteristics of ascorbic acid tablets are

also affected on UV irradiation (Ahmad et al 1973 Jamil et al 1980ab Jamil and

Ahmad 1984)

212 Effect of Various Substances on Photodegradation of Ascorbic Acid

The oxidation-reduction reactions of ascorbic acid in the presence of riboflavin at

pH 8ndash9 under the influence of light have been studied Under these conditions ascorbic

acid is a more active H donor to riboflavin than phenolphthalein (Sibi et al 1953)

Riboflavin has been found to catalyze the photodegradation of ascorbic acid solutions

during exposure to light and air The losses of ascorbic acid are markedly increased by

the presence of Cu2+

and Fe3+

ions under light exposed and unexposed conditions (Sattar

et al 1977) A spectral study of the UV photolysis of ascorbic acid solutions in the

21

presence of riboflavin has shown that the degradation of ascorbic acid is enhanced to the

extent of about 15 (Vaid et al 2005) The influence of DL- methionine on the

photostability of ascorbic acid solutions has also been studied DL- methionine (10 mg

) enhances the photostability of ascorbic acid (40 mg ) in acetate and phosphate

buffers but not in citrate buffer at pH 45 The photoprotective action of DL-methionine

on ascorbic acid appears to be influenced by its concentration pH of the medium and the

buffer species (Asker et al 1985)

The degradation of ascorbic acid solutions on irradiation with simulated sunlight

in the presence of the food dye quinolone yellow (E 104) is enhanced However this

effect is reversed by the addition of mannitol indicating that this dye facilitates the

photogeneration of hydroxyl radicals which may cause degradation of the vitamin The

incorporation of triplet quenchers enhances the stability of substrate solutions suggesting

that the dye acts as a triplet sensitizer to facilitate the reaction (Sidhu and Sugden 1992)

The photostability of ascorbic acid solutions is enhanced by sweetening agents (mannitol

sorbitol sucrose dextrose and Canderal) at 5 wv concentration However the addition

of stoichiometric amounts of hydrogen peroxide as a source of hydroxyl radicals and 2

2rsquo-azobis (2-amidopropane) as a source of hydroperoxyl radicals results in diminished

stability of ascorbic acid solutions The diminished activity may be due to the action of

hydroperoxyl radicals in the presence of hydroxyl radical scavengers (Ho et al 1994)

Metal-complexing agents (eg disodium ethylenediaminetetraacetic acid N-

hydroxylethyl ethylenediaminetetraacetic acid 8-hydroxyquinoline) have a stabilizing

effect on the photolysis of ascorbic acid injectable solutions (Kassem et al 1969ab

22

1972) This may be due to the interaction of these agents with metal ions and other

impurities

213 Photosensitized Oxidation of Ascorbic Acid

In the presence of visible light a photosensitizer such as riboflavin can exhibit

photosensitizing properties through a mixed Type IndashType II mechanism (Yoshimura and

Ohno 1988 Foote 1991 Silva et al 1994 Silva and Quina 2006) as presented below

Type I mechanism (low oxygen concentration)

RF + hυ rarr 1RF rarr

3RF (21)

3RF + SH rarr RF

middot ndash + SH

middot + rarr RFH

middot + S

middot (22)

RFmiddot ndash

+ O2 rarr RF + O2middot ndash

(23)

2RFHmiddot rarr RF + RFH2 (24)

RFH2 + O2 rarr RF + H2O2 (25)

H2O2 + O2middot ndashrarr

ndashOH +

middotOH + O2 (26)

Smiddot and or SH

middot +

+ H2O2 O2middot ndash

rarr Soxid (27)

Type II mechanism (high oxygen concentration)

RF + hυ rarr 1RF rarr

3RF (28)

3RF + O2 rarr RF +

1O2 (29)

SH + 1O2 rarr Soxid (210)

In these reactions RF 1RF and

3RF represent RF in the ground state and in the excited

singlet and triplet states respectively RFmiddot ndash

RFHmiddot and RFH2 are the radical anion the

radical and the reduced form of RF SH is the reduced substrate and SHmiddot

+ S

middot and Soxid

23

represent the intermediate radical cation the radical and the oxidized form of the

substrate respectively

An early study of the riboflavin-sensitized photooxidation of ascorbic acid has

been carried out by flash photolysis (Heelis et al 1981) ESR spectrometry has been

used to investigate the photosensitized formation of ascorbate radicals by riboflavin (Kim

et al 1993) The photochemical behavior of a system consisting of ascorbate ion (AHndash)

and riboflavin has been studied by Mancini et al (2000) and De La Rochette et al (2000

2003) The photosensitized processes were examined as a function of oxygen pressure

and the efficiency of RF induced degradation of AHndash

at various oxygen concentrations

was compared on the basis of the respective initial photosensitization quantum yields

(Table 2)

In this reaction a Type I photosensitization mechanism (Karlsen 1996) implies a

direct electron transfer between AHndash and the RF triplet-excited state followed by the

oxidation of semioxidized ascorbyl radical (AHmiddot) by molecular oxygen or some other

reactive species On the contrary in a Type II photosensitization mechanism singlet

oxygen is produced directly by energy transfer from the RF triplet-excited state to

molecular oxygen and the singlet oxygen then oxidizes the AHndash Thus by irradiating

under increasing oxygen pressure it is possible to control the relative prevalence and

efficiency of Type I or Type II mechanisms The absence of a linear relationship between

the quantum yields of ascorbate degradation and oxygen concentration indicates that the

photosensitization mechanism involved in not exclusively Type II

24

Table 2 Initial quantum yield (φ) for ascorbate (AHndash) degradation during

photosensitization by RF (35 microM) in solutions irradiated at 365 nm and

37ordmC

O2 103 times φ (AH

ndash)a

0

5

20

14 plusmn 06

1670 plusmn 220

1940 plusmn 200

a Data are the mean plusmn SD of three independent experiments

25

In the presence of RF and O2 the quantum yields for degradation of ascorbate ion

have been found to be greater than one suggesting the participation of chain reactions

initiated by the ascorbyl radical as given by the following reactions

3RF + AH

ndash rarr RFmiddot

ndash + AHmiddot (211)

AHmiddot + O2 rarr A + HO2middot (212)

HO2middot + AHndash rarr H2O2 + AHmiddot (213)

The generation of the ascorbyl radical by the reaction between the RF excited-

triplet state and the ascorbate ion (Eq 211) is the only step that requires the absorption of

photons (to form the excited-triplet state of RF) The subsequent reactions (Eqs 212 and

213) are independent of light and lead to further degradation of the ascorbate ion In the

presence of transition metal ions such as Fe3+

in trace amounts in the buffer solution

containing RF and ascorbate ions further oxidation of ascorbate ion could also occur As

a result the reduced form of the metal ion (ie Fe2+

) can be generated by the metal

catalyzed oxidation of ascorbate ion This has been confirmed by the significant decrease

in the AHndash photooxidation quantum yield in the presence of the metal chelator EDTA

which inactivates the trace amounts of iron present in the buffer solution The quantum

yields for the photosensitized oxidation of ascorbate ion are decreased twofold at 20 O2

and fourfold at 5 O2 concentration in the presence of EDTA (Silva and Quina 2006)

Amino acids have been found to affect the photosensitized oxidation of ascorbic acid

(Jung et al 1995)

214 Ascorbic Acid Interaction with Other Water-Soluble Vitamins

The stability of ascorbic acid is reported to be enhanced in syrups containing B-

complex vitamins (Connors et al 1986) This may be due to the increased viscosity of

the syrups inhibiting the oxidation of ascorbic acid The rate of photolysis in solution

26

containing cyanocobalamin and ascorbic acid is reported to decrease with an increase in

pH (Ansari et al 2004) where as use of certain halide salts has been reported to be

beneficial in stabilizing pharmaceutical products and dietary supplements when vitamin

B12 and vitamin C are combined in solution (Ichikawa et al 2005) When a solution of

multivitamins is exposed to light it is reported that organic peroxidases are generated and

the concentration of ascorbic acid decreases (Lavoie et al 2004)

22 ASSAY OF ASCORBIC ACID

Recent accounts of the development and application of analytical methods to the

determination of ascorbic acid in pharmaceuticals biological samples and food materials

are reported in the literature (Rumsey and Levine 2000 Halver and Felton 2001 Moffat

et al 2004 Ball 2006 Sheraz et al 2007 Eitenmiller et al 2008 Salkic and Kubicek

2008) Most of these methods are based on the application of spectrophotometric

fluorimetric and chromatographic techniques to suit the requirements of a particular assay

and are summarized below

221 Spectrophotometric Methods

Spectrophotometric methods are the most widely used methods for the assay of

ascorbic acid in aqueous solution Ascorbic acid exhibits strong absorption in the

ultraviolet region (absorption maxima 243 nm at pH 2 and 265 nm at pH 4ndash10 OrsquoNeil

2001 Moffat et al 2004 British Pharmacopoeia 2009) This is the basis of

spectrophotometric methods for the determination of the vitamins in pure solutions and in

sample preparations where no interference is observed from UV absorbing impurities

The value of A (1 1 cm) at the analytical wavelength of 245 nm (pH 20) is high (695)

which makes the method very sensitive for the determination of mg quantities of the

27

vitamin Treatment of the material to be analyzed with ascorbic acid oxidase is often used

as a blank to correct for the presence of interfering substances in biological samples (Liu

et al 1982) A spectrophotometric method for the determination of ascorbic acid in

pharmaceuticals by background correction (245 nm) has been reported (Verma et al

1991) The direct determination of ascorbic acid in mixtures involves the use of 22prime-

dipyridyl as a colorimetric reagent The method is based on the reduction of Fe (III) by

ascorbic acid to Fe (II) which reacts with 2 2prime-dipyridyl to form a colored complex

(absorption maximum 510 nm) that can be used for quantitative determination (Margolis

and Schmidt 1996) A spectrophotometric method has been developed for the

determination of ascorbic acid and its oxidation product dehydroascorbic acid in

biological samples (Moeslinger et al 1995) A sensitive method has been reported for

the determination of ascorbic acid in pharmaceutical formulations and fruit juices by

interaction with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (Br-PADAP) (Ferreira

et al 1997) A novel UV method has been developed for the analysis of ascorbic acid in

methanol at 245 nm in various formulations (Zeng et al 2005)

Ascorbic acid in aqueous solutions has been assayed at 244 nm (pH ~2) (Ogata

and Kosugi 1969) 245 nm (pH 35) (Blaugh and Hajratwala 1972) 264 nm (pH 7)

(Salkic et al 2007) 265 nm (pH 7) (Hashmi 1973) 275 nm (pH 41 and 70) (Heelis et

al 1981) 265 nm (pH 7) (Al-Meshal and Hassan 1982) 245 nm (pH ~2) (Verma et al

1991) and 265 nm (pH ~7) (Erb et al 2004) Dehydroascorbic acid and 23-

diketogulonic acid do not significantly absorb in this region (Pelletier 1985 Davies et

al 1991 Rumsey and Levine 2000) and therefore do not interfere with the assay of

ascorbic acid in degraded solutions

28

222 Fluorimetric Methods

Fluorimetry is a highly sensitive technique for the determination of fluorescent

compounds or fluorescent derivatives of non-fluorescent compounds The technique has

been used for the detection of microg quantities of ascorbic acid Methods based on

fluorimetric (Kampfenkel et al 1995) and chemiluminescence detection (Zhang and

Chen 2000) provide highly sensitive methods for the determination of ascorbic acid in

plant and other materials

223 Mass Spectrometric Methods

Conventional and isotope mass spectrometric techniques have also been used for

the analysis of ascorbic acid Isotope ratio mass spectrometry is particularly useful and

sensitive when 13

C ascorbic acid is used as a standard in the analysis of complex matrices

(Gensler et al 1995)

224 Chromatographic Methods

High-performance liquid chromatographic (HPLC) methods have extensively

been employed for the determination of ascorbic acid in biological samples These

methods include ion exchange reversed phase and ion-pairing HPLC protocols

Spectrophotometric fluorimetric and electrochemical detection has been used in the

HPLC analysis of ascorbic acid The electrochemical detection is used for the

simultaneous determination of ascorbic acid dehydroascorbic acid and their isomers and

derivatives A number of HPLC methods have been developed for the detection and

determination of ascorbic acid and its oxidation products and derivatives in biological

samples and plant materials (Tsao and Young 1985 Tangney 1988 Dabrowski and

Huiterleitner 1989 Thomson and Trenerry 1995 Kimoto et al 1997 Kall and

29

Anderson 1999 Rumelin et al 1999 Lykkesfeldt 2000 Zhang et al 2000 Pastore et

al 2001 Frenich et al 2005) The limit of detection of ascorbic acid in plasma or urine

with UV detection lies in the range of 100-120 microg (Liau et al 1993 Manoharan and

Schwille 1994) Fluorescence detection of ascorbic acid and dehydroascorbic acid in

plasma and its comparison with coulometric detection has been reported (Tessier et al

1996) A liquid chromatography-diode-array detection (LCndashDAD) method has been

reported for the determination of 10 water-soluble and 10 fat-soluble vitamins including

ascorbic acid in pharmaceutical preparations with a coefficient of variation lt 65

(Konings 2006)

Liquid chromatography methods based on precolumn and o-phenylenediamine

(OPD) derivatization have been used for the determination of total vitamin C and total

isovitamin C in foods and dehydro forms of the vitamin Isoascorbic acid has been used

as an internal standard in the analysis (Speek et al 1985 Vanderslice et al 1990

Dodsun et al 1992 Vanderslice and Higgs 1988 1993 Hagg et al 1994 1995) The

limits of detection of ascorbic acid by HPLC using different detectors are in the range of

16ndash400 microgl (Capellmann and Bolt 1992 Iwase and Ono 1994 Karatepe 2004)

225 Enzymatic Methods

Enzymatic methods using ascorbate oxidase are specific and have the advantage

of selectively measuring the biological activity of ascorbic acid in serum or plasma (Liu

et al 1982) Ascorbate oxidase and OPD derivatization has been used to develop a rapid

automated method for the routine assay of ascorbic acid in serum and plasma The

method has a sample throughput of 100h (Ihara et al 2000)

30

226 Commercial Kits for Clinical Analysis

Commercial kits (eg Immunodiagnostic Germany Biovision USA) are also

used for the determination of ascorbic acid in biological samples (serum or plasma) in

clinical laboratories

227 Analysis in Creams

The general methods for the analysis of active ingredients and excipients in

cosmetic products including creams are described by Salvador and Chisvert (2007)

Ascorbic acid and derivatives in creams have been determined by liquid chromatography

(Irache et al 1993 Varvaresou et al 2006) gas chromatography-mass spectrometry

(Leveque et al 2005) and electrochemical methods (Beissenhirtz et al 2003 Guitton et

al 2007)

CHAPTER III

FORMULATION AND

STABILITY OF CREAM

PREPARATIONS

32

31 FORMULATION OF CREAM PREPARATIONS

Traditionally emulsions have been defined as dispersions of macroscopic droplets

of one liquid in another liquid with a droplet diameter approximately in the range of 05-

100 microm (Becher 1965) According to the definition of International Union of Pure and

Applied Chemistry (IUPAC) (1971) ldquoIn an emulsion liquid droplets and or liquid

crystals are dispersed in a liquidrdquo

Creams are semisolid emulsions intended for external applications They are often

composed of two phases Oil-in-water (ow) emulsions are most useful as water-washable

bases whereas water-in-oil (wo) emulsions are emollient and cleansing agents The

active ingredient is often dissolved in one or both phases thus creating a three-phase

system Patients often prefer a wo cream to an ointment because the cream spreads more

readily is less greasy and the evaporating water soothes the inflamed tissue OW creams

(vanishing creams) rub into the skin the continuous phase evaporates and increases the

concentration of a water-soluble drug in the adhering film The concentration gradient for

drug across the stratum corneum therefore increases promoting percutaneous absorption

(Barry 2002 Betageri and Prabhu 2002)

The various factors involved in the formulation of emulsions and topical products

have been discussed by Block (1996) Barry (2002) and Jain et al (2006) and are briefly

presented in the following sections

311 Choice of Emulsion Type

Oil-in-water emulsions are used for the topical application of water-soluble drugs

mainly for local effect They do not have the greasy texture associated with oily bases

and are therefore pleasant to use and easily washed from skin surfaces Moisturizing

33

creams designed to prevent moisture loss from the skin and thus inhibit drying of the

stratum corneum are more efficient if formulated as ow emulsions which produce a

coherent water-repellent film

312 Choice of Oil Phase

Many emulsions for external use contain oils that are present as carriers for the

active ingredient It must be realized that the type of oil used may also have an effect both

on the viscosity of the product and on the transport of the drug into the skin (Barry

2002) One of the most widely used oils for this type of preparation is liquid paraffin

This is one of a series of hydrocarbons which also includes hard paraffin soft paraffin

and light liquid paraffin They can be used individually or in combination with each other

to control emulsion consistency This will ensure that the product can be spread easily but

will be sufficiently viscous to form a coherent film over the skin The film-forming

capabilities of the emulsion can be further modified by the inclusion of various waxes

such as bees wax carnauba wax or higher fatty alcohols

313 Emulsion Consistency

A consideration of the texture or feel of a product intended for external use is

important A wo preparation will have a greasy texture and often exhibits a higher

apparent viscosity than ow emulsions This fact imparts a feeling of richness to many

cosmetic formulations Oil-in-water emulsions will however feel less greasy or sticky on

application to the skin will be absorbed more readily because of their lower oil content

and can be more easily washed from skin surface Ideally emulsions should exhibit the

rheological properties of plasticity pseudoplasticity and thixotropy Emulsions of high

apparent viscosity for external use (cream) are of a semisolid consistency There are

34

several methods by which the rheological properties of an emulsion can be controlled

(Billany 2002)

314 Choice of Emulsifying Agent

The choice of emulgent to be used would depend on factors such as its

emulsifying ability route of administration and toxicity Most of the non-ionic emulgents

are less irritant and less toxic than their anionic and cationic counter parts Some

emulgents such as the ionic alkali soaps often have a high pH and are thus unsuitable for

application to broken skin Even in normal intact skin with a pH of 55 the application of

such alkaline materials can cause irritation Some emulsifiers in particular wool fat can

cause sensitizing reactions in susceptible people The details of various types of

emulsifying agents are available in the literature (Betageri and Prabhu 2002 Billany

2002 Swarbrick et al 2006)

315 Formulation by the HLB Method

The physically stable emulsions are best achieved by the presence of a condensed

layer of emulgent at the oil water interface and the complex interfacial films formed by a

blend of an oil-soluble emulsifying agent with a water-soluble one produces the most

satisfactory emulsions

It is possible to calculate the relative quantities of the emulgents necessary to

produce the most physically stable emulsions for a particular formulation with water

combination This approach is called the hydrophilic-lipophilic balance (HLB) method

Each surfactant is allocated an HLB number representing the relative properties of the

lipophilic and hydrophilic parts of the molecule High numbers (up to a theoretical

number of 20) therefore indicates a surfactant exhibiting mainly hydrophilic or polar

35

properties whereas low numbers represent lipophilic or non-polar characteristics Each

type of oil requires an emulgent of a particular HLB number in order to ensure a stable

product For an ow emulsion the more polar the oil phase the more polar must be the

emulgent system (Billany 2002 Im-Emsap et al 2002 Swarbrick et al 2006)

316 Concept of Relative Polarity Index

In the ingredient selection in cosmetic formulations a new concept of relative

polarity index (RPI) has been presented (Wiechers 2005) The physicochemical

characteristics of the ingredients determine their skin delivery to a greater extent than the

formulation type The cosmetic formulation cannot change the chemistry of the active

molecule that needs to penetrate to a specific site within the skin However the

formulation type can be selected based on the polarity of the active ingredient and the

desired site of action for the active ingredient For optimum skin delivery the solubility of

the active ingredient needs to be as high as possible (to create a large concentration

gradient) and as small as possible (to create a large partition coefficient) To achieve this

it is necessary to determine the following parameters

The total amount dissolved in the formulation that is available for skin penetration

the higher this amount the more will penetrate until a solution concentration is

reached in the skin therefore a high absolute solubility in the formulation is required

The polarity of the formulation relative to that of the stratum corneum if an active

ingredient dissolves better in the stratum corneum than in the formulation then the

partition of the active ingredient will favour the stratum corneum therefore a low

(relative to that in the stratum corneum) solubility in the formulation is required

(Wiechers 2005)

36

These requirements can be met by considering the concept of RPI (Wiechers

2003 2005) In this systematic approach it is essential to consider the stratum corneum

as another solvent with its own polarity The stratum corneum appears to behave very

similarly to and in a more polar fashion than butanol with respect to its solubilizing

ability for active ingredients (Scheuplein and Blank 1973) The polarity of stratum

corneum as expressed by its octanol water partition coefficient is 63

The relative polarity index may be used to compare the polarity of an active

ingredient with both that of the skin and that of the oil phase of a cosmetic formulation

predominantly consisting of emollients It may be visualized as a vertical line with a high

polarity at the top and a high lipophilicity at the bottom The polarity is expressed as the

log10 of the octanol water coefficient For example the relative polarity index values of

glycerin and isostearyl isostearate are -176 and 2698 respectively (Wiechers 2005) In

order to use the concept of the relative polarity index three numbers (on log10 scale) are

required

The polarity of the stratum corneum is set at 08 However in reality this value will

change with the hydration state of the stratum corneum that is determined in part by

the external relative humidity (Bonwstra et al 2003)

The polarity of the active molecule

The polarity of the formulation

For multiphase or multipolarity systems like emulsions the active ingredient is dissolved

in the phase For example in an ow emulsion where a lipophilic active ingredient is

dissolved in the oil phase it is the polarity of the homogenous mixture of the lipophilic

active ingredient and internal oil For the same lipophilic active in a wo emulsion it is

37

the polarity of the homogenous mixture of the lipophilic active ingredients and external

oil For water-soluble active ingredients it is the polarity of the homogenous mixture of

the hydrophilic active ingredient and the aqueous phase regardless whether it is internal

(wo emulsions) or external (ow emulsions)

Once the active ingredient and the formulation type have been chosen it is

necessary to create the delivery system that will effectively deliver the molecule The

concept of relative polarity index allows the formulator to select the polarity of the phase

in which the active ingredient is incorporated on the basis of its own properties and those

of the stratum corneum In order to achieve maximum delivery the polarity of the active

ingredient and the stratum corneum need to be considered In order to improve the skin

delivery of active ingredients the first step involves selecting a primary emollient with a

polarity close to that of the active ingredient in which it will have a high solubility The

second step is to reduce the solubility of the active ingredient in the primary emollient via

the addition of a secondary emollient with a different polarity and therefore lower

solubility for the active ingredient This approach has shown a 3-4 fold increase in skin

penetration with out increasing the amount of active ingredients in the formulation

(Wiechers 2005)

32 FORMULATION OF ASCORBIC ACID CREAMS

Ascorbic acid is a water-soluble material and is included frequently in skin care

formulations to restore skin health It is very unstable and is easily oxidized in aqueous

solution This vitamin is known to be a reducing agent in biological systems and causes

the reduction of both oxygen- and nitrogen- based free radicals (Higdon and Frei 2002)

It can also act as a co-antioxidant with the tocopheroxyl radical to regenerate alpha-

38

tocopherol (Packer et al 1979 Buettner 1993 Peyrat-Maillard et al 2001) In this

reaction the two vitamins act synergistically Alpha-tocopherol first functions as the

primary antioxidant that reacts with an organic free radical Thereafter ascorbic acid

reacts with the free radical tocopheroxyl to general alpha-tocopherol In physiological

conditions the ascorbyl radical formed by regenerating tocopherol is then converted back

to ascorbate by the redox cycle (Davies et al 1991) The interaction of ascorbic acid

with a redox partner such as alpha-tocopherol has been found useful to slow its oxidation

and prolong its action

The instability of ascorbic acid makes this antioxidant active ingredient a

formulation challenge to deliver to the skin and retain its effective form In addition to its

use in combination with alpha-tocopherol in cream formulations the stability of ascorbic

acid may be improved by its use in the form of a fatty acid ester such as ascorbyl

palmitate Ascorbyl palmitate has been used in thixogel formulations and is typically

incorporated into the mineral oil phase Preliminary experiments have shown that it could

be slowly released from the starch-oil emulsion matrix and act as an antioxidant (Wille

2005)

Various physical and chemical factors involved in the formulation of cream

preparations have been discussed in the above sections For polar and air light sensitive

compounds such as ascorbic acid it is important to consider factors such as the choice of

formulation ingredients polar character of formulation HLB value pH viscosity etc to

achieve stability

39

33 STABILITY OF CREAMS

331 Physical Stability

The most important consideration with respect to pharmaceutical and cosmetic

emulsions (creams) is the stability of the finished product The stability of a

pharmaceutical emulsion is characterized by the absence of coalescence of the internal

phase absence of creaming and maintenance of elegance with respect to appearance

odor color and other physical properties An emulsion is a dynamic system however

any flocculation and resultant creaming represent potential steps towards complete

coalescence of the internal phase In pharmaceutical emulsions creaming results as a lack

of uniformity of drug distribution and poses a problem to the pharmaceutical

compounder Another important factor in the stabilization of emulsions is phase inversion

which involves the change of emulsion type from ow to wo or vice versa and is

considered as a case of instability The four major phenomena associated with the

physical instability of emulsions are flocculation creaming coalescence and breaking

These have been discussed by Garti and Aserin (1996) Im-Emsap et al (2002) and Sinko

(2006)

332 Chemical Stability

The instability of a drug may lead to the loss of its concentration through a

chemical reaction under normal or stress conditions This results in a reduction of the

potency and is a well-recognized cause of poor product quality The degradation of the

drug may make the product esthetically unacceptable if significant changes in color or

odor have occurred The degradation product may also be a toxic substance The various

pathways of chemical degradation of a drug depend on the structural characteristics of the

40

drug and may involve hydrolysis dehydration isomerization and racemization

decarboxylation and elimination oxidation photodegradation drug-excipients and drug-

drug interactions Factors determining the chemical stability of drug substances include

intrinsic factors such as molecular structure of the drug itself and environmental factors

such as temperature light pH buffer species ionic strength oxygen moisture additives

and excipients The application of well-established kinetic principles may throw light on

the role of each variable in altering the kinetics of degradation and to provide valuable

insight into the mechanism of degradation (Baertschi and Alsante 2005 Yoshioka and

Stella 2002 Lachman et al 1986) The chemical stability of individual components

within an emulsion system may be very different from their stability after incorporation

into other formulation types For example many unsaturated oils are prone to oxidation

and their degree of exposure to oxygen may be influenced by factors that affect the extent

of molecular dispersion (eg droplet size) This could be particularly troublesome in

emulsions because emulsification may introduce air into the product and because of the

high interfacial contact area between the phases (Barry 2002) The use of antioxidants

retards oxidation of unsaturated oils minimizes changes in color and texture and prevents

rancidity in the formulation Moreover they can retard the degradation of certain active

ingredients such as vitamin C (Vimaladevi 2005) The stability problems of dispersed

systems and the factors leading to these stability problems have been discussed by

Weiner (1996) and Lu and Flynn (2009)

333 Microbial Stability

Topical bases often contain aqueous and oily phases together with carbohydrates

and proteins and are susceptible to bacterial and fungal attack Microbial growth spoils

41

the formulation and is a potential toxic hazard Therefore topical formulations need

appropriate preservatives to prevent microbial growth and to maintain their quality and

shelf-life (Barry 2002 Arger et al 1996) Cream formulations may contain fats and oils

with high percentage of unsaturated linkages that are susceptible to oxidation degradation

and development of rancidity The addition of antioxidants retards oxidation of fats and

oils minimizes changes in color and texture and prevents rancidity in the formulation

Moreover they can retard the degradation of certain active ingredients such as vitamin C

These aspects in relation to dermatological formulations have been discussed by Barry

(1983 2002) and Vimaladevi 2005)

334 Stability of Ascorbic Acid in Liquid Formulations

Ascorbic acid is very unstable in aqueous solution Different workers have studied

the stability of ascorbic acid in liquid formulations (Connors et al 1986 Austria et al

1997) Its shelf-life can be prolonged by appropriate choice of vehicle and control of

other variables such as pH stabilizers temperature light and oxygen (Table 3)

Similarly the stability of various concentrations of ascorbic acid in solution form may

vary depending upon the type of solvent used (Table 4) (Connors et al 1986 Satoh et

al 2000 Lee et al 2004 Zeng et al 2005)

335 Stability of Ascorbic Acid in Emulsions and Creams

Ascorbic acid exerts several functions on skin such as collagen synthesis

depigmentation and antioxidant activity Ultraviolet radiation generates reactive oxygen

species (ROS) which produce some harmful effects on the skin including photocarcinoma

and photoaging In order to combat these problems ascorbic acid as an antioxidant has

42

Table 3 Effect of vehicles on the stability of ascorbic acid ( ascorbic acid remaining in

solutions after storage at room temperature) (Connors et al 1986)

Storage Time (days) No Vehicle

30 60 90 120 180 240 360

1 Corn Syrup 965 925 920 920 900 860 760

2 Sorbitol 990 990 990 970 960 925 890

3 4 Carboxymethyl

Cellulose

840 680 565 380 ndash ndash ndash

4 Glycerin 100 100 990 990 970 935 920

5 Propylene glycol 995 990 980 945 920 900 900

6 Syrup USP 100 100 980 980 930 900 880

7 Syrup 212 gL 880 810 775 745 645 590 440

8 25 Tragacanth 785 620 510 320 ndash ndash ndash

9 Saturated solution of

Dextrose

990 935 875 800 640 580 510

10 Distilled Water 900 815 745 675 405 185 ndash

11 50 Propylene glycol +

50 Glycerin

980 ndash 960 ndash 933 ndash ndash

12 25 Distilled Water +

75 Sorbo (70 solution

of Sorbitol)

955 954 ndash 942 930 ndash ndash

13 50 Glycerin + 50

Sorbo

982 984 978 ndash ndash 914 ndash

43

Table 4 Stability of various concentrations of ascorbic acid in water propylene glycol

and USP syrup at room temperature ( of ascorbic acid remaining in solution)

(Connors et al 1986)

Storage Time (days) Concentration

(mg ml)

Solvent

30 60 90 120 180 240 360

10 Water 930 840 820 670 515 410 ndash

50 Water 940 920 880 795 605 590 300

100 Water 970 930 910 835 705 680 590

10 Propylene glycol 100 985 980 975 960 920 860

50 Propylene glycol 100 970 980 980 980 965 935

100 Propylene glycol 100 100 100 100 990 100 925

10 Syrup 100 100 980 990 970 960 840

50 Syrup 100 100 100 100 990 100 960

100 Syrup 100 100 100 100 100 100 995

44

been used in various dosage forms and in different concentrations (Darr et al 1996

Gallarate et al 1999 Zhang et al 1999 Pinnell et al 2001 Lee et al 2004 Raschke

et al 2004 Elmore 2005 Farahmand et al 2006 Maia et al 2006) Ascorbic acid has

good photoprotective ability against UVA-mediated phototoxicity (Darr et al 1996) A

variety of formulations containing ascorbic acid or its derivatives have been studied in

order to evaluate their stability and delivery through the skin (Gallarate et al 1999

Zhang et al 1999 Ozer et al 2000 Pinnell et al 2001 Lee et al 2004 Raschke et al

2004 Farahmand et al 2006) Formulations containing derivatives of ascorbic acid are

found to be more stable than ascorbic acid but they do not produce the same effect as that

of the parent compound probably due to the lack of redox properties (Heber et al 2006)

Effective delivery of ascorbic acid through topical preparations is a major factor that

should be critically evaluated as it may be dependent upon the nature or type of the

formulation (Gallarate et al 1999 Pinnell et al 2001) The pH of the formulation

should be on the acidic side (~ pH 35) for effective penetration of the vitamin in the skin

(Pinnell et al 2001) and for its stabilization in the formulation (Gallarate et al 1999)

Some other antioxidants such as alpha-tocopherol ferulic acid and sodium metabisulphite

have also been used in combination with ascorbic acid for the purpose of its stabilization

in topical formulations and to produce some synergistic effects (Darr et al 1996 Lin et

al 2005 Maia et al 2006 Tournas et al 2006) Effect of some rheological properties

such as viscosity and dielectric constant on the stability of ascorbic acid in emulsions has

also been investigated (Connors et al 1986) Viscosity of the medium is an important

factor that should be considered for the purpose of ascorbic acid stability as higher

viscosity formulations have shown some degree of protection (Ozer et al 2000

45

Szymula 2005) Along with other factors formulation type also plays an important role in

the stability of ascorbic acid It is reported that ascorbic acid is more stable in emulsified

system as compared to aqueous solutions (Gallarate et al 1999 Lee et al 2004) In

multiemulsions ascorbic acid is reported to be more stable as compared to simple

emulsions (Gallarate et al 1999 Ozer et al 2000 Lee et al 2004 Farahmand et al

2006)

Ascorbic acid and its derivatives have been used in a variety of cosmetic

formulations as an antioxidant pH adjuster anti-aging and photoprotectant (Elmore

2005) The control of instability of ascorbic acid poses a significant challenge in the

development of cosmetic formulations It is also reported that certain metal ions or

enzyme systems effectively convert ascorbic acidrsquos antioxidant action to pro-oxidant

activity (Elmore 2005) Therefore utilization of an effective antioxidant system is

required to maintain the stability of vitamin C in various preparations (Zhang et al 1999

Pinnell et al 2001 Maia et al 2006) The chemical stability of ascorbic acid has been

studied in emulsions and creams by several workers (Darr et al 1996 Gallarate et al

1999 Lee et al 2004 Raschke et al 2004 Elmore 2005 Farahmand et al 2006)

however there is a lack of information on the photostability of ascorbic acid in cream

formulations

336 Stability Testing of Emulsions

The stability testing of emulsions (creams) may be carried out by performing the

following tests (Billany 2002)

46

3361 Macroscopic examination

The assessment of the physical stability of an emulsion is made by an

examination of the degree of creaming or coalescence occurring over a period of time

This involves the calculation of the ratio of the volume of the creamed or separated part

of the emulsion and the total volume A comparison of these values can be made for

different products

3362 Globule size analysis

An increase in mean globule size with time (coupled with a decrease in globule

numbers) indicates that coalescence is the cause of this behavior This can be used to

compare the rates of coalescence for a variety of emulsion formulations For this purpose

microscopic examination or electronic particle counting devices (coulter counter) or

laser diffraction sizing are widely used

3363 Change in viscosity

Many factors may influence the viscosity of emulsions A change in apparent

viscosity may result from any variation in globule size or number or in the orientation or

migration of emulsifier over a period of time

3264 Accelerated stability tests

In order to compare the relative stabilities of a range of similar products it is

necessary to speed up the processes of creaming and coalescence by storage at elevated

temperatures and then carrying out the tests described in the above sections

337 FDA guidelines for semisolid preparations

According to FDA draft guidelines to the industry (Shah 1997) semisolid

preparations (eg creams) should be evaluated for appearance clarity color

47

homogencity odour pH consistency viscosity particle size distribution (when feasible)

assay degradation products preservative and antioxidant content (if present) microbial

limits sterility and weight loss when appropriate Additionally samples from

production lot or approved products are retained for stability testing in case of product

failure in the field Retained samples can be tested along with returned samples to

ascertain if the problem was manufacturing or storage related Appropriate stability data

should be provided for products supplied in closed-end tubes to support the maximum

anticipated use period during patient use and after the seal is punctured allowing product

contact with the cap cap lever Creams in large containers including tubes should be

assayed by sampling at the surface top middle and bottom of the container In addition

tubes should be sampled near the crimp The objective of stability testing is to determine

whether the product has adequate shelf-life under market and use conditions

48

OBJECT OF PRESENT INVESTIGATION

Ascorbic acid (vitamin C) is extensively used as a single ingredient or in

combination with vitamin B complex and other vitamins in the form of drops injectables

lotions and syrups It is an ingredient of anti-aging cosmetic products alone or along with

alpha-tocopherol (vitamin E) Ascorbic acid exerts several functions on the skin as

collagen synthesis depigmentation and antioxidant activity It protects the signs of

degenerative skin conditions caused by oxy-radical damage In solutions and creams

ascorbic acid is susceptible to air and light and undergoes oxidative degradation to

dehydroascorbic acid and inactive products The degradation is influenced by

temperature viscosity and polarity of the medium and is catalysed by metal ions

particularly Cu+2

Fe+2

and Zn+2

One of the major problems faced in cream preparations is the instability of

ascorbic acid as it may be exposed to light during formulation manufacturing and

storage and the possibility of photochemical degradation can not be neglected The

behaviour of ascorbic acid in light is of particular interest since no systematic kinetic

studies have been conducted on its photodegradation in these preparations under various

conditions The study of the formulation variables such as emulsifier humectants and pH

may throw light on the photostabilization of ascorbic acid in creams

The main object of this investigation is to study the behaviour of ascorbic acid in

cream preparations on exposure to UV light in the pharmaceutically useful pH range An

important aspect of the work is to study the interaction of ascorbic acid with other

vitamins such as riboflavin nicotinamide and alpha-tocopherol and the effect of certain

stabilizers such as citric acid tartaric acid and boric acid on its photodegradation In

49

addition it is intended to study the photolysis of ascorbic acid in organic solvents to

evaluate the effect of solvent characteristics (eg dielectric constant and viscosity) on the

stability of the vitamin The study of all these aspects may provide useful information to

improve the photostability and efficacy of ascorbic acid in cream preparations

An outline of the proposed plan of work is presented as follows

1 To prepare a number of oil-in-water cream formulations based on different

emulsifying agents and humectants containing ascorbic acid alone and in

combination with other vitamins and stabilizing agents

2 To perform photodegradation studies on ascorbic acid in creams using a UV

irradiation source with emission corresponding to the absorption maximum of

ascorbic acid

3 To identify the photoproducts of ascorbic acid in creams using chromatographic

and spectrophotometric methods

4 To apply appropriate and validated analytical methods for the assay of ascorbic

acid alone and in combination with other vitamins and stabilizing agents

5 To study the effect of solvent characteristics such as dielectric constant and

viscosity on the photolysis of ascorbic acid in aqueous and organic solvents

6 To evaluate the kinetics of photodegradation of ascorbic acid and its interactions

with other vitamins (riboflavin nicotinamide and alpha-tocopherol) in creams

7 To evaluate the effect of carbon chain length of the emulsifying agent and the

viscosity of the humectant on the photodegradation of ascorbic acid

50

8 To develop relationships between the rate of photodegradation of ascorbic acid

and the concentration pH carbon chain length of emulsifier viscosity of the

creams

9 To determine the effect of compounds such as citric acid tartaric acid and boric

acid used as stabilizing agents on the rate of photodegradation and stabilization

of ascorbic acid in creams

10 To present reaction schemes for the photodegradation of ascorbic acid and its

interactions with other vitamins

CHAPTER IV

MATERIALS

AND

METHODS

52

41 MATERIALS

Vitamins and Related Compounds

L-Ascorbic Acid vitamin C (5R)-5-[(1S)-12-dihydroxyethyl]-34-dihydroxyfuran-2(5H)-

one Merck

C6H8O6 Mr 1761

Dehydroascorbic Acid L-threo-23-hexodiulosonic acid γ-lactone Sigma

C6H6O6 Mr 1741

23-Diketogulonic Acid

C6H8O7 Mr 192

It was prepared according to the method of Homann and Gaffron (1964) by the

hydrolysis of dehydroascorbic acid

Riboflavin vitamin B2 (310-dihydro-78-dimethyl-10-[(2S3S4R)-2345-

tetrahydroxypentyl] benzopteridine-24-dione) Merck

C17H20N4O6 Mr 3764

Nicotinamide vitamin B3 (pyridine-3-carboxamide) Merck

C6H6N2O Mr 1221

Alpha-Tocopherol vitamin E ((2R)-2578-tetramethyl-2-[(4R8R)-4812-

trimethyltridecyl]-34-dihydro-2H-1-benzopyran-6-ol) Merck

C29H50O2 Mr 4307

Formylmethylflavin (78-dimethyl-10-formylmethylisoalloxazine)

C14H12N4O3 Mr 2843

53

Formylmethylflavin was synthesized according to the method of Fall and Petering

(1956) by the periodic acid oxidation of riboflavin It was recrystallized from absolute

methanol dried in vacuo and stored in the dark in a refrigerator

Lumichrome (78-dimethylalloxazine) Sigma

C12H10N4O2 Mr 2423

It was stored in the dark in a desiccator

Stabilizers

Boric Acid orthoboric acid Merck

H3BO3 Mr 618

Citric Acid 2-hydroxypropane-123-tricarboxylic acid Merck

C6H8O7H2O Mr 2101

L-Tartaric acid [(2R3R)-23-dihydroxybutanedioic acid] Merck

C4H6O6 Mr 1501

Emulsifying Agents

Stearic Acid (95) octadecanoic acid Merck

C18H36O2 Mr 2845

Palmitic Acid hexadecanoic acid Merck

C16H32O2 Mr 2564

Myristic Acid tetradecanoic acid Merck

C14H28O2 Mr 2284

Cetyl alcohol hexadecan-1-ol Merck

C16H34O Mr 2424

54

Humectants

Glycerin glycerol (propane-123-triol) Merck

C3H8O3 Mr 921

Propylene glycol (RS)-propane-12-diol Merck

C3H8O2 Mr 7610

Ethylene glycol ethane-12-diol Merck

C2H6O2 Mr 6207

Potassium Ferrioxalate Actinometry

Potassium Ferrioxalate

K3Fe(C2O4)3 3H2O Mr 4912

Potassium Ferrioxalate was prepared according to the method of Hatchard and

Parker (1956) Three volumes of 15 M potassium oxalate was mixed with one volume of

15 M ferric chloride with vigorous stirring The yellow green precipitate of potassium

ferrioxalate was recrystallized twice from water dried at 45 ordmC and stored in the dark in a

desiccator

Reagents

All the reagents and solvents used were of analytical grade obtained from BDH

Merck

Water

Freshly boiled distilled water was used throughout the work

55

42 METHODS

421 Cream Formulations

On the basis of the various cream formulations reported in the literature (Block

1996 Flynn 2002 Betageri and Prabhu 2002 Vimaladevi 2005 EIRI Board Lu and

Flynn 2009) the following basic formula was used for the preparation of oil-in-water

creams containing ascorbic acid

Oil phase Percentage (ww)

Emulsifier

Myristic palmitic stearic acid

Cetyl alcohol

120

30

Aqueous phase

Humectant

Ethylene glycol propylene glycol glycerin

50

Active ingredient

Ascorbic acid

20 (0114 M)

Neutralizer

Potassium hydroxide

10

Continuous phase

Distilled water

QS

Additional ingredientsa

Vitamins

Riboflavin (Vitamin B2)

Nicotinamide (Vitamin B3)

Alpha-Tocopherol (Vitamin E)

0002ndash001 (053ndash266times10ndash4

M)

028ndash140 (0023ndash0115 M)

017ndash086 (0395ndash200times10ndash2

M)

Stabilizers

Citric acid

Tartaric acid

Boric acid

010ndash040 (0476ndash190times10ndash2

M)

010ndash040 (067ndash266times10ndash2

M)

010ndash040 (0016ndash0065 M)

a The vitamin stabilizer concentrations used were found to be effective in promotion

inhibition of photodegradation of ascorbic acid in cream formulations

56

422 Preparation of Creams

The emulsifiers were melted at 70ndash80 ordmC in a glass jar immersed in a water bath

Ascorbic acid was separately dissolved in a small portion of distilled water Potassium

hydroxide and humectant were dissolved in the remaining portion of water and mixed

with the oily phase with constant stirring until the formation of a thick white mass It was

cooled to ~40 ordmC and the ascorbic acid solution was added The thick mass was mixed

using a mechanical mixer with a glass stirrer at 1000 rpm for 5 minutes The pH of the

cream was adjusted to the desired value and the contents again mixed for 10 minutes at

500 rpm All the creams were prepared under uniform conditions to maintain their

individual physical characteristics and stored at room temperature in airtight glass

containers protected from light

In the case of other vitamins nicotinamide was dissolved along with ascorbic acid

in water and added to the cream Riboflavin or alpha-tocopherol were directly added to

the cream and mixed thoroughly according to the procedure described above

In the case of stabilizing agents (citric tartaric and boric acids) the individual

compounds were dissolved in the ascorbic acid solution and added to the cream followed

by the procedure described above

The details of the various cream formulations used in this study are given in

chapters 5ndash7

57

423 Thin-Layer Chromatography (TLC)

The following TLC systems were used for the separation and identification of

ascorbic acid and photodegradation products

Adsorbent a) Silica gel GF 254 (250-microm) precoated plates

(Merck)

Solvent systems S1 acetic acid-acetone-methanol-benzene

(552070 vv) (Ganshirt and Malzacher 1960)

S2 ethanol-10 acetic acid-water (9010 vv)

(Bolliger and Konig 1969)

S3 acetonitrile-butylnitrile-water (66332 vv)

(Saari et al 1967)

Temperature 25ndash27 ordmC

Location of spots Ascorbic acid UV light 254 nm (Uvitec lamp

UK)

Dehydroascorbic acid Spray with a 3 aqueous

phenylhydrazine hydrochloride solution

424 pH Measurements

The measurements of pH of aqueous solutions and cream formulations were

carried out using an Elmetron LCD display pH meter (modelndashCP501 sensitivity plusmn 001

pH units) (Poland) with a combination electrode The electrode was calibrated

automatically in the desired pH range (25 ordmC) using the following buffer solutions

58

Phthalate pH 4008

Phosphate pH 6865

Disodium tetraborate pH 9180

The electrode was immersed directly into the cream (British Pharmacopoeia

2009) kept for few seconds to equilibrate and the pH adjusted in the range of 40ndash70

with phosphoric acid sodium hydroxide solution

425 Ultraviolet and Visible Spectrometry

The absorbance measurements and spectral determinations were performed on

Shimadzu UVndashVisible recording spectrophotometer (model UVndash1601) using matched

silica cells of 10 mm path length The cells were employed always in the same orientation

using appropriate control solutions in the reference beam The baseline was automatically

corrected by the built-in baseline memory at the initializing period Auto-zero adjustment

was made by a one-touch operation The instrument checked the wavelength calibration

(6561 nm) using the deuterium lamp at the initializing period The absorbance scale was

periodically checked using the following calibration standard (British Pharmacopoeia

2009)

0057ndash0063 gl of potassium dichromate in 0005 M sulphuric acid

The specific absorbance [A(1 1 cm)] of the solution should match the

following values with the stated limit of tolerance

Wavelength

(nm)

Specific absorbance

A (1 1 cm)

Maximum

tolerance

235 1245 1229ndash1262

257 1445 1428ndash1462

313 486 470ndash503

350 1073 1056ndash109

430 159 157ndash161

59

426 Photolysis of Ascorbic Acid

4261 Creams

A 2 g quantity of the cream was uniformly spread on several rectangular glass

plates (5 times 15 cm) covered with a 1 cm tape on each side to give a 1 mm thick layer The

plates were irradiated in a dark chamber using a Philips 30 watt TUV tube (100

emission at 254 nm the wavelength absorbed by ascorbic acid at pH 4ndash7) fixed

horizontally at a distance of 30 cm from the centre of the plates Each plate was removed

at appropriate interval and the cream was subjected to spectrophotometric assay and

chromatographic examination

4262 Aqueous and organic solvents

A 10ndash3

M solution of ascorbic acid (50 ml) prepared in water (pH 70 005 M

phosphate buffer) or in an organic solvent in a 100 ml beaker (Pyrex) was placed in a

water bath maintained at 20 plusmn 1 ordmC The solution was irradiated with the Philips 30 watt

TUV tube in a dark chamber as stated above Samples were withdrawn at appropriate

intervals for assay and chromatography

4263 Storage of creams in dark

In order to determine the stability of various cream formulations in the dark

samples were stored at room temperature in a cupboard protected from light for a period

of three months The samples were analyzed periodically for the content of ascorbic acid

and the presence of any degradation product

427 Measurement of Light Intensity

The potassium ferrioxalate actinometry was used for the measurement of light

intensity of the radiation source employed in this work This actinometer has been

60

developed by Parker (1953) and Hatchard and Parker (1956) and is considered as the

most useful actinometer over a wide range of wavelengths (254ndash577 nm) It has been

used by Holmstrom and Oster (1961) Byrom and Turnbull (1967) McBride and Moore

(1967) Ahmad (1968) Ahmad (1978) Ahmad et al (2004a 2004b 2005 2006a

2006b 2008 2009ab) Fasihullah (1988) Vaid (1998) Ansari (2002) and Ahmad (2009)

for the measurement of light intensity

The irradiation of potassium ferrioxalate solutions in sulphuric acid results in the

reduction of ferric ion to ferrous ion according to the following reaction

2Fe [(C2O4)3]3ndash

rarr 2 Fe (C2O4) + 3 (C2O4)2ndash

+ 2CO2 (31)

The amount of Fe2+

ions formed in the reaction may be determined by

complexation with 110-phenanthroline to give a red colored complex The absorbance of

the complex is measured at 510 nm

428 Procedure

An oxygen free 0006 M solution of potassium ferrioxalate (2947 gl) in 01 N

H2SO4 was placed in the reaction vessel and irradiated with the lamp used for the

photolysis of riboflavin The irradiation was carried out under nitrogen (90ndash120

bubblesminute) which also caused stirring of the solution The temperature of the

reaction vessel was maintained at 25 plusmn 1 ordmC during the reaction

An aliquot of the photolysed solution (1ndash2 ml) was pipetted out at suitable

intervals (up to 30 minutes) into a 10 ml volumetric flask to which was then added 09

ml of N H2SO4 + 1 ml (01) 110-phenanthroline + 05 ml buffer (60 ml N CH3COONa

+ 36 ml N H2SO4 made up to 100 ml with distilled water) The flask was made up

to the mark with distilled water (final pH 35) thoroughly shaken to mix the contents and

61

Fig 3 Spectral power distribution of TUV 30 W tube (Philips)

62

allowed to stand for one hour in the dark to develop the colorndashcomplex The absorbance

of the phenanthrolinendashferrous complex was measured in a 1 cm cell at 510 nm using the

appropriate solution as blank The amount of Fe2+

ions formed was determined from the

calibration graph The calibration graph was constructed in a similar manner using

several dilutions of 1 times 10ndash6

mole ml Fe2+

in 01 N H2SO4 (freshly prepared by dilution

from standardized 01 M FeSO4 in 01 N H2SO4) (Fig 8) The experimental value of the

molar absorptivity of Fe2+

complex as determined from the slope of the calibration graph

is equal to 111 times 104 M

ndash1 cm

ndash1 and is in agreement with the value reported by Parker

(1953) Using the values of the known quantum yield for ferrioxalate actinometer at

different wavelengths (Hatchard and Parker 1956) the number of Fe2+

ions formed

during photolysis the time of exposure and the fraction of the light absorbed by the

length of the actinometer solution employed the light intensity incident just inside the

front window of the photolysis cell can be calculated In the present case total absorption

of the light has been assumed

4281 Calculation

The number of Fe2+

ions formed during photolysis (nFe

2+) is given by the

equation

6023 times 1020

V1 V3 A Σ

n Fe

2+ =

V2 1 ε (32)

where V1 is the volume of the actinometer solution irradiated (ml)

V2 is the volume of the aliquot taken for analysis (ml)

V3 is the final volume to which the aliquot V2 is diluted (ml)

1 is the path length of the spectrophotometer cell used (1 cm)

A is the measured absorbance of the final solution at 510 nm

63

ε is the molar absorptivity of the Fe2+

complex (111 times 104 M

ndash1 cm

ndash1)

The number of quanta absorbed by the actinometer nabs can then be obtained as follows

n Fe

2+

Σ nabs = ф

(33)

where ф is the quantum yield for the Fe2+

formation at the desired wavelength

The number of quanta per second per cell nabs is therefore given by

Σ nabs 6023 times 1020

V1 V3 A nabs =

t =

ф V2 1 ε t (34)

where t is the irradiation time of the actinometer in seconds

The relative spectral energy distribution of the radiation source (Fig 3) shows

100 emission at 254 nm the wavelength used for the photolysis of ascorbic acid (λmax

265 nm at pH 4ndash7) The energy emitted by the radiation source at various wavelengths

can be calculated using the equation

1197 times 105

E (KJ molndash1

) = λ nm

(35)

The quantum efficiency of ferrioxalate actinometer at the wavelength absorbed by

ascorbic acid (ie 254 nm) is high although the sensitivity drops over 450 nm The

average intensity of the TUV tube used in this study was determined as 556 plusmn 012 times

1018

quanta sndash1

429 Viscosity Measurements

The viscosity of the cream formulations was measured with a Brookfield RV

viscometer (Model DV-II + Pro USA) The instrument was calibrated using the

manufacturerrsquos viscosity standard A 200 g quantity of the cream was placed in a beaker

and the spindle (TE) was dipped into the cream It was rotated at a speed of 06 rpm for

64

00

02

04

06

08

10

12

0 2 4 6 8 10 12

Concentration of Fe++

times 105 M

Ab

sorb

an

ce a

t 51

0 n

m

Fig 4 Calibration graph for the determination of K3Fe(C2O4)3

65

one minute and the viscosity was recorded at 25plusmn1 ordmC The test was repeated three times

to account for the experimental variability and the average viscosity was noted

4210 Assay Methods

42101 UV spectrophotometric method for the assay of creams containing ascorbic

acid alone

The creams were thoroughly mixed a quantity of 2 g was accurately weighed and

the assay of ascorbic acid was carried out by the UV method of Zeng et al (2005) In the

case of photodegraded creams (2 g) the material was completely removed from the glass

plate and transferred to a volumetric flask The method involved extraction of ascorbic

acid with methanol (3 times 10 ml) adjustment of the pH of combined methanolic solutions

to 20 (with H3PO4) dilution of the final solution with acidified methanol (pH 20) to 100

ml and measurement of the absorbance at 245 nm using appropriate blank The

concentration of ascorbic acid was calculated using 560 as the value of specific

absorbance [A (1 1 cm)] at the analytical wavelength (Table 5)

The same method was used for the assay of ascorbic acid in creams stored in the

dark and in the presence of individual stabilizing agents (citric tartaric and boric acids)

42102 Iodimetric method for the assay of ascorbic acid in creams containing

riboflavin nicotinamide and alpha-tocopherol

The assay of ascorbic acid in creams in the presence of riboflavin nicotinamide

and alpha-tocopherol was carried out according to the procedure of British

Pharmacopoeia (2009) as follows

The photolysed cream (2 g) was completely scrapped from the glass plate and

transferred to a flask containing 40 ml of distilled water and 10 ml of 1 M sulphuric acid

66

Table 5 Calibration data for ascorbic acid showing linear regression analysisa

λ max 245 nm

Concentration range 01ndash10 times 10ndash4

M (0176ndash1761 mg )

Slope 9920

SE (plusmn) of slope 00114

Intercept 00012

Correlation coefficient 09996

Molar absorptivity (ε) 9920 Mndash1

cmndash1

Specific absorbance [A (1 1 cm)] 560

a Values represent a mean of five determinations

67

was added The solution was titrated with 002 M iodine solution using 1 ml of starch

solution as indicator until a persistent violet-blue color was obtained Each ml of 002 M

iodine solution is equivalent to 352 mg of C6H8O6 The same method was used for the

assay of ascorbic acid in creams stored in the dark

42103 Spectrophotometric method for the assay of ascorbic acid in aqueous and

organic solvents

A 1 ml aliquot of the photolysed solutions of ascorbic acid in water or in an

organic solvent was evaporated to dryness under nitrogen at room temperature and the

residue redissolved in a small volume of methanol The solution was transferred to a 10

ml volumetric flask made up to volume with acidified methanol (pH 20) and the

absorbance measured at 245 nm using an appropriate blank The content of ascorbic acid

in the solutions was determined using 9920 Mndash1

cmndash1

as the value of molar absorptivity at

the analytical wavelength (Table 5)

CHAPTER V

PHOTODEGRADATION OF

ASCORBIC ACID IN

ORGANIC SOLVENTS AND

CREAM FORMULATIONS

69

51 INTRODUCTION

Ascorbic acid (vitamin C) is an essential micronutrient that performs important

metabolic functions (Packer and Fuchs 1999 Davey et al 2000 Johnston et al 2007)

It is an ingredient of anti-aging cosmetic products (Darr et al 1996 Gallarate et al

1999 Traikovich 1999 Zhang et al 1999 Ozer et al 2000 Nusgens et al 2001

Pinnell et al 2001 2003 Lee et al 2004 Raschke et al 2004 Sauermann et al 2004

Elmore 2005 Jentzsch et al 2005 Lin et al 2005 Placzek et al 2005 Carlotti et al

2006 Farahmand et al 2006 Heber et al 2006 Maia et al 2006 Tournas et al 2006)

and exerts several functions on the skin as collagen synthesis depigmentation and

antioxidant activity (Nusgens et al 2001 Spiclin et al 2003) As an antioxidant it

protects skin by neutralizing reactive oxygen species generated on exposure to sunlight

(Shindo et al 1994) In biological systems it reduces both oxygenndash and nitrogenndash based

free radicals (Higdon and Frei 2002) and thus delays the aging process In view of the

instability of ascorbic acid in skin care formulations (Bissett 2006) it is often used in

combination with another redox partner such as alpha-tocopherol (vitamin E) to retard its

oxidation (Wille 2005)

The details of the cream formulations used in this study are given in Table 6 The

results obtained on the photodegradation of ascorbic acid in aqueous organic solvents

and cream formulations are discussed in the following sections

70

Table 6 Composition of cream formulations containing ascorbic acid

Ingredients Cream

No pH

SA PA MA CA AH2 GL PG EG PH DW

1 a 4 + minus minus + + + minus minus + +

b 5 + minus minus + + + minus minus + +

c 6 + minus minus + + + minus minus + +

d 7 + minus minus + + + minus minus + +

2 a 4 minus + minus + + + minus minus + +

b 5 minus + minus + + + minus minus + +

c 6 minus + minus + + + minus minus + +

d 7 minus + minus + + + minus minus + +

3 a 4 minus minus + + + + minus minus + +

b 5 minus minus + + + + minus minus + +

c 6 minus minus + + + + minus minus + +

d 7 minus minus + + + + minus minus + +

4 a 4 + minus minus + + minus + minus + +

b 5 + minus minus + + minus + minus + +

c 6 + minus minus + + minus + minus + +

d 7 + minus minus + + minus + minus + +

5 a 4 minus + minus + + minus + minus + +

b 5 minus + minus + + minus + minus + +

c 6 minus + minus + + minus + minus + +

d 7 minus + minus + + minus + minus + +

6 a 4 minus minus + + + minus + minus + +

b 5 minus minus + + + minus + minus + +

c 6 minus minus + + + minus + minus + +

d 7 minus minus + + + minus + minus + +

7 a 4 + minus minus + + minus minus + + +

b 5 + minus minus + + minus minus + + +

c 6 + minus minus + + minus minus + + +

d 7 + minus minus + + minus minus + + +

8 a 4 minus + minus + + minus minus + + +

b 5 minus + minus + + minus minus + + +

c 6 minus + minus + + minus minus + + +

d 7 minus + minus + + minus minus + + +

9 a 4 minus minus + + + minus minus + + +

b 5 minus minus + + + minus minus + + +

c 6 minus minus + + + minus minus + + +

d 7 minus minus + + + minus minus + + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PG = propylene glycol EG = ethylene glycol

PH = potassium hydroxide DW = distilled water

71

52 PHOTOPRODUCTS OF ASCORBIC ACID

The photolysis of ascorbic acid (AH2) in aqueous and organic solvents and in

cream formulations on UV irradiation leads to the formation of dehydroascorbic acid

(DHA) as detected by TLC along with the undegraded AH2 using the solvent systems A

B and C The identification of DHA was carried out by comparison of the Rf value and

spot color with those of the authentic compound The formation of DHA on

photooxidation of ascorbic acid solutions has previously been reported (Homan and

Gaffron 1964 Sattar et al 1977 Heelis et al 1981 Rozanowska et al 1997 Lavoie et

al 2004) DGA the hydrolysis product of DHA (Homan and Gaffron 1964) could not

be detected under the present experimental conditions

53 SPECTRAL CHARACTERISTICS OF PHOTOLYSED SOLUTIONS

A typical set of the UV absorption spectra of photolysed solutions of AH2 in

methanol is shown in Fig 5 There is a gradual loss of absorbance around 245 nm with

time as a result of the oxidation of the molecule to DHA (Pelletier 1985 Davies et al

1991 Rumsey and Levine 2000) which does not absorb in this region due to the loss of

conjugation Similar absorption changes are observed on the photolysis of AH2 in other

organic solvents and in the methanolic extracts of cream formulations However the

magnitude of these changes varies with the rate of photolysis in a particular solvent or

cream and appears to be a function of the polar character pH and viscosity of the

medium

72

Fig 5 UV absorption spectra of photolysed solutions of ascorbic acid in methanol at

0 40 80 120 160 220 and 300 min

73

54 ASSAY OF ASCORBIC ACID IN CREAMS AND SOLUTIONS

The assay of AH2 in creams and solutions has been carried out in acidified

methanol (pH 20) according to the UV spectrophotometric method of Zeng et al (2005)

Aqueous solutions of AH2 (~pH 2) exhibit absorption maxima at 243 nm (OrsquoNeil 2001

Moffat et al 2004 Sweetman 2009) 244 nm (Ogata and Kosugi 1969) and 245 nm

(Verma et al 1991 Johnston et al 2007) The absorption maxima of AH2 in methanol

and phosphate buffer (pH 25) occur at 245 nm (Zeng et al 2005) Since dilute solutions

of AH2 are highly susceptible to oxidation the pH was adjusted to 20 with phosphoric

acid to convert the molecule to the non-ionized form (99) to minimize degradation

during the assay AH2 in acidified methanol (pH 20) was found to exhibit the absorption

maximum at 245 nm as reported by Zeng et al (2005) The method was also used for the

assay of AH2 in aqueous and organic solvents

The validity of Beerrsquos law relation in the concentration range used was confirmed

prior to the assay The calibration data for AH2 at the analytical wavelength are presented

in Table 5 (Chapter 4) The correlation coefficient (r = 09996) indicates a good linear

relationship over the concentration range employed The values of specific absorbance

and molar absorptivity at 245 nm determined from the slope of the curve are in good

agreement with those reported by previous workers (Davies et al 1991 Johnston et al

2007) The method of Zeng et al (2005) has been found to be satisfactory for the assay of

AH2 in cream formulations and solutions and has been used to study the kinetics of

photolysis reactions The method was validated before its application to the assay of AH2

in photolysed creams The reproducibility of the method was confirmed by the analysis of

known amounts of AH2 in the concentration range likely to be found in photodegraded

74

creams The values of the recoveries of AH2 in creams by the UV spectrophotometric

method are in the range of 90ndash96 The values of RSD for the assays indicate the

precision of the method within plusmn5 (Table 7)

In order to compare the UV spectrophotometric method with the British

Pharmacopoeia iodimetric method (2009) using a dilute iodine solution (002 M) the

creams were simultaneously assayed for AH2 content by the two methods and the results

are reported in Table 8 The statistical evaluation of the accuracy and precision of the two

methods was carried out by the application of the F test and the t test respectively The F

test showed that there is no significant difference between the precision of the two

methods and the calculated value of F is lower than the critical value (F = 639 P = 005)

in each case The t test indicated that the calculated t values are lower than the tabulated t

values (t = 2776 P = 005) suggesting that at 95 confidence level the differences

between the results of the two methods are statistically non-significant Thus the accuracy

and precision of the UV spectrophotometric method is comparable to that of the official

iodimetric method for the assay of AH2 in cream formulations The results of the assays

of AH2 in aqueous organic solvents and cream formulations are reported in Table 9

55 EFFECT OF SOLVENT

The influence of solvent on the rate of degradation of drugs is of considerable

importance to the formulator since the stability of drug species in solution media may be

predicted on the basis of their chemical reactivity The reactivity of drugs in a particular

medium depends to a large extent on solvent characteristics such as the dielectric

constant and viscosity (Connors et al 1986 Yoshioka and Stella 2000 Sinko 2006)

75

Table 7 Recovery of ascorbic acid added to cream formulationsa

Cream

Formulationb

Added

(mg)

Found

(mg)

Recovery

()

RSD

()

1a 400

200

380

183

950

915

21

25

2b 400

200

371

185

928

925

15

25

3c 400

200

375

181

938

905

11

31

4d 400

200

384

189

960

945

13

21

5b 400

200

370

189

925

945

14

26

6c 400

200

369

190

923

950

10

22

7d 400

200

374

182

935

910

17

39

8c 400

200

380

188

950

940

15

33

9d 400

200

367

189

918

945

20

42

a Values expressed as a mean of three to five determinations

b The cream formulations represent combinations of each emulsifier (stearic acid

palmitic acid myristic acid) with each humectant (glycerin propylene glycol ethylene

glycol) to observe the efficiency of methanol to extract AH2 from different creams

(Table 6)

76

Table 8 Assay of ascorbic acid in creams using UV spectrophotometric and iodimetric

methods

Ascorbic acid (mg) Cream

Formulationb Added UV method

a

Iodimetric

methoda

Fcalc tcalc

1a 40

20

380 plusmn 081

183 plusmn 046

375 plusmn 095

185 plusmn 071

138

238

245

104

2b 40

20

371 plusmn 056

185 plusmn 047

373 plusmn 064

193 plusmn 038

130

065

181

200

3c 40

20

375 plusmn 040

181 plusmn 056

374 plusmn 046

183 plusmn 071

132

160

101

223

4d 40

20

384 plusmn 051

189 plusmn 039

381 plusmn 066

190 plusmn 052

167

178

176

231

5b 40

20

370 plusmn 052

189 plusmn 050

372 plusmn 042

185 plusmn 067

065

179

162

125

6c 40

20

369 plusmn 037

190 plusmn 042

371 plusmn 058

188 plusmn 056

245

177

122

197

7d 40

20

374 plusmn 062

182 plusmn 072

370 plusmn 070

184 plusmn 082

127

129

144

168

8c 40

20

380 plusmn 058

188 plusmn 062

375 plusmn 075

192 plusmn 060

167

094

123

162

9d 40

20

367 plusmn 072

189 plusmn 080

365 plusmn 082

187 plusmn 075

149

092

130

203

Theoretical values (P = 005) for F is 639 and for t is 2776

a Mean plusmn SD (n = 5)

b Table 6

77

Table 9 Photodegradation of ascorbic acid in cream formulations

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) pHa 40 50 60 70

0 383 382 384 383

60 374 369 366 361

120 361 354 346 325

180 351 345 325 305

240 345 327 301 284

1

300 336 316 287 264

0 380 383 382 379

60 371 376 362 346

120 359 357 342 320

180 352 345 322 301

240 341 335 299 283

2

300 336 321 291 261

0 384 376 381 385

60 377 367 360 358

120 366 348 334 324

180 356 337 317 305

240 343 320 301 282

3

300 335 307 273 253

78

Table 9 continued

0 377 378 386 372

60 365 361 371 355

120 353 345 347 322

180 344 327 325 298

240 332 320 306 279

4

300 317 303 284 252

0 381 367 372 373

60 372 358 358 353

120 360 337 336 321

180 352 325 320 302

240 341 313 300 284

5

300 327 302 278 256

0 376 386 380 377

60 366 372 350 350

120 353 347 323 316

180 337 334 308 298

240 329 320 291 274

6

300 313 306 267 245

79

Table 9 continued

0 380 372 378 380

60 373 362 350 354

120 358 340 329 321

180 344 328 304 300

240 332 315 292 283

7

300 319 302 272 252

0 380 381 378 361

60 368 364 361 335

120 355 354 340 313

180 342 340 315 281

240 337 331 303 269

8

300 323 314 281 248

0 378 382 370 375

60 370 369 349 342

120 356 347 326 321

180 339 333 298 291

240 326 314 277 271

9

300 313 302 265 242

a The values at pH 40ndash70 represent the formulations a to d of each cream respectively

80

In order to observe the effect of solvent dielectric constant the apparent first-

order rate constants (kobs) for the photolysis of AH2 in alcoholic solvents (Table 10) were

plotted against the dielectric constants of the solvents A linear relationship indicated the

dependence of the rates of photolysis on solvent dielectric constant (Fig 6) This implies

the involvement of a polar intermediate in the reaction to facilitate the formation of the

degradation products as suggested by Ahmad and Tollin (1981) in the case of flavin

electron transfer reactions The effect of solvent polarity has been observed on the

autooxidation of AH2 in organic solvents (Ogata and Kosugi 1969)

Another solvent parameter affecting the rate of a chemical reaction is viscosity

which can greatly influence the stability of oxidisable substances (Wallwork and Grant

1977 Laidler 1987 Fung 1990) A plot of kobs for the photolysis of AH2 against the

reciprocal of solvent viscosity (Table 10) is linear showing that an increase in solvent

viscosity results in a decrease in the rate of photolysis (Fig 7) The viscosity of the liquid

affects the rate at which molecules can diffuse through the solution This in turn may

affect the rate at which a compound can suffer oxidation at the liquid surface This

applies to AH2 and an increase in the viscosity of the medium makes access to air at the

surface more difficult to prevent oxidation (Wallwork and Grant 1977)

56 EFFECT OF CONCENTRATION

In order to observe the effect of concentration (Table 11) on the photostability of

AH2 in a cream using stearic palmitic and myristic acids as emulsifying agents and

glycerin as humectant plots of log concentration versus time were constructed (Fig 8)

and the apparent first-order rate constants were determined (Table 12) A graph of kobs

against concentration of AH2 (Fig 9) exhibited an apparent linear relationship between

81

Table 10 Apparent first-order rate constants (kobs) for the photolysis of ascorbic acid in

water and organic solvents

Solvent Dielectric

Constant (25 ordmC)

Viscosity

(mPas) ndash1

kobs times104

(minndash1

)

Water 785 1000 404

Methanol 326 1838 324

Ethanol 243 0931 316

1-Propanol 201 0514 302

1-Butanol 178 0393 295

82

00

20

40

60

80

0 10 20 30 40 50 60 70 80

Dielectric constant

k (

min

ndash1)

Fig 6 A plot of kobs for photolysis of ascorbic acid against solvent dielectric constant

(times) Water () methanol () ethanol (diams) 1-propanol () 1-butanol

83

00

10

20

30

40

50

00 05 10 15 20

Viscosity (mPas)ndash1

k times

10

4 (m

inndash1)

Fig 7 A plot of kobs for photolysis of ascorbic acid against reciprocal of solvent

viscosity Symbols are as in Fig 6

84

Table 11 Effect of concentration on the photodegradation of ascorbic acid in cream

formulations at pH 60

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) 05 10 15 20 25

0 95 191 290 379 471

60 90 182 277 358 453

120 82 167 260 339 431

180 77 158 239 311 401

240 70 144 225 298 382

1

300 64 134 210 282 363

0 92 186 287 380 472

60 88 175 272 369 453

120 82 160 251 342 429

180 75 152 238 326 405

240 71 144 225 309 392

2

300 65 134 215 289 366

0 94 182 286 376 470

60 87 171 265 352 454

120 78 152 251 337 426

180 69 143 227 315 404

240 62 129 215 290 378

3

300 58 119 195 271 353

85

05

10

15

20

25

06

08

10

12

14

16

18

log

co

nce

ntr

ati

on

(m

g)

a

05

10

15

20

25

06

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

b

05

10

15

20

25

06

08

10

12

14

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

c

Fig 8 Log concentration versus time plots for the photodegradation of ascorbic acid at

various concentrations in creams at pH 60 a) stearic acid b) palmitic acid

c) myristic acid

86

Table 12 Apparent first-order rate constants (kobs) for the photodegradation of various

ascorbic acid concentrations in cream formulations at pH 60

kobs times 103 (min

ndash1)a Cream

Formulationb 05 10 15 20 25

1 133

(0994)

120

(0993)

111

(0995)

101

(0994)

090

(0994)

2 118

(0992)

108

(0994)

098

(0993)

093

(0992)

084

(0994)

3 169

(0994)

144

(0995)

126

(0994)

109

(0993)

097

(0992)

a The values in parenthesis are correlation coefficients

b Table 6

87

Stearic acid

Palmitic acid

Myristic acid

00

05

10

15

20

25

00 05 10 15 20 25

Ascorbic acid concentration ()

kob

s (min

ndash1)

Fig 9 A plot of kobs for photodegradation against ascorbic acid concentrations in cream

formulations

88

the two values Thus the rate of degradation of AH2 is faster at a lower concentration on

exposure to the same intensity of light This may be due to a relatively greater number of

photons available for excitation of the molecule at lower concentration compared to that

at a higher concentration The AH2 concentrations of creams used in this study are within

the range (1ndash15) reported by previous workers for topical applications to skin (Kaplan

et al 1989 Traikovich et al 1999 Nusgens et al 2001 Matsubayashi et al 2003

Espinal-Perez et al 2004 Sauermann et al 2004 Lin et al 2005 Heber et al 2006)

57 EFFECT OF CARBON CHAIN LENGTH OF EMULSIFYING AGENT

The values of kobs for the photodegradation of AH2 (2) in various cream

formulations are reported in Table 13 The first-order plots for the photodegradation of

AH2 at pH 4ndash7 in various cream formulations are shown in Fig 10ndash12 The plots of kobs

against carbon chain length of the emulsifying agents are shown in Fig 13 They indicate

that the photodegradation of AH2 is affected by the emulsifying agent in the order

myristic acid gt stearic acid gt palmitic acid

These acids possess a polar character (Yao et al 2009) and the carbon chain of the acid

may play a part in the photostability of AH2 However the results indicate that in the

presence of palmitic acid AH2 exhibits greater stability as indicated by the plots of kobs

versus the carbon chain length of the emulsifying agents (Fig 13) This could be

predominantly due to the interaction of AH2 with palmitic acid in the cream to impart it

greater stability Ascorbic acid-6-palmitate is known to be an antioxidant in cosmetic

preparations (Lee et al 2009) and food products (Doores 2002)

In view of the above observations it may be suggested that the photodegradation

of AH2 could involve a polar semiquinone intermediate (Johnston et al 2007) which

89

Table 13 First-order rate constants (kobs) for the photodegradation of ascorbic acid in

cream formulations

kobs times 103 (min

ndash1)abc

Cream

Formulation pH 40 50 60 70

1 044

(0992)

064

(0994)

100

(0995)

126

(0995)

2 042

(0992)

060

(0991)

095

(0992)

120

(0995)

3 047

(0993)

069

(0993)

107

(0991)

137

(0995)

4 056

(0993)

072

(0992)

104

(0994)

131

(0993)

5 050

(0991)

067

(0992)

097

(0991)

124

(0992)

6 061

(0992)

079

(0993)

113

(0992)

140

(0994)

7 060

(0992)

071

(0993)

108

(0994)

133

(0992)

8 053

(0991)

062

(0992)

099

(0994)

126

(0993)

9 065

(0991)

081

(0996)

117

(0993)

142

(0995)

a The rate constants at pH 40ndash70 represent the values for formulations a to d of each

cream respectively

b The values in parenthesis are correlation coefficients

c The values of rate constants are relative and depend on specific experimental conditions

including light intensity

The estimated error is plusmn5

90

12

13

14

15

16

17

log

co

nce

ntr

ati

on

(m

g)

1

12

13

14

15

16

17

log

co

nce

ntr

ati

on

(m

g)

2

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

3

Fig 10 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (1ndash3) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

91

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

4

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

5

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

6

Fig 11 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (4ndash6) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

92

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

7

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

8

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

9

Fig 12 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (7ndash9) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

93

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

ko

bs times

10

3 (m

inndash

1)

1-3

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

ko

bs times

10

3 (

min

ndash1)

4-6

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

Carbon chain length

ko

bs times

10

3 (

min

ndash1)

7-9

Fig 13 Plots of kobs for photodegradation of ascorbic acid in creams (1ndash9) against carbon

chain length of emulsifier () Stearic acid () palmitic acid () myristic acid

Humectant used glycerin (1ndash3) propylene glycol (4ndash6) ethylene glycol (7ndash9)

94

depending on the polar character of the medium undergoes oxidation with varying rates

This is similar to the behavior of the photolysis of riboflavin analogs which is dependent

on the polar character of the medium (Ahmad and Tollin 1981) The effect of carbon

chain length on the transdermal delivery of an active ingredient has been discussed (Lu

and Flynn 2009)

58 EFFECT OF VISCOSITY

The plots of rates of AH2 degradation in cream formulations (Table 13) as a

function of carbon chain length (Fig 13) indicate that the rates vary with the humectant

and hence the viscosity of the medium in the order

ethylene glycol gt propylene glycol gt glycerin

This is in agreement with the rate of photolysis of AH2 in organic solvents that

higher the viscosity of the medium lower the rate of photolysis Thus apart from the

carbon chain length of the emulsifier viscosity of the humectant added to the cream

formulation appears to play an important part in the stability of AH2 The stabilizing

effect of viscosity imparting substances on AH2 solutions has been reported (Stone 1969

Kassem et al 1969ab)

59 EFFECT OF pH

The kobsndashpH profiles for the photodegradation of AH2 in various creams (1ndash9) at

pH 4ndash7 (Fig 14) represent a sigmoid type curve indicating the oxidation of the ionized

form (AHndash) of AH2 (pKa 41) (OrsquoNeil 2001) with pH The AH

ndash species appears to be

more susceptible to photooxidation than the non-ionized form (AH2) The behavior of

AH2 on photooxidation in the pH range 4ndash7 is similar to that observed for the chemical

oxidation of AH2 by molecular oxygen (DeRitter 1982) and involves the interaction of

95

Stearic Acid (1)

Palmitic Acid (2)

Myristic Acid (3)

04

06

08

10

12

14

kob

s times

10

3 (m

inndash

1)

Stearic Acid (4)

Palmitic Acid (5)

Myristic Acid (6)

04

06

08

10

12

14

ko

bs times

10

3 (

min

ndash1)

Stearic Acid (7)

Palmitic Acid (8)

Myristic Acid (9)

04

06

08

10

12

14

30 40 50 60 70

pH

ko

bs

times 1

03

(min

ndash1)

Fig 14 The kobsndashpH profiles for the photodegradation of ascorbic acid in creams (1ndash9)

Glycerin

Propylene glycol

Ethylene glycol

96

AH2 with singlet oxygen on UV irradiation (Silva and Quina 2006) The AHndash species

(predominant in the pH range 42ndash70 557ndash999) is more reactive towards singlet

oxygen than its protonated form the AH2 molecule as suggested by Bisby et al (1999)

and therefore the rate of photooxidation is higher in the pH range above 41

corresponding to the pKa1 of AH2 The major goal of a ratendashpH profile is to determine

the optimum pH range for a particular formulation Several workers have studied the

ratendashpH profiles of the chemical oxidation of AH2 in the pH range 2ndash7 (Garrett 1967

Taqui Khan and Martell 1967 Rogers and Yacomeni 1971 Blaugh and Hajratwala

1972 DeRitter 1982 Moura et al 1994) however the kinetics of photooxidation of

AH2 in cream formulations has so far not been reported

510 EFFECT OF REDOX POTENTIAL

The photooxidation of AH2 is also influenced by its redox potential which varies

with pH The greater photostability of AH2 at pH 5ndash6 compared to that at pH 7 and above

is due to its lower rate of oxidationndashreduction in this range (Eordm pH 50 = +0127 V)

(OrsquoNeil 2001) The increase in the rate of photooxidation with pH is due to a

corresponding increase in the redox potential (Eordm pH 70 = +0058 V) (Fasman 1976) of

AH2 and is similar to the photolysis behavior of riboflavin at pH 5ndash6 (Eordm pH 50 = ndash0117

V) (Sinko 2006) compared to that at pH 70 (Eordm pH 70 = ndash 0207 V) (Ahmad et al

2004a Sinko 2006) Since the ionization as well as the redox potentials of AH2 are a

function of pH the rate of photooxidation depends upon the specific species present and

its redox behavior at a particular pH

97

511 PRIMARY PHOTOCHEMICAL REACTIONS IN THE OXIDATION OF

ASCORBIC ACID

A reaction scheme based on general photochemical principles for the important

reactions involved in the photooxidation of ascorbic acid is presented below

0AH2 hv k1

1AH2 (51)

1AH2 k2 Products (52)

1AH2 isc k3

3AH2 (53)

3AH2 k4 Products (54)

0AH

ndash hv k5

1AH

ndash (55)

1AH

ndash k6 Products (56)

1AH

ndash k7

3AH

ndash (57)

3AH

ndash k8 Products (58)

3AH

ndash +

0AH2 k9 AH٠

ndash + AH٠ (59)

2 AH٠ k10 A + AH2 (510)

3AH2 +

3O2 k11

0AH2 +

1O2 (511)

AHndash +

1O2 k12

3AH

ndash +

3O2 (512)

AH٠ + 1O2 k13 AHOO٠ (513)

AHOO٠ k14 A + HO2٠ (514)

AHOO٠ + 0AH2 k15 AH٠ + AHOOH (515)

AHOOH k16 secondary reaction

A + H2O2 (516)

According to this reaction scheme the ground state ascorbic acid species (0AH2

0AH

ndash) each is excited to the lowest singlet state (

1AH2

1AH

ndash) by the absorption of a

quantum of UV light (51 55) These excited states may directly be converted to

98

photoproducts (52 56) or may undergo intersystem crossing (isc) to form the excited

triplet states (53 57) The excited triplet states may then degrade to the photoproducts

(54 58) The monoascorbate triplet (3AH

ndash) may react with the ground state ascorbic

acid to form a monoascorbate radical anion (AH٠ndash) and a monoascorbate radical (AH٠)

(59) Two AH٠ radical species may lead to the formation of an oxidized (A) and a

reduced ascorbic acid molecule (AH2) (510) Ascorbic acid triplet (3AH2) may react with

molecular oxygen (3O2) to yield singlet oxygen (

1O2) (511) which may then react with

monoascorbate anion (AHndash) to form the excited triplet state (

3AH

ndash) (512) or with

monoascorbate radical to form a peroxyl radical (AHOO٠) (513) The peroxyl radical

may yield dehydroascorbic acid (A) (514) or react with ground state ascorbic acid to

give monoascorbate radical and a reduced species AHOOH (515) The reduced species

may give rise to dehydroascorbic acid and hydrogen peroxide (516)

512 DEGRADATION OF ASCORBIC ACID IN THE DARK

In view of the instability of AH2 and to observe its degradation in the dark the

creams were stored in airtight containers at room temperature in a cupboard for a period

of about 3 months and assayed for AH2 content at appropriate intervals The analytical

data (Table 14) were subjected to kinetic treatment (Fig 15ndash17) and the apparent first-

order rate constants for the degradation of AH2 were determined (Table 15) The values

of the rate constants indicate that the degradation of AH2 in the dark is about 70 times

slower than those of the creams exposed to UV irradiation (Table 13) The degradation of

AH2 in creams in the dark is due to chemical oxidation (Section 132) and occurs in the

order of emulsifying agents (Fig 18)

myristic acid gt stearic acid gt palmitic acid

99

Table 14 Degradation of ascorbic acid in the dark in cream formulations

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(days) pHa 40 50 60 70

0 383 382 384 383

10 354 340 313 278

20 309 306 279 245

40 244 209 183 161

60 172 166 131 105

1

80 145 114 81 61

0 380 383 382 379

10 360 343 350 335

20 322 310 301 294

40 266 250 211 186

60 233 211 168 142

2

80 182 153 114 89

0 384 376 381 385

10 368 350 340 318

20 318 273 273 266

40 223 199 172 155

60 174 132 117 84

3

80 122 97 66 54

100

Table 14 continued

0 377 378 386 372

10 350 334 334 318

20 314 268 256 244

40 238 208 182 136

60 179 155 107 94

4

80 128 101 79 59

0 381 367 372 373

10 350 293 300 320

20 299 266 270 263

40 220 191 192 184

60 183 153 139 129

5

80 149 115 87 76

0 376 386 380 377

10 312 320 314 251

20 255 282 226 199

40 175 194 159 131

60 139 128 99 74

6

80 102 81 55 41

101

Table 14 continued

0 380 372 378 380

10 323 330 333 323

20 288 273 276 224

40 212 174 182 146

60 152 133 108 83

7

80 100 82 66 56

0 380 381 378 361

10 333 320 310 310

20 281 266 260 257

40 230 189 171 177

60 156 148 128 111

8

80 123 96 78 66

0 378 382 370 375

10 313 295 281 300

20 256 247 257 203

40 194 178 151 133

60 119 114 88 74

9

80 88 68 49 39

a The values at pH 40ndash70 represent the formulations a to d of each cream respectively

102

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

1

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

2

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

3

Fig 15 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (1ndash3) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

103

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

4

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

5

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

6

Fig 16 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (4ndash6) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

104

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

7

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

8

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

9

Fig 17 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (7ndash9) at pH 40 (times) 50 () 60 () and 70 ()

Palmitic acid

Myristic acid

Stearic acid

105

Table 15 First-order rate constants (kobs) for the degradation of ascorbic acid in cream

formulations in the dark

kobs times 102 (day

ndash1)abc

Cream

Formulation pH 40 50 60 70

1 128

(0991)

152

(0994)

191

(0995)

220

(0994)

2 091

(0992)

110

(0991)

152

(0993)

182

(0992)

3 148

(0991)

176

(0995)

220

(0993)

254

(0995)

4 137

(0992)

161

(0993)

205

(0994)

236

(0995)

5 121

(0991)

141

(0994)

175

(0993)

195

(0993)

6 162

(0992)

194

(0995)

237

(0994)

265

(0994)

7 164

(0994)

189

(0994)

222

(0993)

246

(0996)

8 143

(0994)

167

(0995)

193

(0996)

212

(0993)

9 184

(0995)

208

(0994)

251

(0992)

280

(0996)

a The rate constants at pH 40ndash70 represent the values for formulations a to d of each

cream respectively

b The values in parenthesis are correlation coefficients

c The values of rate constants are relative and depend on specific experimental

conditions

The estimated error is plusmn5

106

pH 4

pH 5

pH 6

pH 7

00

10

20

30

k times

10

2 (d

ayndash

1)

1-3

pH 4

pH 5

pH 6

pH 7

00

10

20

30

k times

10

2 (

da

yndash1)

4-6

pH 4

pH 5

pH 6

pH 7

00

10

20

30

12 14 16 18

Carbon chain length

k times

10

2 (

da

yndash1)

7-9

Fig 18 Plots of kobs for degradation of ascorbic acid in the dark in creams (1ndash9) against

carbon chain length of emulsifier () Stearic acid () palmitic acid

() myristic acid Humectant used glycerin (1ndash3) propylene glycol (4ndash6)

ethylene glycol (7ndash9)

107

Although it is logical to expect a linear relationship between the rate of degradation and

the carbon chain length of the emulsifier due to its polar character (Yao et al 2009) it

has not been observed in the present case The reason for the slowest rate of degradation

of AH2 in the presence of palmitic acid appears to be due to the interaction of AH2 with

palmitic acid (Lee et al 2009) as explained in Section 57

The degradation of AH2 also appears to be affected by the viscosity of the cream

in the order of humectant (Fig 19)

ethylene glycol gt propylene glycol gt glycerin

Thus the presence of glycerin imparts the most stabilizing effect on the degradation of

AH2 This is the same order as observed in the case of photodegradation of AH2 in the

creams The airtight containers used for the storage of creams make the access of air to

the creams difficult to cause chemical oxidation of AH2 However it has been observed

that the degradation of AH2 is highest in the upper layer of the creams compared to that

of the middle and the bottom layers Therefore the creams were thoroughly mixed before

sampling for the assay of AH2 However the scattering in kinetic plots (Fig 15ndash17) is

due to non-uniform distribution of AH2 in degraded creams

The effect of pH on the degradation of AH2 in the creams (Fig 19) shows that the

degradation increases with an increase in pH as observed in the case of photodegradation

of AH2 in the creams This is due to an increase in the ionization and redox potential of

AH2 with pH causing greater oxidation of the molecule and has been discussed in

Sections 59 and 510

108

Stearic Acid (1)

Palmitic Acid (2)

Myristic Acid (3)

00

10

20

30

k times

10

2 (d

ayndash

1)

Glycerin

Stearic Acid (4)

Palmitic Acid (5)

Myristic Acid (6)

00

10

20

30

k times

10

2 (

da

yndash1)

Propylene glycol

Stearic Acid (7)

Palmitic Acid (8)

Myristic Acid (9)

00

10

20

30

30 40 50 60 70

pH

k times

10

2 (d

ayndash

1)

Ethylene glycol

Fig 19 The kobsndashpH profiles for the degradation of ascorbic acid in the dark in creams

(1ndash9)

CHAPTER VI

PHOTOCHEMICAL INTERACTION

OF ASCORBIC ACID WITH

RIBOFLAVIN NICOTINAMIDE

AND ALPHA-TOCOPHEROL IN

CREAM FORMULATIONS

110

61 INTRODUCTION

It is now medically recognized that sagging skin and other signs of degenerative

skin conditions such as wrinkles and age spots are caused primarily by oxy-radical

damage Ascorbic acid can accelerate wound healing protect fatty tissues from oxidative

damage and play an integral role collagen synthesis (Zhang et al 1999) It is used in

cosmetic preparations for its anti-aging depigmentation and antioxidant properties

(Spiclin 2003 Ehrlich et al 2006) It is also used in combination with other vitamins

such as alpha-tocopherol as a co-antioxidant to stabilize cosmetic preparations (Eberlein-

Koumlnig and Ring 2005 Bissett 2006 Murray 2008) Ascorbic acid in the presence of air

or light is known to interact with alpha-tocopherol (Packer et al 2002 Johnston et al

2007) riboflavin (Homann and Gaffron 1964 Sattar et al 1977 Heelis et al 1981

Kim et al 1993 Jung et al 1995 De La Rochette et al 2000 2003 Lavoie et al

2004 Vaid et al 2005 Ahmad and Vaid 2006 Silva and Quina 2006) and

nicotinamide (Bailey et al 1945 Werner et al 1949 Guttman and Brooke 1963

DeRitter 1982) The present work involves a study of the effect of alpha-tocopherol

riboflavin and nicotinamide on the photostability of ascorbic acid in cream formulations

to observe whether the interaction in these formulations leads to the stabilization of

ascorbic acid The chemical structures of nicotinamide (NA) alpha-tocopherol (TP)

riboflavin (RF) formylmethylflavin (FMF) and lumichrome (LC) are shown in Fig 20

The details of the cream formulations used in this study are given in Table 16

The results obtained on the photodegradation of ascorbic acid in cream formulations are

discussed in the following sections

111

Riboflavin

N

N

NH

N

CH2

CH

C OHH

CH OH

CH2OH

N

N

NH

N

CH2

CHO

Formylmethylflavin

N

N

NH

HN

Lumichrome

OH

N

NH2

O

Nicotinamide

O CH3

CH3

CH3

HO

H3C

CH3 CH3 CH3

CH3

Alpha-Tocopherol

O

O

H3C

H3C

H3C

H3C

O

O

H3C

H3C

O

O

Fig 20 Chemical structures of alpha-tocopherol nicotinamide riboflavin

formylmethylflavin and lumichrome

112

Table 16 Composition of cream formulations containing ascorbic acid (2) and other

vitamins

Ingredients Cream

No SA PA MA CA GL AH2 RFa NA

b TP

c PH DW

10 a + minus minus + + + a minus minus + +

b + minus minus + + + b minus minus + +

c + minus minus + + + c minus minus + +

d + minus minus + + + d minus minus + +

e + minus minus + + + e minus minus + +

11 a minus + minus + + + a minus minus + +

b minus + minus + + + b minus minus + +

c minus + minus + + + c minus minus + +

d minus + minus + + + d minus minus + +

e minus + minus + + + e minus minus + +

12 a minus minus + + + + a minus minus + +

b minus minus + + + + b minus minus + +

c minus minus + + + + c minus minus + +

d minus minus + + + + d minus minus + +

e minus minus + + + + e minus minus + +

13 a + minus minus + + + minus a minus + +

b + minus minus + + + minus b minus + +

c + minus minus + + + minus c minus + +

d + minus minus + + + minus d minus + +

e + minus minus + + + minus e minus + +

14 a minus + minus + + + minus a minus + +

b minus + minus + + + minus b minus + +

c minus + minus + + + minus c minus + +

d minus + minus + + + minus d minus + +

e minus + minus + + + minus e minus + +

113

Table 16 continued

15 a minus minus + + + + minus a minus + +

b minus minus + + + + minus b minus + +

c minus minus + + + + minus c minus + +

d minus minus + + + + minus d minus + +

e minus minus + + + + minus e minus + +

16 a + minus minus + + + minus minus a + +

b + minus minus + + + minus minus b + +

c + minus minus + + + minus minus c + +

d + minus minus + + + minus minus d + +

e + minus minus + + + minus minus e + +

17 a minus + minus + + + minus minus a + +

b minus + minus + + + minus minus b + +

c minus + minus + + + minus minus c + +

d minus + minus + + + minus minus d + +

e minus + minus + + + minus minus e + +

18 a minus minus + + + + minus minus a + +

b minus minus + + + + minus minus b + +

c minus minus + + + + minus minus c + +

d minus minus + + + + minus minus d + +

e minus minus + + + + minus minus e + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PH = potassium hydroxide DW = distilled water

RF = riboflavin NA = nicotinamide TP = alpha-tocopherol

a RF(g ) a = 0002 b = 0004 c = 0006 d = 0008 e = 0010

b NA (g ) a = 028 b = 056 c = 084 d = 112 e = 140

c TP (g ) a = 017 b = 034 c = 052 d = 069 e = 086

The molar concentrations of these vitamins are given in Section 421

114

62 ABSORPTION CHARACTERISTICS OF PHOTOLYSED CREAMS

A typical set of the absorption spectra of the methanolic extracts (pH 20) of the

freshly prepared and photolysed creams containing AH2 and TP is shown in Fig 21 AH2

in acidified methanol exhibits absorption maximum at 245 nm (Zeng et al 2005) as

observed in Fig 21 The absorption due to TP at 284 nm (Moffat et al 2004) was

cancelled by using an appropriate blank containing an equivalent concentration of TP

The gradual decrease in absorption at around 245 nm during UV irradiation indicates the

transformation of AH2 to DHA which does not absorb in this region (Davies et al 1991)

as a result of the loss of C3=C2 chromophore Similar spectral changes around 245 nm are

observed in the presence of RF and NA which also strongly absorb in this region A

decrease in the absorption of AH2 around 266 nm in aqueous solution (pH 60) in the

presence of RF has been reported (Vaid et al 2005) The spectral changes and loss of

absorbance in methanolic extracts of creams depends on the rate of photolysis of AH2 in

the presence of these vitamins

63 PHOTOPRODUCTS OF ASCORBIC ACID AND OTHER VITAMINS

The UV irradiation of AH2 in cream formulations (pH 60) in the presence of RF

NA and TP results in the degradation of AH2 and RF and the following photoproducts

have been identified on comparison of their RF values and spot color fluorescence with

those of the authentic compounds

AH2 DHA

RF FMF LC CMF

In the TLC systems used NA and TP did not show the formation of any

degradation product in creams

115

Fig 21 UV absorption spectra of methanolic extracts of photodegraded ascorbic acid in

cream at 0 60 120 180 300 and 480 min

116

The formation of DHA in the photooxidation of AH2 has previously been reported by

many workers (Homann and Gaffron 1964 Sattar et al 1977 Heelis et al 1981

Rozanowska et al 1997 Lavoie et al 2004 Vaid et al 2006) RF is sensitive to light in

aqueous solutions (DeRitter 1982 British Pharmacopoeia 2009 Sweetman 2009) and is

known to form a number of products under aerobic conditions (Treadwell et al 1968

Cairns and Metzler 1971 Schuman Jorns et al 1975 Ahmad and Rapson 1990 Ahmad

and Vaid 2006 Ahmad et al 2004ab 2005 2008 Vaid et al 2006) It has been found

to degrade on UV irradiation in cream formulations to give FMF LC and CMF and these

products have been reported in the photolysis of RF by the workers cited above The

formation of these products may be affected by the interaction of AH2 and RF in creams

(Section 66) NA and TP individually did not appear to form any photoproduct of their

own directly or on interaction with AH2 in creams and may influence the degradation of

AH2 on UV irradiation

64 ASSAY METHOD

In view of the presence of RF (absorption maxima 223 267 373 and 444 nm)

(British Pharmacopoeia 2009) NA (absorption maximum 261 nm) (Moffat et al 2004)

and TP (absorption maximum 284 nm) (Moffat et al 2004) in the cream formulations

and the interference of these vitamins with the absorption of AH2 (absorption maximum

265 nm) (Davies et al 1991) the direct spectrophotometric method cannot be applied for

the determination of AH2 Therefore the iodimetric method (British Pharmacopoeia

2009) was used to determine AH2 in cream formulations The method was validated in

the presence of RF NA and TP before its application to the determination of AH2 in

photodegraded creams The reproducibility of the method has been confirmed by the

117

assay of known concentrations of AH2 in the range present in photodegraded creams The

recovery of AH2 in the creams has been found to be in the range 90ndash96 The values of

RSD indicate that the precision of the method is within plusmn5 (Table 17) and it can be

applied to study the kinetics of AH2 photolysis in cream formulations The assay data on

AH2 in various cream formulations are given in Table 18

65 KINETICS OF PHOTODEGRADATION OF ASCORBIC ACID

Several chemical and physical factors play a role in the photodegradation of AH2

in the presence of other vitamins (RF NA TP) and affect the rate of its degradation in

cream formulations The present work involves the study of photodegradation of AH2 in

cream formulations containing glycerin as humectant as AH2 has been found to be most

stable in these creams (Chapter 5) The apparent first-order rate constants (kobs) for the

photodegradation of AH2 in the presence of other vitamins in cream formulations

derived from the kinetic plots (Fig 22ndash24) are reported in Table 19 The second-order

rate constants (correlation coefficients 0991ndash0996) determined from the slopes of the

graphs of kobs versus vitamin concentration for the individual vitamins (Fig 25) and the

values of k0 determined from the intercept on the vertical axis at zero concentration are

reported in Table 20 The values of k0 give an indication of the effect of other vitamins on

the rate of degradation of AH2 These values are about 13 times lower than the values of

kobs obtained at the highest concentrations of RF and NA indicating that RF and NA both

accelerate the photodegradation of AH2 in creams RF is known to act as a

photosensitizer for the degradation of AH2 (Section 66) and therefore its presence in

creams would accelerate the degradation of AH2 The increase in the rate of

photodegradation of AH2 in the presence of NA has not previously been reported NA

118

Table 17 Recovery of ascorbic acid in cream formulations in the presence of other

vitamins by iodimetric methoda

Cream

Formulationb

Added

(mg )

Found

(mg )

Recovery

()

RSD

()

10e (RF) 400

200

373

187

933

935

29

22

11e (RF) 400

200

379

187

948

935

25

31

12e (RF) 400

200

375

188

938

940

29

28

13e (NA) 400

200

382

191

955

955

23

27

14e (NA) 400

200

380

185

950

925

19

26

15e (NA) 400

200

379

191

948

955

21

17

16e (TP) 400

200

368

183

920

915

29

44

17e (TP) 400

200

391

195

978

975

11

13

18e (TP) 400

200

377

182

943

910

32

37

a Values expressed as a mean of three to five determinations

b The cream formulations represent all the emulsifiers (stearic acid palmitic acid

myristic acid) to observe the efficiency of iodimetric method for the recovery of

ascorbic acid in presence of the highest concentration of vitamins (Table 16)

119

Table 18 Photodegradation of ascorbic acid in cream formulations in the presence of

other vitamins

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) a b C d e

0 373 372 374 372 375

60 362 354 354 360 359

150 342 336 336 332 334

240 315 314 308 310 302

10 (RF)

330 301 291 288 281 282

0 380 379 376 374 374

60 370 366 362 362 361

150 343 337 340 332 328

240 329 323 320 313 310

11 (RF)

330 307 301 294 288 282

0 379 380 375 372 376

60 362 366 361 351 342

150 341 335 319 307 312

240 310 306 295 284 282

12 (RF)

330 285 278 263 254 243

120

Table 18 continued

0 372 370 371 368 365

60 361 358 348 350 349

120 342 343 329 326 330

180 327 325 319 312 308

240 317 309 299 289 285

13 (NA)

300 299 291 283 278 273

0 386 380 375 378 370

60 371 362 365 362 355

120 359 351 343 339 336

200 341 332 325 316 311

14 (NA)

300 313 303 296 294 280

0 375 371 374 370 366

60 362 356 352 352 345

120 343 332 336 326 314

200 323 315 311 295 293

15 (NA)

300 293 283 275 270 259

121

Table 18 continued

0 380 378 380 377 377

60 362 365 369 369 371

120 351 352 360 360 364

180 340 346 349 353 355

240 331 334 343 343 346

16 (TP)

300 320 323 330 332 337

0 383 380 378 380 377

60 372 371 372 373 370

120 363 360 361 366 365

180 348 348 350 356 355

240 341 343 343 348 348

17 (TP)

300 330 332 336 339 341

0 380 383 377 375 373

60 364 370 366 367 366

120 352 356 351 352 351

180 334 338 339 343 342

240 324 328 324 332 330

18 (TP)

300 307 315 317 318 322

122

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

10

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

11

ab

c

de

13

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

12

Fig 22 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing riboflavin (a) 0002 (b) 0004 (c) 0006 (d) 0008

(e) 0010

Stearic acid

Palmitic acid

Myristic acid

123

abcde

13

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

13

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

14

abcde

13

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

15

Fig 23 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing nicotinamide (a) 028 (b) 056 (c) 084 (d) 112 (e) 140

Stearic acid

Palmitic acid

Myristic acid

124

abcde

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

16

abcde

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

17

abcde

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

18

Fig 24 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing alpha-tocopherol (a) 017 (b) 034 (c) 052 (d) 069

(e) 086

Stearic acid

Myristic acid

Palmitic acid

125

Table 19 First-order rate constants (kobs) for the photodegradation of ascorbic acid in the

presence of other vitamins in cream formulations

kobs times 103 (min

ndash1)ab

Cream

formulationd

Other

vitaminc

a b C d e

10 RF 068

(0991)

073

(0996)

079

(0995)

085

(0992)

089

(0995)

11 RF 065

(0992)

070

(0992)

073

(0994)

080

(0995)

086

(0993)

12 RF 087

(0993)

096

(0995)

109

(0993)

116

(0994)

127

(0992)

13 NA 073

(0993)

081

(0992)

088

(0994)

096

(0994)

101

(0993)

14 NA 069

(0992)

074

(0992)

080

(0991)

086

(0995)

094

(0995)

15 NA 083

(0994)

090

(0993)

101

(0993)

109

(0994)

115

(0995)

16 TP 055

(0991)

051

(0994)

046

(0994)

042

(0993)

038

(0991)

17 TP 050

(0995)

045

(0993)

041

(0992)

038

(0995)

034

(0994)

18 TP 070

(0996)

066

(0996)

060

(0994)

055

(0993)

051

(0993)

a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental conditions

including the light intensity

c Vitamin concentrations (andashe) are as given in Table 16

d All the creams contain glycerin as humectant

The estimated error is plusmn5

126

00

05

10

15

00 10 20 30

Riboflavin concentration (M times 104)

kob

s times

10

3 (

min

ndash1)

10-12

00

05

10

15

00 20 40 60 80 100 120

Nicotinamide concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

13-15

00

02

04

06

08

00 04 08 12 16 20

Alpha-Tocopherol concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

16-18

Fig 25 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against individual vitamin concentration in cream formulations (10ndash18)

127

Table 20 First-order rate constants (k0)a for the photodegradation of ascorbic acid in the

absence of other vitamins and second-order rate constants (k) for the

photochemical interaction of ascorbic acid with RF NA and TP

Cream

formulation

Other

vitamin

k0 times 103

(minndash1

)

k

(Mndash1

minndash1

)

Correlation

coefficient

10 RF 062 102 0994

11 RF 059 097 0992

12 RF 077 189 0995

13 NA 066 032 times 10ndash2

0995

14 NA 062 027 times 10ndash2

0993

15 NA 074 037 times 10ndash2

0994

16 TP 059 110 times 10ndash2b

0996

17 TP 053 096 times 10ndash2b

0992

18 TP 075 123 times 10ndash2b

0994

a

The variations in the values of k0 are due to the degradation of AH2 in the presence of

different emulsifying agents in cream formulations

b Values for the inhibition of photodegradation of AH2

128

forms a complex with AH2 (Section 67) and may also act as a photosensitizer for AH2 by

energy transfer in the excited state on UV irradiation The absorption maximum of NA

(261 nm) (Moffat et al 2004) is very close to that of AH2 (265 nm) (Davies et al 1991)

and the possibility of energy transfer in the excited state (Moore 2004) is greater leading

to the photodegradation of AH2

The value of k0 is about 13 times greater than the values of kobs obtained for the

degradation of AH2 in the presence of the highest concentrations of TP in the creams

This indicates that TP has a stabilising effect on the photodegradation of AH2 in the

cream formulations This is in agreement with the view that the TP acts as a redox partner

with AH2 to retard its oxidation (Wille 2005) Thus among the three vitamins studied

only TP appears to have a stabilising effect on photodegradation of AH2 The

photochemical interaction of individual vitamins with AH2 is discussed below

66 INTERACTION OF RIBOFLAVIN WITH ASCORBIC ACID

The interaction of RF with the ascorbate ion (AHndash) may be represented by the

following reactions proposed by Silva and Quina (2006)

RF rarr 1RF (61)

1RF rarr

3RF (62)

3RF + AH

ndash rarr RF

ndashmiddot + AHmiddot (63)

AHmiddot + O2 rarr A + HO2middot (64)

HO2middot + AHndash rarr H2O2 + AHmiddot (65)

RF on the absorption of a quantum of light is promoted to the excited singlet state (1RF)

(61) 1RF may undergo intersystem crossing (isc) to form the excited triplet state (

3RF)

(62) The excited triplet state may react with the ascorbate ion to generate the ascorbyl

hv

isc

129

radical (AH) (63) (Kim et al 1993) The ascorbyl radical reacts with oxygen to give

dehydroascorbic acid (A) and peroxyl radical (HO2) (64) This radical may interact with

ascorbate ion to generate further ascorbyl radicals (65) These radicals may again take

part in the sequence of reactions to form A The role of RF in this reaction is to act as a

photosensitiser in the oxidation of ascorbic acid to A Ascorbic acid is reported to protect

riboflavin in milk under the influence of light by reacting with singlet oxygen (Hall et al

2009) (Section 511)

67 INTERACTION OF NICOTINAMIDE WITH ASCORBIC ACID

NA is known to form a 11 complex with ascorbic acid (Guttman and Brooke

1963 OrsquoNeil 2001 Doores 2002) The complexation of NA and AH2 may result from

the donor-acceptor interaction between AH2 (donor) and NA (acceptor) as observed in

the case of tryptophan and NA (Florence and Attwood 2006) In the presence of light the

interaction may cause reduction of NA (NAH) to form the ascorbyl radical (AH) ((66)-

(68)) which is oxidized to dehydroascorbic acid (A) (69) The NAH may be oxidized to

NA and H2O2 (610)

NA rarr 1NA (66)

1NA rarr

3NA (67)

3NA + AH2 rarr NAH + AHmiddot (68)

2 AH٠ rarr A + AH2 (69)

NAH + O2 rarr NA + H2O2 (610)

The proposed reactions suggest that on photochemical interaction AH2 undergoes

photosensitised oxidation in the presence of NA indicating that the photostability of

ascorbic acid is affected by NA

isc

130

68 INTERACTION OF ΑLPHA-TOCOPHEROL WITH ASCORBIC ACID

TP is an unstable compound and its oxidation by air results in the formation of an

epoxide which than produces a quinone that is inactive (Connors et al 1986) TP is

destroyed by sun light and artificial light containing the wavelengths in the UV region

(Ball 2006) It interacts with other antioxidants such as ascorbic acid (AH2) according to

the following reactions

TPndashO + AH2 rarr TP + AHmiddot (611)

2 AHmiddot rarr A + AH2 (612)

TP + AHmiddot rarr TPndashO + AH2 (613)

The tocopheroxyl radical (TPndashO) reacts with AH2 to regenerate TP and form the

ascorbyl radical (AHmiddot) (611) This radical undergoes further reactions as described in

equations (64) and (65) (Traber 2007) It may also disproportionate back to A and AH2

(612) TP reacts with AHmiddot to produce again the TPndashO radical and AH2 Thus in the

presence of light TP leads to the oxidation of AH2 which is ultimately regenerated in the

reaction (Davies et al 1991) Ascorbic acid has the ability to act as a co-antioxidant with

the TPndashO to regenerate TP (Packer et al 1979 2002) In this manner AH2 and TP act

synergistically to function in a redox cycle and AH2 is stabilized in the cream

formulations and microemulsions (Rozman and Gasperlin 2007 Rozman et al 2009)

69 EFFECT OF CARBON CHAIN LENGTH OF EMULSIFYING AGENT

The graphs of kobs for the photodegradation of AH2 in the presence of RF NA and

TP versus the carbon chain length of emulsifying agents are shown in Fig 26 It appears

that the photodegradation of AH2 in the presence of all the three vitamins in the creams

lies in the order

131

Fig 26 Plots of k for photodegradation of ascorbic acid in creams (10ndash18) against

carbon chain length of emulsifier () Stearic acid () palmitic acid

() myristic acid

00

05

10

15

20

25

k

(Mndash

1 m

inndash

1)

00

05

10

15

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash

1 m

inndash

1)

132

myristic acid gt stearic acid gt palmitic acid

The same order of emulsifying agents has been observed in the absence of the

added vitamins (Section 57) The polar character of these acids (Yao et al 2009) on the

basis of their carbon chain length may play a part in the photostability of AH2 The

greater stability of AH2 in creams in the presence of palmitic acid (Fig 26) may be due to

the interaction of AH2 with palmitic acid as discussed in Section 57 Ascorbic acid-6-

palmitate is known to be an antioxidant in cosmetic preparations (Lee et al 2009) and

food products (Doores 2002)

610 EFFECT OF VISCOSITY OF CREAMS

The plots of kobs for the degradation of AH2 in the presence of the highest

concentration of vitamins versus reciprocal of the viscosity of creams (Table 21) are

linear (Fig 27) and indicate that the increase in cream viscosity leads to a decrease in the

rate of degradation of AH2 The slopes of the plots indicate the effect of viscosity on the

interaction of AH2 with other vitamins in the order

riboflavin gt nicotinamide gt alpha-tocopherol

The relatively slow rate of degradation of AH2 in creams containing palmitic acid may be

due to the interaction of AH2 with the vitamins as well as palmitic acid (Lee et al 2009)

Thus viscosity is an important factor in the stability of AH2 in cream formulations and

may affect its rate of interaction with other vitamins It has been suggested that an

increase in the viscosity of the medium makes access to air at the surface more difficult to

prevent the oxidation of a drug (Wallwork and Grant 1977) This is in agreement with

the photolysis of AH2 in aqueous and organic solvents cream formulations (Chapter 5)

and aerobic oxidation of Ah2 in syrups (Blaug and Hajratwala 1972)

133

Table 21 Average viscosity of cream formulations containing different emulsifying

agents and glycerin as humectant (25 plusmn 1 ordmC) and the photodegradation rate

constants of AH2

Cream No Emulsifying

agent

Viscosityab

(mPa s)

kobs times 103c

10 (RF)

13 (NA)

16 (TP)

Stearic acid 9000 089

101

038

11 (RF)

14 (NA)

17 (TP)

Palmitic acid 8600 086

094

034

12 (RF)

15 (NA)

18 (TP)

Myristic acid 7200 127

115

051

a plusmn10

b Average viscosity of creams containing the individual vitamins (RF NA TP)

c The values have been obtained in the presence of highest concentration of the

vitamins

134

00

05

10

15

20

25

30

100 110 120 130 140

Viscosity (mPa s)ndash1

times 103

kob

s (m

inndash1)

Fig 27 Plots of kobs in the presence of highest concentration of vitamins versus

reciprocal of the viscosity of creams () riboflavin

( ) nicotinamide (- - -- - -) alpha-tocopherol

135

611 DEGRADATION OF ASCORBIC ACID IN THE PRESENCE OF OTHER

VITAMINS IN THE DARK

In order to observe the effect of riboflavin nicotinamide and alpha-tocopherol on

the degradation of AH2 in the creams stored in the dark the AH2 contents of the creams

were assayed at appropriate intervals (Table 22) The apparent first-order rate constants

determined from the kinetic plots (Fig 28) for the degradation of AH2 in the presence of

the highest concentrations of the individual vitamins in cream formulations (10ndash18) are

reported in Table 23 These rate constants indicate that the overall degradation of AH2 in

the presence of the highest concentration of the individual vitamins (RF NA and TP) is

about 70 times slower than that obtained on the exposure of creams to UV irradiation

This decrease in the rate of degradation of AH2 in the creams is the same as observed in

the case of AH2 alone In the absence of light the degradation of AH2 occurs due to

chemical oxidation (Section 132) and does not appear to be affected by the presence of

riboflavin and nicotinamide as indicated by the comparisons of the values of kobs in the

presence and absence of these vitamins (Table 15 and 23) In the presence of alpha-

tocopherol the degradation is slower than that in the presence of riboflavin and

nicotinamide This may be due to some interaction of AH2 and alpha-tocopherol causing

stabilisation of AH2 in the creams

As observed in the case of AH2 degradation alone in creams in the dark the AH2

degradation in the presence of the highest concentrations of other vitamins also occurs in

the same order of emulsifying agents (Fig 29)

myristic acid gt stearic acid gt palmitic acid

136

Table 22 Degradation of ascorbic acid in cream formulations in the dark in presence of

highest concentration of other vitamins

Concentration of ascorbic acid (mg 2g) Cream

No Time (days) 0 10 20 40 60 80

10e (RF) 375 285 233 171 110 69

11e (RF) 374 341 281 221 148 113

12e (RF) 372 259 203 130 89 59

13e (NA) 365 330 255 187 126 81

14e (NA) 370 321 289 219 159 109

15e (NA) 366 289 249 159 110 63

16e (TP) 377 359 321 261 211 159

17e (TP) 377 366 333 275 228 191

18e (TP) 373 361 304 252 200 167

137

02

07

12

17lo

g c

on

cen

tra

tio

n (

mg

)

10-12Riboflavin

02

07

12

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

13-15Nicotinamide

10

12

14

16

18

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

16-18Alpha-Tocopherol

Fig 28 First-order plots for the degradation of ascorbic acid in the dark in presence of

other vitamins using the emulsifying agents (minusminusminusminus) Stearic acid

(minus minusminus minus) palmitic acid (----) myristic acid

138

Table 23 First-order rate constants (kobs) for the degradation of ascorbic acid in presence

of other vitamins in cream formulations in the dark

Cream

formulation

Other

vitaminc

kobs times 102

(dayndash1

)ab

10e RF 204

(0995)

11e RF 156

(0992)

12e RF 222

(0992)

13e NA 189

(0995)

14e NA 151

(0993)

15e NA 214

(0995)

16e TP 100

(0994)

17e TP 088

(0995)

18e TP 105

(0993)

a The values in parenthesis are correlation coefficients and range from 0991ndash0996 due to

some variations in AH2 distribution in the creams

b The values of rate constants are relative and depend on specific experimental

conditions

c Vitamin concentrations (andashe) are as given in Table 16

The estimated error is plusmn5

139

Riboflavin

Nicotinamide

Alpha-Tocopherol

00

10

20

30

12 14 16 18Carbon chain length

ko

bs times

10

2 (

da

yndash1)

Fig 29 Plots of kobs for degradation of ascorbic acid in the dark in creams (10ndash18)

against carbon chain length of the emulsifier () Stearic acid () palmitic acid

() myristic acid

140

This indicates that the rate of degradation of AH2 is slowest in the creams containing

palmitic acid as the emulsifying agent The reason for AH2 degradation in the dark in this

order has already been explained in section 512

CHAPTER VII

STABILIZATION OF

ASCORBIC ACID WITH

CITRIC ACID TARTARIC

ACID AND BORIC ACID IN

CREAM FORMULATIONS

142

71 INTRODUCTION

Ascorbic acid is an ingredient of cosmetic preparations (Section 51) and is

sensitive to light (Rowe et al 2009 Sweetman 2009 British Pharmacopoeia 2009)

degrading to dehydroascorbic acid on UV irradiation by photooxidation (Kitagawa

1968) The photosensitivity of ascorbic acid makes it unstable in pharmaceutical and

cosmetic preparations (DeRitter 1982) The present work is an attempt to study the

photodegradation of ascorbic acid in cream formulations in the presence of certain

compounds (eg citric acid tartaric acid and boric acid) to investigate their role in the

stabilization of the vitamin on exposure to light and in the dark Citric acid and tartaric

acid are used as an antioxidant synergist (Rowe et al 2009) and boric acid is a

complexing agent for hydroxy compounds (Ahmad et al 2009cd)

72 CREAM FORMULATIONS

The details of the various cream formulations used in this study are given in Table

24 and the results obtained on the photodegradation of ascorbic acid in the presence of

stabilizing agents in these formulations are discussed in the following sections

143

Table 24 Composition of cream formulations containing ascorbic acid (2) and

stabilizers

Ingredients Cream

No SA PA MA CA GL PG EG AH2 CTa TA

b BA

c PH DW

19 a + minus minus + + minus minus + a minus minus + +

b + minus minus + + minus minus + b minus minus + +

c + minus minus + + minus minus + c minus minus + +

20 a minus + minus + + minus minus + a minus minus + +

b minus + minus + + minus minus + b minus minus + +

c minus + minus + + minus minus + c minus minus + +

21 a minus minus + + + minus minus + a minus minus + +

b minus minus + + + minus minus + b minus minus + +

c minus minus + + + minus minus + c minus minus + +

22 a + minus minus + + minus minus + minus a minus + +

b + minus minus + + minus minus + minus b minus + +

c + minus minus + + minus minus + minus c minus + +

23 a minus + minus + + minus minus + minus a minus + +

b minus + minus + + minus minus + minus b minus + +

c minus + minus + + minus minus + minus c minus + +

24 a minus minus + + + minus minus + minus a minus + +

b minus minus + + + minus minus + minus b minus + +

c minus minus + + + minus minus + minus c minus + +

25 a + minus minus + + minus minus + minus minus a + +

b + minus minus + + minus minus + minus minus b + +

c + minus minus + + minus minus + minus minus c + +

26 a minus + minus + + minus minus + minus minus a + +

b minus + minus + + minus minus + minus minus b + +

c minus + minus + + minus minus + minus minus c + +

27 a minus minus + + + minus minus + minus minus a + +

b minus minus + + + minus minus + minus minus b + +

c minus minus + + + minus minus + minus minus c + +

144

Table 24 continued

28 a + minus minus + minus + minus + minus minus a + +

b + minus minus + minus + minus + minus minus b + +

c + minus minus + minus + minus + minus minus c + +

29 a minus + minus + minus + minus + minus minus a + +

b minus + minus + minus + minus + minus minus b + +

c minus + minus + minus + minus + minus minus c + +

30 a minus minus + + minus + minus + minus minus a + +

b minus minus + + minus + minus + minus minus b + +

c minus minus + + minus + minus + minus minus c + +

31 a + minus minus + minus minus + + minus minus a + +

b + minus minus + minus minus + + minus minus b + +

c + minus minus + minus minus + + minus minus c + +

32 a minus + minus + minus minus + + minus minus a + +

b minus + minus + minus minus + + minus minus b + +

c minus + minus + minus minus + + minus minus c + +

33 a minus minus + + minus minus + + minus minus a + +

b minus minus + + minus minus + + minus minus b + +

c minus minus + + minus minus + + minus minus c + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PG = propylene glycol EG = ethylene glycol

PH = potassium hydroxide DW = distilled water CT = citric acid TA = tartaric acid

BA = boric acid

a CT (g ) a = 01 b = 02 c = 04

b TA (g ) a = 01 b = 02 c = 04

c BA (g ) a = 01 b = 02 c = 04

145

73 PRODUCTS OF ASCORBIC ACID PHOTODEGRADATION

The photodegradation of AH2 in cream formulations leads to the formation of

DHA as detected by TLC and reported earlier in the photolysis of AH2 in aqueous

solutions (Vaid et al 2006) and cream formulations (Sections 52 and 63) AH2 and

DHA in the methanolic extracts of the degraded creams were identified by comparison of

their Rf and color of the spots with those of the reference standards DHA is also

biologically active (Gardner 1972 Doores 2002) but its further degradation to 23-

diketo-gulonic acid (DGA) results in the loss of vitamin activity (Section 132)

However this product has not been detected in the present cream formulations

Therefore the creams may still possess their biological efficacy

74 SPECTRAL CHANGES IN PHOTODEGRADED CREAMS

In order to observe the spectral changes in photodegraded creams in the presence

of stabilizing agents the absorption spectra of the methanolic extracts of a degraded

cream were determined The spectra show a gradual loss of absorbance around 245 nm

due to the oxidation of AH2 to DHA on UV irradiation and similar to that shown for the

photodegradation of AH2 alone in Fig 5 DHA has negligible absorbance around 245 nm

(Davies et al 1991) and therefore it does not interfere with the absorbance of AH2 in

methanolic solutions The spectral changes and loss of absorbance around 245 nm in

methanolic solution depend on the extent of photooxidation of AH2 in a particular cream

75 ASSAY OF ASCORBIC ACID IN CREAMS

The UV spectrophotometric method (Zeng et al 2005) has previously been

applied to the determination of AH2 in cream formulations (Section 54) The absorbance

of the methanolic extracts of creams containing AH2 during photodegradation was used

146

to determine the concentration of AH2 The method was validated in the presence of citric

acid (CT) tartaric acid (TA) and boric acid (BA) before its application to the evaluation

of the kinetics of AH2 degradation in cream formulations The recovery of AH2 in creams

has been found to be in the range of 90ndash96 and is similar to that reported in Table 7

The reproducibility of the method lies within plusmn5 The assay data on the degradation of

AH2 in various creams in the presence of the stabilizing agents are reported in Table 25

76 KINETICS OF PHOTODEGRADATION

The effect of CT TA and BA as stabilizing agents on the photodegradation of

AH2 was studied by adding 01ndash04 of each compound to the cream formulations (19ndash

33) at pH 60 This concentration range is normally used for the stabilization of drugs in

pharmaceutical preparations (Im-Emsap et al 2002) The apparent first-order rate

constants (kobs) determined from the plots of log concentration versus time (Fig 30ndash34)

are reported in Table 26 The second-order rate constants (k) determined from the plots

of kobs versus concentration of the individual compounds (Fig 35ndash36) are given in Table

27 The values of k indicate the rate of inhibition of photodegradation of AH2 by each

compound

77 EFFECT OF STABILIZING AGENTS

In order to compare the effectiveness of CT TA and BA as stabilizing agents for

AH2 plots of k versus carbon chain length of the emulsifying agents were constructed

(Fig 37) The k values for the interaction of these compounds with AH2 are in the order

citric acid gt tartaric acid gt boric acid

The curves indicate that the highest interaction of these compounds with AH2 is in the

order

147

Table 25 Photodegradation of ascorbic acid in cream formulations in the presence of

stabilizers

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) a b c

0 374 378 379

60 362 362 372

120 349 355 367

210 333 335 349

19 (CT)

300 319 322 336

400 296 309 324

0 381 378 380

60 368 370 369

120 355 363 364

210 344 345 355

20 (CT)

300 328 335 341

400 312 319 331

21 (CT) 0 368 370 374

60 355 356 360

120 340 344 343

210 321 322 333

300 296 299 315

400 272 285 299

148

Table 25 continued

0 375 374 378

60 363 363 368

120 352 354 362

210 329 335 345

22 (TA)

300 307 314 333

400 292 299 313

0 370 377 374

60 364 365 368

120 352 357 357

210 332 344 349

23 (TA)

300 317 330 335

400 301 310 322

24 (TA) 0 376 379 377

60 367 369 368

120 351 348 352

210 325 330 344

300 306 317 326

400 284 294 310

149

Table 25 continued

0 370 375 380

60 356 362 359

120 331 339 344

210 311 318 330

25 (BA)

300 279 288 305

400 260 269 283

0 377 375 370

60 364 363 361

120 351 353 351

210 331 332 337

26 (BA)

300 323 324 325

400 301 307 313

27 (BA) 0 380 377 375

60 369 368 366

120 333 338 341

210 305 313 318

300 292 294 304

400 262 266 281

150

Table 25 continued

0 373 376 378

60 348 349 360

120 329 336 339

210 315 312 323

28 (BA)

300 282 283 299

400 249 264 280

0 370 373 380

60 358 355 367

120 343 346 356

210 325 329 347

29 (BA)

300 307 312 325

400 287 295 315

30 (BA) 0 369 375 372

60 353 358 362

120 321 330 335

210 283 294 303

300 265 281 293

400 242 254 270

151

Table 25 continued

0 374 376 379

60 348 366 352

120 324 340 337

210 303 319 322

31 (BA)

300 275 289 293

400 243 260 275

0 370 374 375

60 355 354 366

120 339 344 345

210 313 319 330

32 (BA)

300 288 297 308

400 261 271 290

33 (BA) 0 377 380 377

60 357 361 367

120 324 335 339

210 288 294 307

300 270 280 293

400 233 248 265

Creams 19ndash27 contain glycerin 28ndash30 contain propylene glycol and 31ndash33 contain

ethylene glycol as humectants

152

a

b

c

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

19

ab

c

14

15

16

log

co

nce

ntr

ati

on

(m

g)

20

a

b

c

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

21

Fig 30 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and citric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

153

a

b

c

14

15

16lo

g c

on

cen

tra

tio

n (

mg

)

22

a

b

c

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

23

a

b

c

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

24

Fig 31 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and tartaric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

154

ab

c

13

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

25

abc

13

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

26

ab

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

27

Fig 32 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and boric acid (a) 01 (b) 02 (c) 04

Palmitic acid

Stearic acid

Myristic acid

155

a

b

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

28

ab

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

29

a

b

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

30

Fig 33 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing propylene glycol and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

156

a

b

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

31

ab

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

32

a

b

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

33

Fig 34 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing ethylene glycol and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

157

Table 26 Apparent first-order rate constants (kobs) for the photodegradation of ascorbic

acid in presence of different stabilizers in cream formulations

kobs times 103 (min

ndash1)ab

Cream

formulation Stabilizer

c

a b c

19 CT 057

(0995)

050

(0992)

041

(0991)

20 CT 049

(0996)

043

(0995)

034

(0993)

21 CT 076

(0995)

067

(0995)

055

(0992)

22 TA 065

(0995)

058

(0995)

046

(0991)

23 TA 054

(0994)

047

(0993)

038

(0994)

24 TA 072

(0996)

063

(0992)

049

(0991)

25 BA 091

(0994)

086

(0995)

071

(0993)

26 BA 055

(0994)

050

(0993)

042

(0993)

27 BA 095

(0995)

089

(0992)

074

(0996)

28 BA 097

(0995)

088

(0992)

075

(0993)

29 BA 064

(0994)

057

(0991)

047

(0993)

30 BA 110

(0994)

100

(0996)

084

(0992)

31 BA 105

(0995)

094

(0994)

078

(0992)

32 BA 088

(0994)

079

(0993)

066

(0993)

33 BA 120

(0995)

108

(0993)

091

(0993) a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental conditions

including the light intensity

c Stabilizer concentrations (andashc) are as given in Table 24

The estimated error is plusmn5

158

00

04

08

12

00 04 08 12 16 20

Citric acid concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

19-21

00

04

08

12

00 05 10 15 20 25

Tartaric acid concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

22-24

Fig 35 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against citric acid (19ndash21) and tartaric acid concentrations (22ndash24) in cream

formulations

159

00

04

08

12k

ob

s times

10

3 (

min

ndash1)

25-27

00

04

08

12

00 20 40 60

ko

bs times

10

3 (

min

ndash1)

28-30

00

04

08

12

00 20 40 60

Boric acid concentration (M times 102)

kob

s times

10

3 (

min

ndash1)

30-33

Fig 36 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against boric acid concentrations in cream formulations (25ndash33)

Propylene glycol

Glycerin

Ethylene glycol

160

Table 27 First-order rate constants (k0)a for the photodegradation of ascorbic acid in the

absence of stabilizers and second-order rate constants (k) for the interaction of

ascorbic acid with CT TA and BA

Cream

formulation Stabilizers

k0 times 103

(minndash1

)

k times 102

(Mndash1

minndash1

)

Correlation

coefficient

19 CT 062 111 0991

20 CT 053 103 0994

21 CT 082 145 0995

22 TA 071 092 0995

23 TA 059 080 0993

24 TA 080 118 0996

25 BA 098 041 0994

26 BA 059 026 0994

27 BA 102 044 0995

28 BA 104 046 0992

29 BA 069 033 0995

30 BA 118 054 0994

31 BA 113 053 0995

32 BA 095 045 0995

33 BA 129 060 0993

a The variations in the values of k0 are due to the presence of different emulsifying agents

in cream formulations

161

00

04

08

12

16

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash1

min

ndash1)

18-33

a

b

e

cd

Fig 37 Plots of k for photodegradation of ascorbic acid in creams (19ndash33) against

carbon chain length of emulsifier () Stearic acid () palmitic acid ()

myristic acid (a) CT (b) TA (c) BA with glycerin (d) BA with propylene

glycol (e) BA with ethylene glycol

162

myristic acid gt stearic acid gt palmitic acid

In the case of myristic acid and stearic acid it may be explained on the basis of the

decreasing polarity (Yao et al 2009) It is interesting to observe the lowest rates of

interaction of these compounds in the creams containing palmitic acid This could be due

to the interaction of AH2 with palmitic acid to form a palmitate derivative in addition to

its interaction with the individual stabilizing agents CT and TA are known to act as

antioxidant synergists (Rowe et al 2009 Sweetman 2009) and in this capacity may

inhibit the photooxidation of AH2 as indicated by the values of the degradation rate

constants in the presence of these compounds The addition of CT to nutritional

supplements is known to inhibit the oxidation of AH2 (Doores 2002) Boric acid forms a

complex with AH2 (Rivlin 2007) and there by may inhibit its degradation Boric acid

may also interact with glycerin added to the creams as a humectant and form a complex

(Rowe et al 2009) This may influence its interaction and stabilizing effect on AH2 in

creams as indicated by the lower k values compared to those in the presence of CT and

TA It has further been observed that the k values for BA are greater in propylene glycol

and ethylene glycol compared to those in glycerin (Table 27) Again this may be due to

greater interaction of BA with glycerin compared to other humectants in the creams

78 DEGRADATION OF ASCORBIC ACID IN PRESENCE OF STABILIZING

AGENTS IN THE DARK

An important factor in the formulation of cosmetic preparations is to ensure the

chemical and photostability of the active ingredient by the use of appropriate stabilizing

agents The choice of these agents would largely depend on the nature and

physicochemical characteristics of the active ingredient AH2 possesses a redox system

163

and can be easily oxidized by air or light In order to observe the effect of CT TA and

BA on the stability of AH2 the cream formulations containing the individual compounds

were stored in the dark for a period of about three months and the rate of degradation of

AH2 was determined The assay data are reported in Table 28 and the kinetic plots are

shown in Fig 38ndash42 The values of apparent first-order rate constants for the degradation

of AH2 in the presence of the stabilizing agents are reported in Table 29 The second

order-rate constants for the interaction of CT TA and BA with AH2 are reported in Table

30 (Fig 43ndash44) The plots of k against the carbon chain length of the emulsifiers are

shown in Fig 45 The kinetic data indicate the same pattern of rates of degradation and

interaction of AH2 with these compounds as observed in the presence of light except that

the rates are much slower in the dark Thus the stabilizing agents are equally effective in

inhibiting the rate of degradation of AH2 in the dark The effect of emulsifying agents and

the humectants on the rate of degradation of AH2 in the presence of the stabilizers has

been discussed in the above Section 77

79 EFFECT OF ADDITIVES ON TRANSMISSION OF ASCORBIC ACID

In order to observe the effect of additives (citric tartaric and boric acids) on the

transmission characteristics of ascorbic acid (0002 mg100 ml) in methanol containing

the highest concentration of the additives (004) used in this study the transmission

spectra were measured It has been found that these additives produce a hypsochromic

shift in the absorption maximum of ascorbic acid This may result in the reduction of the

fraction of light absorbed by ascorbic acid to the extent of about 10 and thus influence

the rate of photodegradation reactions However since all the additives produce similar

effects the rate constants can be considered on a comparative basis

164

Table 28 Degradation of ascorbic acid in cream formulations in the presence of

stabilizers in the dark

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(days) a b c

0 374 378 379

10 355 346 362

20 326 328 342

40 293 297 322

19 (CT)

60 264 269 295

80 241 245 262

0 381 378 380

10 361 364 372

20 339 350 348

40 309 312 330

20 (CT)

60 279 286 301

80 260 266 282

21 (CT) 0 368 370 374

10 342 346 364

20 310 321 348

40 278 282 313

60 249 251 278

80 217 228 249

165

Table 28 continued

0 375 374 378

10 339 344 351

20 317 326 336

40 282 288 306

22 (TA)

60 251 258 280

80 222 235 252

0 370 377 374

10 340 354 355

20 332 336 343

40 297 303 310

23 (TA)

60 266 282 294

80 238 248 267

24 (TA) 0 376 379 377

10 341 339 350

20 306 319 323

40 263 284 279

60 223 241 249

80 196 202 223

166

Table 28 continued

0 370 375 380

10 331 341 334

20 287 289 301

40 225 247 245

25 (BA)

60 189 185 214

80 141 154 170

0 377 375 370

10 355 357 349

20 326 314 324

40 264 267 286

26 (BA)

60 232 238 254

80 189 199 211

27 (BA) 0 380 377 375

10 346 339 337

20 309 288 301

40 233 241 260

60 192 196 211

80 140 147 163

167

Table 28 continued

0 373 376 378

10 314 322 333

20 267 281 305

40 217 233 253

28 (BA)

60 167 177 204

80 122 135 151

0 370 373 380

10 336 329 343

20 283 277 306

40 233 243 267

29 (BA)

60 189 190 217

80 144 154 173

30 (BA) 0 369 375 372

10 308 319 329

20 255 275 310

40 210 226 244

60 158 163 191

80 113 131 147

168

Table 28 continued

0 374 376 379

10 303 311 329

20 266 260 289

40 211 219 239

31 (BA)

60 155 158 178

80 112 121 149

0 370 374 375

10 314 323 339

20 276 280 305

40 222 233 258

32 (BA)

60 172 187 193

80 126 136 162

33 (BA) 0 377 380 377

10 308 306 320

20 254 265 280

40 205 214 237

60 144 155 175

80 107 118 138

169

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

19

abc

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

20

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

21

Fig 38 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing citric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

170

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

22

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

23

ab

c

12

13

14

15

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

24

Fig 39 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing tartaric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

171

a

b

c

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

25

abc

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

26

ab

c

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

27

Fig 40 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing glycerin and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

172

a

b

c

10

12

14

16

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

28

ab

c

10

12

14

16

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

29

a

b

c

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

30

Fig 41 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing propylene glycol and boric acid (a) 01 (b) 02 (c)

04

Palmitic acid

Stearic acid

Myristic acid

173

ab

c

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

31

ab

c

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

32

a

b

c

08

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

33

Fig 42 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing ethylene glycol and boric acid (a) 01 (b) 02 (c)

04

Myristic acid

Palmitic acid

Stearic acid

174

Table 29 Apparent first-order rate constants (kobs) for the degradation of ascorbic acid in

presence of different stabilizers in cream formulations in the dark

kobs times 102 (day

ndash1)ab

Cream

formulation Stabilizer

c

a b c

19 CT 055

(0994)

052

(0992)

044

(0991)

20 CT 048

(0995)

046

(0995)

038

(0992)

21 CT 064

(0994)

061

(0995)

052

(0994)

22 TA 063

(0994)

058

(0995)

049

(0996)

23 TA 054

(0995)

050

(0995)

041

(0994)

24 TA 081

(0995)

075

(0993)

066

(0995)

25 BA 118

(0996)

113

(0994)

097

(0994)

26 BA 087

(0995)

079

(0993)

068

(0994)

27 BA 124

(0995)

114

(0994)

101

(0993)

28 BA 134

(0995)

124

(0996)

110

(0992)

29 BA 116

(0996)

108

(0992)

096

(0995)

30 BA 142

(0993)

131

(0995)

115

(0995)

31 BA 145

(0995)

137

(0992)

117

(0995)

32 BA 130

(0996)

120

(0993)

107

(0994)

33 BA 153

(0995)

141

(0994)

122

(0994) a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental

conditions

c Stabilizer concentrations (andashc) are as given in Table 24

The estimated error is plusmn5

175

176

Table 30 First-order rate constants (k0)a for the degradation of ascorbic acid in the

absence of stabilizers and second-order rate constants (k) for the chemical

interaction of ascorbic acid with CT TA and BA in the dark

Cream

formulation Stabilizers

k0 times 102

(dayndash1

)

k times 102

(Mndash1

dayndash1

)

Correlation

coefficient

19 CT 060 797 0996

20 CT 052 723 0995

21 CT 069 850 0994

22 TA 068 710 0996

23 TA 058 636 0994

24 TA 086 758 0994

25 BA 126 444 0993

26 BA 092 375 0992

27 BA 131 480 0991

28 BA 141 488 0993

29 BA 122 418 0994

30 BA 149 531 0991

31 BA 155 578 0996

32 BA 137 472 0994

33 BA 163 627 0996

a The variations in the values of k0 are due to the presence of different emulsifying agents

in cream formulations

177

00

04

08

12

00 04 08 12 16 20

Citric acid concentration (M times 102)

kob

s times

10

2 (

da

yndash1)

19-21

00

04

08

12

00 05 10 15 20 25

Tartaric acid concentration (M times 102)

kob

s times

10

2 (

da

yndash1)

22-24

Fig 35 Plots of first-order rate constants (kobs) for the degradation of ascorbic acid in the

dark against citric acid (19ndash21) and tartaric acid (22ndash24) concentrations in

cream formulations

178

00

10

20

00 20 40 60

kob

s times

10

3 (

min

ndash1)

25-27

00

10

20

00 20 40 60

kob

s times

10

3 (

min

ndash1)

28-30

00

10

20

00 20 40 60

Boric acid concentration (M times 102)

kob

s times

10

3 (

min

ndash1)

30-33

Fig 44 Plots of first-order rate constants (kobs) for the degradation of ascorbic acid in the

dark against boric acid concentrations in cream formulations (25ndash33)

Glycerin

Propylene glycol

Ethylene glycol

179

00

04

08

12

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash

1 d

ayndash

1)

18-33

b

a

e

dc

Fig 45 Plots of k for degradation of ascorbic acid in the dark in creams (19ndash33) against

carbon chain length of emulsifier () Stearic acid () palmitic acid ()

myristic acid (a) CT (b) TA (c) BA with glycerin (d) BA with propylene

glycol (e) BA with ethylene glycol

CONCLUSIONS

AND

SUGGESTIONS

180

CONCLUSIONS

The main conclusions of the present study on the photodegradation of the ascorbic

acid in organic solvents and cream formulations are as follows

1 Identification of Photodegradation Products

The photodegradation of ascorbic acid in aqueous organic solvents and

laboratory prepared oil-in-water cream preparations on UV irradiation leads to the

formation of dehydroascorbic acid No further degradation products of dehydroascorbic

acid have been detected under the present experimental conditions The product was

identified by comparison of its Rf value and color of the spot with those of the authentic

compound by thin-layer chromatography and spectral changes

2 Assay of Ascorbic Acid

Ascorbic acid in aqueous organic solvents and cream preparations was assayed

in acidified methanolic solutions (pH 20) at 245 nm using a UV spectrophotometric

method Ascorbic acid in combination with other vitamins (riboflavin nicotinamide and

alpha-tocopherol) was assayed by the official iodimetric method due to interference by

these vitamins at the analytical wavelength Both analytical methods were validated

under the experimental conditions employed before their application to the assay of

ascorbic acid The recoveries of ascorbic acid in cream preparations are in the range of

90ndash96 and the reproducibility of both methods are within plusmn5 The F test and the t test

show that there is no significant difference between the precision of the two methods and

therefore these methods can be applied to the assay of ascorbic acid in cream

preparations with comparable results

181

3 Kinetics of Photodegradation

a) Photodegradation of ascorbic acid in organic solvents

Ascorbic acid degradation follows apparent first-order kinetics in aqueous

organic solvents A plot of the first-order rate constants (log kobs) versus solvent dielectric

constant is linear with positive slope indicating an increase in the rate with dielectric

constant On the contrary a plot of kobs verses reciprocal of solvent viscosity is linear with

a positive slope showing a decrease in the rate with solvent viscosity Thus the rate of

photodegradation of ascorbic acid (an oxidizable drug) depends on the solvent

characteristics

b) Photodegradation of ascorbic acid in cream preparations

Ascorbic acid has been found to follow apparent first-order kinetics in cream

preparations and the rate of degradation is affected by the following factors

i Effect of concentration

An apparent linear relationship has been observed between log kobs and

concentration (05ndash25) of ascorbic acid in a cream preparation Thus the rate of

degradation of ascorbic acid appears to be faster at a lower concentration

compared to that of a higher concentration on exposure to the same intensity of

light

ii Effect of carbon chain length of the emulsifying agent

The plots of kobs verses carbon chain length of the emulsifying agent show that the

photodegradation of ascorbic acid is affected in the order myristic acid gt stearic

acid gt palmitic acid This is predominantly due to the interaction of ascorbic acid

with palmitic acid and the carbon chain length (measure of relative polar

182

character) of the emulsifying acid probably does not play a part in the

photodegradation kinetics of ascorbic acid in creams This is evident from the

non-linear relationship between the rate constants for ascorbic acid degradation

and the carbon chain length of the emulsifying acids

iii Effect of viscosity

The values of kobs for the photodegradation of ascorbic acid in cream preparations

are in the order of humectant ethylene glycol gt propylene glycol gt glycerin

showing that the rates of degradation are influenced by the viscosity of the

humectant and decrease with an increase in the viscosity as observed in the case

of organic solvents

iv Effect of pH

The log kndashpH profiles for the photodegradation of ascorbic acid in creams

represent sigmoid type curves indicating an increase in the rate of oxidation of the

molecule with ionization (pH 42ndash70 557ndash999) The AHndash species appears to

be more susceptible to oxidation than the non-ionized molecule in the pH range

studied

v Effect of redox potential

The values of kobs show that the rate of photooxidation of ascorbic acid is

influenced by its redox potential which varies with pH The greater photostability

of ascorbic acid at pH 5ndash6 compared to that at pH 7 and above is due to its lower

rate of oxidation-reduction in the lower range The increase in the rate of

photooxidation with pH is due to a corresponding increase in the redox potential

of ascorbic acid

183

c) Photodegradation of ascorbic acid in the presence of other vitamins (riboflavin

nicotinamide alpha-tocopherol) in cream preparations

The photodegradation of ascorbic acid is affected by the presence of other

vitamins in creams The kinetic data on the photochemical interactions indicate that

riboflavin and nicotinamide act as photosensitizers in the degradation of ascorbic acid

and have an adverse effect on the photostability of the vitamin in creams Whereas

alpha-tocopherol exerts an inhibitory effect on the degradation of ascorbic acid by acting

as a redox partner in the creams Thus a combination of ascorbic acid and alpha-

tocopherol has a synergistic effect on the stabilization of ascorbic acid in creams These

vitamins do not appear to influence the rate of degradation of ascorbic acid in the dark

d) Photodegradation of ascorbic acid in the presence of citric acid tartaric acid and

boric acid in cream preparations

The rate of photodegradation of ascorbic acid in creams has been found to be

inhibited by the addition of compounds such as citric acid tartaric acid and boric acid in

creams These compounds show a stabilizing effect on the photodegradation of ascorbic

acid in the order citric acid gt tartaric acid gt boric acid The lower effect of boric acid

may be due to its interaction with the emulsifying agents and humectants Boric acid

exerts this effect by complex formation with ascorbic acid Citric acid and tartaric acid

are antioxidant synergists and in combination with ascorbic acid may exert a stabilizing

effect on its degradation

184

Salient Features of the Work

In the present work an attempt has been made to study the effects of solvent

characteristics formulation factors particularly the emulsifying agents in terms of the

carbon chain length and humectants in terms of viscosity medium pH drug

concentration redox potential and interactions with other vitamins and stabilizers on the

kinetics of photodegradation of ascorbic acid in cream preparations The study may

provide useful information to improve the photostability and efficacy of ascorbic acid in

cream preparations

SUGGESTIONS

The present work may provide guidelines for a systematic study of the stability of

drug substances in cream ointment preparations and the evaluation of the influence of

formulation variables such as emulsifying agents and humectants concentration pH

polarity viscosity redox potential on the rate of degradation and stabilization of drug

substances This may enable the formulator in the judicious design of formulations that

have improved stability and efficacy for therapeutic use The kinetic parameters may

throw light on the comparative stability of the preparations and help in the choice of

appropriate formulation ingredients

REFERENCES

186

Abe Y Horii H Taniguchi S (1986) A theoretical study of the oxidation mechanism

of triose reductone in reference to L-ascorbic acid Can J Chem 64 360-365

Ahmad I (1968) A Study of the Degradation of Riboflavin and Related Compounds

PhD Thesis University of London London

Ahmad I Rapson HDC (1990) Multicomponent spectrophotometric assay of

riboflavin and photoproducts J Pharm Biomed Anal 8 217-223

Ahmad I Vaid FHM (2006) Photochemistry of flavins in aqueous and organic

solvents In Silva E Edwards AM Eds Flavins Photochemistry and

Photobiology Royal Society of Chemistry Cambridge Chap 2

Ahmad I Tollin G (1981) Solvent effect on flavin electron transfer reactions

Biochemistry 20 5925-5928

Ahmad I Jamil N Zoha SMS (1973) Statistical evaluation of antioxidants used in

the manufacture of ascorbic acid tablets J Sci Univ Kar 2 269-287

Ahmad I Fasihullah Q Vaid FHM (2004b) A study of simultaneous photolysis and

photoaddition reactions of riboflavin in aqueous solution J Photochem Photobiol B

Biol 75 13-20

Ahmad I Fasihullah Q Vaid FHM (2005) Effect of phosphate buffer on

photodegradation reactions of riboflavin in aqueous solution J Photochem

Photobiol B Biol 78 229-234

Ahmad I Fasihullah Q Vaid FHM (2006a) Effect of light intensity and wavelength

on photodegradation reactions of riboflavin in aqueous solution J Photochem

Photobiol B Biol 82 21-27

187

Ahmad I Fasihullah Q Vaid F H M (2006b) Photolysis of formylmethylflavin in

aqueous and organic solvents Photochem Photobiol Sci 5 680-685

Ahmad I Ahmed S Sheraz MA Vaid FHM (2008) Effect of borate buffer on the

photolysis of riboflavin in aqueous solution J Photochem Photobiol B Biol 93 82-

87

Ahmad I Ahmed S Sheraz MA Vaid FHM (2009c) Borate toxicity effect on

drug stability and analytical applications In Chung MP Ed Handbook on

Borates Chemistry Production and Applications Nova Science Publishers Inc

New York in press

Ahmad I Sheraz MA Ahmed S Vaid FHM (2009d) Analytical applications of

borates Mater Sci Res J in press

Ahmad I Fasihullah Q Noor A Ansari IA Ali QNM (2004a) Photolysis of

riboflavin in aqueous solution a kinetic study Int J Pharm 280 199-208

Ahmad I Ahmed S Sheraz MA Aminuddin M Vaid FHM (2009a) Effect of

caffeine complexation on the photolysis of riboflavin in aqueous solution a kinetic

study Chem Pharm Bull in press

Ahmad I Ahmed S Sheraz MA Vaid FHM Ansari IA (2009b) Effect of

divalent ions on photodegradation kinetics and pathways of riboflavin in aqueous

solution Photochem Photobiol Sci accepted

Ahmed S (2009) Effect of Complexing Agents on the Photodegradation of Riboflavin

in Aqueous Solution PhD Thesis Baqai Medical University Karachi

Ahmad T (1978) Studies on the Stability of Sulphacetamide and Degradation Products

PhD Thesis University of Karachi Karachi

188

Ahmad T Ahmad I (1983) The effect of antioxidants on the photolysis of aqueous

sulfacetamide and sulfanilamide solutions Pol J Pharmacol Pharm 35 69-75

Albini A Fasani E Eds (1998) Drugs Photochemistry and Photostability The Royal

Society of Chemistry Cambridge

Allwood MC (1984a) Factors influencing the stability of ascorbic acid in total

parenteral nutrition infusions J Clin Hosp Pharm 9 75-85

Allwood MC (1984b) Compatibility and stability of TPN mixtures in big bags J Clin

Pharm Ther 9 181-198

Allwood MC Kearney MCJ (1998) Compatibility and stability of additives in

parenteral nutrition admixtures Nutrition 14 697-706

Al-Meshal IA Hassan MMA (1982) Ascorbic Acid In Florey K Ed Analytical

Profiles of Drug Substances Vol 11 Academic Press New York pp 45-78

Anderson NH Johnston D McLelland MA Munden P (1991) Photostability

testing of drug substances and drug products in UK pharmaceutical laboratories J

Pharm Biomed Anal 9 443-449

Ansari IA (2002) Photolysis and Interactions of Cyanocobalamin with some B-

Vitamins and Ascorbic Acid in Parenteral Solutions PhD Thesis University of

Karachi Karachi

Ansari IA Vaid Vaid HM Ahmad I (2004) Spectral Study of Photolysis of

Aqueous Cyanocobalamin Solutions in Presence of Vitamins B and C Pak J Pharm

Sci 17 83-93

Arcus CL Zilva SS (1940) The photochemical decomposition of L-ascorbic acid II

Biochem J 34 61-66

189

Arger CB Rupp D Lo P (1996) Preservation of dispersed systems In Liberman

HA Rieger MM Banker GS Eds Pharmaceutical Dosage Forms Disperse

Systems 2nd ed vol1 Marcel Dekker New York Chap9

Asard H May J Smirnoff N Eds (2004) Vitamin C Function and Biochemistry in

Animals and Plants BIOS Scientific Publishers New York

Asker AF Canady D Cobb C (1985) Influence of DL-methionine on the

photostability of ascorbic acid solutions Drug Dev Ind Pharm 11 2109-2125

Ault RG Baird DK Carrington HC Haworth WN Herbert R Hirst EL

Percival EGV Smith F Stacey M (1933) Synthesis of d- and of l-ascorbic acid

and of analogous substances J Chem Soc 1419-1423

Austria R Semenzato A Bettero A (1997) Stability of vitamin C derivatives in

solution and topical formulations J Pharm Biomed Anal 15 795-801

Baertschi SW Alsante KM (2005) Stress testing The chemistry of drug degradation

In Baertschi SW Ed Pharmaceutical Stress testing Predicting Drug Degradation

Taylor amp Francis London Chap 3

Bailey CW Bright JR Jasper JJ (1945) A study of the binary system nicotinamide-

ascorbic acid J Am Chem Soc 67 1184-1186

Ball GFM (2006) Vitamins in Foods Analysis Bioavailability and Stability CRC

Press Boca Raton FL Chap 15

Banhegyi G Marcolongo P Puskas F Fulceri R Mandl J Benedetti A (1998)

Dehydroascorbate and ascorbate transport in rat liver microsomal vesicles J Biol

Chem 273 2758-2762

190

Bardolph EM Taylor RH (1997) Sailors scurvy science and authority J R Soc

Med 90 238

Barel AO Paye M Maibach HI Eds (2001) Handbook of Cosmetic Science and

Technology Marcel Dekker New York

Barry BW (1983) Dermatological Formulations Percutaneous Absorption Marcel

Dekker New York

Barry BW (2002) Transdermal drug delivery In Aulton ME Ed Pharmaceutics

The Science of Dosage Form Design Churchill Livingstone London Chap 33

Basu TK Temple NJ Garg ML (1999) Antioxidants in Human Health and

Disease CABI Publishing Oxford

Becher P (1965) Emulsions Theory and Practice Reinhold New York p 1

Beissenhirtz M Scheller F Lisdat F (2003) Immobilized cytochrome c sensor in

organicaqueous media for the characterization of hydrophilic and hydrophobic

antioxidants Electroanalysis 15 1425-1435

Berlett BS Stadtman ER (1997) Protein oxidation in aging disease and oxidative

stress J Biol Chem 272 20313-20316

Betageri G Prabhu S (2002) Semisolid preparations In Swarbrick J Boylan JC

Eds Encyclopedia of Pharmaceutical Technology 2nd ed Marcel Dekker New

York pp 2436-2457

Bielski BHJ (1982) Chemistry of ascorbic acid radicals In Seib PA Tolbert BM

Eds Ascorbic Acid Chemistry Metabolism and Uses Advances in Chemistry

Series American Chemical Society Washington DC pp 81-100

191

Bielski BHJ Allen AO Schwartz HA (1981) Mechanism of disproportionation of

ascorbate radicals J Am Chem Soc 103 3516-3518

Billany M (2002) Suspensions and emulsions In Aulton ME Ed Pharmaceutics

The Science of Dosage Form Design 2nd ed Churchill Livingstone London Chap

23

Bisby RH Morgan CG Hamblett I Gorman AA (1999) Quenching of singlet

oxygen by trolox C ascorbate and amino acids effect of pH and temperature J Phys

Chem 103 7454-7459

Bissett DL (2006) Anti-aging skin care formulations In Draelos ZD Thaman LA

Eds Cosmetic Formulation of Skin Care Products Taylor amp Francis Group New

York Chap 11

Blaugh SM Hajratwala B (1972) Kinetics of aerobic oxidation of ascorbic acid J

Pharm Sci 61 556-562

Block LH (1996) Pharmaceutical emulsions and microemulsions In Lieberman HA

Rieger MM Banker GS Eds Pharmaceutical Dosage Forms Disperse Systems

Vol 2 2nd ed Chap 2

Bolliger HR Konig A (1969) Water-soluble vitamins In Stahl E Ed Thin-layer

Chromatography Springer-Verlag Berlin pp 304-306

Bonwstra JA De Graaff A Gooris GS Nijsse J Wiechers JW Van Aelst AC

(2003) Water distribution and related morphology in human stratum corneum at

different hydration levels J Invest Dermatol 120 750-758

Bors SR (2005) Scurvy Thomas Dunne Books St Martinrsquos Griffen New York

192

Bors W Buettner GR (1997) The vitamin C radical and its reactions In Packer L

Fuchs J Eds Vitamin C in Health and Disease Marcel Dekker New York Chap

4

Braun L Puskas F Csala M Meszaros G Mandl J Banhegyi G (1997)

Ascorbate as a substrate for glycolysis or gluconeogenesis evidence for an interorgan

ascorbate cycle Free Radic Biol Med 23 804-808

British Pharmacopoeia (2009) Her Majestyrsquos Stationary Office London Electronic

version

Buettner G (1993) The pecking order of free radicals and antioxidants lipid

peroxidation α-tocopherol and ascorbate Arch Biochem Biphys 300 535-543

Buettner GR Schafer FQ (2004) Ascorbate as an antioxidant In Asard H May

JM Smirnoff N Eds Vitamin C Function and Biochemistry in Animals and

Plants BIOS Scientific Publishers Oxford Chap 10

Burke KE (2006) Topical nutritional antioxidants In Draelos ZD Thaman LA

Eds Cosmetic Formulation of Skin Care Products Taylor amp Francis Group New

York Chap 21

Burns JJ Ed (1961) Vitamin C Ann NY Acad Sci 92 1

Byrom P Turnbull JH (1967) Excited states of flavin coenzymes II Anaerobic

oxidation of amino acids by excited riboflavin derivatives Photochem Photobiol 6

125-131

Cadenas E Packer L (2002) Handbook of Antioxidants 2nd ed Marcel Dekker New

York Chap 7-9

193

Capellmann M Bolt HM (1992) Simultaneous determination of ascorbic acid and

dehydroascorbic acid by HPLC with postcolumn derivatisation and fluorometric

detection Fresenius J Anal Chem 342 462-466

Cairns WL Metzler DE (1971) Photochemical degradation of flavins VI A new

photoproduct and its use in studying photolytic mechanism J Am Chem Soc 93

2772-2777

Carlotti ME Ugazio E Sapino S Peira E Gallarate M (2006) Photodegradation

of retinol and anti-aging effectiveness of two commercial emulsions J Cosmet Sci

57 261-277

Carpenter K (1986) The History of Scurvy and Vitamin C Cambridge University Press

Cambridge

Carr AC Zhu BZ Frei B (2000) Potential antiatherogenic mechanisms of ascorbate

(vitamin C) and alpha-tocopherol (vitamin E) Circ Res 87 349-354

Carstensen JT (1972) Theory of Pharmaceutical Systems Academic Press New York

pp 211-214

Chaudhri SK Jain NK (2009) History of cosmetics Asian J Pharm 3 164-167

Chessex P Lovoic JC Roulean T Brochu P St Louis P Levy E Al Varez F

(2002) Photooxidation of parenteral multivitamins induced hepatic steatosis in a

neonatal guinea pig model of intravenous infusion Pediatr Res 52 958-963

Connors KA Amidon GL Stella VJ (1986) Chemical Stability of Pharmaceuticals

A Handbook for Pharmacists 2nd ed Wiley New York pp 105-106 142 208-220

194

Dabrowski K Huiterleitner S (1989) Application of a simultaneous assay of ascorbic

acid dehydroascorbic acid and ascorbic sulfate in biological matter Analyst

(London) 114 83-87

Dale JK Booth RE (1976) In Hoover JE Ed Dispensing of Medication 8th ed

Mack Easton Pennsylvania pp 479-481

Darr D Dunston S Faust H Pinnell S (1996) Effectiveness of antioxidants (vitamin

C and E) with and without sunscreens as topical photoprotectants Acta Derm

Venereol 76 264-268

Davey MW Montagu MV Inze D Sanmartin M Kanellis A Smirnoff N

Benzie IJJ Strain JJ Favell D Fletcher J (2000) Plant L-ascorbic acid

chemistry function metabolism bioavailability and effect of processing J Sci Food

Agr 80 825-860

Davies MB Austin J Partridge DA (1991) Vitamin C Its Chemistry and

Biochemistry The Royal Society of Chemistry Cambridge

De La Rochette A Birlouez-Aragon I Silva E Morliere P (2003) Advanced

glycation endproducts as UVA photosensitizers of tryptophan and ascorbic acid

consequences for the lens Biochem Biophys Acta 1621 235-241

De La Rochette A Silva E Birlouez-Aragon I Mancini M Edwards AM

Morliere P (2000) Riboflavin photodegradation and photosensitization effects are

highly dependent on oxygen and ascorbate concentration Photochem Photobiol 72

815-820

195

De Vries H Beijersbergen Van Henegouwen GMJ Huff FA (1984) Photochemical

decomposition of chloramphenicol in a 025 eye drop and in a therapeutic

intraocular concentration Int J Pharm 20 265-271

Dean RT Fu S Stocker R Davies MJ (1997) Biochemistry and pathology of

radicalmediated protein oxidation Biochem J 324 1-18

DeRitter E (1982) Vitamins in pharmaceutical formulations J Pharm Sci 71 1073-

1096

Deutsch JC (1998a) Ascorbic acid oxidation by hydrogen peroxide Anal Biochem

255 1-7

Deutsch JC (1998b)Spontaneous hydrolysis and dehydration of dehydroascorbic acid

in aqueous solution Anal Biochem 260 223-229

Deutsch JC (1998c)oxygen-accepting antioxidants which arise during ascorbate

oxidation Anal Biochem 265 238-245

Dodsun KY Young ER Soliman AGM (1992) Determination of total vitamin C

in various food matrices by liquid chromatography and fluorescence detection J

AOAC Int 75 887-891

Dollery C Ed (1999) Therapeutic Drugs 2nd ed Vol 1 Churchill Livingstone

London pp A213-A216

Doores S (2002) pH control agents and acidulants In Branen LA Davidson PM

Salminen S Thorngate JH Eds Food Additives Marcel Dekker New York p

637

Eberlein-Koumlnig B Ring J (2005) Relevance of vitamins C and E in cutaneous

photoprotection J Cosmet Dermatol 4 4-9

196

Ehrlich M Rao J Pabby A Goldman MP (2006) Improvement in the appearance

of wrinkles with topical transforming growth factor beta(1) and l-ascorbic acid

Dermatol Surg 32 618-625

EIRI Board (2007) Profitable Small Scale Manufacture of Cosmetics Engineers India

Research Institute Nai Sarak Delhi

Eitenmiller RR Ye L Landen WOJr (2008) Vitamin Analysis for the Health and

Food Sciences 2nd ed CRC Press Boca Raton FL Chap 5

Elia S (2009) Nutrition In Kumar P Clark M Eds Kumar and Clarkrsquos Clinical

Medicine 7th ed Elsevier Limited Chap 5

Elmore AR (2005) Final report of the safety assessment of L-ascorbic acid calcium

ascorbate magnesium ascorbate magnesium ascorbyl phosphate sodium ascorbate

and sodium ascorbyl phosphate as used in cosmetics Int J Toxicol 24 51-111

Erb C Nau-Staudt K Flammer J Nau W (2004) Ascorbic acid as a free radical

scavenger in porcine and bovine aqueous humour Ophthal Res 36 38-42

Espinal-Perez LE Moncada B Castanedo-Cazares JP (2004) A double-blind

randomized trial of 5 ascorbic acid vs 4 hydroquinone in melasma Int J

Dermatol 43 604-607

Fall HH Petering HG (1956) Metabolic inhibitors 1 67-Dimethyl-9-

formylmethylisoalloxazine67-dimethyl-9-(2-hydroxyethyl)-isoalloxazine and

derivatives J Am Chem Soc 78 377-381

Farahmand S Tajerzadeh H Farboud ES (2006) Formulation and evaluation of a

vitamin C multiple emulsion Pharm Dev Technol 11 255-261

197

Fasani E Albini A (2005) Photostability Stress Testing Predicting Drug Degradation

Taylor amp Francis London Chap 10

Fasihullah Q (1988) Photolysis of Flavins in Aqueous and Organic Solvents PhD

Thesis University of Karachi Karachi

Fasman GD Ed (1976) CRC Handbook of Biochemistry and Molecular Biology 3rd

ed Physical Chemical Data Vol 1 CRC Press Cleveland Ohio pp 122-130

Ferreira SLC Bandeira MLSF Lemos VA Dos Santos HC Spinola Costa

AC De Jesus DS (1997) Sensitive spectrophotometric determination of ascorbic

acid in fruit juices and pharmaceutical formulations using 2-(5-bromo-2-pyridylazo)-

5-diethylaminophenol (Br-PADAP) Anal Bioanal Chem 357 1174-1178

Fessenden RW Verma NC (1978) A time-resolved electron spin resonance study of

the oxidation of ascorbic acid by hydroxyl radical Biophys J 24 413-430

Finholt P Paulssen RB Higuchi T (1963) Rate of anaerobic degradation of ascorbic

acid in aqueous solution J Pharm Sci 52 948-954

Florence AT Attwood D (2006) Drug Stability Physicochemical Principles of

Pharmacy 4th ed Pharmaceutical Press London p 411

Flynn GL (2002) Cutaneous and transdermal delivery-processes and systems of

delivery In Banker GS and Rhodes CT Eds Modern Pharmaceutics Marcel

Dekker New York Chap 8

Foote CS (1991) Definition of Type I and Type II Photosensitized oxidation

Photochem Photobiol 54 659

Fornaro A Coicher N (1998) L-ascorbic acid complexation and redox reactions with

some transition metal ions Quni Nova 21 642-650

198

Fraga CG Motchink PA Wyrobek AJ Rempel DM Ames BN (1996)

Smoking and low antioxidant levels increase oxidative damage to sperm DNA

Mutat Res 351 199-203

Fraga CG Motchnik PA Shigenaga MK Helbock HJ Jacob RA Ames BN

(1991) Ascorbic acid protects against endogenous oxidative DNA damage in human

sperm Proc Natl Acad Sci USA 88 11003-11006

Frenich AG Torres ME Vega AB Vidal JL Bolanos PP (2005) Determination

of ascorbic acid and carotenoids in food commodities by liquid chromatography with

mass spectrometry detection J Agric Food Chem 53 7371-7376

Fung HL (1990) Drug stability In Banker GS Rhodes CT Eds Modern

Pharmaceutics 2nd ed Marcel Dekker New York Chap 6

Gallarate M Carlotti ME Trotta M Bovo S (1999) On the stability of ascorbic

acid in emulsified systems for topical and cosmetic use Int J Pharm 188 233-241

Ganshirt H Malzacher A (1960) Separation of several vitamins of the B group and C

by chromatography Naturwiss 47 279-280

Gardner WH (1972) Acidulants in food processing In Furia TE Ed Handbook of

Food Additives 2nd ed Vol 1 CRC Press Cleveland OH pp 225ndash270

Garrett ER (1967) Kinetics and mechanisms in stability of drugs In Bean HS

Beckett AH Carless JE Eds Advances in Pharmaceutical Sciences Vol 2

Academic Press London Chap 1

Garti N Aserin A (1996) Pharmaceutical emulsions double emulsions and

microemulsions In Benita S Ed Microencapsulation Methods and Industrial

Applications Marcel Dekker New York pp 411-534

199

Gensler M Rossmann A Schmidt HL (1995) Detection of added L-ascorbic acid in

fruit juice by isotope ratio mass spectrometry J Agric Food Chem 43 2662-2666

Gibbons E Allwood MC Neal T Hardy G (2001) Degradation of dehydroascorbic

acid in parenteral nutrition mixtures J Pharm Biomed Anal 25 605-611

Guitton C Gros P Comtat M (2007) Direct electrochemical measurements in dermo-

cosmetic creams In Alegret Merkoci Eds Comprehensive Analytical Chemistry

Elsevier Amsterdam 49 e59-e68

Guttman DE Brooke D (1963) Solution phase interaction of nicotinamide with

ascorbic acid J Pharm Sci 52 941-945

Gyorgy P Pearson WN Eds (1967) Vitamin Methods Vol VII Academic Press

New York pp 27-49

Hagg M Ylikoski S Kumpulainen J (1994) Vitamin C and α- and β-carotene in

vegetables consumed in Finland during 1988-1989 and 1992-1993 J Food Compos

Anal 7 252-259

Hagg M Ylikoski S Kumpulainen A (1995) Vitamin C content in fruits and berries

consumed in Finland J Food Compos Anal 8 12-20

Hall NK Chapman TM Kim HJ Min DB (2009) Antioxidant mechanisms of

Trolox and ascorbic acid on the oxidation of riboflavin in milk under light Food

Chem in press doi 101016jfoodchem200905017

Halliwell B (1996) Vitamin C antioxidant or pro-oxidant in vivo Free Radic Res 25

439-454

200

Halliwell B Whiteman M (1997) Antioxidant and prooxidant properties of vitamin C

In Packer L Fuchs J Eds Vitamin C in Health and Disease Marcel Dekker New

York pp 25-94

Halver JE Felton SP (2001) Analytical enigmas in assaying for vitamers C In

Dabrowski K Ed Ascorbic Acid in Aquatic Organisms CRC Press Boca Raton

Chap 3

Hanck A Ed (1982) Vitamin C New Clinical Applications in Immunology Lipid

Metabolism and Cancer Hans Huber Bern

Hashmi MH (1973) Assay of Vitamins in Pharmaceutical Preparations John Wiley

New York Chap 13

Hatchard CG Parker CA (1956) A new sensitive chemical actinometer II Potassium

ferrioxalate as a standard chemical actinometer Proc R Soc London Ser A A235

518-536

Heber GK Markovic B Hayes A (2006) An immunohistological study of anhydrous

topical ascorbic acid compositions on ex vivo human skin J Cosmet Dermatol 5

150-156

Heelis PF Parsons BJ Phillips GO McKellar JF (1981) The flavin sensitized

photooxidation of ascorbic acid a continuous and flash photolysis study Photochem

Photobiol 37 7-13

Henry JA Ed (1997) The New Guide to Medicines and Drugs The British Medical

Association 4th ed Dorling Kindersley London p 434

Hickey S Roberts H (2004) Ascorbate-The Science of Vitamin C Electronic Version

(ISBN 1-4116-0724-4)

201

Higdon JV Frei B (2002) The Antioxidant Vitamin C and E AOAC Press Arlington

VA pp 1-16

Hirst EL Zilva SS (1933) Ascorbic acid as the anti-scorbutic factor Biochem J 27

1271-1278

Ho AHL Puri A Sugden JK (1994) Effect of sweetening agents on the light

stability of aqueous solutions of L-ascorbic acid Int J Pharm 107 199-203

Holmstrom B Oster G (1961) Riboflavin as an electron donor in photochemical

reactions J Am Chem Soc 83 1867-1871

Homann P Gaffron H (1964) Photochemistry and metal catalyzes studies in a flavin

sensitized oxidation of ascorbic acid Photochem Photobiol 3 499-515

Hughes DE (1985) Irreversible reaction kinetics of the aerobic oxidation of ascorbic

acid Anal Chem 57 555-558

Humbert PG Haftek M Creidi P Lapiegravere C Nusgens B Richard A Schmitt D

Rougier A Zahouani H (2003) Topical ascorbic acid on photoaged skin Clinical

topographical and ultrastructural evaluation double-blind study vs placebo Exp

Dermatol 12 237-244

ICH Harmonized Tripartite Guideline (1997) QIB Photostability Testing of New Drug

Substances and Products Federal Register 62 27115-27122

Ichikawa M Ide N Shiraishi S Ono K (2005) Effect of various halide salts on the

incompatibility of cyanocobalamin and ascorbic acid in aqueous solution Chem

Pharm Bull 53 688-690

202

Ihara H Shino Y Aoki Y Hashizume N Minegishi N (2000) A simple and rapid

method for the routine assay of total ascorbic acid in serum and plasma using

ascorbate oxidase and o-phenylenediamine J Nutr Sci Vitaminol 46 321-324

Im-Emsap W Paeratakul O Siepmann J (2002) Disperse Systems In Banker GS

Rhodes CT Ed Modern Pharmaceutics Maecel Dekker New York Chap 9

Irache JM Ezpeleta I Vega FA (1993) HPLC determination of antioxidant

synergists and ascorbic acid in some fatty pharmaceuticals cosmetics and food

Chromatographia 35 232-23

Iwase H Ono I (1994) Determination of ascorbic acid in human plasma by high-

performance liquid chromatography with electrochemical detection using a

hydroxyapatite cartridge for precolumn deproteinization J Chromatogr B Biomed

Appl 655 195-200

Jain S Tiwari AK Jain NK (2006) Topical products In Jain NK Ed

Pharmaceutical Product Development CBS Publishers New Delhi Chap 7

Jamil N Ahmad I (1984) Moisture content of ascorbic acid tablets stored at various

temperatures and humidities Pak J Sci Ind Res 27 252

Jamil N Ahmad I Beg AE (1980a) The effect of UV and tungsten light on physical

characteristics of ascorbic acid tablets Laboratory prepared samples IV Kar Univ

J Sci 8 77-87

Jamil N Ahmad I Beg AE (1980b) The effect of UV and tungsten light on physical

characteristics of ascorbic acid tablets Commercial samples III Kar Univ J Sci 8

117-126

203

Jentzsch A Aikens P (2005) RetiSTARTM

for cosmetic formulations-stabilized retinol

In Rosen MR Ed Delivery System Handbook for Personal Care and Cosmetic

Products Technology Applications and Formulations William Andrew Publishing

Norwich NY pp 862-866

Johnston CS Steinberg FM Rucker RB (2007) Ascorbic Acid In Zempleni J

Rucker RB McCormick DB Suttie JW (Eds) Handbook of Vitamins 4th ed

CRC Press Boca Raton FL Chap 15

Jung MY Kim SM Kim SY (1995) Riboflavin-sensitized photooxidation of

ascorbic acid kinetics and amino acid effects Food Chem 53 397-403

Kall MA Anderson CJ (1999) Improved method for simultaneous determination of

ascorbic acid and dehydroascorbic acid isoascorbic acid and dehydroisoascorbic acid

in food and biological samples J Chromatogr B Biomed Sci Appl 730 101-111

Kampfenkel K Van Montagu M Inze D (1995) Extraction and determination of

ascorbate and dehydroascorbate from plant tissue Anal Biochem 225 165-167

Kaplan DL Moloney SJ Troy WR Dickens MS Pinnell SR (1989) A new

stabilized ascorbic acid solution percutaneous absorption and effect on relative

collagen synthesis J Cutan Aging Cosmet Derm I 115-121

Karatepe M (2004) Simultaneous determination of ascorbic acid and free

malondialdehyde in human serum by HPLC-UV LC GC (North America) 22 362-

365

Karlsen J (1996) Light-activated drugs and drug formulations in drug targeting In

Tonneson HH Ed The Photostability of Drugs and Formulations Taylor amp

Francis London Chap 8

204

Kassem MA Kassem AA Ammar HO (1969a) Studies on the stability of

injectable L-ascorbic acid solutions I Effect of pH solvent light and container

Pharm Acta Helv 44 611-623

Kassem MA Kassem AA Ammar HO (1969b) Studies on the stability of

injectable L-ascorbic acid solutions II Effect of metal ions and oxygen content of

solvent water Pharm Acta Helv 44 667-675

Kassem MA Kassem AA Ammar HO (1972) Studies on the stability of injectable

L-ascorbic acid solutions 3 Effect of metal-complexing agents Pharm Acta Helv

47 89-97

Kaushansky K Kipps TJ (2006) Hematopoietic agents growth factors minerals and

vitamins In Brunton L Ed Goodman amp Gilmans The Pharmacologic Basis of

Therapeutics 11th ed digital ed McGraw-Hill Companies USA Chap 53

Kearney MCJ Allwood MC Martin H Neal T Hardy G (1998) The influence

of amino acid source on the stability of ascorbic acid in TPN mixtures Nutrition 14

173-178

Kim H Kirshenbaum LJ Rosenithol I Riesz P (1993) Photosensitized formation

of ascorbate radicals by riboflavin an ESR study Photochem Photobiol 57 777-

784

Kimoto K Terada S Yamaguchi T (1997) Analysis of ascorbic acid

dehydroascorbic acid and transformation products by ion-pairing high-performance

liquid chromatography with multi-wavelength ultraviolet and electrochemical

detection Methods Enzymol 279 3-12

205

King CG Burns JJ Ed (1975) Second Conference on Vitamin C Ann N Y Acad

Sci 258

Kitagawa Y (1968) The effects of ultraviolet rays on vitamin C Eiyo To Shokuryo 20

416-421 through CA (1968) 68 111560f

Klick S Muijselaar PG Waterval J EichingerT Korn C Gerding TK Debets

AJ de Griend CS van den Beld C Somsen GW De Jong GJ (2005)Toward

a generic approach for stress testing of drug substances and drug products Pharm

Tech 29 48-66

Konings EJ (2006) Water-soluble vitamins J AOAC Int 89 285-288

Kwon BM Foote CS (1988) Chemistry of singlet oxygen 50 Hydroperoxide

intermediates in the photooxygenation of ascorbic acid J Am Chem Soc 110

6582-6583

Kwon BM Foote CS Khan SI (1989) Photooxygenation of ascorbic acid

derivatives and model compounds J Am Chem Soc 111 1854-1860

Laborie S Lavoie JC Chessex P (1998) Paradoxical role of ascorbic acid and

riboflavin in solutions of total parenteral nutrition implication in photoinduced

peroxide generation Pediatr Res 43 601-601

Laborie S Lavoie JC Pineault M Chessex P (1999) Protecting solutions of

parenteral nutrition from peroxidation JPEN 23 104-108

Laborie S Lavoie JC Pineault M Chessex P (2000) Contribution of multivitamin

air and light in the generation of peroxides in adult and neonatal parenteral nutrition

solutions Ann Pharmacother 34440-445

206

Laborie S Lavoie JC Rouleau T Chessex P (2002) Multivitamin solutions for

enteral supplementation a source of peroxides Nutrition 18 470-473s

Lachman L DeLuca P Akers MJ (1986) Kinetic principles and stability testing In

Lachman L Liberman HA Kanig JL Eds The Theory and Practice of

Industrial Pharmacy 3rd ed Lea amp Febiger Philadelphia Chap 26

Laidler KJ (1987) Chemical Kinetics 3rd ed Harper and Row New York pp 183-

185

Lambert WE de Leenheer A (1992) In Nollet LML Ed Food Analysis by HPLC

Marcel Dekker New York Chap 10

Lavoie JC Chessex P Rouleau T Migneault D Comte B (2004) Light-induced

byproducts of vitamin C in multivitamin solutions Clin Chem 50 135-140

Lee JS Kim JW Han SH Chang IS Kang HH Lee OS Oh SG Suh KD

(2004) The stabilization of L-ascorbic acid in aqueous solution and water-in-oil-in-

water double emulsion by controlling pH and electrolyte concentration J Cosmet

Sci 55 1-12

Lee KM Kwon JY Lee KW Lee HJ (2009) Ascorbic acid 6-palmitate

suppresses gap-junctional intercellular communication through phosphorylation of

connexin 43 via activation of the MEK-ERK pathway Mutat Res 660 51-56

Leacutevecircque N Mac-Mary S Muret P Makki S Aubin F Kantelip JP Heusegravele C

SS Humbert P (2005) Evaluation of a sunscreen photoprotective effect by ascorbic

acid assessment in human dermis using microdialysis and gas chromatography mass

spectrometry Exp Dermatol 14 176-181

207

Levine M (1986) New concepts in the biology and biochemistry of ascorbic acid N

Engl J Med 314 892-902

Lewin S (1976) Vitamin C Its Molecular Biology and Medical Potential Academic

Press New York

Liau LS Lee BL New AL Ong CN (1993) Determination of plasma ascorbic

acid by high-performance liquid chromatography with ultraviolet and electrochemical

detection J Chromatogr 612 63-70

Lin JY Selim MA Shea CR Grichnik JM Omar MM Monteiro-Riviere NA

Pinnell SR (2003) UV photoprotection by combination topical antioxidants vitamin

C and vitamin E J Am Acad Dermatol 48 866-874

Lin FH Lin JY Gupta RD Tournas JA Burch JA Selim MA Monteiro-

Riviere NA Grichnik JM Zielinski J Pinnell SR (2005) Ferulic acid

stabilizes a solution of vitamins C and E and doubles its photoprotection of skin J

Invest Dermatol 125 826-832

Lindahl T (1993) Instability and decay of the primary structure of DNA Nature 362

709-715

Litner CJ (1973) Drug Product Stability In Cooper MS Ed Quality Control in

Pharmaceutical Industry Vol 2 Academic Press New York p 185

Liu TZ Chin N Kiser MD Bigler WN (1982) Specific spectrophotometry of

ascorbic acid in serum or plasma by use of ascorbate oxidase Clin Chem 28 2225-

2228

Lu GW Flynn GL (2009) Cutaneous and transdermal delivery-processes and

systems of delivery In Florence AT Siepmann J Eds Modern Pharmaceutics-

208

Applications and Advances Vol 2 5th ed Informa Healthcare Inc New York

Chap 3

Lykkesfeldt J (2000) Determination of ascorbic acid and dehydroascorbic acid in

biological samples by high-performance liquid chromatography using subtraction

methods reliable reduction with tris [2-carboxyethyl] phosphine hydrochloride Anal

Biochem 282 89-93

Macek TJ (1960) Stability problems with some vitamins in pharmaceuticals Am J

Pharm 132 433-455

Mader WJ (1961) Vitamins In Higuchi T Brochmann-Hanssen E Eds

Pharmaceutical Analysis Interscience New York pp 689-693

Maggi N Pasqualucci CR Ballotta R Sensi P (1966) Rifampicin a new orally

active rifamycin Chemotherapy 11 285-292

Maia AM Baby AR Pinto CA Yasaka WJ Suenaga E Kaneko TM Velasco

MV (2006) Influence of sodium metabisulfite and glutathione on the stability of

vitamin C in OW emulsion and extemporaneous aqueous gel Int J Pharm 322

130-135

Mancini M Edwards AM Becker MI De Ioannes A Silva E (2000) Reactivity

of monoclonal antibodies against a tryptophan-riboflavin adduct toward irradiated

and non-irradiated bovine-eye-lens protein fractions an indicator of in vivo visible

light mediated phototransformations J Photochem Photobiol B Biol 55 9-15

Manoharan M Schwille PO (1994) Measurement of ascorbic acid in human plasma

and urine by high performance liquid chromatography Results in healthy subjects

209

and patients with idiopathic calcium urolithiasis J Chromatogr B Biomed Appl

654 134-139

Margolis SA Schmidt HL (1996) Measurement of ascorbic acid in human plasma

and serum stability intralaboratory repeatability and interlaboratory reproducibility

Clin Chem 42 1257-1262

Mason P (2007) Dietary Supplements 3rd ed Pharmaceutical Press pp 330-339

Massad WA Bertolotti S Garcia N (2004) Kinetics and mechanism of the vitamin

B2-sensitized photooxidation of isoproterenol Photochem Photobiol 79 428-433

Matsubayashi T Sakaeda T Kita T Kurimoto Y Nakamura T Nishiguchi K

Fujita T Kamiyama F Yamamoto A Okumura K (2003) Intradermal

concentration of hydroquinone after application of hydroquinone ointments is higher

than its cytotoxic concentration Biol Pharm Bull 26 1365-1367

McBride MM Moore WM (1967) The photochemistry of riboflavin II

Polarographic studies on the hydroxyethyl and formylmethyl analogues of riboflavin

Photochem Photobiol 6 103-113

McDowell LR (2000) Vitamins in Animal and Human Nutrition 2nd ed Iowa State

University Press Chap 15

Meister A (1994) Glutathione-ascorbic acid antioxidant system in animals J Biol

Chem 269 9397-9400

Meister A (1995) Mitochondrial changes associated with glutathione deficiency

Biochim Biophys Acta 1271 35-42

Min DB Boff JM (2002) Chemistry and reaction of singlet oxygen in foods

CRFSFS 1 58-72

210

Moeslinger T Brunner M Volf I Spieckermann PG (1995) Spectrophotometric

determination of ascorbic acid and dehydroascorbic acid Clin Chem 41 1177-1181

Moffat AC Osselton MD Widdop B (2004) Clarkersquos Analysis of Drugs and

Poisons 3rd ed Pharmaceutical Press London pp 649-650

Moore DE (2004) Standardization of kinetic studies of photodegradation reactions In

Tonnesen HH Ed Photostability of Drugs and Drug Formulations 2nd ed CRC

Press Boca Raton FL Chap 3

Moura T Gaudy D Jacob M Cassanas G (1994) pH influence on the stability of

ascorbic acid spray-drying solutions Pharm Acta Helv 69 77-80

Murray JC Burch JA Streilein RD Iannacchione MA Hall RP Pinnell SR

(2008) A topical antioxidant solution containing vitamins C and E stabilized by

ferulic acid provides protection for human skin against damage caused by ultraviolet

irradiation J Am Acad Dermatol 59 418-425

Mushran SP Agarwal MC (1977) Mechanistic studies on the oxidation of ascorbic

acid 5 J Sci Res (India) 36 274-283

Nerlo H Palak W Tomaszek J (1968) Stabilization of vitamin preparations II

Syrup of vitamin D3 and its stabilization Acta Polon Pharm 25 183-188

Niki E Noguchi N (1997) Protection of human low-density lipoprotein from oxidative

modification by vitamin C In Packer L Fuchs J Eds Vitamin C in Health and

Disease Marcel Dekker New York pp 183-192

Nishikimi M Fukuyama R Minoshima S Shimizu N Yagi K (1994) Cloning and

chromosomal mapping of the human nonfunctional gene for L-gulono-gamma-

211

lactone oxidase the enzyme for L-ascorbic acid biosynthesis missing in man J Biol

Chem 269 13685-13688

Njus D Wigle M Kelly PM Kipp BH Schlegel HB (2001) Mechanism of

ascorbic acid oxidation by Cytochrome B561 Biochemistry 40 11905-11911

Nusgens BV Humbert P Rougier A Colige AC Haftek M Lambert CA

Richard A Creidi P Lapiegravere CM (2001) Topically applied vitamin C enhances

the mRNA level of collagens I and III their processing enzymes and tissue inhibitor

of matrix metalloproteinase 1 in the human dermis J Invest Dermatol 116 853-859

Ogata Y Kosugi Y (1969) Solvent effect on the autooxidation of L-ascorbic acid

Tetrahedron 25 1055-1062

OrsquoKeefe T (2001) Ascorbic acid and stable ascorbate esters as sources of vitamin C in

aquaculture feeds ASA Technical Bulletin AQ48 wwwsoyaquaorg

OrsquoNeil MJ Ed (2001) The Merck Index 13th ed Merck amp Co Inc Rahway NJ

USA Electronic version

Ortega GR Delgado JN (1998) Vitamins and related compounds In Wilson and

Gisvoldrsquos Textbook of Organic Medicinal and Pharmaceutical Chemistry 10th ed

Lippincott-Raven Philadelphia Chap 27

Ortwerth BJ Monnier VM (1997) Protein glycation by the oxidation products of

ascorbic acid In Packer L Fuchs J Eds Vitamin C in Health and Disease Marcel

Dekker New York pp 123ndash142

Ozer O Muguet V Roy E Grossiord JL Seiller M (2000) Stability study of

WOW viscosified multiple emulsions Drug Dev Ind Pharm 26 1185-1189

Packer I Fuchs J (1999) Vitamin C in Health and Disease Marcel Dekker New York

212

Packer JE Slater TF Wilson RL (1979) Direct observation of a free radical

interaction between vitamin E and vitamin C Nature 278 737-738

Packer L Traber MG Kraemer K Frei B (2002) The Antioxidant Vitamins C and

E AOCS Press Illinois

Padayatty SJ Katz A Wang Y Eck P Kwon O Lee JH Chen S Corpe C

Dutta A Dutta SK Levine M (2003) Vitamin C as an antioxidant Evaluation of

its role in disease prevention J Am Coll Nutr 22 18-35

Parker CA (1953) A new sensitive chemical actinometer I Some trials with potassium

ferrioxalate Proc Roy Soc (Lond) A220 104-116

Parker JN Parker PM (2003) Antioxidants ICON Group International

Pastore P Rizzetto T Curcuruto O Cin MD Zaramella A Martin D (2001)

Characterization of dehydroascorbic acid solutions by liquid chromatography mass

spectrometry Rapid Commun Mass Spectrom 15 2051-2057

Pelletier O (1985) In Augustin J Klein BP Becker D Venugopal PB Eds

Methods of Vitamins Assay Wiley New York pp 303-347

Peyrat-Maillard MN Bonnely S Rondini L Berset C (2001) Effect of vitamin E

and vitamin C on the antioxidant activity of malt rootlets extracts Lebensmittel-

Wissenschaft Und-Technological 34 176-182

Pinnell SR (2003) Cutaneous photodamage oxidative stress and topical antioxidant

protection J Am Acad Dermatol 48 1-19

Pinnell SR Madey DL (1998) Topical vitamin C in skin care Aesthet Surg J 18

468-470

213

Pinnell SR Yang H Omar M Monteiro-Riviere N DeBuys HV Walker LC

Wang Y Levine M (2001) Topical L-ascorbic acid percutaneous absorption

studies Dermatol Surg 27 137-142

Placzek M Gaube S Kerkmann U Gilbertz KP Herzinger T Haen E Przybilla

B (2005) Ultraviolet B-induced DNA damage in human epidermis is modified by

the antioxidants ascorbic acid and D-alpha-tocopherol J Invest Dermatol 124 304-

307

Puskas F Gergely PJr Banki K Perl A (2000) Stimulation of the pentose

phosphate pathway and glutathione levels by dehydroascorbate the oxidized form of

vitamin C FASEB J 14 1352-1361

Puskas F Gergely P Niland B Banki K Perl A (2002) Differential regulation of

hydrogen peroxide and Fas-dependent apoptosis pathways by dehydroascorbate the

oxidized form of vitamin C Antioxid Redox Signal 4 357-369

Racz I (1989) Drug Formulation Wiley New York pp 121-122

Ramu A Mehta MM (2000) The riboflavin-mediated photooxidation of doxorubicin

Cancer Chemoth Pharm 46 449-458

Raschke T Koop U Duumlsing HJ Filbry A Sauermann K Jaspers S Wenck H

Wittern KP (2004) Topical activity of ascorbic acid from in vitro optimization to in

vivo efficacy Skin Pharmacol Physiol 17 200-206

Reed RA Harmon P Manas D Wasylaschuk W Galli C Biddell R Bergquist

PA Hunke W Templeton AC Ip D (2003) The role of excipients and package

components in the photostability of liquid formulations PDA J Pharm Sci Tech 57

351-368

214

Reichstein T Griissner A Oppcnuuer R (1933) Synthese der d- und l-Ascorbinsaumlure

(C-Vitamin) Helv Chim Acta 16 1019-1033

Riehl J Maupin C Layloff T (1995) On the choice of a photolysis source for the

photostability testing of pharmaceuticals Pharmacop Forum 21 1654-1663

Rivlin RS (2007) Riboflavin (vitamin B2) In Zempleni J Rucker RB McCormick

DB Suttie JW Eds Handbook of Vitamins 4th ed Taylor amp Francis CRC

Press Boca Raton FL p 241

Rogers AR Yacomeni JA (1971) The effect of pH on the aerobic degradation of

ascorbic acid solutions J Pharm Pharmacol 23 218S

Rosen MR Ed (2005) Delivery System Handbook for Personal Care and Cosmetic

Products-Technology Applications and Formulations William Andrew Publishing

Norwich NY

Rosenberg HR (1945) Chemistry and Physiology of the Vitamins Interscience New

York pp 289-338

Roth HJ Eger K Troschutz R (1991) Pharmaceutical Chemistry Vol 2 Drug

Analysis Ellis Horwood London pp 282-288

Rowe RC Sheskey PJ Quinn ME (2009) Handbook of Pharmaceutical Excipients

6th ed Pharmaceutical Press London pp 43-47 181-183 731-732

Roacutezanowska M Bober A Burke JM Sarna T (1997) The role of retinal pigment

epithelium melanin in photoinduced oxidation of ascorbate Photochem Photobiol

65 472-479

Rozman B Gasperlin M (2007) Stability of vitamins C and E in topical

microemulsions for combined antioxidant therapy Drug Deliv 14 235-245

215

Rozman B Gasperlin M Tinois-Tessoneaud E Pirot F Falson F (2009)

Simultaneous absorption of vitamins C and E from topical microemulsions using

reconstructed human epidermis as a skin model Eur J Pharm Biopharm 72 69-75

Ruumlmelin A Fauth U Halmagyi M (1999) Determination of ascorbic acid in plasma

and urine by high performance liquid chromatography with ultraviolet detection Clin

Chem Lab Med 37 533-536

Rumsey SC Levine M (2000) Vitamin C In Song WO Beecher GR

Eitenmiller RR Eds Modern Analytical Methodologies in Fat- and Water-Soluble

Vitamins Wiley New York Chap 13

Saari JC Baker EM Sauberlich HE (1967) Thin-layer chromatographic separation

of the oxidative degradation products of ascorbic acid Anal Biochem 18 173-177

Salkić M Keran H Jašić M (2007) Direct spectrophotometric determination of l-

ascorbic acid in the presence of potassium cyanide Agric Conspec Sci 72 371-375

Salvador A Chisvert A (2007) Analysis of Cosmetic Products Elsevier Amsterdam

Sattar A DeMan JM Alexander JC (1977) Light-induced degradation of vitamins

II Kinetic studies on ascorbic acid decomposition in solution Can Inst Food Sci

Technol J 10 65-68

Satoh K Sakagami H Terasaka H Ida Y Fujisawa S (2000) Interaction between

hydroquinone and ascorbic acid derivatives quenching effects of organic solvents

Antican Res 20 1577-1581

Sauberlich HE (1994a) Pharmacology of vitamin C Annu Rev Nutr 14 371-391

Sauberlich HE (1994b) Vitamin C and Cancer In Kritchevsky D Carroll K Eds

Nutrition and Disease Update Cancer AOCS Press Champaign IL pp 111-172

216

Sauermann K Jaspers S Koop U Wenck H (2004) Topically applied vitamin C

increases the density of dermal papillae in aged human skin BMC Dermatol 4 13

Sawicka J (1991) The influence of excipients and technological process on

cholecalciferol stability and its liberation from tablets Pharmazie 46 519-521

Scheuplein RJ Blank IH (1973) Mechanism of percutaneous absorption IV

Penetration of non-electrolytes (alcohols) from aqueous solutions and from pure

liquids J Invest Dermatol 60 286-296

Schick B (1943) Scurvy and pine needles Science 98 241-242

Schuman Jorns M Schollnhammer G Hemmerich P (1975) Intramolecular addition

of the riboflavin side chain Anion-catalysed neutral photochemistry Eur J

Biochem 57 35-48

Seib PA Tolbert BM (1982) Ascorbic Acid Chemistry Metabolism and Uses

Advances in Chemistry Series 200 American Chemical Society Washington DC

Sequeira F Vozone C (2000) Photostability studies of drug substances and products

Practical implications of the ICH guideline Pharm Tech 24 30-35

Shacter E (2000) Quantification and significance of protein oxidation in biological

samples Drug Metab Rev 32 307-326

Shah VP (1997) FDA Guidance for Industry Document Nonsterile Semisolid Dosage

Forms Centre for Drug Evaluation and Research Rockville MD pp 1-40

Sheraz MA Ahmed S Ahmad S (2007) Developments in the clinical and food

analysis of vitamin C J Baqai Med Univ 10 19-24

217

Shindo Y Witt E Han D Packer L (1994) Dose-response effect of acute ultraviolet

irradiation on antioxidants and molecular markers of oxidation in murine epidermis

and dermis J Invest Dermatol 102 470-475

Sibi M Batcu A Gheorghiu I (1953) Vitamin oxidation-reduction effect II Effect of

light on the oxidation-reduction reactions of lactoflavin Studii cercetari stiinf

(Romania) 4 291-305 through CA (1956) 50 14816g

Sidhu DS Sugden JK (1992) Effect of food dyes in the photostability of aqueous

solutions of L-ascorbic acid Int J Pharm 83 263-266

Silva E Quina FH (2006) Photoinduced processes in the eye lens do flavins really

play a role In Silva E Edwards AM Eds Flavins Photochemisry and

Photobiology The Royal Society of Chemistry Cambridge Chap 7

Silva E Ugarte P Andrade A Edwards AM (1994) Riboflavin-sensitized photo-

processes of tryptophan J Photochem Photobiol B Biol 23 43-48

Sim AK (1972) Ascorbic acid-A scurvy past and present Chem Indust 8 160-165

Singh S (2006) Stability testing during product development In Jain NK ED

Pharmaceutical Product Development CBS Publishers New Delhi Chap 9

Singh S Bakshi M (2000) Guidance on conduct of stress testing to determine inherent

stability of drugs Pharm Tech 24 1-14

Sinko PJ (2006) Chemical Kinetics and Stability Martinrsquos Physical Pharmacy and

Pharmaceutical Sciences 5th ed Lippincott Williams amp Wilkins Baltimore Chap

8 15 18

218

Smith JL Canham JE Kirkland WD Wells PA (1988) Effect of intralipid amino

acids container temperature and duration of storage on vitamin stability in total

parenteral nutrition admixtures J Parenter Enteral Nutr 12 478-483

Speek AJ Schrijver J Schreurs WHP (1984) Fluorimetric determination of total

vitamin C and total isovitamin C in food stuffs and beverages by high-performance

liquid chromatography with precolumn derivatization J Agric Food Chem 32 352-

355Spratley JE Hellstroumlm SO Mattsson CK Pais-Clemente M (2001)

Topical ascorbic acid reduces myringosclerosis in perforated tympanic membranes A

study in the rat Ann Otol Rhinol Laryngol 110 585-591

Spiclin P Homer M Zupancic A Gasperlin M (2003) Sodium ascorbyl phosphate

in topical microemulsions Int J Pharm 256 65-73

Stadtman ER (1991) Ascorbic acid and oxidative inactivation of proteins Am J Clin

Nutr 54 1125S-1128S

Stone I (1969) The Healing Factor Vitamin C Against Disease Grosset and Dunlap

New York

Sugden JK (1985) Photostability of cosmetic materials Int J Cosmet Sci 7 165-173

Swarbrick J Rubino JT Rubino OP (2006) Coarse Dispersions In Hendrickson

R Ed Remington The Science and Practice of Pharmacy 21st ed Lippincott

Williams amp Wilkins Baltimore Chap22

Sweetman SC Ed (2009) Martindale The Complete Drug Reference 36th ed

Pharmaceutical Press London Electronic version

219

Szent-Gyoumlrgi A (1928) Observation on function of peroxidase systems and chemistry

of adrenal cortex description of new carbohydrate derivative Biochem J 22 1387-

1389

Szymula M (2005) The influence of ascorbic acid on the rheological properties of the

microemulsion region of the SDSpentanolwater system J Cosmet Sci 56 267-

277

Taqui Khan MM Martell AE (1967) Metal ion and metal chelate catalyzed oxidation

of ascorbic acid by molecular oxygen 1 Cupric acid and ferric ion catalyzed

oxidation J Am Chem Soc 89 4176-4185

Tangney CC (1988) Analysis of vitamin C in biological samples with an emphasis on

recent chromatographic techniques Prog Clin Biol Res 259 331-362

Tatsumi K Ichikawa H Wada S (1992) Flavin-sensitized photooxidation of

substituted phenols in natural water J Contam Hydrol 9 207-219

Tessier F Birlouez-Aragon I Tjani C Guilland JC (1996) Validation of a

micromethod for determining oxidized and reduced vitamin C in plasma by HPLC-

fluorescence Int J Vitam Nutr Res 66 166-170

Thatcher SR Mansfield RK Miller RB Davis CW Baertschi SW (2001a)

Pharmaceutical photostability a technical and practical interpretation of the ICH

guideline and its application to pharmaceutical stability Part 1 Pharm Tech 25 98-

110

Thatcher SR Mansfield RK Miller RB Davis CW Baertschi SW (2001b)

Pharmaceutical photostability a technical and practical interpretation of the ICH

220

guideline and its application to pharmaceutical stability Part 2 Pharm Tech 25 50-

62

Thiele JJ Dreher F Packer L (2000) Antioxidant defense systems in skin In

Elsner P Maibach HI Eds Cosmeceuticals- Drugs vs Cosmetics Marcel

Dekker New York Chap 11

Thomas DP (1997) Sailors scurvy and science J R Soc Med 90 50-54

Thomson CO Trenerry VC (1995) A rapid method for the determination of total L-

ascorbic acid in fruits and vegetables by micellar electrokinetic capillary

chromatography Food Chem 53 43-50

Tonnesen HH (1991) Photochemical degradation of components in drug formulations

1 An approach to the standardization of degradation studies Pharmazie 46 263-265

Tonnesen HH (2002) Photodecomposition of Drugs In Swarbrick J Boylan JC

Eds Encyclopedia of Pharmaceutical Technology Vol 3 Marcel Dekker New

York pp 2197-2203

Tonnesen HH Ed (2004) The Photostability of Drugs and Drug Formulations

Culinary and Hospitality Industry Publications Services Weimex TX

Tonnesen HH Karlsen J (1997) A comment on photostability testing according to

ICH guideline calibration of light sources PharmEuropa 9 735-736

Tonnesen HH Moore DE (1993) Photochemical degradation of components in drug

formulations Pharm Technol Int 5 27-33

Tournas JA Lin FH Burch JA Selim MA Monteiro-Riviere NA Zielinski

JE Pinnell SR (2006) Ubiquinone idebenone and kinetin provide ineffective

221

photoprotection to skin when compared to a topical antioxidant combination of

vitamins C and E with ferulic acid J Invest Dermatol 126 1185-1187

Traber MG (2007) Vitamin E In Zempleni J Rucker RB McCormick DB

Suttie JW Eds Handbook of Vitamins 4th ed Taylor amp Francis CRC Press

Boca Raton FL Chap 4

Traikovich SS (1999) Use of topical ascorbic acid and its effect on photodamaged skin

topography Arch Otolaryngol Head Neck Surg 125 1091-1098

Treadwell GE Cairns WL Metzler DE (1968) Photochemical degradation of

flavins V Chromatographic studies of the products of photolysis of riboflavin J

Chromatogr 35 376-388

Truswell AS (2003) ABC of Nutrition 4th ed BMJ Publishing Group Chap 10

Tsao CS Young M (1985) Analysis of ascorbic acid derivatives by high-performance

liquid chromatography with electrochemical detection J Chromatogr 13 855-856

Underberg WJM (1978) Oxidative degradation of pharmaceutically important

phenothiazines III Kinetics and mechanism of promethazine oxidation J Pharm Sci

67 1133-1138

Urso ML Clarkson PM (2003) Oxidative stress exercise and antioxidant

supplementation Toxicology 189 41-54

Vaid FHM (1998) Photolysis and Interaction of Thiamine Hydrochloride with

Riboflavin in Aqueous Solution PhD Thesis University of Karachi Karachi

Vaid FHM Shaikh RH Ansari IA Ahmad I (2005) Spectral study of the

photolysis of aqueous thiamine hydrochloride and ascorbic acid solution in the

presence and absence of riboflavin J Chem Soc Pak 27 227-232

222

Vaid F H M Shaikh RH Ansari I A Ahmad I (2006) Chromatographic study of

the photolysis of aqueous thiamine hydrochloride and ascorbic acid solutions in the

presence and absence of riboflavin J Chem Soc Pak 28 481-464

Valvani SC (2000) Industrial stability testing in the United States and computerization

of stability data In Carstensen JT Rhodes CT Eds Drug Stability Principles

and Practices 3rd

ed Marcel Dekker New York pp 534-536

Vanderslice JT Higgs DJ (1988) Chromatographic separation of ascorbic acid

isoascorbic acid dehydroascorbic acid and dehydroisoascorbic acid and their

quantitation in food products J Micronutr Anal 4 109-118

Vanderslice JT Higgs DJ Hayes JM Block G (1990) Ascorbic acid and

dehydroascorbic acid content of foods-as-eaten J Food Comp Anal 3 105-118

Vanderslice JT Higgs DJ (1993) Quantitative determination of ascorbic acid

dehydroascorbic acid by HPLC in foods and other matrices J Nutr Biochem 4 184-

190

Varvaresou A Tsirivas E Iakovou K Gikas E Papathomas Z Vonaparti A

Panderi I (2006) Development and validation of a reversed-phase ion-pair liquid

chromatography method for the determination of magnesium ascorbyl phosphate and

melatonin in cosmetic creams Anal Chim Acta 573-574 284-290

Verma KK Jain A Verma A Chaurasia A (1991) Spectrophotometric

determination of ascorbic acid in pharmaceuticals by background correction and flow

injection Analyst (London) 116 641-645

Vimaladevi M (2005) Textbook of Cosmetics CBS Publishers New Dehli Chap 2

and 17

223

Wallwork SC Grant DJW (1977) Physical Chemistry for Students of Pharmacy and

Biology 3rd ed Longmann New York pp 502-505

Waugh WA King CG (1932) Isolation and identification of vitamin C J Biol

Chem 97 325-331

Wechtersbach L Cigic B (2007) Reduction of dehydroascorbic acid at low pH J

Biochem Biophys Methods 70 767-772

Weiner N (1996) Introduction In Liberman HA Rieger MM Banker GS Eds

Pharmaceutical Dosage Forms Disperse Systems 2nd ed vol1 Marcel Dekker

New York Chap1

Werner W (1949) The reaction of l-ascorbic and d-isoascorbic acid with nicotinic acid

and its amide J Org Chem 14 22-26

Wiechers JW (2003) Formulating for Efficacy Proceedings of the 2003 IFSCC

Conference Seoul Korea

Wiechers JW (2005) Optimizing skin delivery of active ingredients from emulsions

from theory to practice In Rosen MR Ed Delivery System Handbook for

Personal Care and Cosmetic Products-Technology Applications and Formulations

William Andrew Inc Norwich NY Chap 20

Wille JJ (2005) Thixogel-Novel topical delivery systems for hydrophobic plant actives

In Rosen MR Ed Delivery System Handbook for Personal Care and Cosmetic

Products-Technology Applications and Formulations William Andrew Inc

Norwich NY Chap 36

224

Wright JL (1986) Vitamin A monograph In Connors KA Amidon GL Stella

VJ Eds Chemical Stability of Pharmaceuticals A Handbook for Pharmacists 2nd

ed Wiley New York pp 791-803

Yao AA Wathelet B Thonart P (2009) Effect of protective compounds on the

survival electrolyte leakage and lipid degradation of freeze-dried Weissella

paramesenteroides LC11 during storage J Microbiol Biotechnol 19 810-817

Yeh SY Lach JL (1961) Stability of morphine in aqueous solutions III Kinetics of

morphine degradation in aqueous solution J Pharm Sci 50 35-42

Yoshimura A Ohno T (1988) Lumiflavin-sensitized photooxygenation of indole

Photochem Photobiol 48 561-565

Yoshioka S Stella VJ (2000) Stability of Drugs and Dosage Forms Kluwer

Academic Plenum Publishers New York Chap 2

Zaeslein C (1982) Vitamins in the Field of Medicine Hoffman La Roche Basle pp

89-96

Zeng W Martinuzzi F MacGregor A (2005) Development and application of a novel

UV method for the analysis of ascorbic acid J Pharm Biomed Anal 36 1107-1111

Zhang GF Chen HY (2000) Chemiluminescence studies of the oxidation of ascorbic

acid with copper (II) catalyzed by halide anions and its applications to the

determination of halide anions and ascorbic acid Anal Sci 16 1317-1321

Zhang L Lerner S Rustrum WV Hofmann GA (1999) Electroporation-mediated

topical delivery of vitamin C for cosmetic applications Bioelectrochem Bioenerg

48 453-461

225

Zilva SS (1932) The non-specificity of the phenolindophenol reducing capacity of

lemon juice and its fractions as a measure of their antiscorbutic activity Biochem J

26 1624-1627

226

AUTHORrsquoS PUBLICATIONS

The author obtained his B Pharm degree in 2003 and joined the post graduate

program securing an M Phil degree in Pharmaceutics in 2006 from Baqai Medical

University He is a co-author of following publications

CHAPTER IN BOOK

1 Chapter on ldquoBorate Toxicity Effect on Drug Stability and Analytical

Applicationsrdquo by Iqbal Ahmad Sofia Ahmed MA Sheraz and Faiyaz H M

Vaid In Handbook on Borates Chemistry Production and Applications (MP

Chung Ed) Nova Science Publishers Inc NY USA (in press)

PAPERS PUBLISHED

INTERNATIONAL

2 Iqbal Ahmad Sofia Ahmed MA Sheraz and Faiyaz HM Vaid ldquoEffect of Borate

Buffer on the Photolysis of Riboflavin in Aqueous Solutionrdquo Journal of

Photochemistry and Photobiology B Biology 93 82-87 (2008)

3 Iqbal Ahmad Sofia Ahmed MA Sheraz M Aminuddin and Faiyaz HM Vaid

ldquoEffect of Caffeine Complexation on the Photolysis of Riboflavin in Aqueous

Solution A Kinetic Studyrdquo Chemical and Pharmaceutical Bulletin 57 (2009)

published online September 14 2009

4 Iqbal Ahmad MA Sheraz Sofia Ahmed and Faiyaz HM Vaid ldquoAnalytical

Applications of Boratesrdquo Materials Science Research Journal (in press)

5 Iqbal Ahmad Sofia Ahmed MA Sheraz Kefi Iqbal and Faiyaz HM Vaid

ldquoPharmacological Aspects of Boratesrdquo International Journal of Medical and

Biological Frontiers (in press)

6 Iqbal Ahmad Sofia Ahmed MA Sheraz Faiyaz HM Vaid and Izhar A Ansari

ldquoEffect of Divalent Ions on Photodegradation Kinetics and Pathways of

Riboflavin in Aqueous Solutionrdquo Photochemical and Photobiological Sciences

accepted

227

NATIONAL

7 Sofia Ahmed MA Sheraz and Iqbal Ahmad ldquoAdvances in Antioxidant Activity of

Vitamin Erdquo Journal of Baqai Medical University 10 13-18 (2007)

8 MA Sheraz Sofia Ahmed and Iqbal Ahmad ldquoDevelopments in the Clinical and

Food Analysis of Vitamin Crdquo Journal of Baqai Medical University 10 19-24

(2007)

9 A Azmi SNH Naqvi M Usman MA Sheraz and Sofia Ahmed ldquoPancreatic

Glucagon in Certain Ungulates Comparative Study of Extraction and

Bioassayrdquo Pakistan Journal of Entomology 20 23-28 (2005)

10 Iqbal Ahmad Sofia Ahmed MA Sheraz Faiyaz HM Vaid and S Hasan

ldquoAdvances in Biochemical Functions and the Photochemistry of Flavins and

Flavoproteinsrdquo Pakistan Journal of Pharmaceutical Sciences in press

11 MA Sheraz Sofia Ahmed and Iqbal Ahmad ldquoEffect of Borates on the Stability of

Chemical and Pharmaceutical Compoundsrdquo Journal of Baqai Medical University

accepted

PAPERS SUBMITTED

12 Iqbal Ahmad MA Sheraz Sofia Ahmed Riaz H Shaikh and Faiyaz HM Vaid

ldquoPhotostability of Ascorbic Acid in Organic Solvents and Cream Formulationsrdquo

Chemical and Pharmaceutical Bulletin

13 Iqbal Ahmad MA Sheraz Sofia Ahmed Riaz H Shaikh and Faiyaz HM Vaid

ldquoPhotochemical Interaction of Ascorbic Acid with Riboflavin Nicotinamide and

Alpha-Tocopherol in Cream Formulationsrdquo Journal of Cosmetic Science

14 Iqbal Ahmad Kefi Iqbal Sofia Ahmed MA Sheraz ldquoApplications of Laser Flash

Photolysis Spectroscopy and Electron Microscopy in Photopolymerization and

Development of Glass Ionomer Dental Cementsrdquo Materials Science Research

Journal

15 Sofia Ahmed MA Sheraz M Aminuddin I Ahmad and Faiyaz HM Vaid ldquoA

Rapid Titrimetric Assay for Quantitation of Vitamin B1 in Neat and

Pharmaceutical Preparationsrdquo Pakistan Journal of Pharmaceutical Sciences

• 01 SZ-786
• 02 SZ-title
• 03 SZ-Certificate
• 04 SZ-Abstract
• 05 SZ-Acknowledgement
• 06 SZ-Dedication
• 07 SZ-Contents
• 08 SZ-Chapter 1
• 09 SZ-Chapter 2
• 10 SZ-Chapter 3
• 11 SZ-Object of Present Investigation
• 12 SZ-Chapter 4
• 13 SZ-Chapter 5
• 14 SZ-Chapter 6
• 15 SZ-Chapter 7
• 16 SZ-Conclusion
• 17 SZ-References
• 18 SZ-Authors Publications

vi

ACKNOWLEDGMENTS

I am highly grateful to All Mighty Allah who guided me in all difficulties and

provided me strength to overcome the problems during this work

Words are confined and inefficacious to express my immense gratitude to my

respectable supervisor Prof Dr Iqbal Ahmad Department of Pharmaceutical

Chemistry for his guidance encouragement keen interest and above all giving his

valuable time suggestions and attention His personality has been a source of constant

inspiration through out my research work

I would like to extend my sincere thanks to Prof Lt Gen (R) Dr Syed Azhar

Ahmed Vice Chancellor Baqai Medical University for his personal interest and

constant encouragement through out the study

It is my great desire to express my gratitude to Prof Dr Syed Fazal Hussain

CEO Baqai Institute of Pharmaceutical Sciences for his cooperation and attention and

providing all the facilities of the Institute at my disposal during the research work

I am also thankful to Mrs Shaukat Khalid Dean Faculty of Pharmaceutical

Sciences for her support during the study

I feel honored to express my sincere thanks and indebtedness to Prof Dr

Khursheed Ali Khan Department of Pharmaceutics Prof Dr Aminuddin Department

of Pharmaceutical Chemistry and Dr Faiyaz H M Vaid Chairman Department of

Pharmaceutical Chemistry Faculty of Pharmacy University of Karachi who helped me

selflessly with their invaluable suggestions through out the research work

vii

I feel immense pleasure to pay my sincere and special thanks to Ms Sofia

Ahmed Assistant Professor and In charge Department of Pharmaceutics who lent all

sort of cooperation and spared no effort in helping me during this work

Special thanks are due to Mr Saif-ur-Rehman Khatak Deputy Drug Controller

for his cooperation and help during this study

I acknowledge with sincere thanks the contribution of Tabros Pharmaceutical

Industry Karachi for providing me the opportunity to use their facilities for certain

measurements without which the completion of this work would not have been possible

I highly appreciate the technical services rendered by Mr Anees Mr Wajahat

and Mr Sajjad in pursuance of this study

I am very grateful to Mrs Prof Dr Iqbal Ahmad for her kindness and generous

hospitality during my innumerable visits to their residence

Last but not the least I would like to express my immense indebtedness to My

Gracious Parents Beloved Brothers and Sisters for their moral support kindness and

encouragement throughout my career

I am also thankful to all my students for their affectionate feelings

M A S

viii

To

My Beloved Parents amp

Late Prof Dr S Sabir Ali for their interest and endless support

ix

CONTENTS

Chapter Page

ABSTRACT iv

ACKNOWLEDGEMENTS vi

I INTRODUCTION 1

11 HISTORICAL BACKGROUND 2

12 PHYSICOCHEMICAL CHARACTERISTICS OF

ASCORBIC ACID

2

13 CHEMISTRY OF ASCORBIC ACID 3

131 Nomenclature and Structure 3

132 Chemical Stability 3

14 BIOCHEMICAL FUNCTIONS 7

15 ANTIOXIDANT ACTIVITY 8

16 PHOTOSTABILITY OF DRUGS 9

17 KINETIC TREATMENTS OF PHOTOCHEMICAL

REACTIONS

12

18 LITERATURE ON ASCORBIC ACID 15

II PHOTODEGRADATION REACTIONS AND ASSAY OF

ASCORBIC ACID

17

21 PHOTODEGRADATION REACTIONS 18

211 Photodegradation of Ascorbic Acid 18

212 Effect of Various Substances on Photodegradation of Ascorbic

Acid

20

213 Photosensitized Oxidation of Ascorbic Acid 22

214 Ascorbic Acid Interaction with Other Water-Soluble Vitamins 25

22 ASSAY OF ASCORBIC ACID 26

221 Spectrophotometric Methods 26

222 Fluorimetric Methods 28

x

223 Mass spectrometric Methods 28

224 Chromatographic Methods 28

225 Enzymatic Methods 29

226 Commercial Kits for Clinical Analysis 30

227 Analysis in Creams 30

III FORMULATION AND STABILITY OF CREAM

PREPARATIONS

31

31 FORMULATION OF CREAM PREPARATIONS 32

311 Choice of Emulsion Type 32

312 Choice of Oil Phase 33

313 Emulsion Consistency 33

314 Choice of Emulsifying Agent 34

315 Formulation by the HLB Method 34

316 Concept of Relative Polarity Index 35

32 FORMULATION OF ASCORBIC ACID CREAMS 37

33 STABILITY OF CREAMS 39

331 Physical Stability 39

332 Chemical Stability 39

333 Microbial Stability 40

334 Stability of Ascorbic Acid in Liquid Formulations 41

335 Stability of Ascorbic Acid in Emulsions and Creams 41

336 Stability Testing of Emulsions 45

3361 Macroscopic examination 46

3362 Globule size analysis 46

3363 Change in viscosity 46

3364 Accelerated stability tests 46

337 FDA Guidelines for Semisolid Preparations 46

xi

OBJECT OF PRESENT INVESTIGATION 48

IV MATERIALS AND METHODS 51

41 MATERIALS 52

42 METHODS 55

421 Cream Formulations 55

422 Preparation of Creams 56

423 Thin-Layer Chromatography 57

424 pH Measurements 57

425 Ultraviolet and Visible Spectrometry 58

426 Photolysis of Ascorbic Acid 59

4261 Creams 59

4262 Aqueous and organic solvents 59

4263 Storage of creams in dark 59

427 Measurement of Light Intensity 59

428 Procedure 60

4281 Calculation 62

429 Viscosity Measurements 63

4210 Assay method 65

42101 UV spectrophotometric method for the assay of creams

containing ascorbic acid alone

65

42102 Iodimetric method for the assay of ascorbic acid in creams

containing riboflavin nicotinamide and alpha-tocopherol 65

42103 Spectrophotometric method for the assay of ascorbic acid in

aqueous and organic solvents

67

V PHOTODEGRADATION OF ASCORBIC ACID IN

ORGANIC SOLVENTS AND CREAM FORMULATIONS

68

51 INTRODUCTION 69

52 PHOTOPRODUCTS OF ASCORBIC ACID 71

xii

53 SPECTRAL CHARACTERISTICS OF PHOTOLYSED

SOLUTIONS

71

54 ASSAY OF ASCORBIC ACID IN CREAMS AND

SOLUTIONS

73

55 EFFECT OF SOLVENT 74

56 EFFECT OF CONCENTRATION 80

57 EFFECT OF CARBON CHAIN LENGTH OF

EMULSIFYING AGENT

88

58 EFFECT OF VISCOSITY 94

59 EFFECT OF pH 94

510 EFFECT OF REDOX POTENTIAL 96

511 PRIMARY PHOTOCHEMICAL REACTIONS IN THE

OXIDATION OF ASCORBIC ACID

97

512 DEGRADATION OF ASCORBIC ACID IN THE DARK 98

VI PHOTOCHEMICAL INTERACTION OF ASCORBIC

ACID WITH RIBOFLAVIN NICOTINAMIDE AND

ALPHA-TOCOPHEROL IN CREAM FORMULATIONS

109

61 INTRODUCTION 110

62 ABSORPTION CHARACTERISTICS OF PHOTOLYSED

CREAMS

114

63 PHOTOPRODUCTS OF ASCORBIC ACID AND OTHER

VITAMINS

114

64 ASSAY METHOD 116

65 KINETICS OF PHOTODEGRADATION OF ASCORBIC

ACID

117

66 INTERACTION OF RIBOFLAVIN WITH ASCORBIC ACID 128

67 INTERACTION OF NICOTINAMIDE WITH ASCORBIC

ACID

129

68 INTERACTION OF ΑLPHA-TOCOPHEROL WITH

ASCORBIC ACID

130

69 EFFECT OF CARBON CHAIN LENGTH OF

EMULSIFYING AGENT

130

xiii

610 EFFECT OF VISCOSITY OF CREAMS 132

611 DEGRADATION OF ASCORBIC ACID IN THE PRESENCE

OF OTHER VITAMINS IN THE DARK

135

VII STABILIZATION OF ASCORBIC ACID WITH CITRIC

ACID TARTARIC ACID AND BORIC ACID IN CREAM

FORMULATIONS

141

71 INTRODUCTION 142

72 CREAM FORMULATIONS 142

73 PRODUCTS OF ASCORBIC ACID

PHOTODEGRADATION

145

74 SPECTRAL CHANGES IN PHOTODEGRADED CREAMS 145

75 ASSAY OF ASCORBIC ACID IN CREAMS 145

76 KINETICS OF PHOTODEGRADATION 146

77 EFFECT OF STABILIZING AGENTS 146

78 DEGRADATION OF ASCORBIC ACID IN PRESENCE OF

STABILIZING AGENTS IN THE DARK

162

79 EFFECT OF ADDITIVES ON TRANSMISSION OF

ASCORBIC ACID

163

CONCLUSIONS AND SUGGESTIONS 179

CONCLUSIONS 180

SUGGESTIONS 184

REFERENCES 185

AUTHORrsquoS PUBLICATIONS 226

CHAPTER I

INTRODUCTION

2

11 HISTORICAL BACKGROUND

The disease scurvy which now is known as a condition due to a deficiency of

ascorbic acid in the diet has considerable historical significance (Schick 1943

Carpenter 1986 Bardolph and Taylor 1997 Thomas 1997 Bors 2005) Zilva (1932)

isolated the antiscorbutic activity factor from a crude fraction of lemon and showed that

the activity was destroyed by oxidation and protected by reducing agents Waugh and

King (1932) isolated crystalline vitamin C from lemon juice and showed it to be the

antiscorbutic factor Szent-Gyorgyi (1928) had isolated the same factor from pepper in

connection with his biological oxidation-reduction studies Hirst and Zilva (1933)

identified the antiscorbutic factor as ascorbic acid Early work on the chemical

identification and elucidation of the structure of ascorbic acid has been well documented

(Carpenter 1986) The first synthesis of L-ascorbic acid was achieved almost

simultaneously by Ault et al (1933) and Reichstein et al (1933)

Plants and most animals synthesize their own vitamin C but humans lack this

ability due to the deficiency in an enzyme L-gulono-gamma-lactone oxidase that

catalyzes the terminal step in ascorbic acid biosynthesis (Nishikimi et al 1994)

Therefore humans obtain this vitamin from diet and or vitamin supplements to not only

avoid the development of scurvy but also for overall well being (Stone 1969 Lewin

1976 Davies et al 1991) The minimal daily requirement for ascorbic acid in healthy

adults is 40ndash60 mg (Truswell 2003 Mason 2007 Eitenmiller et al 2008 Elia 2009)

12 PHYSICOCHEMICAL CHARACTERISTICS OF ASCORBIC ACID

The important physicochemical characteristics of ascorbic acid (Table 1) involved

in its identification and degradation are described by many authors (Connors et al 1986

3

OrsquoNeil 2001 Moffat et al 2004 Sinko 2006 Johnston et al 2007) The most

important chemical property of ascorbic acid is the reversible oxidation to semidehydro-

L-ascorbic acid and further oxidation to dehydro-L-ascorbic acid This property is the

basis for its physiological activity In addition the proton on oxygenndash3 is acidic (pKa1 =

417) which contributes to the acidic nature of ascorbic acid (1)

13 CHEMISTRY OF ASCORBIC ACID

131 Nomenclature and Structure

The IUPAC-IUB Commission on Biochemical Nomenclature changed the name

vitamin C (2-oxo-L-theo-hexono-4-lactone-23-enediol) to ascorbic acid or L-ascorbic

acid in 1965 (Johnston et al 2007) The chemical structure of ascorbic acid (1) is

HO OH

O

OHHO

H

(1)

O

The molecule has a near planar five-membered ring with two chiral centers

which contain four stereoisomers

132 Chemical Stability

Ascorbic acid is sensitive to air and light and is kept in a well-closed container

protected from light (British Pharmacopoeia 2009) The degradation reactions of

ascorbic acid in aqueous solution depend on a number of factors such as pH temperature

presence of oxygen or metal It is not very stable in aqueous media at room

temperature and undergoes oxidative degradation to dehydroascorbic acid and

4

Table 1 Physicochemical characteristics of ascorbic acid

Empirical formula C6H8O6

Molar mass 17613

Crystalline form Monoclinic mix of platelets and needles

Melting point 190 to 192 degC

[α]25

+205deg to +215deg

pH

5 mg ml

50 mg ml

~3

~2

pKa 417 1157 (20deg)

Redox potential

(dehydroascorbic acid ascorbate)

(H+ ascorbate

ndash)

ndash174 mV

+282 mV

Solubility g ml

Water

Ethanol absolute

Ether chloroform benzene

033

002

Insoluble

UV spectrum

Absorption maximum [A(1 1 cm)]

pH 20

pH 70

245 nm [695]

265 nm [940]

Infrared spectrum

Principal peaks (Nujol mull)

1026 (CminusOH str) 1111(CminusOminusC str) 1312

(minusCminusOminus str) 1653 (C=O str) 990 (C=C str)

cmndash1

Mass spectrum

Principal ions at mz

29 41 39 42 69 116 167 168

D

5

23-diketogulonic acid The stability of ascorbic acid and dehydroascorbic acid can be

improved by lowering the pH below 2 (Wechtersbach and Cigic 2007) Above pH 7

alkali-catalyzed degradation by cleavage at Cndash1 or Cndash2 results in a number of

compounds mainly monondash dindash and tricarboxylic acids (Connors et al 1986 Bors and

Buettner 1997 Halliwell and Whiteman 1997) The oxidative degradation of ascorbic

acid and dehydroascorbic acid in parenteral nutrition mixtures is catalyzed by trace

elements particularly copper (Allwood 1984ab Allwood et al 1992 Allwood and

Kearney 1998 Kearney et al 1998 Gibbons et al 2001) Stabilized ascorbic acid

preparations in hydroalcoholic vehicle (Kaplan et al 1989) and aquaculture feeds

(OrsquoKeefe 2001) have been reported The various oxidation products of ascorbic acid are

shown in Fig 1

It is interesting to note that in addition to redox and acid-base properties ascorbic

acid can exist as a free radical (Bielski et al 1981 Bielski 1982 Halliwell 1996 Bors

and Buettner 1997) The ascorbate radical anion is an important intermediate in the

reactions involving oxidants and ascorbic acidrsquos antioxidant activity Rate constants for

the generation of ascorbate radicals are in the range of 104ndash10

8 s

ndash1 When ascorbate

radicals are generated by oxyanions the rate constants are of the order of 104ndash10

7 s

ndash1

when generated by halide radicals 106ndash10

8 s

ndash1 and when generated by tocopherols and

flavonoids radicals 106ndash10

8 s

ndash1 (Bielski 1982 Halliwell and Whiteman 1997) The

ascorbate radicals decay usually by disproportionation However a change in ionic

strength or pH can influence the rate of dismutation of ascorbic acid Certain oxyanions

such as phosphates accelerate dismutation (Bielski et al 1981) The acceleration is

attributed to the activity of various protonated forms of phosphate to donate a proton

6

Fig 1 Oxidation products of ascorbic acid

O

OHOH

H

OO

OHOH

H

OO

OHOH

H

O

Ascorbyl radical anion

(interm ediate)

Ascorbic acid

(1)

-e- -2H

+

+e- +2H

+

-e-

+e-

Dehydroascorbic acid

(2)

23-diketo-L-gulonic acid

O xalic acid

+

L-Threonic acid

L-Xylose

+

C O 2

CO 2

L-Xylonic acid

+

L-Lyxonic acid CO 2

HO OH O O-

O O

7

efficiently to the ascorbate radical particularly the dimer form of ascorbate

The unusual stability of the ascorbate radical in biological systems dictates that

accessory enzymatic systems be made available to reduce the potential transient

accumulation of ascorbate radical The excess ascorbate radicals may initiate a chain of

free-radicals reactions In plants NADHmonodehydroascorbate reductase maintains

ascorbic acid in its reduced form NADHmonodehydroascorbate reductase plays a major

role in stress related responses in plants Glutathione dehydroascorbate reductase serves

this purpose in animal tissues Such enzymes keep ascorbic acid operating at maximum

efficiency so that other enzyme systems may take advantage of the univalent redox

cycling capacity of ascorbate (Asard et al 2004 Johnston et al 2007)

The anaerobic degradation of ascorbic acid has been studied by Finholt et al

(1963) Under these conditions the molecule is dehydrated and hydrolyzed in aqueous

solution to give furfural and carbon dioxide The rate of degradation is maximum at pH

41 corresponding to the pKa of ascorbic acid This has been suggested due to the

formation of a saltndashacid complex in solution The reaction is dependent on buffer

concentration but has relatively small effect of ionic strength

14 BIOCHEMICAL FUNCTIONS

Ascorbic acid plays an essential role in the activities of several enzymes It is vital

for the growth and maintenance of healthy bones teeth gums ligaments and blood

vessels It is important for the manufacture of certain neurotransmitters and adrenal

hormones Ascorbic acid is required for the utilization of folic acid and the absorption of

iron It is also necessary for normal immune responses to infection and for wound healing

(Henry 1997)

8

Ascorbic acid deprivation and scurvy include a range of signs and symptoms that

involves defects in specific enzymatic processes (Johnston et al 2007) The

administration of ascorbic acid improves most of the signs of chemically induced

glutathione (L-γ-glutamyl-L-cysteine-glycine GSH) deficiency (Meister 1994) The

effect is very pronounced in newborn rats which do not efficiently synthesize ascorbic

acid in contrast to adult rats and guinea pigs When L-buthionine-(SR)-sulphoxime is

administered in addition to the loss in GSH there is a marked increase in

dehydroascorbic acid This has led to the hypothesis that GSH is very important to

dehydroascorbic acid reduction and as a sequence to ascorbic acid recycling (Meister

1995)

Ascorbic acid also possesses pro-oxidant properties and may cause apoptosis

lymphoid and myeloid cells It has been shown that dehydroascorbic acid also stimulates

the antioxidant defenses in some cells by preferentially importing dehydroascorbate over

ascorbate (Braun et al 1997 Banhegyi et al 1998 Puskas et al 2000 2002)

15 ANTIOXIDANT ACTIVITY

Ascorbic acid is known to readily scavenge reactive oxygen and nitrogen species

such as superoxide and hydroperoxyl radicals aqueous peroxyl radicals singlet oxygen

ozone peroxynitrite nitrogen dioxide nitroxide radicals and hypochlorous acid Excess

of such products has been associated with lipids (Niki and Noguchi 1997 Carr et al

2000 Urso and Clarkson 2003) DNA (Fraga et al 1991 1996 Lindahl 1993) and

protein oxidation (Stadtman 1991 Berlett and Stadtman 1997 Dean et al 1997

Ortwerth and Monnier 1997 Padayatty et al 2003)

9

The electron donor character of ascorbate may be responsible for many of its

known biological functions Inspite of the availability of ascorbic acid to influence the

production of hydroxyl and alkoxyl radicals it remains uncertain whether this is the

principal effect or mechanism that occurs in vivo There is a good evidence that ascorbic

acid protects lipids in biological fluids as an antioxidant (Johnston et al 2007) A

detailed account of the function of ascorbate as an antioxidant and its reactions with

reactive nitrogen species and singlet oxygen has been reported by Packer et al (2002)

and Buettner and Schafer (2004)

Ascorbic acid (Eordm ndash0115 V pH 52 Sinko 2006) has been used as an antioxidant

for the stabilization of drugs with a higher oxidation potential These drugs include

morphine (Yeh and Lach 1961) vitamin A (Wright 1986) rifampin (Maggi et al

1966) cholecalciferol (Nerlo et al 1968 Sawicka 1991) promethazine (Underberg

1978) and sulphacetamide and sulphanilamide (Ahmad and Ahmad 1983)

16 PHOTOSTABILITY OF DRUGS

Many drug substances are sensitive to light (British Pharmacopoeia 2009) and

may degrade in pharmaceutical formulations to inactive or toxic compounds This could

make a product therapeutically inactive while in use by the patients The

photodegradation (photolysis) of drug substances may occur not only during storage but

also during the use of the product It may involve several mechanisms including

oxidation reduction hydrolysis decarboxylation isomerization rearrangement and other

reactions Normal sunlight or room light may cause substantial degradation of drug

molecules The study of degradation of drug substances under the action of UVvisible

light is relevant to the process of drug development for several reasons such as

10

Exposure to light can influence the stability of a drug formulation resulting in the

loss of potency

Inappropriate exposure to light of the raw material or the final product can lead to

the formation of toxic photoproducts that are dangerous to health

Information about the stability of drug substances and formulations is needed to

predict the shelf-life of the final product (Tonnesen and Moore 1993)

The development of light-activated drugs involves activation of the compound

through photochemical reactions (Tonnesen 1991)

Adverse effects due to the formation of minor degradation products during

storage and administration have been reported (de Vries et al 1984) The drugs

substances may also cause light-induced side effects after administration to the patient by

interaction with endogenous substances The study of the photochemical properties of

drug substances and formulated products is an integral part of formulation development

to ensure the safety and efficacy of the product

The photodegradation of drug substances occurs as a result of the absorption of

radiation energy by a molecule (A) to produce an excited state species (A) (11) The

absorbed energy can be lost either by a radiative process involving fluorescence or

phosphorescence (12) or by a physical or chemical radiationless process The physical

process results in the loss of energy as heat (13) or by collisional quenching (14) The

chemical decay leads to the formation of a new species (15) The whole process is

represented as

11

A A (11)

A A + hυprime (12)

A A + heat (13)

A + A 2A (14)

A product (s) (15)

According to the Stark-Einstein law the absorption of one quantum of radiation

results in the formation of one excited molecule which may take part in several

photochemical processes [Eqs (11)ndash(15)] The quantum yield φ for any one of these

processes is defined by

Number of molecules undergoing the photochemical process φ =

Number of quanta absorbed

Considering a pure photochemical reaction the quantum yield has a value of 0ndash1

however if A is a radical that can take part in a free-radical chain reaction so that the

absorption of energy simply initiates the reaction then each quantum of energy may

result in the decomposition of molecules and φ may appear to be greater than 1 (Connors

et al 1986)

Detailed information on the photostability and photodegradation of drug

substances including vitamins alone or in solid or liquid formulations is available in the

reviews published by DeRitter (1982) Albini and Fasani (1998) Sequeira and Vozone

(2000) Tonnesen (2002 2004) Yoshioka and Stella (2002) Min and Boff (2002) Reed

et al (2003) Fasani and Albini (2005) and Sinko (2006) The photostability of cosmetic

materials has been reviewed by Sugden (1985) Important aspects dealing with the

photostability testing of drug substances have been dealt by Anderson et al (1991)

k1

k2

k3

k4

hυ

12

Tonnesen and Moore (1993) Tonnesen and Karlsen (1997) Riehl et al (1995) ICH

(1997) Singh and Bakshi (2000) Valvani (2000) Thatcher et al (2001ab) Fasani and

Albini (2005) Klick et al (2005) Singh (2006) and Ahmad and Vaid (2006)

17 KINETIC TREATMENT OF PHOTOCHEMICAL REACTIONS

The kinetic treatment of photochemical reactions with reference to the

photostability of drug substances has been considered by Moore (2004) and is presented

in this section

The photostability testing of a drug substance at the preformulation stage involves

a study of the drugrsquos rate of degradation in solution on exposure to light for a period of

time The value of the degradation rate constant depends very much on the design of the

experimental conditions (eg concentration solvent pH irradiation source oxygen

content) The factors that determine the rate of a photochemical reaction are simply the

rate at which the radiation is absorbed by the test sample (ie the number N of photons

absorbed per second) and the efficiency of the photochemical process (ie the quantum

yield of the reaction φ) For a monochromatic photon source the number of photons

absorbed depends upon the intensity of the photon source and the absorbance at that

wavelength of the absorbing species The rate of a photochemical reaction is defined as

Rate = number of molecules transformed per second = N φ (16)

In the first instance the rate can be determined for a homogeneous liquid sample

in which the only photon absorption is due to the drug molecule undergoing

transformation with the restriction that the concentration is low so that the drug does not

absorb all of the available radiation in the wavelength range corresponding to its

13

absorption spectrum The value of N can be derived at a particular wavelength λ and is

given by

Nλ = Iλ ndash It = Iλ (1 ndash 10ndashA

) (17)

where Iλ and It are the incident and transmitted radiation intensities respectively and A is

the absorbance of the sample at the wavelength of irradiation This expression can be

expanded as a power series

Nλ = 2303 Iλ (A + A22 + A

36 + hellip) (18)

When the absorbance is low (Alt 002) the second- and higher-order terms are negligible

and the expression simplifies to the first term in Eq 18 Given the Beerrsquos law relation

between absorbance and concentration N can be seen to be directly proportional to

concentration

Nλ = 2303 Iλ A = 2303 Iλ ελ b C (19)

where ελ is the molar absorptivity at wavelength λ C the molar concentration of the

absorbing species and b the optical path length of the reaction vessel Now Iλ and ελ vary

with wavelength so the expression must be integrated over the relevant wavelength range

where each has a non-zero value

N = 2303 b C int (Iλ ελ) dλ integrated from λ1 to λ2 (110)

Thus

Rate = 2303 b C φ int (Iλ ελ) dλ (111)

Now the overlap integral (int Iλ ελ dλ) is a constant for a particular combination of photon

source and absorbing substance b is determined by the reaction vessel chosen and φ is a

characteristic of the reaction Thus by grouping the constant terms into an overall

constant k1 the expression is simplified to a first-order kinetic equation

14

Rate = ndashd [Drug] dt = k1C (112)

The integrated form of Eq 112 can be expressed in exponential form (Eq 113) or

logarithmic form (Eq 114)

[Drug]t = [Drug]0 endashk1t

(113)

ln [Drug]t = ln [Drug]0 ndash k1t (114)

Verification of first-order kinetics is obtained when a plot of the logarithm of the

concentration of drug remaining is linear with slope equal to (ndashk1)

Eq 112 predicts that a photodegradation reaction studied at low concentrations in

solution will follow first-order kinetics however the rate constant derived from a study

performed in one laboratory will not be the same as that found in another The reason for

this is the inherent difficulty in reproducing exactly the experimental arrangement of

photon source and sample irradiation geometry Therefore the relative values of the rate

constants are useful in a given experimental arrangement for making comparisons of

degradation of the absorbing substance in different formulations eg those containing

ingredients designed to inhibit the photoreaction The use of rate constants is helpful for

comparative purposes when studying a number of different reaction mixtures under the

same irradiation conditions such as the effect of pH on the degradation of a drug

However the reaction order and numerical values of the rate constants are relative to the

specific conditions used

15

18 LITERATURE ON ASCORBIC ACID

A large number of reviews have been published on various aspects of ascorbic

acid A list of important reviews is given below

Chemistry biochemical functions and related aspects

Rosenberg (1945) Burns (1961) King and Burns (1975) Sim (1972) Hanck

(1982) Zaeslein (1982) Seib and Tolbert (1982) Carpenter (1986) Levine

(1986) Davies et al (1991) Halliwell and Whiteman (1997) Ortega and Delgado

(1998) Asard et al (2004) Hickey and Roberts (2004) Johnston et al (2007)

Eitenmiller (2008)

Chemical and pharmaceutical stability

Macek (1960) Garrett (1967) Carstensen (1972) Dale and Booth (1976) Hashmi

(1973) Litner (1973) DeRitter (1982) Allwood (1984ab) Allwood and Kearney

(1998) Connors et al (1986) Smith et al (1988) Racz (1989) Roth et al 1991

Ball (2006) Eitenmiller et al (2008) Sweetman (2009)

Methods of assay and chromatography

Mader (1961) Gyorgy and Pearson (1967) Bolliger and Konig (1969) Hashmi

(1973) Al-Meshal and Hassan (1982) Pelletier (1985) Lambert and deLeenheer

(1992) Halver and Felton (2001) Moffat et al (2004) Ball (2006) Eitenmiller et

al (2008)

Pharmacology and related aspects

Levine (1986) Dollery (1999) Sauberlich (1994ab) McDowell (2000)

Kaushansky and Kipps (2006) Sweetman (2009)

16

Antioxidant activity

Basu et al (1999) Shacter (2000) Thiele et al (2000) Cadenas and Packer

(2002) Packer et al (2002) Padayathy et al (2003) Parker and Parker (2003)

Burke (2006) Johnston et al (2007)

Cosmetic Preparations

Barel et al (2001) Salvador and Chisvert (2007) Rosen (2005) Bissett (2006)

Chaudhri and Jain (2009)

CHAPTER II

PHOTODEGRADATION

REACTIONS AND ASSAY

OF ASCORBIC ACID

18

21 PHOTODEGRADATION REACTIONS

211 Photodegradation of Ascorbic Acid

Aqueous ascorbic acid (1) solutions are degraded by UV light to give

dehydroascorbic acid (2) (Arcus and Zilva 1940) Ascorbic acid degradation at a

concentration of 52 and 50 mg on UV irradiation for 2 hours gave a loss of 43 and 8

respectively Dehydroascorbic acid solutions are more stable to UV light than the

ascorbic acid (Kitagawa 1968) In many natural products the vitamin is oxidized on

exposure to air and light (OrsquoNeil 2001) When solutions of multivitamin preparations are

exposed to light H2O2 as well as organic peroxides are generated and specific

byproducts that differ from dehydroascorbic acid and 23-diketogulonic acid (3) are

produced (Lavoie et al 2004)

In aqueous neutral or alkaline solution ascorbic acid (1) undergoes chemical or

photochemical oxidation to dehydroascorbic acid (2) which upon saponification of the

lactone ring under the influence of the base water produces 23-diketo-L-gulonic acid (an

α szlig- diketogulonic acid) (3) This acid undergoes further oxidation to oxalic acid (4) and

L-threonic acid (5) (Racz 1989) (Fig 2a) At room temperature oxalic acid (4) is also

formed along with threonolactone (6) by photochemical degradation of ascorbic acid (1)

in the presence of singlet oxygen (1O2) (Silva and Quina 2006) (Fig 2a) The low-

temperature photooxygenation of ascorbic acid (1) gives a mixture of unstable

hydroperoxide ketones (7) and (8) which on standing interconvert and cyclize to

hydroperoxyhemiketal (9) The hydroperoxyhemiketal breaks down on warming to

produce the oxalate esters of threonic acid (10) (Fig 2b) (Kwon and Foote 1988)

19

COOH

COOH

O

OHHO

O

HOH2C

HO2

O

O

HO

OO

O O2H

OHHO

O

HOH2C

OH

O

O

OH

O2H

OO

HO O2CCO2H

(1)hv

room temperature

(4)(6)

(1)hv

85 oC

(7)

(a)

(8)

+

cyclization

(9)

ring cleavage

(b)

(10)

(2)

OH O

OHHO

OH O O

(3)

OH OH

OH

OH O

O

OH

1O2 [O]

+

(5)

COOH

COOH

(4)

+

OH

Fig 2 Photooxidation of ascorbic acid at room and low temperature

20

An important consideration in the stability of ascorbic acid in total parenteral

nutrition (TPN) solutions is the generation of hydrogen peroxide in the presence of light

(Laborie et al 1998 1999 2000 2002 Chessex et al 2002) This may result from the

oxidation of ascorbate anion by molecular oxygen (Homann and Gaffron 1964 Taqui

Khan and Martell 1967 Mushran and Agarwal 1977 Hughes 1985 De La Rochette et

al 2000) leading to further degradation of ascorbic acid (Deutsch 1998a 1998b

1998c) The kinetics and mechanism of oxidation reactions of ascorbic acid have been

studied by several workers (Taqui Khan and Martell 1967 Ogata and Kosugi 1969

Blaugh and Hajratwala 1972 Fessenden and Verma 1978 Abe et al 1986 Kwon et al

1989 Fornaro and Coicher 1998 Njus et al 2001)

The photostability of various ascorbic acid tablets on exposure to UV light has

been studied and the influence of antioxidants and moisture on the potency loss of

ascorbic acid has been evaluated The physical characteristics of ascorbic acid tablets are

also affected on UV irradiation (Ahmad et al 1973 Jamil et al 1980ab Jamil and

Ahmad 1984)

212 Effect of Various Substances on Photodegradation of Ascorbic Acid

The oxidation-reduction reactions of ascorbic acid in the presence of riboflavin at

pH 8ndash9 under the influence of light have been studied Under these conditions ascorbic

acid is a more active H donor to riboflavin than phenolphthalein (Sibi et al 1953)

Riboflavin has been found to catalyze the photodegradation of ascorbic acid solutions

during exposure to light and air The losses of ascorbic acid are markedly increased by

the presence of Cu2+

and Fe3+

ions under light exposed and unexposed conditions (Sattar

et al 1977) A spectral study of the UV photolysis of ascorbic acid solutions in the

21

presence of riboflavin has shown that the degradation of ascorbic acid is enhanced to the

extent of about 15 (Vaid et al 2005) The influence of DL- methionine on the

photostability of ascorbic acid solutions has also been studied DL- methionine (10 mg

) enhances the photostability of ascorbic acid (40 mg ) in acetate and phosphate

buffers but not in citrate buffer at pH 45 The photoprotective action of DL-methionine

on ascorbic acid appears to be influenced by its concentration pH of the medium and the

buffer species (Asker et al 1985)

The degradation of ascorbic acid solutions on irradiation with simulated sunlight

in the presence of the food dye quinolone yellow (E 104) is enhanced However this

effect is reversed by the addition of mannitol indicating that this dye facilitates the

photogeneration of hydroxyl radicals which may cause degradation of the vitamin The

incorporation of triplet quenchers enhances the stability of substrate solutions suggesting

that the dye acts as a triplet sensitizer to facilitate the reaction (Sidhu and Sugden 1992)

The photostability of ascorbic acid solutions is enhanced by sweetening agents (mannitol

sorbitol sucrose dextrose and Canderal) at 5 wv concentration However the addition

of stoichiometric amounts of hydrogen peroxide as a source of hydroxyl radicals and 2

2rsquo-azobis (2-amidopropane) as a source of hydroperoxyl radicals results in diminished

stability of ascorbic acid solutions The diminished activity may be due to the action of

hydroperoxyl radicals in the presence of hydroxyl radical scavengers (Ho et al 1994)

Metal-complexing agents (eg disodium ethylenediaminetetraacetic acid N-

hydroxylethyl ethylenediaminetetraacetic acid 8-hydroxyquinoline) have a stabilizing

effect on the photolysis of ascorbic acid injectable solutions (Kassem et al 1969ab

22

1972) This may be due to the interaction of these agents with metal ions and other

impurities

213 Photosensitized Oxidation of Ascorbic Acid

In the presence of visible light a photosensitizer such as riboflavin can exhibit

photosensitizing properties through a mixed Type IndashType II mechanism (Yoshimura and

Ohno 1988 Foote 1991 Silva et al 1994 Silva and Quina 2006) as presented below

Type I mechanism (low oxygen concentration)

RF + hυ rarr 1RF rarr

3RF (21)

3RF + SH rarr RF

middot ndash + SH

middot + rarr RFH

middot + S

middot (22)

RFmiddot ndash

+ O2 rarr RF + O2middot ndash

(23)

2RFHmiddot rarr RF + RFH2 (24)

RFH2 + O2 rarr RF + H2O2 (25)

H2O2 + O2middot ndashrarr

ndashOH +

middotOH + O2 (26)

Smiddot and or SH

middot +

+ H2O2 O2middot ndash

rarr Soxid (27)

Type II mechanism (high oxygen concentration)

RF + hυ rarr 1RF rarr

3RF (28)

3RF + O2 rarr RF +

1O2 (29)

SH + 1O2 rarr Soxid (210)

In these reactions RF 1RF and

3RF represent RF in the ground state and in the excited

singlet and triplet states respectively RFmiddot ndash

RFHmiddot and RFH2 are the radical anion the

radical and the reduced form of RF SH is the reduced substrate and SHmiddot

+ S

middot and Soxid

23

represent the intermediate radical cation the radical and the oxidized form of the

substrate respectively

An early study of the riboflavin-sensitized photooxidation of ascorbic acid has

been carried out by flash photolysis (Heelis et al 1981) ESR spectrometry has been

used to investigate the photosensitized formation of ascorbate radicals by riboflavin (Kim

et al 1993) The photochemical behavior of a system consisting of ascorbate ion (AHndash)

and riboflavin has been studied by Mancini et al (2000) and De La Rochette et al (2000

2003) The photosensitized processes were examined as a function of oxygen pressure

and the efficiency of RF induced degradation of AHndash

at various oxygen concentrations

was compared on the basis of the respective initial photosensitization quantum yields

(Table 2)

In this reaction a Type I photosensitization mechanism (Karlsen 1996) implies a

direct electron transfer between AHndash and the RF triplet-excited state followed by the

oxidation of semioxidized ascorbyl radical (AHmiddot) by molecular oxygen or some other

reactive species On the contrary in a Type II photosensitization mechanism singlet

oxygen is produced directly by energy transfer from the RF triplet-excited state to

molecular oxygen and the singlet oxygen then oxidizes the AHndash Thus by irradiating

under increasing oxygen pressure it is possible to control the relative prevalence and

efficiency of Type I or Type II mechanisms The absence of a linear relationship between

the quantum yields of ascorbate degradation and oxygen concentration indicates that the

photosensitization mechanism involved in not exclusively Type II

24

Table 2 Initial quantum yield (φ) for ascorbate (AHndash) degradation during

photosensitization by RF (35 microM) in solutions irradiated at 365 nm and

37ordmC

O2 103 times φ (AH

ndash)a

0

5

20

14 plusmn 06

1670 plusmn 220

1940 plusmn 200

a Data are the mean plusmn SD of three independent experiments

25

In the presence of RF and O2 the quantum yields for degradation of ascorbate ion

have been found to be greater than one suggesting the participation of chain reactions

initiated by the ascorbyl radical as given by the following reactions

3RF + AH

ndash rarr RFmiddot

ndash + AHmiddot (211)

AHmiddot + O2 rarr A + HO2middot (212)

HO2middot + AHndash rarr H2O2 + AHmiddot (213)

The generation of the ascorbyl radical by the reaction between the RF excited-

triplet state and the ascorbate ion (Eq 211) is the only step that requires the absorption of

photons (to form the excited-triplet state of RF) The subsequent reactions (Eqs 212 and

213) are independent of light and lead to further degradation of the ascorbate ion In the

presence of transition metal ions such as Fe3+

in trace amounts in the buffer solution

containing RF and ascorbate ions further oxidation of ascorbate ion could also occur As

a result the reduced form of the metal ion (ie Fe2+

) can be generated by the metal

catalyzed oxidation of ascorbate ion This has been confirmed by the significant decrease

in the AHndash photooxidation quantum yield in the presence of the metal chelator EDTA

which inactivates the trace amounts of iron present in the buffer solution The quantum

yields for the photosensitized oxidation of ascorbate ion are decreased twofold at 20 O2

and fourfold at 5 O2 concentration in the presence of EDTA (Silva and Quina 2006)

Amino acids have been found to affect the photosensitized oxidation of ascorbic acid

(Jung et al 1995)

214 Ascorbic Acid Interaction with Other Water-Soluble Vitamins

The stability of ascorbic acid is reported to be enhanced in syrups containing B-

complex vitamins (Connors et al 1986) This may be due to the increased viscosity of

the syrups inhibiting the oxidation of ascorbic acid The rate of photolysis in solution

26

containing cyanocobalamin and ascorbic acid is reported to decrease with an increase in

pH (Ansari et al 2004) where as use of certain halide salts has been reported to be

beneficial in stabilizing pharmaceutical products and dietary supplements when vitamin

B12 and vitamin C are combined in solution (Ichikawa et al 2005) When a solution of

multivitamins is exposed to light it is reported that organic peroxidases are generated and

the concentration of ascorbic acid decreases (Lavoie et al 2004)

22 ASSAY OF ASCORBIC ACID

Recent accounts of the development and application of analytical methods to the

determination of ascorbic acid in pharmaceuticals biological samples and food materials

are reported in the literature (Rumsey and Levine 2000 Halver and Felton 2001 Moffat

et al 2004 Ball 2006 Sheraz et al 2007 Eitenmiller et al 2008 Salkic and Kubicek

2008) Most of these methods are based on the application of spectrophotometric

fluorimetric and chromatographic techniques to suit the requirements of a particular assay

and are summarized below

221 Spectrophotometric Methods

Spectrophotometric methods are the most widely used methods for the assay of

ascorbic acid in aqueous solution Ascorbic acid exhibits strong absorption in the

ultraviolet region (absorption maxima 243 nm at pH 2 and 265 nm at pH 4ndash10 OrsquoNeil

2001 Moffat et al 2004 British Pharmacopoeia 2009) This is the basis of

spectrophotometric methods for the determination of the vitamins in pure solutions and in

sample preparations where no interference is observed from UV absorbing impurities

The value of A (1 1 cm) at the analytical wavelength of 245 nm (pH 20) is high (695)

which makes the method very sensitive for the determination of mg quantities of the

27

vitamin Treatment of the material to be analyzed with ascorbic acid oxidase is often used

as a blank to correct for the presence of interfering substances in biological samples (Liu

et al 1982) A spectrophotometric method for the determination of ascorbic acid in

pharmaceuticals by background correction (245 nm) has been reported (Verma et al

1991) The direct determination of ascorbic acid in mixtures involves the use of 22prime-

dipyridyl as a colorimetric reagent The method is based on the reduction of Fe (III) by

ascorbic acid to Fe (II) which reacts with 2 2prime-dipyridyl to form a colored complex

(absorption maximum 510 nm) that can be used for quantitative determination (Margolis

and Schmidt 1996) A spectrophotometric method has been developed for the

determination of ascorbic acid and its oxidation product dehydroascorbic acid in

biological samples (Moeslinger et al 1995) A sensitive method has been reported for

the determination of ascorbic acid in pharmaceutical formulations and fruit juices by

interaction with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (Br-PADAP) (Ferreira

et al 1997) A novel UV method has been developed for the analysis of ascorbic acid in

methanol at 245 nm in various formulations (Zeng et al 2005)

Ascorbic acid in aqueous solutions has been assayed at 244 nm (pH ~2) (Ogata

and Kosugi 1969) 245 nm (pH 35) (Blaugh and Hajratwala 1972) 264 nm (pH 7)

(Salkic et al 2007) 265 nm (pH 7) (Hashmi 1973) 275 nm (pH 41 and 70) (Heelis et

al 1981) 265 nm (pH 7) (Al-Meshal and Hassan 1982) 245 nm (pH ~2) (Verma et al

1991) and 265 nm (pH ~7) (Erb et al 2004) Dehydroascorbic acid and 23-

diketogulonic acid do not significantly absorb in this region (Pelletier 1985 Davies et

al 1991 Rumsey and Levine 2000) and therefore do not interfere with the assay of

ascorbic acid in degraded solutions

28

222 Fluorimetric Methods

Fluorimetry is a highly sensitive technique for the determination of fluorescent

compounds or fluorescent derivatives of non-fluorescent compounds The technique has

been used for the detection of microg quantities of ascorbic acid Methods based on

fluorimetric (Kampfenkel et al 1995) and chemiluminescence detection (Zhang and

Chen 2000) provide highly sensitive methods for the determination of ascorbic acid in

plant and other materials

223 Mass Spectrometric Methods

Conventional and isotope mass spectrometric techniques have also been used for

the analysis of ascorbic acid Isotope ratio mass spectrometry is particularly useful and

sensitive when 13

C ascorbic acid is used as a standard in the analysis of complex matrices

(Gensler et al 1995)

224 Chromatographic Methods

High-performance liquid chromatographic (HPLC) methods have extensively

been employed for the determination of ascorbic acid in biological samples These

methods include ion exchange reversed phase and ion-pairing HPLC protocols

Spectrophotometric fluorimetric and electrochemical detection has been used in the

HPLC analysis of ascorbic acid The electrochemical detection is used for the

simultaneous determination of ascorbic acid dehydroascorbic acid and their isomers and

derivatives A number of HPLC methods have been developed for the detection and

determination of ascorbic acid and its oxidation products and derivatives in biological

samples and plant materials (Tsao and Young 1985 Tangney 1988 Dabrowski and

Huiterleitner 1989 Thomson and Trenerry 1995 Kimoto et al 1997 Kall and

29

Anderson 1999 Rumelin et al 1999 Lykkesfeldt 2000 Zhang et al 2000 Pastore et

al 2001 Frenich et al 2005) The limit of detection of ascorbic acid in plasma or urine

with UV detection lies in the range of 100-120 microg (Liau et al 1993 Manoharan and

Schwille 1994) Fluorescence detection of ascorbic acid and dehydroascorbic acid in

plasma and its comparison with coulometric detection has been reported (Tessier et al

1996) A liquid chromatography-diode-array detection (LCndashDAD) method has been

reported for the determination of 10 water-soluble and 10 fat-soluble vitamins including

ascorbic acid in pharmaceutical preparations with a coefficient of variation lt 65

(Konings 2006)

Liquid chromatography methods based on precolumn and o-phenylenediamine

(OPD) derivatization have been used for the determination of total vitamin C and total

isovitamin C in foods and dehydro forms of the vitamin Isoascorbic acid has been used

as an internal standard in the analysis (Speek et al 1985 Vanderslice et al 1990

Dodsun et al 1992 Vanderslice and Higgs 1988 1993 Hagg et al 1994 1995) The

limits of detection of ascorbic acid by HPLC using different detectors are in the range of

16ndash400 microgl (Capellmann and Bolt 1992 Iwase and Ono 1994 Karatepe 2004)

225 Enzymatic Methods

Enzymatic methods using ascorbate oxidase are specific and have the advantage

of selectively measuring the biological activity of ascorbic acid in serum or plasma (Liu

et al 1982) Ascorbate oxidase and OPD derivatization has been used to develop a rapid

automated method for the routine assay of ascorbic acid in serum and plasma The

method has a sample throughput of 100h (Ihara et al 2000)

30

226 Commercial Kits for Clinical Analysis

Commercial kits (eg Immunodiagnostic Germany Biovision USA) are also

used for the determination of ascorbic acid in biological samples (serum or plasma) in

clinical laboratories

227 Analysis in Creams

The general methods for the analysis of active ingredients and excipients in

cosmetic products including creams are described by Salvador and Chisvert (2007)

Ascorbic acid and derivatives in creams have been determined by liquid chromatography

(Irache et al 1993 Varvaresou et al 2006) gas chromatography-mass spectrometry

(Leveque et al 2005) and electrochemical methods (Beissenhirtz et al 2003 Guitton et

al 2007)

CHAPTER III

FORMULATION AND

STABILITY OF CREAM

PREPARATIONS

32

31 FORMULATION OF CREAM PREPARATIONS

Traditionally emulsions have been defined as dispersions of macroscopic droplets

of one liquid in another liquid with a droplet diameter approximately in the range of 05-

100 microm (Becher 1965) According to the definition of International Union of Pure and

Applied Chemistry (IUPAC) (1971) ldquoIn an emulsion liquid droplets and or liquid

crystals are dispersed in a liquidrdquo

Creams are semisolid emulsions intended for external applications They are often

composed of two phases Oil-in-water (ow) emulsions are most useful as water-washable

bases whereas water-in-oil (wo) emulsions are emollient and cleansing agents The

active ingredient is often dissolved in one or both phases thus creating a three-phase

system Patients often prefer a wo cream to an ointment because the cream spreads more

readily is less greasy and the evaporating water soothes the inflamed tissue OW creams

(vanishing creams) rub into the skin the continuous phase evaporates and increases the

concentration of a water-soluble drug in the adhering film The concentration gradient for

drug across the stratum corneum therefore increases promoting percutaneous absorption

(Barry 2002 Betageri and Prabhu 2002)

The various factors involved in the formulation of emulsions and topical products

have been discussed by Block (1996) Barry (2002) and Jain et al (2006) and are briefly

presented in the following sections

311 Choice of Emulsion Type

Oil-in-water emulsions are used for the topical application of water-soluble drugs

mainly for local effect They do not have the greasy texture associated with oily bases

and are therefore pleasant to use and easily washed from skin surfaces Moisturizing

33

creams designed to prevent moisture loss from the skin and thus inhibit drying of the

stratum corneum are more efficient if formulated as ow emulsions which produce a

coherent water-repellent film

312 Choice of Oil Phase

Many emulsions for external use contain oils that are present as carriers for the

active ingredient It must be realized that the type of oil used may also have an effect both

on the viscosity of the product and on the transport of the drug into the skin (Barry

2002) One of the most widely used oils for this type of preparation is liquid paraffin

This is one of a series of hydrocarbons which also includes hard paraffin soft paraffin

and light liquid paraffin They can be used individually or in combination with each other

to control emulsion consistency This will ensure that the product can be spread easily but

will be sufficiently viscous to form a coherent film over the skin The film-forming

capabilities of the emulsion can be further modified by the inclusion of various waxes

such as bees wax carnauba wax or higher fatty alcohols

313 Emulsion Consistency

A consideration of the texture or feel of a product intended for external use is

important A wo preparation will have a greasy texture and often exhibits a higher

apparent viscosity than ow emulsions This fact imparts a feeling of richness to many

cosmetic formulations Oil-in-water emulsions will however feel less greasy or sticky on

application to the skin will be absorbed more readily because of their lower oil content

and can be more easily washed from skin surface Ideally emulsions should exhibit the

rheological properties of plasticity pseudoplasticity and thixotropy Emulsions of high

apparent viscosity for external use (cream) are of a semisolid consistency There are

34

several methods by which the rheological properties of an emulsion can be controlled

(Billany 2002)

314 Choice of Emulsifying Agent

The choice of emulgent to be used would depend on factors such as its

emulsifying ability route of administration and toxicity Most of the non-ionic emulgents

are less irritant and less toxic than their anionic and cationic counter parts Some

emulgents such as the ionic alkali soaps often have a high pH and are thus unsuitable for

application to broken skin Even in normal intact skin with a pH of 55 the application of

such alkaline materials can cause irritation Some emulsifiers in particular wool fat can

cause sensitizing reactions in susceptible people The details of various types of

emulsifying agents are available in the literature (Betageri and Prabhu 2002 Billany

2002 Swarbrick et al 2006)

315 Formulation by the HLB Method

The physically stable emulsions are best achieved by the presence of a condensed

layer of emulgent at the oil water interface and the complex interfacial films formed by a

blend of an oil-soluble emulsifying agent with a water-soluble one produces the most

satisfactory emulsions

It is possible to calculate the relative quantities of the emulgents necessary to

produce the most physically stable emulsions for a particular formulation with water

combination This approach is called the hydrophilic-lipophilic balance (HLB) method

Each surfactant is allocated an HLB number representing the relative properties of the

lipophilic and hydrophilic parts of the molecule High numbers (up to a theoretical

number of 20) therefore indicates a surfactant exhibiting mainly hydrophilic or polar

35

properties whereas low numbers represent lipophilic or non-polar characteristics Each

type of oil requires an emulgent of a particular HLB number in order to ensure a stable

product For an ow emulsion the more polar the oil phase the more polar must be the

emulgent system (Billany 2002 Im-Emsap et al 2002 Swarbrick et al 2006)

316 Concept of Relative Polarity Index

In the ingredient selection in cosmetic formulations a new concept of relative

polarity index (RPI) has been presented (Wiechers 2005) The physicochemical

characteristics of the ingredients determine their skin delivery to a greater extent than the

formulation type The cosmetic formulation cannot change the chemistry of the active

molecule that needs to penetrate to a specific site within the skin However the

formulation type can be selected based on the polarity of the active ingredient and the

desired site of action for the active ingredient For optimum skin delivery the solubility of

the active ingredient needs to be as high as possible (to create a large concentration

gradient) and as small as possible (to create a large partition coefficient) To achieve this

it is necessary to determine the following parameters

The total amount dissolved in the formulation that is available for skin penetration

the higher this amount the more will penetrate until a solution concentration is

reached in the skin therefore a high absolute solubility in the formulation is required

The polarity of the formulation relative to that of the stratum corneum if an active

ingredient dissolves better in the stratum corneum than in the formulation then the

partition of the active ingredient will favour the stratum corneum therefore a low

(relative to that in the stratum corneum) solubility in the formulation is required

(Wiechers 2005)

36

These requirements can be met by considering the concept of RPI (Wiechers

2003 2005) In this systematic approach it is essential to consider the stratum corneum

as another solvent with its own polarity The stratum corneum appears to behave very

similarly to and in a more polar fashion than butanol with respect to its solubilizing

ability for active ingredients (Scheuplein and Blank 1973) The polarity of stratum

corneum as expressed by its octanol water partition coefficient is 63

The relative polarity index may be used to compare the polarity of an active

ingredient with both that of the skin and that of the oil phase of a cosmetic formulation

predominantly consisting of emollients It may be visualized as a vertical line with a high

polarity at the top and a high lipophilicity at the bottom The polarity is expressed as the

log10 of the octanol water coefficient For example the relative polarity index values of

glycerin and isostearyl isostearate are -176 and 2698 respectively (Wiechers 2005) In

order to use the concept of the relative polarity index three numbers (on log10 scale) are

required

The polarity of the stratum corneum is set at 08 However in reality this value will

change with the hydration state of the stratum corneum that is determined in part by

the external relative humidity (Bonwstra et al 2003)

The polarity of the active molecule

The polarity of the formulation

For multiphase or multipolarity systems like emulsions the active ingredient is dissolved

in the phase For example in an ow emulsion where a lipophilic active ingredient is

dissolved in the oil phase it is the polarity of the homogenous mixture of the lipophilic

active ingredient and internal oil For the same lipophilic active in a wo emulsion it is

37

the polarity of the homogenous mixture of the lipophilic active ingredients and external

oil For water-soluble active ingredients it is the polarity of the homogenous mixture of

the hydrophilic active ingredient and the aqueous phase regardless whether it is internal

(wo emulsions) or external (ow emulsions)

Once the active ingredient and the formulation type have been chosen it is

necessary to create the delivery system that will effectively deliver the molecule The

concept of relative polarity index allows the formulator to select the polarity of the phase

in which the active ingredient is incorporated on the basis of its own properties and those

of the stratum corneum In order to achieve maximum delivery the polarity of the active

ingredient and the stratum corneum need to be considered In order to improve the skin

delivery of active ingredients the first step involves selecting a primary emollient with a

polarity close to that of the active ingredient in which it will have a high solubility The

second step is to reduce the solubility of the active ingredient in the primary emollient via

the addition of a secondary emollient with a different polarity and therefore lower

solubility for the active ingredient This approach has shown a 3-4 fold increase in skin

penetration with out increasing the amount of active ingredients in the formulation

(Wiechers 2005)

32 FORMULATION OF ASCORBIC ACID CREAMS

Ascorbic acid is a water-soluble material and is included frequently in skin care

formulations to restore skin health It is very unstable and is easily oxidized in aqueous

solution This vitamin is known to be a reducing agent in biological systems and causes

the reduction of both oxygen- and nitrogen- based free radicals (Higdon and Frei 2002)

It can also act as a co-antioxidant with the tocopheroxyl radical to regenerate alpha-

38

tocopherol (Packer et al 1979 Buettner 1993 Peyrat-Maillard et al 2001) In this

reaction the two vitamins act synergistically Alpha-tocopherol first functions as the

primary antioxidant that reacts with an organic free radical Thereafter ascorbic acid

reacts with the free radical tocopheroxyl to general alpha-tocopherol In physiological

conditions the ascorbyl radical formed by regenerating tocopherol is then converted back

to ascorbate by the redox cycle (Davies et al 1991) The interaction of ascorbic acid

with a redox partner such as alpha-tocopherol has been found useful to slow its oxidation

and prolong its action

The instability of ascorbic acid makes this antioxidant active ingredient a

formulation challenge to deliver to the skin and retain its effective form In addition to its

use in combination with alpha-tocopherol in cream formulations the stability of ascorbic

acid may be improved by its use in the form of a fatty acid ester such as ascorbyl

palmitate Ascorbyl palmitate has been used in thixogel formulations and is typically

incorporated into the mineral oil phase Preliminary experiments have shown that it could

be slowly released from the starch-oil emulsion matrix and act as an antioxidant (Wille

2005)

Various physical and chemical factors involved in the formulation of cream

preparations have been discussed in the above sections For polar and air light sensitive

compounds such as ascorbic acid it is important to consider factors such as the choice of

formulation ingredients polar character of formulation HLB value pH viscosity etc to

achieve stability

39

33 STABILITY OF CREAMS

331 Physical Stability

The most important consideration with respect to pharmaceutical and cosmetic

emulsions (creams) is the stability of the finished product The stability of a

pharmaceutical emulsion is characterized by the absence of coalescence of the internal

phase absence of creaming and maintenance of elegance with respect to appearance

odor color and other physical properties An emulsion is a dynamic system however

any flocculation and resultant creaming represent potential steps towards complete

coalescence of the internal phase In pharmaceutical emulsions creaming results as a lack

of uniformity of drug distribution and poses a problem to the pharmaceutical

compounder Another important factor in the stabilization of emulsions is phase inversion

which involves the change of emulsion type from ow to wo or vice versa and is

considered as a case of instability The four major phenomena associated with the

physical instability of emulsions are flocculation creaming coalescence and breaking

These have been discussed by Garti and Aserin (1996) Im-Emsap et al (2002) and Sinko

(2006)

332 Chemical Stability

The instability of a drug may lead to the loss of its concentration through a

chemical reaction under normal or stress conditions This results in a reduction of the

potency and is a well-recognized cause of poor product quality The degradation of the

drug may make the product esthetically unacceptable if significant changes in color or

odor have occurred The degradation product may also be a toxic substance The various

pathways of chemical degradation of a drug depend on the structural characteristics of the

40

drug and may involve hydrolysis dehydration isomerization and racemization

decarboxylation and elimination oxidation photodegradation drug-excipients and drug-

drug interactions Factors determining the chemical stability of drug substances include

intrinsic factors such as molecular structure of the drug itself and environmental factors

such as temperature light pH buffer species ionic strength oxygen moisture additives

and excipients The application of well-established kinetic principles may throw light on

the role of each variable in altering the kinetics of degradation and to provide valuable

insight into the mechanism of degradation (Baertschi and Alsante 2005 Yoshioka and

Stella 2002 Lachman et al 1986) The chemical stability of individual components

within an emulsion system may be very different from their stability after incorporation

into other formulation types For example many unsaturated oils are prone to oxidation

and their degree of exposure to oxygen may be influenced by factors that affect the extent

of molecular dispersion (eg droplet size) This could be particularly troublesome in

emulsions because emulsification may introduce air into the product and because of the

high interfacial contact area between the phases (Barry 2002) The use of antioxidants

retards oxidation of unsaturated oils minimizes changes in color and texture and prevents

rancidity in the formulation Moreover they can retard the degradation of certain active

ingredients such as vitamin C (Vimaladevi 2005) The stability problems of dispersed

systems and the factors leading to these stability problems have been discussed by

Weiner (1996) and Lu and Flynn (2009)

333 Microbial Stability

Topical bases often contain aqueous and oily phases together with carbohydrates

and proteins and are susceptible to bacterial and fungal attack Microbial growth spoils

41

the formulation and is a potential toxic hazard Therefore topical formulations need

appropriate preservatives to prevent microbial growth and to maintain their quality and

shelf-life (Barry 2002 Arger et al 1996) Cream formulations may contain fats and oils

with high percentage of unsaturated linkages that are susceptible to oxidation degradation

and development of rancidity The addition of antioxidants retards oxidation of fats and

oils minimizes changes in color and texture and prevents rancidity in the formulation

Moreover they can retard the degradation of certain active ingredients such as vitamin C

These aspects in relation to dermatological formulations have been discussed by Barry

(1983 2002) and Vimaladevi 2005)

334 Stability of Ascorbic Acid in Liquid Formulations

Ascorbic acid is very unstable in aqueous solution Different workers have studied

the stability of ascorbic acid in liquid formulations (Connors et al 1986 Austria et al

1997) Its shelf-life can be prolonged by appropriate choice of vehicle and control of

other variables such as pH stabilizers temperature light and oxygen (Table 3)

Similarly the stability of various concentrations of ascorbic acid in solution form may

vary depending upon the type of solvent used (Table 4) (Connors et al 1986 Satoh et

al 2000 Lee et al 2004 Zeng et al 2005)

335 Stability of Ascorbic Acid in Emulsions and Creams

Ascorbic acid exerts several functions on skin such as collagen synthesis

depigmentation and antioxidant activity Ultraviolet radiation generates reactive oxygen

species (ROS) which produce some harmful effects on the skin including photocarcinoma

and photoaging In order to combat these problems ascorbic acid as an antioxidant has

42

Table 3 Effect of vehicles on the stability of ascorbic acid ( ascorbic acid remaining in

solutions after storage at room temperature) (Connors et al 1986)

Storage Time (days) No Vehicle

30 60 90 120 180 240 360

1 Corn Syrup 965 925 920 920 900 860 760

2 Sorbitol 990 990 990 970 960 925 890

3 4 Carboxymethyl

Cellulose

840 680 565 380 ndash ndash ndash

4 Glycerin 100 100 990 990 970 935 920

5 Propylene glycol 995 990 980 945 920 900 900

6 Syrup USP 100 100 980 980 930 900 880

7 Syrup 212 gL 880 810 775 745 645 590 440

8 25 Tragacanth 785 620 510 320 ndash ndash ndash

9 Saturated solution of

Dextrose

990 935 875 800 640 580 510

10 Distilled Water 900 815 745 675 405 185 ndash

11 50 Propylene glycol +

50 Glycerin

980 ndash 960 ndash 933 ndash ndash

12 25 Distilled Water +

75 Sorbo (70 solution

of Sorbitol)

955 954 ndash 942 930 ndash ndash

13 50 Glycerin + 50

Sorbo

982 984 978 ndash ndash 914 ndash

43

Table 4 Stability of various concentrations of ascorbic acid in water propylene glycol

and USP syrup at room temperature ( of ascorbic acid remaining in solution)

(Connors et al 1986)

Storage Time (days) Concentration

(mg ml)

Solvent

30 60 90 120 180 240 360

10 Water 930 840 820 670 515 410 ndash

50 Water 940 920 880 795 605 590 300

100 Water 970 930 910 835 705 680 590

10 Propylene glycol 100 985 980 975 960 920 860

50 Propylene glycol 100 970 980 980 980 965 935

100 Propylene glycol 100 100 100 100 990 100 925

10 Syrup 100 100 980 990 970 960 840

50 Syrup 100 100 100 100 990 100 960

100 Syrup 100 100 100 100 100 100 995

44

been used in various dosage forms and in different concentrations (Darr et al 1996

Gallarate et al 1999 Zhang et al 1999 Pinnell et al 2001 Lee et al 2004 Raschke

et al 2004 Elmore 2005 Farahmand et al 2006 Maia et al 2006) Ascorbic acid has

good photoprotective ability against UVA-mediated phototoxicity (Darr et al 1996) A

variety of formulations containing ascorbic acid or its derivatives have been studied in

order to evaluate their stability and delivery through the skin (Gallarate et al 1999

Zhang et al 1999 Ozer et al 2000 Pinnell et al 2001 Lee et al 2004 Raschke et al

2004 Farahmand et al 2006) Formulations containing derivatives of ascorbic acid are

found to be more stable than ascorbic acid but they do not produce the same effect as that

of the parent compound probably due to the lack of redox properties (Heber et al 2006)

Effective delivery of ascorbic acid through topical preparations is a major factor that

should be critically evaluated as it may be dependent upon the nature or type of the

formulation (Gallarate et al 1999 Pinnell et al 2001) The pH of the formulation

should be on the acidic side (~ pH 35) for effective penetration of the vitamin in the skin

(Pinnell et al 2001) and for its stabilization in the formulation (Gallarate et al 1999)

Some other antioxidants such as alpha-tocopherol ferulic acid and sodium metabisulphite

have also been used in combination with ascorbic acid for the purpose of its stabilization

in topical formulations and to produce some synergistic effects (Darr et al 1996 Lin et

al 2005 Maia et al 2006 Tournas et al 2006) Effect of some rheological properties

such as viscosity and dielectric constant on the stability of ascorbic acid in emulsions has

also been investigated (Connors et al 1986) Viscosity of the medium is an important

factor that should be considered for the purpose of ascorbic acid stability as higher

viscosity formulations have shown some degree of protection (Ozer et al 2000

45

Szymula 2005) Along with other factors formulation type also plays an important role in

the stability of ascorbic acid It is reported that ascorbic acid is more stable in emulsified

system as compared to aqueous solutions (Gallarate et al 1999 Lee et al 2004) In

multiemulsions ascorbic acid is reported to be more stable as compared to simple

emulsions (Gallarate et al 1999 Ozer et al 2000 Lee et al 2004 Farahmand et al

2006)

Ascorbic acid and its derivatives have been used in a variety of cosmetic

formulations as an antioxidant pH adjuster anti-aging and photoprotectant (Elmore

2005) The control of instability of ascorbic acid poses a significant challenge in the

development of cosmetic formulations It is also reported that certain metal ions or

enzyme systems effectively convert ascorbic acidrsquos antioxidant action to pro-oxidant

activity (Elmore 2005) Therefore utilization of an effective antioxidant system is

required to maintain the stability of vitamin C in various preparations (Zhang et al 1999

Pinnell et al 2001 Maia et al 2006) The chemical stability of ascorbic acid has been

studied in emulsions and creams by several workers (Darr et al 1996 Gallarate et al

1999 Lee et al 2004 Raschke et al 2004 Elmore 2005 Farahmand et al 2006)

however there is a lack of information on the photostability of ascorbic acid in cream

formulations

336 Stability Testing of Emulsions

The stability testing of emulsions (creams) may be carried out by performing the

following tests (Billany 2002)

46

3361 Macroscopic examination

The assessment of the physical stability of an emulsion is made by an

examination of the degree of creaming or coalescence occurring over a period of time

This involves the calculation of the ratio of the volume of the creamed or separated part

of the emulsion and the total volume A comparison of these values can be made for

different products

3362 Globule size analysis

An increase in mean globule size with time (coupled with a decrease in globule

numbers) indicates that coalescence is the cause of this behavior This can be used to

compare the rates of coalescence for a variety of emulsion formulations For this purpose

microscopic examination or electronic particle counting devices (coulter counter) or

laser diffraction sizing are widely used

3363 Change in viscosity

Many factors may influence the viscosity of emulsions A change in apparent

viscosity may result from any variation in globule size or number or in the orientation or

migration of emulsifier over a period of time

3264 Accelerated stability tests

In order to compare the relative stabilities of a range of similar products it is

necessary to speed up the processes of creaming and coalescence by storage at elevated

temperatures and then carrying out the tests described in the above sections

337 FDA guidelines for semisolid preparations

According to FDA draft guidelines to the industry (Shah 1997) semisolid

preparations (eg creams) should be evaluated for appearance clarity color

47

homogencity odour pH consistency viscosity particle size distribution (when feasible)

assay degradation products preservative and antioxidant content (if present) microbial

limits sterility and weight loss when appropriate Additionally samples from

production lot or approved products are retained for stability testing in case of product

failure in the field Retained samples can be tested along with returned samples to

ascertain if the problem was manufacturing or storage related Appropriate stability data

should be provided for products supplied in closed-end tubes to support the maximum

anticipated use period during patient use and after the seal is punctured allowing product

contact with the cap cap lever Creams in large containers including tubes should be

assayed by sampling at the surface top middle and bottom of the container In addition

tubes should be sampled near the crimp The objective of stability testing is to determine

whether the product has adequate shelf-life under market and use conditions

48

OBJECT OF PRESENT INVESTIGATION

Ascorbic acid (vitamin C) is extensively used as a single ingredient or in

combination with vitamin B complex and other vitamins in the form of drops injectables

lotions and syrups It is an ingredient of anti-aging cosmetic products alone or along with

alpha-tocopherol (vitamin E) Ascorbic acid exerts several functions on the skin as

collagen synthesis depigmentation and antioxidant activity It protects the signs of

degenerative skin conditions caused by oxy-radical damage In solutions and creams

ascorbic acid is susceptible to air and light and undergoes oxidative degradation to

dehydroascorbic acid and inactive products The degradation is influenced by

temperature viscosity and polarity of the medium and is catalysed by metal ions

particularly Cu+2

Fe+2

and Zn+2

One of the major problems faced in cream preparations is the instability of

ascorbic acid as it may be exposed to light during formulation manufacturing and

storage and the possibility of photochemical degradation can not be neglected The

behaviour of ascorbic acid in light is of particular interest since no systematic kinetic

studies have been conducted on its photodegradation in these preparations under various

conditions The study of the formulation variables such as emulsifier humectants and pH

may throw light on the photostabilization of ascorbic acid in creams

The main object of this investigation is to study the behaviour of ascorbic acid in

cream preparations on exposure to UV light in the pharmaceutically useful pH range An

important aspect of the work is to study the interaction of ascorbic acid with other

vitamins such as riboflavin nicotinamide and alpha-tocopherol and the effect of certain

stabilizers such as citric acid tartaric acid and boric acid on its photodegradation In

49

addition it is intended to study the photolysis of ascorbic acid in organic solvents to

evaluate the effect of solvent characteristics (eg dielectric constant and viscosity) on the

stability of the vitamin The study of all these aspects may provide useful information to

improve the photostability and efficacy of ascorbic acid in cream preparations

An outline of the proposed plan of work is presented as follows

1 To prepare a number of oil-in-water cream formulations based on different

emulsifying agents and humectants containing ascorbic acid alone and in

combination with other vitamins and stabilizing agents

2 To perform photodegradation studies on ascorbic acid in creams using a UV

irradiation source with emission corresponding to the absorption maximum of

ascorbic acid

3 To identify the photoproducts of ascorbic acid in creams using chromatographic

and spectrophotometric methods

4 To apply appropriate and validated analytical methods for the assay of ascorbic

acid alone and in combination with other vitamins and stabilizing agents

5 To study the effect of solvent characteristics such as dielectric constant and

viscosity on the photolysis of ascorbic acid in aqueous and organic solvents

6 To evaluate the kinetics of photodegradation of ascorbic acid and its interactions

with other vitamins (riboflavin nicotinamide and alpha-tocopherol) in creams

7 To evaluate the effect of carbon chain length of the emulsifying agent and the

viscosity of the humectant on the photodegradation of ascorbic acid

50

8 To develop relationships between the rate of photodegradation of ascorbic acid

and the concentration pH carbon chain length of emulsifier viscosity of the

creams

9 To determine the effect of compounds such as citric acid tartaric acid and boric

acid used as stabilizing agents on the rate of photodegradation and stabilization

of ascorbic acid in creams

10 To present reaction schemes for the photodegradation of ascorbic acid and its

interactions with other vitamins

CHAPTER IV

MATERIALS

AND

METHODS

52

41 MATERIALS

Vitamins and Related Compounds

L-Ascorbic Acid vitamin C (5R)-5-[(1S)-12-dihydroxyethyl]-34-dihydroxyfuran-2(5H)-

one Merck

C6H8O6 Mr 1761

Dehydroascorbic Acid L-threo-23-hexodiulosonic acid γ-lactone Sigma

C6H6O6 Mr 1741

23-Diketogulonic Acid

C6H8O7 Mr 192

It was prepared according to the method of Homann and Gaffron (1964) by the

hydrolysis of dehydroascorbic acid

Riboflavin vitamin B2 (310-dihydro-78-dimethyl-10-[(2S3S4R)-2345-

tetrahydroxypentyl] benzopteridine-24-dione) Merck

C17H20N4O6 Mr 3764

Nicotinamide vitamin B3 (pyridine-3-carboxamide) Merck

C6H6N2O Mr 1221

Alpha-Tocopherol vitamin E ((2R)-2578-tetramethyl-2-[(4R8R)-4812-

trimethyltridecyl]-34-dihydro-2H-1-benzopyran-6-ol) Merck

C29H50O2 Mr 4307

Formylmethylflavin (78-dimethyl-10-formylmethylisoalloxazine)

C14H12N4O3 Mr 2843

53

Formylmethylflavin was synthesized according to the method of Fall and Petering

(1956) by the periodic acid oxidation of riboflavin It was recrystallized from absolute

methanol dried in vacuo and stored in the dark in a refrigerator

Lumichrome (78-dimethylalloxazine) Sigma

C12H10N4O2 Mr 2423

It was stored in the dark in a desiccator

Stabilizers

Boric Acid orthoboric acid Merck

H3BO3 Mr 618

Citric Acid 2-hydroxypropane-123-tricarboxylic acid Merck

C6H8O7H2O Mr 2101

L-Tartaric acid [(2R3R)-23-dihydroxybutanedioic acid] Merck

C4H6O6 Mr 1501

Emulsifying Agents

Stearic Acid (95) octadecanoic acid Merck

C18H36O2 Mr 2845

Palmitic Acid hexadecanoic acid Merck

C16H32O2 Mr 2564

Myristic Acid tetradecanoic acid Merck

C14H28O2 Mr 2284

Cetyl alcohol hexadecan-1-ol Merck

C16H34O Mr 2424

54

Humectants

Glycerin glycerol (propane-123-triol) Merck

C3H8O3 Mr 921

Propylene glycol (RS)-propane-12-diol Merck

C3H8O2 Mr 7610

Ethylene glycol ethane-12-diol Merck

C2H6O2 Mr 6207

Potassium Ferrioxalate Actinometry

Potassium Ferrioxalate

K3Fe(C2O4)3 3H2O Mr 4912

Potassium Ferrioxalate was prepared according to the method of Hatchard and

Parker (1956) Three volumes of 15 M potassium oxalate was mixed with one volume of

15 M ferric chloride with vigorous stirring The yellow green precipitate of potassium

ferrioxalate was recrystallized twice from water dried at 45 ordmC and stored in the dark in a

desiccator

Reagents

All the reagents and solvents used were of analytical grade obtained from BDH

Merck

Water

Freshly boiled distilled water was used throughout the work

55

42 METHODS

421 Cream Formulations

On the basis of the various cream formulations reported in the literature (Block

1996 Flynn 2002 Betageri and Prabhu 2002 Vimaladevi 2005 EIRI Board Lu and

Flynn 2009) the following basic formula was used for the preparation of oil-in-water

creams containing ascorbic acid

Oil phase Percentage (ww)

Emulsifier

Myristic palmitic stearic acid

Cetyl alcohol

120

30

Aqueous phase

Humectant

Ethylene glycol propylene glycol glycerin

50

Active ingredient

Ascorbic acid

20 (0114 M)

Neutralizer

Potassium hydroxide

10

Continuous phase

Distilled water

QS

Additional ingredientsa

Vitamins

Riboflavin (Vitamin B2)

Nicotinamide (Vitamin B3)

Alpha-Tocopherol (Vitamin E)

0002ndash001 (053ndash266times10ndash4

M)

028ndash140 (0023ndash0115 M)

017ndash086 (0395ndash200times10ndash2

M)

Stabilizers

Citric acid

Tartaric acid

Boric acid

010ndash040 (0476ndash190times10ndash2

M)

010ndash040 (067ndash266times10ndash2

M)

010ndash040 (0016ndash0065 M)

a The vitamin stabilizer concentrations used were found to be effective in promotion

inhibition of photodegradation of ascorbic acid in cream formulations

56

422 Preparation of Creams

The emulsifiers were melted at 70ndash80 ordmC in a glass jar immersed in a water bath

Ascorbic acid was separately dissolved in a small portion of distilled water Potassium

hydroxide and humectant were dissolved in the remaining portion of water and mixed

with the oily phase with constant stirring until the formation of a thick white mass It was

cooled to ~40 ordmC and the ascorbic acid solution was added The thick mass was mixed

using a mechanical mixer with a glass stirrer at 1000 rpm for 5 minutes The pH of the

cream was adjusted to the desired value and the contents again mixed for 10 minutes at

500 rpm All the creams were prepared under uniform conditions to maintain their

individual physical characteristics and stored at room temperature in airtight glass

containers protected from light

In the case of other vitamins nicotinamide was dissolved along with ascorbic acid

in water and added to the cream Riboflavin or alpha-tocopherol were directly added to

the cream and mixed thoroughly according to the procedure described above

In the case of stabilizing agents (citric tartaric and boric acids) the individual

compounds were dissolved in the ascorbic acid solution and added to the cream followed

by the procedure described above

The details of the various cream formulations used in this study are given in

chapters 5ndash7

57

423 Thin-Layer Chromatography (TLC)

The following TLC systems were used for the separation and identification of

ascorbic acid and photodegradation products

Adsorbent a) Silica gel GF 254 (250-microm) precoated plates

(Merck)

Solvent systems S1 acetic acid-acetone-methanol-benzene

(552070 vv) (Ganshirt and Malzacher 1960)

S2 ethanol-10 acetic acid-water (9010 vv)

(Bolliger and Konig 1969)

S3 acetonitrile-butylnitrile-water (66332 vv)

(Saari et al 1967)

Temperature 25ndash27 ordmC

Location of spots Ascorbic acid UV light 254 nm (Uvitec lamp

UK)

Dehydroascorbic acid Spray with a 3 aqueous

phenylhydrazine hydrochloride solution

424 pH Measurements

The measurements of pH of aqueous solutions and cream formulations were

carried out using an Elmetron LCD display pH meter (modelndashCP501 sensitivity plusmn 001

pH units) (Poland) with a combination electrode The electrode was calibrated

automatically in the desired pH range (25 ordmC) using the following buffer solutions

58

Phthalate pH 4008

Phosphate pH 6865

Disodium tetraborate pH 9180

The electrode was immersed directly into the cream (British Pharmacopoeia

2009) kept for few seconds to equilibrate and the pH adjusted in the range of 40ndash70

with phosphoric acid sodium hydroxide solution

425 Ultraviolet and Visible Spectrometry

The absorbance measurements and spectral determinations were performed on

Shimadzu UVndashVisible recording spectrophotometer (model UVndash1601) using matched

silica cells of 10 mm path length The cells were employed always in the same orientation

using appropriate control solutions in the reference beam The baseline was automatically

corrected by the built-in baseline memory at the initializing period Auto-zero adjustment

was made by a one-touch operation The instrument checked the wavelength calibration

(6561 nm) using the deuterium lamp at the initializing period The absorbance scale was

periodically checked using the following calibration standard (British Pharmacopoeia

2009)

0057ndash0063 gl of potassium dichromate in 0005 M sulphuric acid

The specific absorbance [A(1 1 cm)] of the solution should match the

following values with the stated limit of tolerance

Wavelength

(nm)

Specific absorbance

A (1 1 cm)

Maximum

tolerance

235 1245 1229ndash1262

257 1445 1428ndash1462

313 486 470ndash503

350 1073 1056ndash109

430 159 157ndash161

59

426 Photolysis of Ascorbic Acid

4261 Creams

A 2 g quantity of the cream was uniformly spread on several rectangular glass

plates (5 times 15 cm) covered with a 1 cm tape on each side to give a 1 mm thick layer The

plates were irradiated in a dark chamber using a Philips 30 watt TUV tube (100

emission at 254 nm the wavelength absorbed by ascorbic acid at pH 4ndash7) fixed

horizontally at a distance of 30 cm from the centre of the plates Each plate was removed

at appropriate interval and the cream was subjected to spectrophotometric assay and

chromatographic examination

4262 Aqueous and organic solvents

A 10ndash3

M solution of ascorbic acid (50 ml) prepared in water (pH 70 005 M

phosphate buffer) or in an organic solvent in a 100 ml beaker (Pyrex) was placed in a

water bath maintained at 20 plusmn 1 ordmC The solution was irradiated with the Philips 30 watt

TUV tube in a dark chamber as stated above Samples were withdrawn at appropriate

intervals for assay and chromatography

4263 Storage of creams in dark

In order to determine the stability of various cream formulations in the dark

samples were stored at room temperature in a cupboard protected from light for a period

of three months The samples were analyzed periodically for the content of ascorbic acid

and the presence of any degradation product

427 Measurement of Light Intensity

The potassium ferrioxalate actinometry was used for the measurement of light

intensity of the radiation source employed in this work This actinometer has been

60

developed by Parker (1953) and Hatchard and Parker (1956) and is considered as the

most useful actinometer over a wide range of wavelengths (254ndash577 nm) It has been

used by Holmstrom and Oster (1961) Byrom and Turnbull (1967) McBride and Moore

(1967) Ahmad (1968) Ahmad (1978) Ahmad et al (2004a 2004b 2005 2006a

2006b 2008 2009ab) Fasihullah (1988) Vaid (1998) Ansari (2002) and Ahmad (2009)

for the measurement of light intensity

The irradiation of potassium ferrioxalate solutions in sulphuric acid results in the

reduction of ferric ion to ferrous ion according to the following reaction

2Fe [(C2O4)3]3ndash

rarr 2 Fe (C2O4) + 3 (C2O4)2ndash

+ 2CO2 (31)

The amount of Fe2+

ions formed in the reaction may be determined by

complexation with 110-phenanthroline to give a red colored complex The absorbance of

the complex is measured at 510 nm

428 Procedure

An oxygen free 0006 M solution of potassium ferrioxalate (2947 gl) in 01 N

H2SO4 was placed in the reaction vessel and irradiated with the lamp used for the

photolysis of riboflavin The irradiation was carried out under nitrogen (90ndash120

bubblesminute) which also caused stirring of the solution The temperature of the

reaction vessel was maintained at 25 plusmn 1 ordmC during the reaction

An aliquot of the photolysed solution (1ndash2 ml) was pipetted out at suitable

intervals (up to 30 minutes) into a 10 ml volumetric flask to which was then added 09

ml of N H2SO4 + 1 ml (01) 110-phenanthroline + 05 ml buffer (60 ml N CH3COONa

+ 36 ml N H2SO4 made up to 100 ml with distilled water) The flask was made up

to the mark with distilled water (final pH 35) thoroughly shaken to mix the contents and

61

Fig 3 Spectral power distribution of TUV 30 W tube (Philips)

62

allowed to stand for one hour in the dark to develop the colorndashcomplex The absorbance

of the phenanthrolinendashferrous complex was measured in a 1 cm cell at 510 nm using the

appropriate solution as blank The amount of Fe2+

ions formed was determined from the

calibration graph The calibration graph was constructed in a similar manner using

several dilutions of 1 times 10ndash6

mole ml Fe2+

in 01 N H2SO4 (freshly prepared by dilution

from standardized 01 M FeSO4 in 01 N H2SO4) (Fig 8) The experimental value of the

molar absorptivity of Fe2+

complex as determined from the slope of the calibration graph

is equal to 111 times 104 M

ndash1 cm

ndash1 and is in agreement with the value reported by Parker

(1953) Using the values of the known quantum yield for ferrioxalate actinometer at

different wavelengths (Hatchard and Parker 1956) the number of Fe2+

ions formed

during photolysis the time of exposure and the fraction of the light absorbed by the

length of the actinometer solution employed the light intensity incident just inside the

front window of the photolysis cell can be calculated In the present case total absorption

of the light has been assumed

4281 Calculation

The number of Fe2+

ions formed during photolysis (nFe

2+) is given by the

equation

6023 times 1020

V1 V3 A Σ

n Fe

2+ =

V2 1 ε (32)

where V1 is the volume of the actinometer solution irradiated (ml)

V2 is the volume of the aliquot taken for analysis (ml)

V3 is the final volume to which the aliquot V2 is diluted (ml)

1 is the path length of the spectrophotometer cell used (1 cm)

A is the measured absorbance of the final solution at 510 nm

63

ε is the molar absorptivity of the Fe2+

complex (111 times 104 M

ndash1 cm

ndash1)

The number of quanta absorbed by the actinometer nabs can then be obtained as follows

n Fe

2+

Σ nabs = ф

(33)

where ф is the quantum yield for the Fe2+

formation at the desired wavelength

The number of quanta per second per cell nabs is therefore given by

Σ nabs 6023 times 1020

V1 V3 A nabs =

t =

ф V2 1 ε t (34)

where t is the irradiation time of the actinometer in seconds

The relative spectral energy distribution of the radiation source (Fig 3) shows

100 emission at 254 nm the wavelength used for the photolysis of ascorbic acid (λmax

265 nm at pH 4ndash7) The energy emitted by the radiation source at various wavelengths

can be calculated using the equation

1197 times 105

E (KJ molndash1

) = λ nm

(35)

The quantum efficiency of ferrioxalate actinometer at the wavelength absorbed by

ascorbic acid (ie 254 nm) is high although the sensitivity drops over 450 nm The

average intensity of the TUV tube used in this study was determined as 556 plusmn 012 times

1018

quanta sndash1

429 Viscosity Measurements

The viscosity of the cream formulations was measured with a Brookfield RV

viscometer (Model DV-II + Pro USA) The instrument was calibrated using the

manufacturerrsquos viscosity standard A 200 g quantity of the cream was placed in a beaker

and the spindle (TE) was dipped into the cream It was rotated at a speed of 06 rpm for

64

00

02

04

06

08

10

12

0 2 4 6 8 10 12

Concentration of Fe++

times 105 M

Ab

sorb

an

ce a

t 51

0 n

m

Fig 4 Calibration graph for the determination of K3Fe(C2O4)3

65

one minute and the viscosity was recorded at 25plusmn1 ordmC The test was repeated three times

to account for the experimental variability and the average viscosity was noted

4210 Assay Methods

42101 UV spectrophotometric method for the assay of creams containing ascorbic

acid alone

The creams were thoroughly mixed a quantity of 2 g was accurately weighed and

the assay of ascorbic acid was carried out by the UV method of Zeng et al (2005) In the

case of photodegraded creams (2 g) the material was completely removed from the glass

plate and transferred to a volumetric flask The method involved extraction of ascorbic

acid with methanol (3 times 10 ml) adjustment of the pH of combined methanolic solutions

to 20 (with H3PO4) dilution of the final solution with acidified methanol (pH 20) to 100

ml and measurement of the absorbance at 245 nm using appropriate blank The

concentration of ascorbic acid was calculated using 560 as the value of specific

absorbance [A (1 1 cm)] at the analytical wavelength (Table 5)

The same method was used for the assay of ascorbic acid in creams stored in the

dark and in the presence of individual stabilizing agents (citric tartaric and boric acids)

42102 Iodimetric method for the assay of ascorbic acid in creams containing

riboflavin nicotinamide and alpha-tocopherol

The assay of ascorbic acid in creams in the presence of riboflavin nicotinamide

and alpha-tocopherol was carried out according to the procedure of British

Pharmacopoeia (2009) as follows

The photolysed cream (2 g) was completely scrapped from the glass plate and

transferred to a flask containing 40 ml of distilled water and 10 ml of 1 M sulphuric acid

66

Table 5 Calibration data for ascorbic acid showing linear regression analysisa

λ max 245 nm

Concentration range 01ndash10 times 10ndash4

M (0176ndash1761 mg )

Slope 9920

SE (plusmn) of slope 00114

Intercept 00012

Correlation coefficient 09996

Molar absorptivity (ε) 9920 Mndash1

cmndash1

Specific absorbance [A (1 1 cm)] 560

a Values represent a mean of five determinations

67

was added The solution was titrated with 002 M iodine solution using 1 ml of starch

solution as indicator until a persistent violet-blue color was obtained Each ml of 002 M

iodine solution is equivalent to 352 mg of C6H8O6 The same method was used for the

assay of ascorbic acid in creams stored in the dark

42103 Spectrophotometric method for the assay of ascorbic acid in aqueous and

organic solvents

A 1 ml aliquot of the photolysed solutions of ascorbic acid in water or in an

organic solvent was evaporated to dryness under nitrogen at room temperature and the

residue redissolved in a small volume of methanol The solution was transferred to a 10

ml volumetric flask made up to volume with acidified methanol (pH 20) and the

absorbance measured at 245 nm using an appropriate blank The content of ascorbic acid

in the solutions was determined using 9920 Mndash1

cmndash1

as the value of molar absorptivity at

the analytical wavelength (Table 5)

CHAPTER V

PHOTODEGRADATION OF

ASCORBIC ACID IN

ORGANIC SOLVENTS AND

CREAM FORMULATIONS

69

51 INTRODUCTION

Ascorbic acid (vitamin C) is an essential micronutrient that performs important

metabolic functions (Packer and Fuchs 1999 Davey et al 2000 Johnston et al 2007)

It is an ingredient of anti-aging cosmetic products (Darr et al 1996 Gallarate et al

1999 Traikovich 1999 Zhang et al 1999 Ozer et al 2000 Nusgens et al 2001

Pinnell et al 2001 2003 Lee et al 2004 Raschke et al 2004 Sauermann et al 2004

Elmore 2005 Jentzsch et al 2005 Lin et al 2005 Placzek et al 2005 Carlotti et al

2006 Farahmand et al 2006 Heber et al 2006 Maia et al 2006 Tournas et al 2006)

and exerts several functions on the skin as collagen synthesis depigmentation and

antioxidant activity (Nusgens et al 2001 Spiclin et al 2003) As an antioxidant it

protects skin by neutralizing reactive oxygen species generated on exposure to sunlight

(Shindo et al 1994) In biological systems it reduces both oxygenndash and nitrogenndash based

free radicals (Higdon and Frei 2002) and thus delays the aging process In view of the

instability of ascorbic acid in skin care formulations (Bissett 2006) it is often used in

combination with another redox partner such as alpha-tocopherol (vitamin E) to retard its

oxidation (Wille 2005)

The details of the cream formulations used in this study are given in Table 6 The

results obtained on the photodegradation of ascorbic acid in aqueous organic solvents

and cream formulations are discussed in the following sections

70

Table 6 Composition of cream formulations containing ascorbic acid

Ingredients Cream

No pH

SA PA MA CA AH2 GL PG EG PH DW

1 a 4 + minus minus + + + minus minus + +

b 5 + minus minus + + + minus minus + +

c 6 + minus minus + + + minus minus + +

d 7 + minus minus + + + minus minus + +

2 a 4 minus + minus + + + minus minus + +

b 5 minus + minus + + + minus minus + +

c 6 minus + minus + + + minus minus + +

d 7 minus + minus + + + minus minus + +

3 a 4 minus minus + + + + minus minus + +

b 5 minus minus + + + + minus minus + +

c 6 minus minus + + + + minus minus + +

d 7 minus minus + + + + minus minus + +

4 a 4 + minus minus + + minus + minus + +

b 5 + minus minus + + minus + minus + +

c 6 + minus minus + + minus + minus + +

d 7 + minus minus + + minus + minus + +

5 a 4 minus + minus + + minus + minus + +

b 5 minus + minus + + minus + minus + +

c 6 minus + minus + + minus + minus + +

d 7 minus + minus + + minus + minus + +

6 a 4 minus minus + + + minus + minus + +

b 5 minus minus + + + minus + minus + +

c 6 minus minus + + + minus + minus + +

d 7 minus minus + + + minus + minus + +

7 a 4 + minus minus + + minus minus + + +

b 5 + minus minus + + minus minus + + +

c 6 + minus minus + + minus minus + + +

d 7 + minus minus + + minus minus + + +

8 a 4 minus + minus + + minus minus + + +

b 5 minus + minus + + minus minus + + +

c 6 minus + minus + + minus minus + + +

d 7 minus + minus + + minus minus + + +

9 a 4 minus minus + + + minus minus + + +

b 5 minus minus + + + minus minus + + +

c 6 minus minus + + + minus minus + + +

d 7 minus minus + + + minus minus + + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PG = propylene glycol EG = ethylene glycol

PH = potassium hydroxide DW = distilled water

71

52 PHOTOPRODUCTS OF ASCORBIC ACID

The photolysis of ascorbic acid (AH2) in aqueous and organic solvents and in

cream formulations on UV irradiation leads to the formation of dehydroascorbic acid

(DHA) as detected by TLC along with the undegraded AH2 using the solvent systems A

B and C The identification of DHA was carried out by comparison of the Rf value and

spot color with those of the authentic compound The formation of DHA on

photooxidation of ascorbic acid solutions has previously been reported (Homan and

Gaffron 1964 Sattar et al 1977 Heelis et al 1981 Rozanowska et al 1997 Lavoie et

al 2004) DGA the hydrolysis product of DHA (Homan and Gaffron 1964) could not

be detected under the present experimental conditions

53 SPECTRAL CHARACTERISTICS OF PHOTOLYSED SOLUTIONS

A typical set of the UV absorption spectra of photolysed solutions of AH2 in

methanol is shown in Fig 5 There is a gradual loss of absorbance around 245 nm with

time as a result of the oxidation of the molecule to DHA (Pelletier 1985 Davies et al

1991 Rumsey and Levine 2000) which does not absorb in this region due to the loss of

conjugation Similar absorption changes are observed on the photolysis of AH2 in other

organic solvents and in the methanolic extracts of cream formulations However the

magnitude of these changes varies with the rate of photolysis in a particular solvent or

cream and appears to be a function of the polar character pH and viscosity of the

medium

72

Fig 5 UV absorption spectra of photolysed solutions of ascorbic acid in methanol at

0 40 80 120 160 220 and 300 min

73

54 ASSAY OF ASCORBIC ACID IN CREAMS AND SOLUTIONS

The assay of AH2 in creams and solutions has been carried out in acidified

methanol (pH 20) according to the UV spectrophotometric method of Zeng et al (2005)

Aqueous solutions of AH2 (~pH 2) exhibit absorption maxima at 243 nm (OrsquoNeil 2001

Moffat et al 2004 Sweetman 2009) 244 nm (Ogata and Kosugi 1969) and 245 nm

(Verma et al 1991 Johnston et al 2007) The absorption maxima of AH2 in methanol

and phosphate buffer (pH 25) occur at 245 nm (Zeng et al 2005) Since dilute solutions

of AH2 are highly susceptible to oxidation the pH was adjusted to 20 with phosphoric

acid to convert the molecule to the non-ionized form (99) to minimize degradation

during the assay AH2 in acidified methanol (pH 20) was found to exhibit the absorption

maximum at 245 nm as reported by Zeng et al (2005) The method was also used for the

assay of AH2 in aqueous and organic solvents

The validity of Beerrsquos law relation in the concentration range used was confirmed

prior to the assay The calibration data for AH2 at the analytical wavelength are presented

in Table 5 (Chapter 4) The correlation coefficient (r = 09996) indicates a good linear

relationship over the concentration range employed The values of specific absorbance

and molar absorptivity at 245 nm determined from the slope of the curve are in good

agreement with those reported by previous workers (Davies et al 1991 Johnston et al

2007) The method of Zeng et al (2005) has been found to be satisfactory for the assay of

AH2 in cream formulations and solutions and has been used to study the kinetics of

photolysis reactions The method was validated before its application to the assay of AH2

in photolysed creams The reproducibility of the method was confirmed by the analysis of

known amounts of AH2 in the concentration range likely to be found in photodegraded

74

creams The values of the recoveries of AH2 in creams by the UV spectrophotometric

method are in the range of 90ndash96 The values of RSD for the assays indicate the

precision of the method within plusmn5 (Table 7)

In order to compare the UV spectrophotometric method with the British

Pharmacopoeia iodimetric method (2009) using a dilute iodine solution (002 M) the

creams were simultaneously assayed for AH2 content by the two methods and the results

are reported in Table 8 The statistical evaluation of the accuracy and precision of the two

methods was carried out by the application of the F test and the t test respectively The F

test showed that there is no significant difference between the precision of the two

methods and the calculated value of F is lower than the critical value (F = 639 P = 005)

in each case The t test indicated that the calculated t values are lower than the tabulated t

values (t = 2776 P = 005) suggesting that at 95 confidence level the differences

between the results of the two methods are statistically non-significant Thus the accuracy

and precision of the UV spectrophotometric method is comparable to that of the official

iodimetric method for the assay of AH2 in cream formulations The results of the assays

of AH2 in aqueous organic solvents and cream formulations are reported in Table 9

55 EFFECT OF SOLVENT

The influence of solvent on the rate of degradation of drugs is of considerable

importance to the formulator since the stability of drug species in solution media may be

predicted on the basis of their chemical reactivity The reactivity of drugs in a particular

medium depends to a large extent on solvent characteristics such as the dielectric

constant and viscosity (Connors et al 1986 Yoshioka and Stella 2000 Sinko 2006)

75

Table 7 Recovery of ascorbic acid added to cream formulationsa

Cream

Formulationb

Added

(mg)

Found

(mg)

Recovery

()

RSD

()

1a 400

200

380

183

950

915

21

25

2b 400

200

371

185

928

925

15

25

3c 400

200

375

181

938

905

11

31

4d 400

200

384

189

960

945

13

21

5b 400

200

370

189

925

945

14

26

6c 400

200

369

190

923

950

10

22

7d 400

200

374

182

935

910

17

39

8c 400

200

380

188

950

940

15

33

9d 400

200

367

189

918

945

20

42

a Values expressed as a mean of three to five determinations

b The cream formulations represent combinations of each emulsifier (stearic acid

palmitic acid myristic acid) with each humectant (glycerin propylene glycol ethylene

glycol) to observe the efficiency of methanol to extract AH2 from different creams

(Table 6)

76

Table 8 Assay of ascorbic acid in creams using UV spectrophotometric and iodimetric

methods

Ascorbic acid (mg) Cream

Formulationb Added UV method

a

Iodimetric

methoda

Fcalc tcalc

1a 40

20

380 plusmn 081

183 plusmn 046

375 plusmn 095

185 plusmn 071

138

238

245

104

2b 40

20

371 plusmn 056

185 plusmn 047

373 plusmn 064

193 plusmn 038

130

065

181

200

3c 40

20

375 plusmn 040

181 plusmn 056

374 plusmn 046

183 plusmn 071

132

160

101

223

4d 40

20

384 plusmn 051

189 plusmn 039

381 plusmn 066

190 plusmn 052

167

178

176

231

5b 40

20

370 plusmn 052

189 plusmn 050

372 plusmn 042

185 plusmn 067

065

179

162

125

6c 40

20

369 plusmn 037

190 plusmn 042

371 plusmn 058

188 plusmn 056

245

177

122

197

7d 40

20

374 plusmn 062

182 plusmn 072

370 plusmn 070

184 plusmn 082

127

129

144

168

8c 40

20

380 plusmn 058

188 plusmn 062

375 plusmn 075

192 plusmn 060

167

094

123

162

9d 40

20

367 plusmn 072

189 plusmn 080

365 plusmn 082

187 plusmn 075

149

092

130

203

Theoretical values (P = 005) for F is 639 and for t is 2776

a Mean plusmn SD (n = 5)

b Table 6

77

Table 9 Photodegradation of ascorbic acid in cream formulations

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) pHa 40 50 60 70

0 383 382 384 383

60 374 369 366 361

120 361 354 346 325

180 351 345 325 305

240 345 327 301 284

1

300 336 316 287 264

0 380 383 382 379

60 371 376 362 346

120 359 357 342 320

180 352 345 322 301

240 341 335 299 283

2

300 336 321 291 261

0 384 376 381 385

60 377 367 360 358

120 366 348 334 324

180 356 337 317 305

240 343 320 301 282

3

300 335 307 273 253

78

Table 9 continued

0 377 378 386 372

60 365 361 371 355

120 353 345 347 322

180 344 327 325 298

240 332 320 306 279

4

300 317 303 284 252

0 381 367 372 373

60 372 358 358 353

120 360 337 336 321

180 352 325 320 302

240 341 313 300 284

5

300 327 302 278 256

0 376 386 380 377

60 366 372 350 350

120 353 347 323 316

180 337 334 308 298

240 329 320 291 274

6

300 313 306 267 245

79

Table 9 continued

0 380 372 378 380

60 373 362 350 354

120 358 340 329 321

180 344 328 304 300

240 332 315 292 283

7

300 319 302 272 252

0 380 381 378 361

60 368 364 361 335

120 355 354 340 313

180 342 340 315 281

240 337 331 303 269

8

300 323 314 281 248

0 378 382 370 375

60 370 369 349 342

120 356 347 326 321

180 339 333 298 291

240 326 314 277 271

9

300 313 302 265 242

a The values at pH 40ndash70 represent the formulations a to d of each cream respectively

80

In order to observe the effect of solvent dielectric constant the apparent first-

order rate constants (kobs) for the photolysis of AH2 in alcoholic solvents (Table 10) were

plotted against the dielectric constants of the solvents A linear relationship indicated the

dependence of the rates of photolysis on solvent dielectric constant (Fig 6) This implies

the involvement of a polar intermediate in the reaction to facilitate the formation of the

degradation products as suggested by Ahmad and Tollin (1981) in the case of flavin

electron transfer reactions The effect of solvent polarity has been observed on the

autooxidation of AH2 in organic solvents (Ogata and Kosugi 1969)

Another solvent parameter affecting the rate of a chemical reaction is viscosity

which can greatly influence the stability of oxidisable substances (Wallwork and Grant

1977 Laidler 1987 Fung 1990) A plot of kobs for the photolysis of AH2 against the

reciprocal of solvent viscosity (Table 10) is linear showing that an increase in solvent

viscosity results in a decrease in the rate of photolysis (Fig 7) The viscosity of the liquid

affects the rate at which molecules can diffuse through the solution This in turn may

affect the rate at which a compound can suffer oxidation at the liquid surface This

applies to AH2 and an increase in the viscosity of the medium makes access to air at the

surface more difficult to prevent oxidation (Wallwork and Grant 1977)

56 EFFECT OF CONCENTRATION

In order to observe the effect of concentration (Table 11) on the photostability of

AH2 in a cream using stearic palmitic and myristic acids as emulsifying agents and

glycerin as humectant plots of log concentration versus time were constructed (Fig 8)

and the apparent first-order rate constants were determined (Table 12) A graph of kobs

against concentration of AH2 (Fig 9) exhibited an apparent linear relationship between

81

Table 10 Apparent first-order rate constants (kobs) for the photolysis of ascorbic acid in

water and organic solvents

Solvent Dielectric

Constant (25 ordmC)

Viscosity

(mPas) ndash1

kobs times104

(minndash1

)

Water 785 1000 404

Methanol 326 1838 324

Ethanol 243 0931 316

1-Propanol 201 0514 302

1-Butanol 178 0393 295

82

00

20

40

60

80

0 10 20 30 40 50 60 70 80

Dielectric constant

k (

min

ndash1)

Fig 6 A plot of kobs for photolysis of ascorbic acid against solvent dielectric constant

(times) Water () methanol () ethanol (diams) 1-propanol () 1-butanol

83

00

10

20

30

40

50

00 05 10 15 20

Viscosity (mPas)ndash1

k times

10

4 (m

inndash1)

Fig 7 A plot of kobs for photolysis of ascorbic acid against reciprocal of solvent

viscosity Symbols are as in Fig 6

84

Table 11 Effect of concentration on the photodegradation of ascorbic acid in cream

formulations at pH 60

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) 05 10 15 20 25

0 95 191 290 379 471

60 90 182 277 358 453

120 82 167 260 339 431

180 77 158 239 311 401

240 70 144 225 298 382

1

300 64 134 210 282 363

0 92 186 287 380 472

60 88 175 272 369 453

120 82 160 251 342 429

180 75 152 238 326 405

240 71 144 225 309 392

2

300 65 134 215 289 366

0 94 182 286 376 470

60 87 171 265 352 454

120 78 152 251 337 426

180 69 143 227 315 404

240 62 129 215 290 378

3

300 58 119 195 271 353

85

05

10

15

20

25

06

08

10

12

14

16

18

log

co

nce

ntr

ati

on

(m

g)

a

05

10

15

20

25

06

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

b

05

10

15

20

25

06

08

10

12

14

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

c

Fig 8 Log concentration versus time plots for the photodegradation of ascorbic acid at

various concentrations in creams at pH 60 a) stearic acid b) palmitic acid

c) myristic acid

86

Table 12 Apparent first-order rate constants (kobs) for the photodegradation of various

ascorbic acid concentrations in cream formulations at pH 60

kobs times 103 (min

ndash1)a Cream

Formulationb 05 10 15 20 25

1 133

(0994)

120

(0993)

111

(0995)

101

(0994)

090

(0994)

2 118

(0992)

108

(0994)

098

(0993)

093

(0992)

084

(0994)

3 169

(0994)

144

(0995)

126

(0994)

109

(0993)

097

(0992)

a The values in parenthesis are correlation coefficients

b Table 6

87

Stearic acid

Palmitic acid

Myristic acid

00

05

10

15

20

25

00 05 10 15 20 25

Ascorbic acid concentration ()

kob

s (min

ndash1)

Fig 9 A plot of kobs for photodegradation against ascorbic acid concentrations in cream

formulations

88

the two values Thus the rate of degradation of AH2 is faster at a lower concentration on

exposure to the same intensity of light This may be due to a relatively greater number of

photons available for excitation of the molecule at lower concentration compared to that

at a higher concentration The AH2 concentrations of creams used in this study are within

the range (1ndash15) reported by previous workers for topical applications to skin (Kaplan

et al 1989 Traikovich et al 1999 Nusgens et al 2001 Matsubayashi et al 2003

Espinal-Perez et al 2004 Sauermann et al 2004 Lin et al 2005 Heber et al 2006)

57 EFFECT OF CARBON CHAIN LENGTH OF EMULSIFYING AGENT

The values of kobs for the photodegradation of AH2 (2) in various cream

formulations are reported in Table 13 The first-order plots for the photodegradation of

AH2 at pH 4ndash7 in various cream formulations are shown in Fig 10ndash12 The plots of kobs

against carbon chain length of the emulsifying agents are shown in Fig 13 They indicate

that the photodegradation of AH2 is affected by the emulsifying agent in the order

myristic acid gt stearic acid gt palmitic acid

These acids possess a polar character (Yao et al 2009) and the carbon chain of the acid

may play a part in the photostability of AH2 However the results indicate that in the

presence of palmitic acid AH2 exhibits greater stability as indicated by the plots of kobs

versus the carbon chain length of the emulsifying agents (Fig 13) This could be

predominantly due to the interaction of AH2 with palmitic acid in the cream to impart it

greater stability Ascorbic acid-6-palmitate is known to be an antioxidant in cosmetic

preparations (Lee et al 2009) and food products (Doores 2002)

In view of the above observations it may be suggested that the photodegradation

of AH2 could involve a polar semiquinone intermediate (Johnston et al 2007) which

89

Table 13 First-order rate constants (kobs) for the photodegradation of ascorbic acid in

cream formulations

kobs times 103 (min

ndash1)abc

Cream

Formulation pH 40 50 60 70

1 044

(0992)

064

(0994)

100

(0995)

126

(0995)

2 042

(0992)

060

(0991)

095

(0992)

120

(0995)

3 047

(0993)

069

(0993)

107

(0991)

137

(0995)

4 056

(0993)

072

(0992)

104

(0994)

131

(0993)

5 050

(0991)

067

(0992)

097

(0991)

124

(0992)

6 061

(0992)

079

(0993)

113

(0992)

140

(0994)

7 060

(0992)

071

(0993)

108

(0994)

133

(0992)

8 053

(0991)

062

(0992)

099

(0994)

126

(0993)

9 065

(0991)

081

(0996)

117

(0993)

142

(0995)

a The rate constants at pH 40ndash70 represent the values for formulations a to d of each

cream respectively

b The values in parenthesis are correlation coefficients

c The values of rate constants are relative and depend on specific experimental conditions

including light intensity

The estimated error is plusmn5

90

12

13

14

15

16

17

log

co

nce

ntr

ati

on

(m

g)

1

12

13

14

15

16

17

log

co

nce

ntr

ati

on

(m

g)

2

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

3

Fig 10 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (1ndash3) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

91

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

4

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

5

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

6

Fig 11 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (4ndash6) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

92

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

7

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

8

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

9

Fig 12 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (7ndash9) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

93

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

ko

bs times

10

3 (m

inndash

1)

1-3

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

ko

bs times

10

3 (

min

ndash1)

4-6

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

Carbon chain length

ko

bs times

10

3 (

min

ndash1)

7-9

Fig 13 Plots of kobs for photodegradation of ascorbic acid in creams (1ndash9) against carbon

chain length of emulsifier () Stearic acid () palmitic acid () myristic acid

Humectant used glycerin (1ndash3) propylene glycol (4ndash6) ethylene glycol (7ndash9)

94

depending on the polar character of the medium undergoes oxidation with varying rates

This is similar to the behavior of the photolysis of riboflavin analogs which is dependent

on the polar character of the medium (Ahmad and Tollin 1981) The effect of carbon

chain length on the transdermal delivery of an active ingredient has been discussed (Lu

and Flynn 2009)

58 EFFECT OF VISCOSITY

The plots of rates of AH2 degradation in cream formulations (Table 13) as a

function of carbon chain length (Fig 13) indicate that the rates vary with the humectant

and hence the viscosity of the medium in the order

ethylene glycol gt propylene glycol gt glycerin

This is in agreement with the rate of photolysis of AH2 in organic solvents that

higher the viscosity of the medium lower the rate of photolysis Thus apart from the

carbon chain length of the emulsifier viscosity of the humectant added to the cream

formulation appears to play an important part in the stability of AH2 The stabilizing

effect of viscosity imparting substances on AH2 solutions has been reported (Stone 1969

Kassem et al 1969ab)

59 EFFECT OF pH

The kobsndashpH profiles for the photodegradation of AH2 in various creams (1ndash9) at

pH 4ndash7 (Fig 14) represent a sigmoid type curve indicating the oxidation of the ionized

form (AHndash) of AH2 (pKa 41) (OrsquoNeil 2001) with pH The AH

ndash species appears to be

more susceptible to photooxidation than the non-ionized form (AH2) The behavior of

AH2 on photooxidation in the pH range 4ndash7 is similar to that observed for the chemical

oxidation of AH2 by molecular oxygen (DeRitter 1982) and involves the interaction of

95

Stearic Acid (1)

Palmitic Acid (2)

Myristic Acid (3)

04

06

08

10

12

14

kob

s times

10

3 (m

inndash

1)

Stearic Acid (4)

Palmitic Acid (5)

Myristic Acid (6)

04

06

08

10

12

14

ko

bs times

10

3 (

min

ndash1)

Stearic Acid (7)

Palmitic Acid (8)

Myristic Acid (9)

04

06

08

10

12

14

30 40 50 60 70

pH

ko

bs

times 1

03

(min

ndash1)

Fig 14 The kobsndashpH profiles for the photodegradation of ascorbic acid in creams (1ndash9)

Glycerin

Propylene glycol

Ethylene glycol

96

AH2 with singlet oxygen on UV irradiation (Silva and Quina 2006) The AHndash species

(predominant in the pH range 42ndash70 557ndash999) is more reactive towards singlet

oxygen than its protonated form the AH2 molecule as suggested by Bisby et al (1999)

and therefore the rate of photooxidation is higher in the pH range above 41

corresponding to the pKa1 of AH2 The major goal of a ratendashpH profile is to determine

the optimum pH range for a particular formulation Several workers have studied the

ratendashpH profiles of the chemical oxidation of AH2 in the pH range 2ndash7 (Garrett 1967

Taqui Khan and Martell 1967 Rogers and Yacomeni 1971 Blaugh and Hajratwala

1972 DeRitter 1982 Moura et al 1994) however the kinetics of photooxidation of

AH2 in cream formulations has so far not been reported

510 EFFECT OF REDOX POTENTIAL

The photooxidation of AH2 is also influenced by its redox potential which varies

with pH The greater photostability of AH2 at pH 5ndash6 compared to that at pH 7 and above

is due to its lower rate of oxidationndashreduction in this range (Eordm pH 50 = +0127 V)

(OrsquoNeil 2001) The increase in the rate of photooxidation with pH is due to a

corresponding increase in the redox potential (Eordm pH 70 = +0058 V) (Fasman 1976) of

AH2 and is similar to the photolysis behavior of riboflavin at pH 5ndash6 (Eordm pH 50 = ndash0117

V) (Sinko 2006) compared to that at pH 70 (Eordm pH 70 = ndash 0207 V) (Ahmad et al

2004a Sinko 2006) Since the ionization as well as the redox potentials of AH2 are a

function of pH the rate of photooxidation depends upon the specific species present and

its redox behavior at a particular pH

97

511 PRIMARY PHOTOCHEMICAL REACTIONS IN THE OXIDATION OF

ASCORBIC ACID

A reaction scheme based on general photochemical principles for the important

reactions involved in the photooxidation of ascorbic acid is presented below

0AH2 hv k1

1AH2 (51)

1AH2 k2 Products (52)

1AH2 isc k3

3AH2 (53)

3AH2 k4 Products (54)

0AH

ndash hv k5

1AH

ndash (55)

1AH

ndash k6 Products (56)

1AH

ndash k7

3AH

ndash (57)

3AH

ndash k8 Products (58)

3AH

ndash +

0AH2 k9 AH٠

ndash + AH٠ (59)

2 AH٠ k10 A + AH2 (510)

3AH2 +

3O2 k11

0AH2 +

1O2 (511)

AHndash +

1O2 k12

3AH

ndash +

3O2 (512)

AH٠ + 1O2 k13 AHOO٠ (513)

AHOO٠ k14 A + HO2٠ (514)

AHOO٠ + 0AH2 k15 AH٠ + AHOOH (515)

AHOOH k16 secondary reaction

A + H2O2 (516)

According to this reaction scheme the ground state ascorbic acid species (0AH2

0AH

ndash) each is excited to the lowest singlet state (

1AH2

1AH

ndash) by the absorption of a

quantum of UV light (51 55) These excited states may directly be converted to

98

photoproducts (52 56) or may undergo intersystem crossing (isc) to form the excited

triplet states (53 57) The excited triplet states may then degrade to the photoproducts

(54 58) The monoascorbate triplet (3AH

ndash) may react with the ground state ascorbic

acid to form a monoascorbate radical anion (AH٠ndash) and a monoascorbate radical (AH٠)

(59) Two AH٠ radical species may lead to the formation of an oxidized (A) and a

reduced ascorbic acid molecule (AH2) (510) Ascorbic acid triplet (3AH2) may react with

molecular oxygen (3O2) to yield singlet oxygen (

1O2) (511) which may then react with

monoascorbate anion (AHndash) to form the excited triplet state (

3AH

ndash) (512) or with

monoascorbate radical to form a peroxyl radical (AHOO٠) (513) The peroxyl radical

may yield dehydroascorbic acid (A) (514) or react with ground state ascorbic acid to

give monoascorbate radical and a reduced species AHOOH (515) The reduced species

may give rise to dehydroascorbic acid and hydrogen peroxide (516)

512 DEGRADATION OF ASCORBIC ACID IN THE DARK

In view of the instability of AH2 and to observe its degradation in the dark the

creams were stored in airtight containers at room temperature in a cupboard for a period

of about 3 months and assayed for AH2 content at appropriate intervals The analytical

data (Table 14) were subjected to kinetic treatment (Fig 15ndash17) and the apparent first-

order rate constants for the degradation of AH2 were determined (Table 15) The values

of the rate constants indicate that the degradation of AH2 in the dark is about 70 times

slower than those of the creams exposed to UV irradiation (Table 13) The degradation of

AH2 in creams in the dark is due to chemical oxidation (Section 132) and occurs in the

order of emulsifying agents (Fig 18)

myristic acid gt stearic acid gt palmitic acid

99

Table 14 Degradation of ascorbic acid in the dark in cream formulations

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(days) pHa 40 50 60 70

0 383 382 384 383

10 354 340 313 278

20 309 306 279 245

40 244 209 183 161

60 172 166 131 105

1

80 145 114 81 61

0 380 383 382 379

10 360 343 350 335

20 322 310 301 294

40 266 250 211 186

60 233 211 168 142

2

80 182 153 114 89

0 384 376 381 385

10 368 350 340 318

20 318 273 273 266

40 223 199 172 155

60 174 132 117 84

3

80 122 97 66 54

100

Table 14 continued

0 377 378 386 372

10 350 334 334 318

20 314 268 256 244

40 238 208 182 136

60 179 155 107 94

4

80 128 101 79 59

0 381 367 372 373

10 350 293 300 320

20 299 266 270 263

40 220 191 192 184

60 183 153 139 129

5

80 149 115 87 76

0 376 386 380 377

10 312 320 314 251

20 255 282 226 199

40 175 194 159 131

60 139 128 99 74

6

80 102 81 55 41

101

Table 14 continued

0 380 372 378 380

10 323 330 333 323

20 288 273 276 224

40 212 174 182 146

60 152 133 108 83

7

80 100 82 66 56

0 380 381 378 361

10 333 320 310 310

20 281 266 260 257

40 230 189 171 177

60 156 148 128 111

8

80 123 96 78 66

0 378 382 370 375

10 313 295 281 300

20 256 247 257 203

40 194 178 151 133

60 119 114 88 74

9

80 88 68 49 39

a The values at pH 40ndash70 represent the formulations a to d of each cream respectively

102

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

1

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

2

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

3

Fig 15 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (1ndash3) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

103

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

4

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

5

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

6

Fig 16 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (4ndash6) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

104

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

7

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

8

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

9

Fig 17 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (7ndash9) at pH 40 (times) 50 () 60 () and 70 ()

Palmitic acid

Myristic acid

Stearic acid

105

Table 15 First-order rate constants (kobs) for the degradation of ascorbic acid in cream

formulations in the dark

kobs times 102 (day

ndash1)abc

Cream

Formulation pH 40 50 60 70

1 128

(0991)

152

(0994)

191

(0995)

220

(0994)

2 091

(0992)

110

(0991)

152

(0993)

182

(0992)

3 148

(0991)

176

(0995)

220

(0993)

254

(0995)

4 137

(0992)

161

(0993)

205

(0994)

236

(0995)

5 121

(0991)

141

(0994)

175

(0993)

195

(0993)

6 162

(0992)

194

(0995)

237

(0994)

265

(0994)

7 164

(0994)

189

(0994)

222

(0993)

246

(0996)

8 143

(0994)

167

(0995)

193

(0996)

212

(0993)

9 184

(0995)

208

(0994)

251

(0992)

280

(0996)

a The rate constants at pH 40ndash70 represent the values for formulations a to d of each

cream respectively

b The values in parenthesis are correlation coefficients

c The values of rate constants are relative and depend on specific experimental

conditions

The estimated error is plusmn5

106

pH 4

pH 5

pH 6

pH 7

00

10

20

30

k times

10

2 (d

ayndash

1)

1-3

pH 4

pH 5

pH 6

pH 7

00

10

20

30

k times

10

2 (

da

yndash1)

4-6

pH 4

pH 5

pH 6

pH 7

00

10

20

30

12 14 16 18

Carbon chain length

k times

10

2 (

da

yndash1)

7-9

Fig 18 Plots of kobs for degradation of ascorbic acid in the dark in creams (1ndash9) against

carbon chain length of emulsifier () Stearic acid () palmitic acid

() myristic acid Humectant used glycerin (1ndash3) propylene glycol (4ndash6)

ethylene glycol (7ndash9)

107

Although it is logical to expect a linear relationship between the rate of degradation and

the carbon chain length of the emulsifier due to its polar character (Yao et al 2009) it

has not been observed in the present case The reason for the slowest rate of degradation

of AH2 in the presence of palmitic acid appears to be due to the interaction of AH2 with

palmitic acid (Lee et al 2009) as explained in Section 57

The degradation of AH2 also appears to be affected by the viscosity of the cream

in the order of humectant (Fig 19)

ethylene glycol gt propylene glycol gt glycerin

Thus the presence of glycerin imparts the most stabilizing effect on the degradation of

AH2 This is the same order as observed in the case of photodegradation of AH2 in the

creams The airtight containers used for the storage of creams make the access of air to

the creams difficult to cause chemical oxidation of AH2 However it has been observed

that the degradation of AH2 is highest in the upper layer of the creams compared to that

of the middle and the bottom layers Therefore the creams were thoroughly mixed before

sampling for the assay of AH2 However the scattering in kinetic plots (Fig 15ndash17) is

due to non-uniform distribution of AH2 in degraded creams

The effect of pH on the degradation of AH2 in the creams (Fig 19) shows that the

degradation increases with an increase in pH as observed in the case of photodegradation

of AH2 in the creams This is due to an increase in the ionization and redox potential of

AH2 with pH causing greater oxidation of the molecule and has been discussed in

Sections 59 and 510

108

Stearic Acid (1)

Palmitic Acid (2)

Myristic Acid (3)

00

10

20

30

k times

10

2 (d

ayndash

1)

Glycerin

Stearic Acid (4)

Palmitic Acid (5)

Myristic Acid (6)

00

10

20

30

k times

10

2 (

da

yndash1)

Propylene glycol

Stearic Acid (7)

Palmitic Acid (8)

Myristic Acid (9)

00

10

20

30

30 40 50 60 70

pH

k times

10

2 (d

ayndash

1)

Ethylene glycol

Fig 19 The kobsndashpH profiles for the degradation of ascorbic acid in the dark in creams

(1ndash9)

CHAPTER VI

PHOTOCHEMICAL INTERACTION

OF ASCORBIC ACID WITH

RIBOFLAVIN NICOTINAMIDE

AND ALPHA-TOCOPHEROL IN

CREAM FORMULATIONS

110

61 INTRODUCTION

It is now medically recognized that sagging skin and other signs of degenerative

skin conditions such as wrinkles and age spots are caused primarily by oxy-radical

damage Ascorbic acid can accelerate wound healing protect fatty tissues from oxidative

damage and play an integral role collagen synthesis (Zhang et al 1999) It is used in

cosmetic preparations for its anti-aging depigmentation and antioxidant properties

(Spiclin 2003 Ehrlich et al 2006) It is also used in combination with other vitamins

such as alpha-tocopherol as a co-antioxidant to stabilize cosmetic preparations (Eberlein-

Koumlnig and Ring 2005 Bissett 2006 Murray 2008) Ascorbic acid in the presence of air

or light is known to interact with alpha-tocopherol (Packer et al 2002 Johnston et al

2007) riboflavin (Homann and Gaffron 1964 Sattar et al 1977 Heelis et al 1981

Kim et al 1993 Jung et al 1995 De La Rochette et al 2000 2003 Lavoie et al

2004 Vaid et al 2005 Ahmad and Vaid 2006 Silva and Quina 2006) and

nicotinamide (Bailey et al 1945 Werner et al 1949 Guttman and Brooke 1963

DeRitter 1982) The present work involves a study of the effect of alpha-tocopherol

riboflavin and nicotinamide on the photostability of ascorbic acid in cream formulations

to observe whether the interaction in these formulations leads to the stabilization of

ascorbic acid The chemical structures of nicotinamide (NA) alpha-tocopherol (TP)

riboflavin (RF) formylmethylflavin (FMF) and lumichrome (LC) are shown in Fig 20

The details of the cream formulations used in this study are given in Table 16

The results obtained on the photodegradation of ascorbic acid in cream formulations are

discussed in the following sections

111

Riboflavin

N

N

NH

N

CH2

CH

C OHH

CH OH

CH2OH

N

N

NH

N

CH2

CHO

Formylmethylflavin

N

N

NH

HN

Lumichrome

OH

N

NH2

O

Nicotinamide

O CH3

CH3

CH3

HO

H3C

CH3 CH3 CH3

CH3

Alpha-Tocopherol

O

O

H3C

H3C

H3C

H3C

O

O

H3C

H3C

O

O

Fig 20 Chemical structures of alpha-tocopherol nicotinamide riboflavin

formylmethylflavin and lumichrome

112

Table 16 Composition of cream formulations containing ascorbic acid (2) and other

vitamins

Ingredients Cream

No SA PA MA CA GL AH2 RFa NA

b TP

c PH DW

10 a + minus minus + + + a minus minus + +

b + minus minus + + + b minus minus + +

c + minus minus + + + c minus minus + +

d + minus minus + + + d minus minus + +

e + minus minus + + + e minus minus + +

11 a minus + minus + + + a minus minus + +

b minus + minus + + + b minus minus + +

c minus + minus + + + c minus minus + +

d minus + minus + + + d minus minus + +

e minus + minus + + + e minus minus + +

12 a minus minus + + + + a minus minus + +

b minus minus + + + + b minus minus + +

c minus minus + + + + c minus minus + +

d minus minus + + + + d minus minus + +

e minus minus + + + + e minus minus + +

13 a + minus minus + + + minus a minus + +

b + minus minus + + + minus b minus + +

c + minus minus + + + minus c minus + +

d + minus minus + + + minus d minus + +

e + minus minus + + + minus e minus + +

14 a minus + minus + + + minus a minus + +

b minus + minus + + + minus b minus + +

c minus + minus + + + minus c minus + +

d minus + minus + + + minus d minus + +

e minus + minus + + + minus e minus + +

113

Table 16 continued

15 a minus minus + + + + minus a minus + +

b minus minus + + + + minus b minus + +

c minus minus + + + + minus c minus + +

d minus minus + + + + minus d minus + +

e minus minus + + + + minus e minus + +

16 a + minus minus + + + minus minus a + +

b + minus minus + + + minus minus b + +

c + minus minus + + + minus minus c + +

d + minus minus + + + minus minus d + +

e + minus minus + + + minus minus e + +

17 a minus + minus + + + minus minus a + +

b minus + minus + + + minus minus b + +

c minus + minus + + + minus minus c + +

d minus + minus + + + minus minus d + +

e minus + minus + + + minus minus e + +

18 a minus minus + + + + minus minus a + +

b minus minus + + + + minus minus b + +

c minus minus + + + + minus minus c + +

d minus minus + + + + minus minus d + +

e minus minus + + + + minus minus e + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PH = potassium hydroxide DW = distilled water

RF = riboflavin NA = nicotinamide TP = alpha-tocopherol

a RF(g ) a = 0002 b = 0004 c = 0006 d = 0008 e = 0010

b NA (g ) a = 028 b = 056 c = 084 d = 112 e = 140

c TP (g ) a = 017 b = 034 c = 052 d = 069 e = 086

The molar concentrations of these vitamins are given in Section 421

114

62 ABSORPTION CHARACTERISTICS OF PHOTOLYSED CREAMS

A typical set of the absorption spectra of the methanolic extracts (pH 20) of the

freshly prepared and photolysed creams containing AH2 and TP is shown in Fig 21 AH2

in acidified methanol exhibits absorption maximum at 245 nm (Zeng et al 2005) as

observed in Fig 21 The absorption due to TP at 284 nm (Moffat et al 2004) was

cancelled by using an appropriate blank containing an equivalent concentration of TP

The gradual decrease in absorption at around 245 nm during UV irradiation indicates the

transformation of AH2 to DHA which does not absorb in this region (Davies et al 1991)

as a result of the loss of C3=C2 chromophore Similar spectral changes around 245 nm are

observed in the presence of RF and NA which also strongly absorb in this region A

decrease in the absorption of AH2 around 266 nm in aqueous solution (pH 60) in the

presence of RF has been reported (Vaid et al 2005) The spectral changes and loss of

absorbance in methanolic extracts of creams depends on the rate of photolysis of AH2 in

the presence of these vitamins

63 PHOTOPRODUCTS OF ASCORBIC ACID AND OTHER VITAMINS

The UV irradiation of AH2 in cream formulations (pH 60) in the presence of RF

NA and TP results in the degradation of AH2 and RF and the following photoproducts

have been identified on comparison of their RF values and spot color fluorescence with

those of the authentic compounds

AH2 DHA

RF FMF LC CMF

In the TLC systems used NA and TP did not show the formation of any

degradation product in creams

115

Fig 21 UV absorption spectra of methanolic extracts of photodegraded ascorbic acid in

cream at 0 60 120 180 300 and 480 min

116

The formation of DHA in the photooxidation of AH2 has previously been reported by

many workers (Homann and Gaffron 1964 Sattar et al 1977 Heelis et al 1981

Rozanowska et al 1997 Lavoie et al 2004 Vaid et al 2006) RF is sensitive to light in

aqueous solutions (DeRitter 1982 British Pharmacopoeia 2009 Sweetman 2009) and is

known to form a number of products under aerobic conditions (Treadwell et al 1968

Cairns and Metzler 1971 Schuman Jorns et al 1975 Ahmad and Rapson 1990 Ahmad

and Vaid 2006 Ahmad et al 2004ab 2005 2008 Vaid et al 2006) It has been found

to degrade on UV irradiation in cream formulations to give FMF LC and CMF and these

products have been reported in the photolysis of RF by the workers cited above The

formation of these products may be affected by the interaction of AH2 and RF in creams

(Section 66) NA and TP individually did not appear to form any photoproduct of their

own directly or on interaction with AH2 in creams and may influence the degradation of

AH2 on UV irradiation

64 ASSAY METHOD

In view of the presence of RF (absorption maxima 223 267 373 and 444 nm)

(British Pharmacopoeia 2009) NA (absorption maximum 261 nm) (Moffat et al 2004)

and TP (absorption maximum 284 nm) (Moffat et al 2004) in the cream formulations

and the interference of these vitamins with the absorption of AH2 (absorption maximum

265 nm) (Davies et al 1991) the direct spectrophotometric method cannot be applied for

the determination of AH2 Therefore the iodimetric method (British Pharmacopoeia

2009) was used to determine AH2 in cream formulations The method was validated in

the presence of RF NA and TP before its application to the determination of AH2 in

photodegraded creams The reproducibility of the method has been confirmed by the

117

assay of known concentrations of AH2 in the range present in photodegraded creams The

recovery of AH2 in the creams has been found to be in the range 90ndash96 The values of

RSD indicate that the precision of the method is within plusmn5 (Table 17) and it can be

applied to study the kinetics of AH2 photolysis in cream formulations The assay data on

AH2 in various cream formulations are given in Table 18

65 KINETICS OF PHOTODEGRADATION OF ASCORBIC ACID

Several chemical and physical factors play a role in the photodegradation of AH2

in the presence of other vitamins (RF NA TP) and affect the rate of its degradation in

cream formulations The present work involves the study of photodegradation of AH2 in

cream formulations containing glycerin as humectant as AH2 has been found to be most

stable in these creams (Chapter 5) The apparent first-order rate constants (kobs) for the

photodegradation of AH2 in the presence of other vitamins in cream formulations

derived from the kinetic plots (Fig 22ndash24) are reported in Table 19 The second-order

rate constants (correlation coefficients 0991ndash0996) determined from the slopes of the

graphs of kobs versus vitamin concentration for the individual vitamins (Fig 25) and the

values of k0 determined from the intercept on the vertical axis at zero concentration are

reported in Table 20 The values of k0 give an indication of the effect of other vitamins on

the rate of degradation of AH2 These values are about 13 times lower than the values of

kobs obtained at the highest concentrations of RF and NA indicating that RF and NA both

accelerate the photodegradation of AH2 in creams RF is known to act as a

photosensitizer for the degradation of AH2 (Section 66) and therefore its presence in

creams would accelerate the degradation of AH2 The increase in the rate of

photodegradation of AH2 in the presence of NA has not previously been reported NA

118

Table 17 Recovery of ascorbic acid in cream formulations in the presence of other

vitamins by iodimetric methoda

Cream

Formulationb

Added

(mg )

Found

(mg )

Recovery

()

RSD

()

10e (RF) 400

200

373

187

933

935

29

22

11e (RF) 400

200

379

187

948

935

25

31

12e (RF) 400

200

375

188

938

940

29

28

13e (NA) 400

200

382

191

955

955

23

27

14e (NA) 400

200

380

185

950

925

19

26

15e (NA) 400

200

379

191

948

955

21

17

16e (TP) 400

200

368

183

920

915

29

44

17e (TP) 400

200

391

195

978

975

11

13

18e (TP) 400

200

377

182

943

910

32

37

a Values expressed as a mean of three to five determinations

b The cream formulations represent all the emulsifiers (stearic acid palmitic acid

myristic acid) to observe the efficiency of iodimetric method for the recovery of

ascorbic acid in presence of the highest concentration of vitamins (Table 16)

119

Table 18 Photodegradation of ascorbic acid in cream formulations in the presence of

other vitamins

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) a b C d e

0 373 372 374 372 375

60 362 354 354 360 359

150 342 336 336 332 334

240 315 314 308 310 302

10 (RF)

330 301 291 288 281 282

0 380 379 376 374 374

60 370 366 362 362 361

150 343 337 340 332 328

240 329 323 320 313 310

11 (RF)

330 307 301 294 288 282

0 379 380 375 372 376

60 362 366 361 351 342

150 341 335 319 307 312

240 310 306 295 284 282

12 (RF)

330 285 278 263 254 243

120

Table 18 continued

0 372 370 371 368 365

60 361 358 348 350 349

120 342 343 329 326 330

180 327 325 319 312 308

240 317 309 299 289 285

13 (NA)

300 299 291 283 278 273

0 386 380 375 378 370

60 371 362 365 362 355

120 359 351 343 339 336

200 341 332 325 316 311

14 (NA)

300 313 303 296 294 280

0 375 371 374 370 366

60 362 356 352 352 345

120 343 332 336 326 314

200 323 315 311 295 293

15 (NA)

300 293 283 275 270 259

121

Table 18 continued

0 380 378 380 377 377

60 362 365 369 369 371

120 351 352 360 360 364

180 340 346 349 353 355

240 331 334 343 343 346

16 (TP)

300 320 323 330 332 337

0 383 380 378 380 377

60 372 371 372 373 370

120 363 360 361 366 365

180 348 348 350 356 355

240 341 343 343 348 348

17 (TP)

300 330 332 336 339 341

0 380 383 377 375 373

60 364 370 366 367 366

120 352 356 351 352 351

180 334 338 339 343 342

240 324 328 324 332 330

18 (TP)

300 307 315 317 318 322

122

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

10

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

11

ab

c

de

13

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

12

Fig 22 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing riboflavin (a) 0002 (b) 0004 (c) 0006 (d) 0008

(e) 0010

Stearic acid

Palmitic acid

Myristic acid

123

abcde

13

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

13

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

14

abcde

13

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

15

Fig 23 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing nicotinamide (a) 028 (b) 056 (c) 084 (d) 112 (e) 140

Stearic acid

Palmitic acid

Myristic acid

124

abcde

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

16

abcde

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

17

abcde

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

18

Fig 24 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing alpha-tocopherol (a) 017 (b) 034 (c) 052 (d) 069

(e) 086

Stearic acid

Myristic acid

Palmitic acid

125

Table 19 First-order rate constants (kobs) for the photodegradation of ascorbic acid in the

presence of other vitamins in cream formulations

kobs times 103 (min

ndash1)ab

Cream

formulationd

Other

vitaminc

a b C d e

10 RF 068

(0991)

073

(0996)

079

(0995)

085

(0992)

089

(0995)

11 RF 065

(0992)

070

(0992)

073

(0994)

080

(0995)

086

(0993)

12 RF 087

(0993)

096

(0995)

109

(0993)

116

(0994)

127

(0992)

13 NA 073

(0993)

081

(0992)

088

(0994)

096

(0994)

101

(0993)

14 NA 069

(0992)

074

(0992)

080

(0991)

086

(0995)

094

(0995)

15 NA 083

(0994)

090

(0993)

101

(0993)

109

(0994)

115

(0995)

16 TP 055

(0991)

051

(0994)

046

(0994)

042

(0993)

038

(0991)

17 TP 050

(0995)

045

(0993)

041

(0992)

038

(0995)

034

(0994)

18 TP 070

(0996)

066

(0996)

060

(0994)

055

(0993)

051

(0993)

a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental conditions

including the light intensity

c Vitamin concentrations (andashe) are as given in Table 16

d All the creams contain glycerin as humectant

The estimated error is plusmn5

126

00

05

10

15

00 10 20 30

Riboflavin concentration (M times 104)

kob

s times

10

3 (

min

ndash1)

10-12

00

05

10

15

00 20 40 60 80 100 120

Nicotinamide concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

13-15

00

02

04

06

08

00 04 08 12 16 20

Alpha-Tocopherol concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

16-18

Fig 25 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against individual vitamin concentration in cream formulations (10ndash18)

127

Table 20 First-order rate constants (k0)a for the photodegradation of ascorbic acid in the

absence of other vitamins and second-order rate constants (k) for the

photochemical interaction of ascorbic acid with RF NA and TP

Cream

formulation

Other

vitamin

k0 times 103

(minndash1

)

k

(Mndash1

minndash1

)

Correlation

coefficient

10 RF 062 102 0994

11 RF 059 097 0992

12 RF 077 189 0995

13 NA 066 032 times 10ndash2

0995

14 NA 062 027 times 10ndash2

0993

15 NA 074 037 times 10ndash2

0994

16 TP 059 110 times 10ndash2b

0996

17 TP 053 096 times 10ndash2b

0992

18 TP 075 123 times 10ndash2b

0994

a

The variations in the values of k0 are due to the degradation of AH2 in the presence of

different emulsifying agents in cream formulations

b Values for the inhibition of photodegradation of AH2

128

forms a complex with AH2 (Section 67) and may also act as a photosensitizer for AH2 by

energy transfer in the excited state on UV irradiation The absorption maximum of NA

(261 nm) (Moffat et al 2004) is very close to that of AH2 (265 nm) (Davies et al 1991)

and the possibility of energy transfer in the excited state (Moore 2004) is greater leading

to the photodegradation of AH2

The value of k0 is about 13 times greater than the values of kobs obtained for the

degradation of AH2 in the presence of the highest concentrations of TP in the creams

This indicates that TP has a stabilising effect on the photodegradation of AH2 in the

cream formulations This is in agreement with the view that the TP acts as a redox partner

with AH2 to retard its oxidation (Wille 2005) Thus among the three vitamins studied

only TP appears to have a stabilising effect on photodegradation of AH2 The

photochemical interaction of individual vitamins with AH2 is discussed below

66 INTERACTION OF RIBOFLAVIN WITH ASCORBIC ACID

The interaction of RF with the ascorbate ion (AHndash) may be represented by the

following reactions proposed by Silva and Quina (2006)

RF rarr 1RF (61)

1RF rarr

3RF (62)

3RF + AH

ndash rarr RF

ndashmiddot + AHmiddot (63)

AHmiddot + O2 rarr A + HO2middot (64)

HO2middot + AHndash rarr H2O2 + AHmiddot (65)

RF on the absorption of a quantum of light is promoted to the excited singlet state (1RF)

(61) 1RF may undergo intersystem crossing (isc) to form the excited triplet state (

3RF)

(62) The excited triplet state may react with the ascorbate ion to generate the ascorbyl

hv

isc

129

radical (AH) (63) (Kim et al 1993) The ascorbyl radical reacts with oxygen to give

dehydroascorbic acid (A) and peroxyl radical (HO2) (64) This radical may interact with

ascorbate ion to generate further ascorbyl radicals (65) These radicals may again take

part in the sequence of reactions to form A The role of RF in this reaction is to act as a

photosensitiser in the oxidation of ascorbic acid to A Ascorbic acid is reported to protect

riboflavin in milk under the influence of light by reacting with singlet oxygen (Hall et al

2009) (Section 511)

67 INTERACTION OF NICOTINAMIDE WITH ASCORBIC ACID

NA is known to form a 11 complex with ascorbic acid (Guttman and Brooke

1963 OrsquoNeil 2001 Doores 2002) The complexation of NA and AH2 may result from

the donor-acceptor interaction between AH2 (donor) and NA (acceptor) as observed in

the case of tryptophan and NA (Florence and Attwood 2006) In the presence of light the

interaction may cause reduction of NA (NAH) to form the ascorbyl radical (AH) ((66)-

(68)) which is oxidized to dehydroascorbic acid (A) (69) The NAH may be oxidized to

NA and H2O2 (610)

NA rarr 1NA (66)

1NA rarr

3NA (67)

3NA + AH2 rarr NAH + AHmiddot (68)

2 AH٠ rarr A + AH2 (69)

NAH + O2 rarr NA + H2O2 (610)

The proposed reactions suggest that on photochemical interaction AH2 undergoes

photosensitised oxidation in the presence of NA indicating that the photostability of

ascorbic acid is affected by NA

isc

130

68 INTERACTION OF ΑLPHA-TOCOPHEROL WITH ASCORBIC ACID

TP is an unstable compound and its oxidation by air results in the formation of an

epoxide which than produces a quinone that is inactive (Connors et al 1986) TP is

destroyed by sun light and artificial light containing the wavelengths in the UV region

(Ball 2006) It interacts with other antioxidants such as ascorbic acid (AH2) according to

the following reactions

TPndashO + AH2 rarr TP + AHmiddot (611)

2 AHmiddot rarr A + AH2 (612)

TP + AHmiddot rarr TPndashO + AH2 (613)

The tocopheroxyl radical (TPndashO) reacts with AH2 to regenerate TP and form the

ascorbyl radical (AHmiddot) (611) This radical undergoes further reactions as described in

equations (64) and (65) (Traber 2007) It may also disproportionate back to A and AH2

(612) TP reacts with AHmiddot to produce again the TPndashO radical and AH2 Thus in the

presence of light TP leads to the oxidation of AH2 which is ultimately regenerated in the

reaction (Davies et al 1991) Ascorbic acid has the ability to act as a co-antioxidant with

the TPndashO to regenerate TP (Packer et al 1979 2002) In this manner AH2 and TP act

synergistically to function in a redox cycle and AH2 is stabilized in the cream

formulations and microemulsions (Rozman and Gasperlin 2007 Rozman et al 2009)

69 EFFECT OF CARBON CHAIN LENGTH OF EMULSIFYING AGENT

The graphs of kobs for the photodegradation of AH2 in the presence of RF NA and

TP versus the carbon chain length of emulsifying agents are shown in Fig 26 It appears

that the photodegradation of AH2 in the presence of all the three vitamins in the creams

lies in the order

131

Fig 26 Plots of k for photodegradation of ascorbic acid in creams (10ndash18) against

carbon chain length of emulsifier () Stearic acid () palmitic acid

() myristic acid

00

05

10

15

20

25

k

(Mndash

1 m

inndash

1)

00

05

10

15

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash

1 m

inndash

1)

132

myristic acid gt stearic acid gt palmitic acid

The same order of emulsifying agents has been observed in the absence of the

added vitamins (Section 57) The polar character of these acids (Yao et al 2009) on the

basis of their carbon chain length may play a part in the photostability of AH2 The

greater stability of AH2 in creams in the presence of palmitic acid (Fig 26) may be due to

the interaction of AH2 with palmitic acid as discussed in Section 57 Ascorbic acid-6-

palmitate is known to be an antioxidant in cosmetic preparations (Lee et al 2009) and

food products (Doores 2002)

610 EFFECT OF VISCOSITY OF CREAMS

The plots of kobs for the degradation of AH2 in the presence of the highest

concentration of vitamins versus reciprocal of the viscosity of creams (Table 21) are

linear (Fig 27) and indicate that the increase in cream viscosity leads to a decrease in the

rate of degradation of AH2 The slopes of the plots indicate the effect of viscosity on the

interaction of AH2 with other vitamins in the order

riboflavin gt nicotinamide gt alpha-tocopherol

The relatively slow rate of degradation of AH2 in creams containing palmitic acid may be

due to the interaction of AH2 with the vitamins as well as palmitic acid (Lee et al 2009)

Thus viscosity is an important factor in the stability of AH2 in cream formulations and

may affect its rate of interaction with other vitamins It has been suggested that an

increase in the viscosity of the medium makes access to air at the surface more difficult to

prevent the oxidation of a drug (Wallwork and Grant 1977) This is in agreement with

the photolysis of AH2 in aqueous and organic solvents cream formulations (Chapter 5)

and aerobic oxidation of Ah2 in syrups (Blaug and Hajratwala 1972)

133

Table 21 Average viscosity of cream formulations containing different emulsifying

agents and glycerin as humectant (25 plusmn 1 ordmC) and the photodegradation rate

constants of AH2

Cream No Emulsifying

agent

Viscosityab

(mPa s)

kobs times 103c

10 (RF)

13 (NA)

16 (TP)

Stearic acid 9000 089

101

038

11 (RF)

14 (NA)

17 (TP)

Palmitic acid 8600 086

094

034

12 (RF)

15 (NA)

18 (TP)

Myristic acid 7200 127

115

051

a plusmn10

b Average viscosity of creams containing the individual vitamins (RF NA TP)

c The values have been obtained in the presence of highest concentration of the

vitamins

134

00

05

10

15

20

25

30

100 110 120 130 140

Viscosity (mPa s)ndash1

times 103

kob

s (m

inndash1)

Fig 27 Plots of kobs in the presence of highest concentration of vitamins versus

reciprocal of the viscosity of creams () riboflavin

( ) nicotinamide (- - -- - -) alpha-tocopherol

135

611 DEGRADATION OF ASCORBIC ACID IN THE PRESENCE OF OTHER

VITAMINS IN THE DARK

In order to observe the effect of riboflavin nicotinamide and alpha-tocopherol on

the degradation of AH2 in the creams stored in the dark the AH2 contents of the creams

were assayed at appropriate intervals (Table 22) The apparent first-order rate constants

determined from the kinetic plots (Fig 28) for the degradation of AH2 in the presence of

the highest concentrations of the individual vitamins in cream formulations (10ndash18) are

reported in Table 23 These rate constants indicate that the overall degradation of AH2 in

the presence of the highest concentration of the individual vitamins (RF NA and TP) is

about 70 times slower than that obtained on the exposure of creams to UV irradiation

This decrease in the rate of degradation of AH2 in the creams is the same as observed in

the case of AH2 alone In the absence of light the degradation of AH2 occurs due to

chemical oxidation (Section 132) and does not appear to be affected by the presence of

riboflavin and nicotinamide as indicated by the comparisons of the values of kobs in the

presence and absence of these vitamins (Table 15 and 23) In the presence of alpha-

tocopherol the degradation is slower than that in the presence of riboflavin and

nicotinamide This may be due to some interaction of AH2 and alpha-tocopherol causing

stabilisation of AH2 in the creams

As observed in the case of AH2 degradation alone in creams in the dark the AH2

degradation in the presence of the highest concentrations of other vitamins also occurs in

the same order of emulsifying agents (Fig 29)

myristic acid gt stearic acid gt palmitic acid

136

Table 22 Degradation of ascorbic acid in cream formulations in the dark in presence of

highest concentration of other vitamins

Concentration of ascorbic acid (mg 2g) Cream

No Time (days) 0 10 20 40 60 80

10e (RF) 375 285 233 171 110 69

11e (RF) 374 341 281 221 148 113

12e (RF) 372 259 203 130 89 59

13e (NA) 365 330 255 187 126 81

14e (NA) 370 321 289 219 159 109

15e (NA) 366 289 249 159 110 63

16e (TP) 377 359 321 261 211 159

17e (TP) 377 366 333 275 228 191

18e (TP) 373 361 304 252 200 167

137

02

07

12

17lo

g c

on

cen

tra

tio

n (

mg

)

10-12Riboflavin

02

07

12

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

13-15Nicotinamide

10

12

14

16

18

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

16-18Alpha-Tocopherol

Fig 28 First-order plots for the degradation of ascorbic acid in the dark in presence of

other vitamins using the emulsifying agents (minusminusminusminus) Stearic acid

(minus minusminus minus) palmitic acid (----) myristic acid

138

Table 23 First-order rate constants (kobs) for the degradation of ascorbic acid in presence

of other vitamins in cream formulations in the dark

Cream

formulation

Other

vitaminc

kobs times 102

(dayndash1

)ab

10e RF 204

(0995)

11e RF 156

(0992)

12e RF 222

(0992)

13e NA 189

(0995)

14e NA 151

(0993)

15e NA 214

(0995)

16e TP 100

(0994)

17e TP 088

(0995)

18e TP 105

(0993)

a The values in parenthesis are correlation coefficients and range from 0991ndash0996 due to

some variations in AH2 distribution in the creams

b The values of rate constants are relative and depend on specific experimental

conditions

c Vitamin concentrations (andashe) are as given in Table 16

The estimated error is plusmn5

139

Riboflavin

Nicotinamide

Alpha-Tocopherol

00

10

20

30

12 14 16 18Carbon chain length

ko

bs times

10

2 (

da

yndash1)

Fig 29 Plots of kobs for degradation of ascorbic acid in the dark in creams (10ndash18)

against carbon chain length of the emulsifier () Stearic acid () palmitic acid

() myristic acid

140

This indicates that the rate of degradation of AH2 is slowest in the creams containing

palmitic acid as the emulsifying agent The reason for AH2 degradation in the dark in this

order has already been explained in section 512

CHAPTER VII

STABILIZATION OF

ASCORBIC ACID WITH

CITRIC ACID TARTARIC

ACID AND BORIC ACID IN

CREAM FORMULATIONS

142

71 INTRODUCTION

Ascorbic acid is an ingredient of cosmetic preparations (Section 51) and is

sensitive to light (Rowe et al 2009 Sweetman 2009 British Pharmacopoeia 2009)

degrading to dehydroascorbic acid on UV irradiation by photooxidation (Kitagawa

1968) The photosensitivity of ascorbic acid makes it unstable in pharmaceutical and

cosmetic preparations (DeRitter 1982) The present work is an attempt to study the

photodegradation of ascorbic acid in cream formulations in the presence of certain

compounds (eg citric acid tartaric acid and boric acid) to investigate their role in the

stabilization of the vitamin on exposure to light and in the dark Citric acid and tartaric

acid are used as an antioxidant synergist (Rowe et al 2009) and boric acid is a

complexing agent for hydroxy compounds (Ahmad et al 2009cd)

72 CREAM FORMULATIONS

The details of the various cream formulations used in this study are given in Table

24 and the results obtained on the photodegradation of ascorbic acid in the presence of

stabilizing agents in these formulations are discussed in the following sections

143

Table 24 Composition of cream formulations containing ascorbic acid (2) and

stabilizers

Ingredients Cream

No SA PA MA CA GL PG EG AH2 CTa TA

b BA

c PH DW

19 a + minus minus + + minus minus + a minus minus + +

b + minus minus + + minus minus + b minus minus + +

c + minus minus + + minus minus + c minus minus + +

20 a minus + minus + + minus minus + a minus minus + +

b minus + minus + + minus minus + b minus minus + +

c minus + minus + + minus minus + c minus minus + +

21 a minus minus + + + minus minus + a minus minus + +

b minus minus + + + minus minus + b minus minus + +

c minus minus + + + minus minus + c minus minus + +

22 a + minus minus + + minus minus + minus a minus + +

b + minus minus + + minus minus + minus b minus + +

c + minus minus + + minus minus + minus c minus + +

23 a minus + minus + + minus minus + minus a minus + +

b minus + minus + + minus minus + minus b minus + +

c minus + minus + + minus minus + minus c minus + +

24 a minus minus + + + minus minus + minus a minus + +

b minus minus + + + minus minus + minus b minus + +

c minus minus + + + minus minus + minus c minus + +

25 a + minus minus + + minus minus + minus minus a + +

b + minus minus + + minus minus + minus minus b + +

c + minus minus + + minus minus + minus minus c + +

26 a minus + minus + + minus minus + minus minus a + +

b minus + minus + + minus minus + minus minus b + +

c minus + minus + + minus minus + minus minus c + +

27 a minus minus + + + minus minus + minus minus a + +

b minus minus + + + minus minus + minus minus b + +

c minus minus + + + minus minus + minus minus c + +

144

Table 24 continued

28 a + minus minus + minus + minus + minus minus a + +

b + minus minus + minus + minus + minus minus b + +

c + minus minus + minus + minus + minus minus c + +

29 a minus + minus + minus + minus + minus minus a + +

b minus + minus + minus + minus + minus minus b + +

c minus + minus + minus + minus + minus minus c + +

30 a minus minus + + minus + minus + minus minus a + +

b minus minus + + minus + minus + minus minus b + +

c minus minus + + minus + minus + minus minus c + +

31 a + minus minus + minus minus + + minus minus a + +

b + minus minus + minus minus + + minus minus b + +

c + minus minus + minus minus + + minus minus c + +

32 a minus + minus + minus minus + + minus minus a + +

b minus + minus + minus minus + + minus minus b + +

c minus + minus + minus minus + + minus minus c + +

33 a minus minus + + minus minus + + minus minus a + +

b minus minus + + minus minus + + minus minus b + +

c minus minus + + minus minus + + minus minus c + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PG = propylene glycol EG = ethylene glycol

PH = potassium hydroxide DW = distilled water CT = citric acid TA = tartaric acid

BA = boric acid

a CT (g ) a = 01 b = 02 c = 04

b TA (g ) a = 01 b = 02 c = 04

c BA (g ) a = 01 b = 02 c = 04

145

73 PRODUCTS OF ASCORBIC ACID PHOTODEGRADATION

The photodegradation of AH2 in cream formulations leads to the formation of

DHA as detected by TLC and reported earlier in the photolysis of AH2 in aqueous

solutions (Vaid et al 2006) and cream formulations (Sections 52 and 63) AH2 and

DHA in the methanolic extracts of the degraded creams were identified by comparison of

their Rf and color of the spots with those of the reference standards DHA is also

biologically active (Gardner 1972 Doores 2002) but its further degradation to 23-

diketo-gulonic acid (DGA) results in the loss of vitamin activity (Section 132)

However this product has not been detected in the present cream formulations

Therefore the creams may still possess their biological efficacy

74 SPECTRAL CHANGES IN PHOTODEGRADED CREAMS

In order to observe the spectral changes in photodegraded creams in the presence

of stabilizing agents the absorption spectra of the methanolic extracts of a degraded

cream were determined The spectra show a gradual loss of absorbance around 245 nm

due to the oxidation of AH2 to DHA on UV irradiation and similar to that shown for the

photodegradation of AH2 alone in Fig 5 DHA has negligible absorbance around 245 nm

(Davies et al 1991) and therefore it does not interfere with the absorbance of AH2 in

methanolic solutions The spectral changes and loss of absorbance around 245 nm in

methanolic solution depend on the extent of photooxidation of AH2 in a particular cream

75 ASSAY OF ASCORBIC ACID IN CREAMS

The UV spectrophotometric method (Zeng et al 2005) has previously been

applied to the determination of AH2 in cream formulations (Section 54) The absorbance

of the methanolic extracts of creams containing AH2 during photodegradation was used

146

to determine the concentration of AH2 The method was validated in the presence of citric

acid (CT) tartaric acid (TA) and boric acid (BA) before its application to the evaluation

of the kinetics of AH2 degradation in cream formulations The recovery of AH2 in creams

has been found to be in the range of 90ndash96 and is similar to that reported in Table 7

The reproducibility of the method lies within plusmn5 The assay data on the degradation of

AH2 in various creams in the presence of the stabilizing agents are reported in Table 25

76 KINETICS OF PHOTODEGRADATION

The effect of CT TA and BA as stabilizing agents on the photodegradation of

AH2 was studied by adding 01ndash04 of each compound to the cream formulations (19ndash

33) at pH 60 This concentration range is normally used for the stabilization of drugs in

pharmaceutical preparations (Im-Emsap et al 2002) The apparent first-order rate

constants (kobs) determined from the plots of log concentration versus time (Fig 30ndash34)

are reported in Table 26 The second-order rate constants (k) determined from the plots

of kobs versus concentration of the individual compounds (Fig 35ndash36) are given in Table

27 The values of k indicate the rate of inhibition of photodegradation of AH2 by each

compound

77 EFFECT OF STABILIZING AGENTS

In order to compare the effectiveness of CT TA and BA as stabilizing agents for

AH2 plots of k versus carbon chain length of the emulsifying agents were constructed

(Fig 37) The k values for the interaction of these compounds with AH2 are in the order

citric acid gt tartaric acid gt boric acid

The curves indicate that the highest interaction of these compounds with AH2 is in the

order

147

Table 25 Photodegradation of ascorbic acid in cream formulations in the presence of

stabilizers

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) a b c

0 374 378 379

60 362 362 372

120 349 355 367

210 333 335 349

19 (CT)

300 319 322 336

400 296 309 324

0 381 378 380

60 368 370 369

120 355 363 364

210 344 345 355

20 (CT)

300 328 335 341

400 312 319 331

21 (CT) 0 368 370 374

60 355 356 360

120 340 344 343

210 321 322 333

300 296 299 315

400 272 285 299

148

Table 25 continued

0 375 374 378

60 363 363 368

120 352 354 362

210 329 335 345

22 (TA)

300 307 314 333

400 292 299 313

0 370 377 374

60 364 365 368

120 352 357 357

210 332 344 349

23 (TA)

300 317 330 335

400 301 310 322

24 (TA) 0 376 379 377

60 367 369 368

120 351 348 352

210 325 330 344

300 306 317 326

400 284 294 310

149

Table 25 continued

0 370 375 380

60 356 362 359

120 331 339 344

210 311 318 330

25 (BA)

300 279 288 305

400 260 269 283

0 377 375 370

60 364 363 361

120 351 353 351

210 331 332 337

26 (BA)

300 323 324 325

400 301 307 313

27 (BA) 0 380 377 375

60 369 368 366

120 333 338 341

210 305 313 318

300 292 294 304

400 262 266 281

150

Table 25 continued

0 373 376 378

60 348 349 360

120 329 336 339

210 315 312 323

28 (BA)

300 282 283 299

400 249 264 280

0 370 373 380

60 358 355 367

120 343 346 356

210 325 329 347

29 (BA)

300 307 312 325

400 287 295 315

30 (BA) 0 369 375 372

60 353 358 362

120 321 330 335

210 283 294 303

300 265 281 293

400 242 254 270

151

Table 25 continued

0 374 376 379

60 348 366 352

120 324 340 337

210 303 319 322

31 (BA)

300 275 289 293

400 243 260 275

0 370 374 375

60 355 354 366

120 339 344 345

210 313 319 330

32 (BA)

300 288 297 308

400 261 271 290

33 (BA) 0 377 380 377

60 357 361 367

120 324 335 339

210 288 294 307

300 270 280 293

400 233 248 265

Creams 19ndash27 contain glycerin 28ndash30 contain propylene glycol and 31ndash33 contain

ethylene glycol as humectants

152

a

b

c

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

19

ab

c

14

15

16

log

co

nce

ntr

ati

on

(m

g)

20

a

b

c

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

21

Fig 30 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and citric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

153

a

b

c

14

15

16lo

g c

on

cen

tra

tio

n (

mg

)

22

a

b

c

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

23

a

b

c

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

24

Fig 31 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and tartaric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

154

ab

c

13

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

25

abc

13

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

26

ab

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

27

Fig 32 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and boric acid (a) 01 (b) 02 (c) 04

Palmitic acid

Stearic acid

Myristic acid

155

a

b

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

28

ab

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

29

a

b

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

30

Fig 33 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing propylene glycol and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

156

a

b

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

31

ab

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

32

a

b

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

33

Fig 34 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing ethylene glycol and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

157

Table 26 Apparent first-order rate constants (kobs) for the photodegradation of ascorbic

acid in presence of different stabilizers in cream formulations

kobs times 103 (min

ndash1)ab

Cream

formulation Stabilizer

c

a b c

19 CT 057

(0995)

050

(0992)

041

(0991)

20 CT 049

(0996)

043

(0995)

034

(0993)

21 CT 076

(0995)

067

(0995)

055

(0992)

22 TA 065

(0995)

058

(0995)

046

(0991)

23 TA 054

(0994)

047

(0993)

038

(0994)

24 TA 072

(0996)

063

(0992)

049

(0991)

25 BA 091

(0994)

086

(0995)

071

(0993)

26 BA 055

(0994)

050

(0993)

042

(0993)

27 BA 095

(0995)

089

(0992)

074

(0996)

28 BA 097

(0995)

088

(0992)

075

(0993)

29 BA 064

(0994)

057

(0991)

047

(0993)

30 BA 110

(0994)

100

(0996)

084

(0992)

31 BA 105

(0995)

094

(0994)

078

(0992)

32 BA 088

(0994)

079

(0993)

066

(0993)

33 BA 120

(0995)

108

(0993)

091

(0993) a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental conditions

including the light intensity

c Stabilizer concentrations (andashc) are as given in Table 24

The estimated error is plusmn5

158

00

04

08

12

00 04 08 12 16 20

Citric acid concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

19-21

00

04

08

12

00 05 10 15 20 25

Tartaric acid concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

22-24

Fig 35 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against citric acid (19ndash21) and tartaric acid concentrations (22ndash24) in cream

formulations

159

00

04

08

12k

ob

s times

10

3 (

min

ndash1)

25-27

00

04

08

12

00 20 40 60

ko

bs times

10

3 (

min

ndash1)

28-30

00

04

08

12

00 20 40 60

Boric acid concentration (M times 102)

kob

s times

10

3 (

min

ndash1)

30-33

Fig 36 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against boric acid concentrations in cream formulations (25ndash33)

Propylene glycol

Glycerin

Ethylene glycol

160

Table 27 First-order rate constants (k0)a for the photodegradation of ascorbic acid in the

absence of stabilizers and second-order rate constants (k) for the interaction of

ascorbic acid with CT TA and BA

Cream

formulation Stabilizers

k0 times 103

(minndash1

)

k times 102

(Mndash1

minndash1

)

Correlation

coefficient

19 CT 062 111 0991

20 CT 053 103 0994

21 CT 082 145 0995

22 TA 071 092 0995

23 TA 059 080 0993

24 TA 080 118 0996

25 BA 098 041 0994

26 BA 059 026 0994

27 BA 102 044 0995

28 BA 104 046 0992

29 BA 069 033 0995

30 BA 118 054 0994

31 BA 113 053 0995

32 BA 095 045 0995

33 BA 129 060 0993

a The variations in the values of k0 are due to the presence of different emulsifying agents

in cream formulations

161

00

04

08

12

16

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash1

min

ndash1)

18-33

a

b

e

cd

Fig 37 Plots of k for photodegradation of ascorbic acid in creams (19ndash33) against

carbon chain length of emulsifier () Stearic acid () palmitic acid ()

myristic acid (a) CT (b) TA (c) BA with glycerin (d) BA with propylene

glycol (e) BA with ethylene glycol

162

myristic acid gt stearic acid gt palmitic acid

In the case of myristic acid and stearic acid it may be explained on the basis of the

decreasing polarity (Yao et al 2009) It is interesting to observe the lowest rates of

interaction of these compounds in the creams containing palmitic acid This could be due

to the interaction of AH2 with palmitic acid to form a palmitate derivative in addition to

its interaction with the individual stabilizing agents CT and TA are known to act as

antioxidant synergists (Rowe et al 2009 Sweetman 2009) and in this capacity may

inhibit the photooxidation of AH2 as indicated by the values of the degradation rate

constants in the presence of these compounds The addition of CT to nutritional

supplements is known to inhibit the oxidation of AH2 (Doores 2002) Boric acid forms a

complex with AH2 (Rivlin 2007) and there by may inhibit its degradation Boric acid

may also interact with glycerin added to the creams as a humectant and form a complex

(Rowe et al 2009) This may influence its interaction and stabilizing effect on AH2 in

creams as indicated by the lower k values compared to those in the presence of CT and

TA It has further been observed that the k values for BA are greater in propylene glycol

and ethylene glycol compared to those in glycerin (Table 27) Again this may be due to

greater interaction of BA with glycerin compared to other humectants in the creams

78 DEGRADATION OF ASCORBIC ACID IN PRESENCE OF STABILIZING

AGENTS IN THE DARK

An important factor in the formulation of cosmetic preparations is to ensure the

chemical and photostability of the active ingredient by the use of appropriate stabilizing

agents The choice of these agents would largely depend on the nature and

physicochemical characteristics of the active ingredient AH2 possesses a redox system

163

and can be easily oxidized by air or light In order to observe the effect of CT TA and

BA on the stability of AH2 the cream formulations containing the individual compounds

were stored in the dark for a period of about three months and the rate of degradation of

AH2 was determined The assay data are reported in Table 28 and the kinetic plots are

shown in Fig 38ndash42 The values of apparent first-order rate constants for the degradation

of AH2 in the presence of the stabilizing agents are reported in Table 29 The second

order-rate constants for the interaction of CT TA and BA with AH2 are reported in Table

30 (Fig 43ndash44) The plots of k against the carbon chain length of the emulsifiers are

shown in Fig 45 The kinetic data indicate the same pattern of rates of degradation and

interaction of AH2 with these compounds as observed in the presence of light except that

the rates are much slower in the dark Thus the stabilizing agents are equally effective in

inhibiting the rate of degradation of AH2 in the dark The effect of emulsifying agents and

the humectants on the rate of degradation of AH2 in the presence of the stabilizers has

been discussed in the above Section 77

79 EFFECT OF ADDITIVES ON TRANSMISSION OF ASCORBIC ACID

In order to observe the effect of additives (citric tartaric and boric acids) on the

transmission characteristics of ascorbic acid (0002 mg100 ml) in methanol containing

the highest concentration of the additives (004) used in this study the transmission

spectra were measured It has been found that these additives produce a hypsochromic

shift in the absorption maximum of ascorbic acid This may result in the reduction of the

fraction of light absorbed by ascorbic acid to the extent of about 10 and thus influence

the rate of photodegradation reactions However since all the additives produce similar

effects the rate constants can be considered on a comparative basis

164

Table 28 Degradation of ascorbic acid in cream formulations in the presence of

stabilizers in the dark

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(days) a b c

0 374 378 379

10 355 346 362

20 326 328 342

40 293 297 322

19 (CT)

60 264 269 295

80 241 245 262

0 381 378 380

10 361 364 372

20 339 350 348

40 309 312 330

20 (CT)

60 279 286 301

80 260 266 282

21 (CT) 0 368 370 374

10 342 346 364

20 310 321 348

40 278 282 313

60 249 251 278

80 217 228 249

165

Table 28 continued

0 375 374 378

10 339 344 351

20 317 326 336

40 282 288 306

22 (TA)

60 251 258 280

80 222 235 252

0 370 377 374

10 340 354 355

20 332 336 343

40 297 303 310

23 (TA)

60 266 282 294

80 238 248 267

24 (TA) 0 376 379 377

10 341 339 350

20 306 319 323

40 263 284 279

60 223 241 249

80 196 202 223

166

Table 28 continued

0 370 375 380

10 331 341 334

20 287 289 301

40 225 247 245

25 (BA)

60 189 185 214

80 141 154 170

0 377 375 370

10 355 357 349

20 326 314 324

40 264 267 286

26 (BA)

60 232 238 254

80 189 199 211

27 (BA) 0 380 377 375

10 346 339 337

20 309 288 301

40 233 241 260

60 192 196 211

80 140 147 163

167

Table 28 continued

0 373 376 378

10 314 322 333

20 267 281 305

40 217 233 253

28 (BA)

60 167 177 204

80 122 135 151

0 370 373 380

10 336 329 343

20 283 277 306

40 233 243 267

29 (BA)

60 189 190 217

80 144 154 173

30 (BA) 0 369 375 372

10 308 319 329

20 255 275 310

40 210 226 244

60 158 163 191

80 113 131 147

168

Table 28 continued

0 374 376 379

10 303 311 329

20 266 260 289

40 211 219 239

31 (BA)

60 155 158 178

80 112 121 149

0 370 374 375

10 314 323 339

20 276 280 305

40 222 233 258

32 (BA)

60 172 187 193

80 126 136 162

33 (BA) 0 377 380 377

10 308 306 320

20 254 265 280

40 205 214 237

60 144 155 175

80 107 118 138

169

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

19

abc

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

20

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

21

Fig 38 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing citric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

170

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

22

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

23

ab

c

12

13

14

15

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

24

Fig 39 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing tartaric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

171

a

b

c

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

25

abc

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

26

ab

c

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

27

Fig 40 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing glycerin and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

172

a

b

c

10

12

14

16

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

28

ab

c

10

12

14

16

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

29

a

b

c

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

30

Fig 41 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing propylene glycol and boric acid (a) 01 (b) 02 (c)

04

Palmitic acid

Stearic acid

Myristic acid

173

ab

c

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

31

ab

c

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

32

a

b

c

08

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

33

Fig 42 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing ethylene glycol and boric acid (a) 01 (b) 02 (c)

04

Myristic acid

Palmitic acid

Stearic acid

174

Table 29 Apparent first-order rate constants (kobs) for the degradation of ascorbic acid in

presence of different stabilizers in cream formulations in the dark

kobs times 102 (day

ndash1)ab

Cream

formulation Stabilizer

c

a b c

19 CT 055

(0994)

052

(0992)

044

(0991)

20 CT 048

(0995)

046

(0995)

038

(0992)

21 CT 064

(0994)

061

(0995)

052

(0994)

22 TA 063

(0994)

058

(0995)

049

(0996)

23 TA 054

(0995)

050

(0995)

041

(0994)

24 TA 081

(0995)

075

(0993)

066

(0995)

25 BA 118

(0996)

113

(0994)

097

(0994)

26 BA 087

(0995)

079

(0993)

068

(0994)

27 BA 124

(0995)

114

(0994)

101

(0993)

28 BA 134

(0995)

124

(0996)

110

(0992)

29 BA 116

(0996)

108

(0992)

096

(0995)

30 BA 142

(0993)

131

(0995)

115

(0995)

31 BA 145

(0995)

137

(0992)

117

(0995)

32 BA 130

(0996)

120

(0993)

107

(0994)

33 BA 153

(0995)

141

(0994)

122

(0994) a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental

conditions

c Stabilizer concentrations (andashc) are as given in Table 24

The estimated error is plusmn5

175

176

Table 30 First-order rate constants (k0)a for the degradation of ascorbic acid in the

absence of stabilizers and second-order rate constants (k) for the chemical

interaction of ascorbic acid with CT TA and BA in the dark

Cream

formulation Stabilizers

k0 times 102

(dayndash1

)

k times 102

(Mndash1

dayndash1

)

Correlation

coefficient

19 CT 060 797 0996

20 CT 052 723 0995

21 CT 069 850 0994

22 TA 068 710 0996

23 TA 058 636 0994

24 TA 086 758 0994

25 BA 126 444 0993

26 BA 092 375 0992

27 BA 131 480 0991

28 BA 141 488 0993

29 BA 122 418 0994

30 BA 149 531 0991

31 BA 155 578 0996

32 BA 137 472 0994

33 BA 163 627 0996

a The variations in the values of k0 are due to the presence of different emulsifying agents

in cream formulations

177

00

04

08

12

00 04 08 12 16 20

Citric acid concentration (M times 102)

kob

s times

10

2 (

da

yndash1)

19-21

00

04

08

12

00 05 10 15 20 25

Tartaric acid concentration (M times 102)

kob

s times

10

2 (

da

yndash1)

22-24

Fig 35 Plots of first-order rate constants (kobs) for the degradation of ascorbic acid in the

dark against citric acid (19ndash21) and tartaric acid (22ndash24) concentrations in

cream formulations

178

00

10

20

00 20 40 60

kob

s times

10

3 (

min

ndash1)

25-27

00

10

20

00 20 40 60

kob

s times

10

3 (

min

ndash1)

28-30

00

10

20

00 20 40 60

Boric acid concentration (M times 102)

kob

s times

10

3 (

min

ndash1)

30-33

Fig 44 Plots of first-order rate constants (kobs) for the degradation of ascorbic acid in the

dark against boric acid concentrations in cream formulations (25ndash33)

Glycerin

Propylene glycol

Ethylene glycol

179

00

04

08

12

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash

1 d

ayndash

1)

18-33

b

a

e

dc

Fig 45 Plots of k for degradation of ascorbic acid in the dark in creams (19ndash33) against

carbon chain length of emulsifier () Stearic acid () palmitic acid ()

myristic acid (a) CT (b) TA (c) BA with glycerin (d) BA with propylene

glycol (e) BA with ethylene glycol

CONCLUSIONS

AND

SUGGESTIONS

180

CONCLUSIONS

The main conclusions of the present study on the photodegradation of the ascorbic

acid in organic solvents and cream formulations are as follows

1 Identification of Photodegradation Products

The photodegradation of ascorbic acid in aqueous organic solvents and

laboratory prepared oil-in-water cream preparations on UV irradiation leads to the

formation of dehydroascorbic acid No further degradation products of dehydroascorbic

acid have been detected under the present experimental conditions The product was

identified by comparison of its Rf value and color of the spot with those of the authentic

compound by thin-layer chromatography and spectral changes

2 Assay of Ascorbic Acid

Ascorbic acid in aqueous organic solvents and cream preparations was assayed

in acidified methanolic solutions (pH 20) at 245 nm using a UV spectrophotometric

method Ascorbic acid in combination with other vitamins (riboflavin nicotinamide and

alpha-tocopherol) was assayed by the official iodimetric method due to interference by

these vitamins at the analytical wavelength Both analytical methods were validated

under the experimental conditions employed before their application to the assay of

ascorbic acid The recoveries of ascorbic acid in cream preparations are in the range of

90ndash96 and the reproducibility of both methods are within plusmn5 The F test and the t test

show that there is no significant difference between the precision of the two methods and

therefore these methods can be applied to the assay of ascorbic acid in cream

preparations with comparable results

181

3 Kinetics of Photodegradation

a) Photodegradation of ascorbic acid in organic solvents

Ascorbic acid degradation follows apparent first-order kinetics in aqueous

organic solvents A plot of the first-order rate constants (log kobs) versus solvent dielectric

constant is linear with positive slope indicating an increase in the rate with dielectric

constant On the contrary a plot of kobs verses reciprocal of solvent viscosity is linear with

a positive slope showing a decrease in the rate with solvent viscosity Thus the rate of

photodegradation of ascorbic acid (an oxidizable drug) depends on the solvent

characteristics

b) Photodegradation of ascorbic acid in cream preparations

Ascorbic acid has been found to follow apparent first-order kinetics in cream

preparations and the rate of degradation is affected by the following factors

i Effect of concentration

An apparent linear relationship has been observed between log kobs and

concentration (05ndash25) of ascorbic acid in a cream preparation Thus the rate of

degradation of ascorbic acid appears to be faster at a lower concentration

compared to that of a higher concentration on exposure to the same intensity of

light

ii Effect of carbon chain length of the emulsifying agent

The plots of kobs verses carbon chain length of the emulsifying agent show that the

photodegradation of ascorbic acid is affected in the order myristic acid gt stearic

acid gt palmitic acid This is predominantly due to the interaction of ascorbic acid

with palmitic acid and the carbon chain length (measure of relative polar

182

character) of the emulsifying acid probably does not play a part in the

photodegradation kinetics of ascorbic acid in creams This is evident from the

non-linear relationship between the rate constants for ascorbic acid degradation

and the carbon chain length of the emulsifying acids

iii Effect of viscosity

The values of kobs for the photodegradation of ascorbic acid in cream preparations

are in the order of humectant ethylene glycol gt propylene glycol gt glycerin

showing that the rates of degradation are influenced by the viscosity of the

humectant and decrease with an increase in the viscosity as observed in the case

of organic solvents

iv Effect of pH

The log kndashpH profiles for the photodegradation of ascorbic acid in creams

represent sigmoid type curves indicating an increase in the rate of oxidation of the

molecule with ionization (pH 42ndash70 557ndash999) The AHndash species appears to

be more susceptible to oxidation than the non-ionized molecule in the pH range

studied

v Effect of redox potential

The values of kobs show that the rate of photooxidation of ascorbic acid is

influenced by its redox potential which varies with pH The greater photostability

of ascorbic acid at pH 5ndash6 compared to that at pH 7 and above is due to its lower

rate of oxidation-reduction in the lower range The increase in the rate of

photooxidation with pH is due to a corresponding increase in the redox potential

of ascorbic acid

183

c) Photodegradation of ascorbic acid in the presence of other vitamins (riboflavin

nicotinamide alpha-tocopherol) in cream preparations

The photodegradation of ascorbic acid is affected by the presence of other

vitamins in creams The kinetic data on the photochemical interactions indicate that

riboflavin and nicotinamide act as photosensitizers in the degradation of ascorbic acid

and have an adverse effect on the photostability of the vitamin in creams Whereas

alpha-tocopherol exerts an inhibitory effect on the degradation of ascorbic acid by acting

as a redox partner in the creams Thus a combination of ascorbic acid and alpha-

tocopherol has a synergistic effect on the stabilization of ascorbic acid in creams These

vitamins do not appear to influence the rate of degradation of ascorbic acid in the dark

d) Photodegradation of ascorbic acid in the presence of citric acid tartaric acid and

boric acid in cream preparations

The rate of photodegradation of ascorbic acid in creams has been found to be

inhibited by the addition of compounds such as citric acid tartaric acid and boric acid in

creams These compounds show a stabilizing effect on the photodegradation of ascorbic

acid in the order citric acid gt tartaric acid gt boric acid The lower effect of boric acid

may be due to its interaction with the emulsifying agents and humectants Boric acid

exerts this effect by complex formation with ascorbic acid Citric acid and tartaric acid

are antioxidant synergists and in combination with ascorbic acid may exert a stabilizing

effect on its degradation

184

Salient Features of the Work

In the present work an attempt has been made to study the effects of solvent

characteristics formulation factors particularly the emulsifying agents in terms of the

carbon chain length and humectants in terms of viscosity medium pH drug

concentration redox potential and interactions with other vitamins and stabilizers on the

kinetics of photodegradation of ascorbic acid in cream preparations The study may

provide useful information to improve the photostability and efficacy of ascorbic acid in

cream preparations

SUGGESTIONS

The present work may provide guidelines for a systematic study of the stability of

drug substances in cream ointment preparations and the evaluation of the influence of

formulation variables such as emulsifying agents and humectants concentration pH

polarity viscosity redox potential on the rate of degradation and stabilization of drug

substances This may enable the formulator in the judicious design of formulations that

have improved stability and efficacy for therapeutic use The kinetic parameters may

throw light on the comparative stability of the preparations and help in the choice of

appropriate formulation ingredients

REFERENCES

186

Abe Y Horii H Taniguchi S (1986) A theoretical study of the oxidation mechanism

of triose reductone in reference to L-ascorbic acid Can J Chem 64 360-365

Ahmad I (1968) A Study of the Degradation of Riboflavin and Related Compounds

PhD Thesis University of London London

Ahmad I Rapson HDC (1990) Multicomponent spectrophotometric assay of

riboflavin and photoproducts J Pharm Biomed Anal 8 217-223

Ahmad I Vaid FHM (2006) Photochemistry of flavins in aqueous and organic

solvents In Silva E Edwards AM Eds Flavins Photochemistry and

Photobiology Royal Society of Chemistry Cambridge Chap 2

Ahmad I Tollin G (1981) Solvent effect on flavin electron transfer reactions

Biochemistry 20 5925-5928

Ahmad I Jamil N Zoha SMS (1973) Statistical evaluation of antioxidants used in

the manufacture of ascorbic acid tablets J Sci Univ Kar 2 269-287

Ahmad I Fasihullah Q Vaid FHM (2004b) A study of simultaneous photolysis and

photoaddition reactions of riboflavin in aqueous solution J Photochem Photobiol B

Biol 75 13-20

Ahmad I Fasihullah Q Vaid FHM (2005) Effect of phosphate buffer on

photodegradation reactions of riboflavin in aqueous solution J Photochem

Photobiol B Biol 78 229-234

Ahmad I Fasihullah Q Vaid FHM (2006a) Effect of light intensity and wavelength

on photodegradation reactions of riboflavin in aqueous solution J Photochem

Photobiol B Biol 82 21-27

187

Ahmad I Fasihullah Q Vaid F H M (2006b) Photolysis of formylmethylflavin in

aqueous and organic solvents Photochem Photobiol Sci 5 680-685

Ahmad I Ahmed S Sheraz MA Vaid FHM (2008) Effect of borate buffer on the

photolysis of riboflavin in aqueous solution J Photochem Photobiol B Biol 93 82-

87

Ahmad I Ahmed S Sheraz MA Vaid FHM (2009c) Borate toxicity effect on

drug stability and analytical applications In Chung MP Ed Handbook on

Borates Chemistry Production and Applications Nova Science Publishers Inc

New York in press

Ahmad I Sheraz MA Ahmed S Vaid FHM (2009d) Analytical applications of

borates Mater Sci Res J in press

Ahmad I Fasihullah Q Noor A Ansari IA Ali QNM (2004a) Photolysis of

riboflavin in aqueous solution a kinetic study Int J Pharm 280 199-208

Ahmad I Ahmed S Sheraz MA Aminuddin M Vaid FHM (2009a) Effect of

caffeine complexation on the photolysis of riboflavin in aqueous solution a kinetic

study Chem Pharm Bull in press

Ahmad I Ahmed S Sheraz MA Vaid FHM Ansari IA (2009b) Effect of

divalent ions on photodegradation kinetics and pathways of riboflavin in aqueous

solution Photochem Photobiol Sci accepted

Ahmed S (2009) Effect of Complexing Agents on the Photodegradation of Riboflavin

in Aqueous Solution PhD Thesis Baqai Medical University Karachi

Ahmad T (1978) Studies on the Stability of Sulphacetamide and Degradation Products

PhD Thesis University of Karachi Karachi

188

Ahmad T Ahmad I (1983) The effect of antioxidants on the photolysis of aqueous

sulfacetamide and sulfanilamide solutions Pol J Pharmacol Pharm 35 69-75

Albini A Fasani E Eds (1998) Drugs Photochemistry and Photostability The Royal

Society of Chemistry Cambridge

Allwood MC (1984a) Factors influencing the stability of ascorbic acid in total

parenteral nutrition infusions J Clin Hosp Pharm 9 75-85

Allwood MC (1984b) Compatibility and stability of TPN mixtures in big bags J Clin

Pharm Ther 9 181-198

Allwood MC Kearney MCJ (1998) Compatibility and stability of additives in

parenteral nutrition admixtures Nutrition 14 697-706

Al-Meshal IA Hassan MMA (1982) Ascorbic Acid In Florey K Ed Analytical

Profiles of Drug Substances Vol 11 Academic Press New York pp 45-78

Anderson NH Johnston D McLelland MA Munden P (1991) Photostability

testing of drug substances and drug products in UK pharmaceutical laboratories J

Pharm Biomed Anal 9 443-449

Ansari IA (2002) Photolysis and Interactions of Cyanocobalamin with some B-

Vitamins and Ascorbic Acid in Parenteral Solutions PhD Thesis University of

Karachi Karachi

Ansari IA Vaid Vaid HM Ahmad I (2004) Spectral Study of Photolysis of

Aqueous Cyanocobalamin Solutions in Presence of Vitamins B and C Pak J Pharm

Sci 17 83-93

Arcus CL Zilva SS (1940) The photochemical decomposition of L-ascorbic acid II

Biochem J 34 61-66

189

Arger CB Rupp D Lo P (1996) Preservation of dispersed systems In Liberman

HA Rieger MM Banker GS Eds Pharmaceutical Dosage Forms Disperse

Systems 2nd ed vol1 Marcel Dekker New York Chap9

Asard H May J Smirnoff N Eds (2004) Vitamin C Function and Biochemistry in

Animals and Plants BIOS Scientific Publishers New York

Asker AF Canady D Cobb C (1985) Influence of DL-methionine on the

photostability of ascorbic acid solutions Drug Dev Ind Pharm 11 2109-2125

Ault RG Baird DK Carrington HC Haworth WN Herbert R Hirst EL

Percival EGV Smith F Stacey M (1933) Synthesis of d- and of l-ascorbic acid

and of analogous substances J Chem Soc 1419-1423

Austria R Semenzato A Bettero A (1997) Stability of vitamin C derivatives in

solution and topical formulations J Pharm Biomed Anal 15 795-801

Baertschi SW Alsante KM (2005) Stress testing The chemistry of drug degradation

In Baertschi SW Ed Pharmaceutical Stress testing Predicting Drug Degradation

Taylor amp Francis London Chap 3

Bailey CW Bright JR Jasper JJ (1945) A study of the binary system nicotinamide-

ascorbic acid J Am Chem Soc 67 1184-1186

Ball GFM (2006) Vitamins in Foods Analysis Bioavailability and Stability CRC

Press Boca Raton FL Chap 15

Banhegyi G Marcolongo P Puskas F Fulceri R Mandl J Benedetti A (1998)

Dehydroascorbate and ascorbate transport in rat liver microsomal vesicles J Biol

Chem 273 2758-2762

190

Bardolph EM Taylor RH (1997) Sailors scurvy science and authority J R Soc

Med 90 238

Barel AO Paye M Maibach HI Eds (2001) Handbook of Cosmetic Science and

Technology Marcel Dekker New York

Barry BW (1983) Dermatological Formulations Percutaneous Absorption Marcel

Dekker New York

Barry BW (2002) Transdermal drug delivery In Aulton ME Ed Pharmaceutics

The Science of Dosage Form Design Churchill Livingstone London Chap 33

Basu TK Temple NJ Garg ML (1999) Antioxidants in Human Health and

Disease CABI Publishing Oxford

Becher P (1965) Emulsions Theory and Practice Reinhold New York p 1

Beissenhirtz M Scheller F Lisdat F (2003) Immobilized cytochrome c sensor in

organicaqueous media for the characterization of hydrophilic and hydrophobic

antioxidants Electroanalysis 15 1425-1435

Berlett BS Stadtman ER (1997) Protein oxidation in aging disease and oxidative

stress J Biol Chem 272 20313-20316

Betageri G Prabhu S (2002) Semisolid preparations In Swarbrick J Boylan JC

Eds Encyclopedia of Pharmaceutical Technology 2nd ed Marcel Dekker New

York pp 2436-2457

Bielski BHJ (1982) Chemistry of ascorbic acid radicals In Seib PA Tolbert BM

Eds Ascorbic Acid Chemistry Metabolism and Uses Advances in Chemistry

Series American Chemical Society Washington DC pp 81-100

191

Bielski BHJ Allen AO Schwartz HA (1981) Mechanism of disproportionation of

ascorbate radicals J Am Chem Soc 103 3516-3518

Billany M (2002) Suspensions and emulsions In Aulton ME Ed Pharmaceutics

The Science of Dosage Form Design 2nd ed Churchill Livingstone London Chap

23

Bisby RH Morgan CG Hamblett I Gorman AA (1999) Quenching of singlet

oxygen by trolox C ascorbate and amino acids effect of pH and temperature J Phys

Chem 103 7454-7459

Bissett DL (2006) Anti-aging skin care formulations In Draelos ZD Thaman LA

Eds Cosmetic Formulation of Skin Care Products Taylor amp Francis Group New

York Chap 11

Blaugh SM Hajratwala B (1972) Kinetics of aerobic oxidation of ascorbic acid J

Pharm Sci 61 556-562

Block LH (1996) Pharmaceutical emulsions and microemulsions In Lieberman HA

Rieger MM Banker GS Eds Pharmaceutical Dosage Forms Disperse Systems

Vol 2 2nd ed Chap 2

Bolliger HR Konig A (1969) Water-soluble vitamins In Stahl E Ed Thin-layer

Chromatography Springer-Verlag Berlin pp 304-306

Bonwstra JA De Graaff A Gooris GS Nijsse J Wiechers JW Van Aelst AC

(2003) Water distribution and related morphology in human stratum corneum at

different hydration levels J Invest Dermatol 120 750-758

Bors SR (2005) Scurvy Thomas Dunne Books St Martinrsquos Griffen New York

192

Bors W Buettner GR (1997) The vitamin C radical and its reactions In Packer L

Fuchs J Eds Vitamin C in Health and Disease Marcel Dekker New York Chap

4

Braun L Puskas F Csala M Meszaros G Mandl J Banhegyi G (1997)

Ascorbate as a substrate for glycolysis or gluconeogenesis evidence for an interorgan

ascorbate cycle Free Radic Biol Med 23 804-808

British Pharmacopoeia (2009) Her Majestyrsquos Stationary Office London Electronic

version

Buettner G (1993) The pecking order of free radicals and antioxidants lipid

peroxidation α-tocopherol and ascorbate Arch Biochem Biphys 300 535-543

Buettner GR Schafer FQ (2004) Ascorbate as an antioxidant In Asard H May

JM Smirnoff N Eds Vitamin C Function and Biochemistry in Animals and

Plants BIOS Scientific Publishers Oxford Chap 10

Burke KE (2006) Topical nutritional antioxidants In Draelos ZD Thaman LA

Eds Cosmetic Formulation of Skin Care Products Taylor amp Francis Group New

York Chap 21

Burns JJ Ed (1961) Vitamin C Ann NY Acad Sci 92 1

Byrom P Turnbull JH (1967) Excited states of flavin coenzymes II Anaerobic

oxidation of amino acids by excited riboflavin derivatives Photochem Photobiol 6

125-131

Cadenas E Packer L (2002) Handbook of Antioxidants 2nd ed Marcel Dekker New

York Chap 7-9

193

Capellmann M Bolt HM (1992) Simultaneous determination of ascorbic acid and

dehydroascorbic acid by HPLC with postcolumn derivatisation and fluorometric

detection Fresenius J Anal Chem 342 462-466

Cairns WL Metzler DE (1971) Photochemical degradation of flavins VI A new

photoproduct and its use in studying photolytic mechanism J Am Chem Soc 93

2772-2777

Carlotti ME Ugazio E Sapino S Peira E Gallarate M (2006) Photodegradation

of retinol and anti-aging effectiveness of two commercial emulsions J Cosmet Sci

57 261-277

Carpenter K (1986) The History of Scurvy and Vitamin C Cambridge University Press

Cambridge

Carr AC Zhu BZ Frei B (2000) Potential antiatherogenic mechanisms of ascorbate

(vitamin C) and alpha-tocopherol (vitamin E) Circ Res 87 349-354

Carstensen JT (1972) Theory of Pharmaceutical Systems Academic Press New York

pp 211-214

Chaudhri SK Jain NK (2009) History of cosmetics Asian J Pharm 3 164-167

Chessex P Lovoic JC Roulean T Brochu P St Louis P Levy E Al Varez F

(2002) Photooxidation of parenteral multivitamins induced hepatic steatosis in a

neonatal guinea pig model of intravenous infusion Pediatr Res 52 958-963

Connors KA Amidon GL Stella VJ (1986) Chemical Stability of Pharmaceuticals

A Handbook for Pharmacists 2nd ed Wiley New York pp 105-106 142 208-220

194

Dabrowski K Huiterleitner S (1989) Application of a simultaneous assay of ascorbic

acid dehydroascorbic acid and ascorbic sulfate in biological matter Analyst

(London) 114 83-87

Dale JK Booth RE (1976) In Hoover JE Ed Dispensing of Medication 8th ed

Mack Easton Pennsylvania pp 479-481

Darr D Dunston S Faust H Pinnell S (1996) Effectiveness of antioxidants (vitamin

C and E) with and without sunscreens as topical photoprotectants Acta Derm

Venereol 76 264-268

Davey MW Montagu MV Inze D Sanmartin M Kanellis A Smirnoff N

Benzie IJJ Strain JJ Favell D Fletcher J (2000) Plant L-ascorbic acid

chemistry function metabolism bioavailability and effect of processing J Sci Food

Agr 80 825-860

Davies MB Austin J Partridge DA (1991) Vitamin C Its Chemistry and

Biochemistry The Royal Society of Chemistry Cambridge

De La Rochette A Birlouez-Aragon I Silva E Morliere P (2003) Advanced

glycation endproducts as UVA photosensitizers of tryptophan and ascorbic acid

consequences for the lens Biochem Biophys Acta 1621 235-241

De La Rochette A Silva E Birlouez-Aragon I Mancini M Edwards AM

Morliere P (2000) Riboflavin photodegradation and photosensitization effects are

highly dependent on oxygen and ascorbate concentration Photochem Photobiol 72

815-820

195

De Vries H Beijersbergen Van Henegouwen GMJ Huff FA (1984) Photochemical

decomposition of chloramphenicol in a 025 eye drop and in a therapeutic

intraocular concentration Int J Pharm 20 265-271

Dean RT Fu S Stocker R Davies MJ (1997) Biochemistry and pathology of

radicalmediated protein oxidation Biochem J 324 1-18

DeRitter E (1982) Vitamins in pharmaceutical formulations J Pharm Sci 71 1073-

1096

Deutsch JC (1998a) Ascorbic acid oxidation by hydrogen peroxide Anal Biochem

255 1-7

Deutsch JC (1998b)Spontaneous hydrolysis and dehydration of dehydroascorbic acid

in aqueous solution Anal Biochem 260 223-229

Deutsch JC (1998c)oxygen-accepting antioxidants which arise during ascorbate

oxidation Anal Biochem 265 238-245

Dodsun KY Young ER Soliman AGM (1992) Determination of total vitamin C

in various food matrices by liquid chromatography and fluorescence detection J

AOAC Int 75 887-891

Dollery C Ed (1999) Therapeutic Drugs 2nd ed Vol 1 Churchill Livingstone

London pp A213-A216

Doores S (2002) pH control agents and acidulants In Branen LA Davidson PM

Salminen S Thorngate JH Eds Food Additives Marcel Dekker New York p

637

Eberlein-Koumlnig B Ring J (2005) Relevance of vitamins C and E in cutaneous

photoprotection J Cosmet Dermatol 4 4-9

196

Ehrlich M Rao J Pabby A Goldman MP (2006) Improvement in the appearance

of wrinkles with topical transforming growth factor beta(1) and l-ascorbic acid

Dermatol Surg 32 618-625

EIRI Board (2007) Profitable Small Scale Manufacture of Cosmetics Engineers India

Research Institute Nai Sarak Delhi

Eitenmiller RR Ye L Landen WOJr (2008) Vitamin Analysis for the Health and

Food Sciences 2nd ed CRC Press Boca Raton FL Chap 5

Elia S (2009) Nutrition In Kumar P Clark M Eds Kumar and Clarkrsquos Clinical

Medicine 7th ed Elsevier Limited Chap 5

Elmore AR (2005) Final report of the safety assessment of L-ascorbic acid calcium

ascorbate magnesium ascorbate magnesium ascorbyl phosphate sodium ascorbate

and sodium ascorbyl phosphate as used in cosmetics Int J Toxicol 24 51-111

Erb C Nau-Staudt K Flammer J Nau W (2004) Ascorbic acid as a free radical

scavenger in porcine and bovine aqueous humour Ophthal Res 36 38-42

Espinal-Perez LE Moncada B Castanedo-Cazares JP (2004) A double-blind

randomized trial of 5 ascorbic acid vs 4 hydroquinone in melasma Int J

Dermatol 43 604-607

Fall HH Petering HG (1956) Metabolic inhibitors 1 67-Dimethyl-9-

formylmethylisoalloxazine67-dimethyl-9-(2-hydroxyethyl)-isoalloxazine and

derivatives J Am Chem Soc 78 377-381

Farahmand S Tajerzadeh H Farboud ES (2006) Formulation and evaluation of a

vitamin C multiple emulsion Pharm Dev Technol 11 255-261

197

Fasani E Albini A (2005) Photostability Stress Testing Predicting Drug Degradation

Taylor amp Francis London Chap 10

Fasihullah Q (1988) Photolysis of Flavins in Aqueous and Organic Solvents PhD

Thesis University of Karachi Karachi

Fasman GD Ed (1976) CRC Handbook of Biochemistry and Molecular Biology 3rd

ed Physical Chemical Data Vol 1 CRC Press Cleveland Ohio pp 122-130

Ferreira SLC Bandeira MLSF Lemos VA Dos Santos HC Spinola Costa

AC De Jesus DS (1997) Sensitive spectrophotometric determination of ascorbic

acid in fruit juices and pharmaceutical formulations using 2-(5-bromo-2-pyridylazo)-

5-diethylaminophenol (Br-PADAP) Anal Bioanal Chem 357 1174-1178

Fessenden RW Verma NC (1978) A time-resolved electron spin resonance study of

the oxidation of ascorbic acid by hydroxyl radical Biophys J 24 413-430

Finholt P Paulssen RB Higuchi T (1963) Rate of anaerobic degradation of ascorbic

acid in aqueous solution J Pharm Sci 52 948-954

Florence AT Attwood D (2006) Drug Stability Physicochemical Principles of

Pharmacy 4th ed Pharmaceutical Press London p 411

Flynn GL (2002) Cutaneous and transdermal delivery-processes and systems of

delivery In Banker GS and Rhodes CT Eds Modern Pharmaceutics Marcel

Dekker New York Chap 8

Foote CS (1991) Definition of Type I and Type II Photosensitized oxidation

Photochem Photobiol 54 659

Fornaro A Coicher N (1998) L-ascorbic acid complexation and redox reactions with

some transition metal ions Quni Nova 21 642-650

198

Fraga CG Motchink PA Wyrobek AJ Rempel DM Ames BN (1996)

Smoking and low antioxidant levels increase oxidative damage to sperm DNA

Mutat Res 351 199-203

Fraga CG Motchnik PA Shigenaga MK Helbock HJ Jacob RA Ames BN

(1991) Ascorbic acid protects against endogenous oxidative DNA damage in human

sperm Proc Natl Acad Sci USA 88 11003-11006

Frenich AG Torres ME Vega AB Vidal JL Bolanos PP (2005) Determination

of ascorbic acid and carotenoids in food commodities by liquid chromatography with

mass spectrometry detection J Agric Food Chem 53 7371-7376

Fung HL (1990) Drug stability In Banker GS Rhodes CT Eds Modern

Pharmaceutics 2nd ed Marcel Dekker New York Chap 6

Gallarate M Carlotti ME Trotta M Bovo S (1999) On the stability of ascorbic

acid in emulsified systems for topical and cosmetic use Int J Pharm 188 233-241

Ganshirt H Malzacher A (1960) Separation of several vitamins of the B group and C

by chromatography Naturwiss 47 279-280

Gardner WH (1972) Acidulants in food processing In Furia TE Ed Handbook of

Food Additives 2nd ed Vol 1 CRC Press Cleveland OH pp 225ndash270

Garrett ER (1967) Kinetics and mechanisms in stability of drugs In Bean HS

Beckett AH Carless JE Eds Advances in Pharmaceutical Sciences Vol 2

Academic Press London Chap 1

Garti N Aserin A (1996) Pharmaceutical emulsions double emulsions and

microemulsions In Benita S Ed Microencapsulation Methods and Industrial

Applications Marcel Dekker New York pp 411-534

199

Gensler M Rossmann A Schmidt HL (1995) Detection of added L-ascorbic acid in

fruit juice by isotope ratio mass spectrometry J Agric Food Chem 43 2662-2666

Gibbons E Allwood MC Neal T Hardy G (2001) Degradation of dehydroascorbic

acid in parenteral nutrition mixtures J Pharm Biomed Anal 25 605-611

Guitton C Gros P Comtat M (2007) Direct electrochemical measurements in dermo-

cosmetic creams In Alegret Merkoci Eds Comprehensive Analytical Chemistry

Elsevier Amsterdam 49 e59-e68

Guttman DE Brooke D (1963) Solution phase interaction of nicotinamide with

ascorbic acid J Pharm Sci 52 941-945

Gyorgy P Pearson WN Eds (1967) Vitamin Methods Vol VII Academic Press

New York pp 27-49

Hagg M Ylikoski S Kumpulainen J (1994) Vitamin C and α- and β-carotene in

vegetables consumed in Finland during 1988-1989 and 1992-1993 J Food Compos

Anal 7 252-259

Hagg M Ylikoski S Kumpulainen A (1995) Vitamin C content in fruits and berries

consumed in Finland J Food Compos Anal 8 12-20

Hall NK Chapman TM Kim HJ Min DB (2009) Antioxidant mechanisms of

Trolox and ascorbic acid on the oxidation of riboflavin in milk under light Food

Chem in press doi 101016jfoodchem200905017

Halliwell B (1996) Vitamin C antioxidant or pro-oxidant in vivo Free Radic Res 25

439-454

200

Halliwell B Whiteman M (1997) Antioxidant and prooxidant properties of vitamin C

In Packer L Fuchs J Eds Vitamin C in Health and Disease Marcel Dekker New

York pp 25-94

Halver JE Felton SP (2001) Analytical enigmas in assaying for vitamers C In

Dabrowski K Ed Ascorbic Acid in Aquatic Organisms CRC Press Boca Raton

Chap 3

Hanck A Ed (1982) Vitamin C New Clinical Applications in Immunology Lipid

Metabolism and Cancer Hans Huber Bern

Hashmi MH (1973) Assay of Vitamins in Pharmaceutical Preparations John Wiley

New York Chap 13

Hatchard CG Parker CA (1956) A new sensitive chemical actinometer II Potassium

ferrioxalate as a standard chemical actinometer Proc R Soc London Ser A A235

518-536

Heber GK Markovic B Hayes A (2006) An immunohistological study of anhydrous

topical ascorbic acid compositions on ex vivo human skin J Cosmet Dermatol 5

150-156

Heelis PF Parsons BJ Phillips GO McKellar JF (1981) The flavin sensitized

photooxidation of ascorbic acid a continuous and flash photolysis study Photochem

Photobiol 37 7-13

Henry JA Ed (1997) The New Guide to Medicines and Drugs The British Medical

Association 4th ed Dorling Kindersley London p 434

Hickey S Roberts H (2004) Ascorbate-The Science of Vitamin C Electronic Version

(ISBN 1-4116-0724-4)

201

Higdon JV Frei B (2002) The Antioxidant Vitamin C and E AOAC Press Arlington

VA pp 1-16

Hirst EL Zilva SS (1933) Ascorbic acid as the anti-scorbutic factor Biochem J 27

1271-1278

Ho AHL Puri A Sugden JK (1994) Effect of sweetening agents on the light

stability of aqueous solutions of L-ascorbic acid Int J Pharm 107 199-203

Holmstrom B Oster G (1961) Riboflavin as an electron donor in photochemical

reactions J Am Chem Soc 83 1867-1871

Homann P Gaffron H (1964) Photochemistry and metal catalyzes studies in a flavin

sensitized oxidation of ascorbic acid Photochem Photobiol 3 499-515

Hughes DE (1985) Irreversible reaction kinetics of the aerobic oxidation of ascorbic

acid Anal Chem 57 555-558

Humbert PG Haftek M Creidi P Lapiegravere C Nusgens B Richard A Schmitt D

Rougier A Zahouani H (2003) Topical ascorbic acid on photoaged skin Clinical

topographical and ultrastructural evaluation double-blind study vs placebo Exp

Dermatol 12 237-244

ICH Harmonized Tripartite Guideline (1997) QIB Photostability Testing of New Drug

Substances and Products Federal Register 62 27115-27122

Ichikawa M Ide N Shiraishi S Ono K (2005) Effect of various halide salts on the

incompatibility of cyanocobalamin and ascorbic acid in aqueous solution Chem

Pharm Bull 53 688-690

202

Ihara H Shino Y Aoki Y Hashizume N Minegishi N (2000) A simple and rapid

method for the routine assay of total ascorbic acid in serum and plasma using

ascorbate oxidase and o-phenylenediamine J Nutr Sci Vitaminol 46 321-324

Im-Emsap W Paeratakul O Siepmann J (2002) Disperse Systems In Banker GS

Rhodes CT Ed Modern Pharmaceutics Maecel Dekker New York Chap 9

Irache JM Ezpeleta I Vega FA (1993) HPLC determination of antioxidant

synergists and ascorbic acid in some fatty pharmaceuticals cosmetics and food

Chromatographia 35 232-23

Iwase H Ono I (1994) Determination of ascorbic acid in human plasma by high-

performance liquid chromatography with electrochemical detection using a

hydroxyapatite cartridge for precolumn deproteinization J Chromatogr B Biomed

Appl 655 195-200

Jain S Tiwari AK Jain NK (2006) Topical products In Jain NK Ed

Pharmaceutical Product Development CBS Publishers New Delhi Chap 7

Jamil N Ahmad I (1984) Moisture content of ascorbic acid tablets stored at various

temperatures and humidities Pak J Sci Ind Res 27 252

Jamil N Ahmad I Beg AE (1980a) The effect of UV and tungsten light on physical

characteristics of ascorbic acid tablets Laboratory prepared samples IV Kar Univ

J Sci 8 77-87

Jamil N Ahmad I Beg AE (1980b) The effect of UV and tungsten light on physical

characteristics of ascorbic acid tablets Commercial samples III Kar Univ J Sci 8

117-126

203

Jentzsch A Aikens P (2005) RetiSTARTM

for cosmetic formulations-stabilized retinol

In Rosen MR Ed Delivery System Handbook for Personal Care and Cosmetic

Products Technology Applications and Formulations William Andrew Publishing

Norwich NY pp 862-866

Johnston CS Steinberg FM Rucker RB (2007) Ascorbic Acid In Zempleni J

Rucker RB McCormick DB Suttie JW (Eds) Handbook of Vitamins 4th ed

CRC Press Boca Raton FL Chap 15

Jung MY Kim SM Kim SY (1995) Riboflavin-sensitized photooxidation of

ascorbic acid kinetics and amino acid effects Food Chem 53 397-403

Kall MA Anderson CJ (1999) Improved method for simultaneous determination of

ascorbic acid and dehydroascorbic acid isoascorbic acid and dehydroisoascorbic acid

in food and biological samples J Chromatogr B Biomed Sci Appl 730 101-111

Kampfenkel K Van Montagu M Inze D (1995) Extraction and determination of

ascorbate and dehydroascorbate from plant tissue Anal Biochem 225 165-167

Kaplan DL Moloney SJ Troy WR Dickens MS Pinnell SR (1989) A new

stabilized ascorbic acid solution percutaneous absorption and effect on relative

collagen synthesis J Cutan Aging Cosmet Derm I 115-121

Karatepe M (2004) Simultaneous determination of ascorbic acid and free

malondialdehyde in human serum by HPLC-UV LC GC (North America) 22 362-

365

Karlsen J (1996) Light-activated drugs and drug formulations in drug targeting In

Tonneson HH Ed The Photostability of Drugs and Formulations Taylor amp

Francis London Chap 8

204

Kassem MA Kassem AA Ammar HO (1969a) Studies on the stability of

injectable L-ascorbic acid solutions I Effect of pH solvent light and container

Pharm Acta Helv 44 611-623

Kassem MA Kassem AA Ammar HO (1969b) Studies on the stability of

injectable L-ascorbic acid solutions II Effect of metal ions and oxygen content of

solvent water Pharm Acta Helv 44 667-675

Kassem MA Kassem AA Ammar HO (1972) Studies on the stability of injectable

L-ascorbic acid solutions 3 Effect of metal-complexing agents Pharm Acta Helv

47 89-97

Kaushansky K Kipps TJ (2006) Hematopoietic agents growth factors minerals and

vitamins In Brunton L Ed Goodman amp Gilmans The Pharmacologic Basis of

Therapeutics 11th ed digital ed McGraw-Hill Companies USA Chap 53

Kearney MCJ Allwood MC Martin H Neal T Hardy G (1998) The influence

of amino acid source on the stability of ascorbic acid in TPN mixtures Nutrition 14

173-178

Kim H Kirshenbaum LJ Rosenithol I Riesz P (1993) Photosensitized formation

of ascorbate radicals by riboflavin an ESR study Photochem Photobiol 57 777-

784

Kimoto K Terada S Yamaguchi T (1997) Analysis of ascorbic acid

dehydroascorbic acid and transformation products by ion-pairing high-performance

liquid chromatography with multi-wavelength ultraviolet and electrochemical

detection Methods Enzymol 279 3-12

205

King CG Burns JJ Ed (1975) Second Conference on Vitamin C Ann N Y Acad

Sci 258

Kitagawa Y (1968) The effects of ultraviolet rays on vitamin C Eiyo To Shokuryo 20

416-421 through CA (1968) 68 111560f

Klick S Muijselaar PG Waterval J EichingerT Korn C Gerding TK Debets

AJ de Griend CS van den Beld C Somsen GW De Jong GJ (2005)Toward

a generic approach for stress testing of drug substances and drug products Pharm

Tech 29 48-66

Konings EJ (2006) Water-soluble vitamins J AOAC Int 89 285-288

Kwon BM Foote CS (1988) Chemistry of singlet oxygen 50 Hydroperoxide

intermediates in the photooxygenation of ascorbic acid J Am Chem Soc 110

6582-6583

Kwon BM Foote CS Khan SI (1989) Photooxygenation of ascorbic acid

derivatives and model compounds J Am Chem Soc 111 1854-1860

Laborie S Lavoie JC Chessex P (1998) Paradoxical role of ascorbic acid and

riboflavin in solutions of total parenteral nutrition implication in photoinduced

peroxide generation Pediatr Res 43 601-601

Laborie S Lavoie JC Pineault M Chessex P (1999) Protecting solutions of

parenteral nutrition from peroxidation JPEN 23 104-108

Laborie S Lavoie JC Pineault M Chessex P (2000) Contribution of multivitamin

air and light in the generation of peroxides in adult and neonatal parenteral nutrition

solutions Ann Pharmacother 34440-445

206

Laborie S Lavoie JC Rouleau T Chessex P (2002) Multivitamin solutions for

enteral supplementation a source of peroxides Nutrition 18 470-473s

Lachman L DeLuca P Akers MJ (1986) Kinetic principles and stability testing In

Lachman L Liberman HA Kanig JL Eds The Theory and Practice of

Industrial Pharmacy 3rd ed Lea amp Febiger Philadelphia Chap 26

Laidler KJ (1987) Chemical Kinetics 3rd ed Harper and Row New York pp 183-

185

Lambert WE de Leenheer A (1992) In Nollet LML Ed Food Analysis by HPLC

Marcel Dekker New York Chap 10

Lavoie JC Chessex P Rouleau T Migneault D Comte B (2004) Light-induced

byproducts of vitamin C in multivitamin solutions Clin Chem 50 135-140

Lee JS Kim JW Han SH Chang IS Kang HH Lee OS Oh SG Suh KD

(2004) The stabilization of L-ascorbic acid in aqueous solution and water-in-oil-in-

water double emulsion by controlling pH and electrolyte concentration J Cosmet

Sci 55 1-12

Lee KM Kwon JY Lee KW Lee HJ (2009) Ascorbic acid 6-palmitate

suppresses gap-junctional intercellular communication through phosphorylation of

connexin 43 via activation of the MEK-ERK pathway Mutat Res 660 51-56

Leacutevecircque N Mac-Mary S Muret P Makki S Aubin F Kantelip JP Heusegravele C

SS Humbert P (2005) Evaluation of a sunscreen photoprotective effect by ascorbic

acid assessment in human dermis using microdialysis and gas chromatography mass

spectrometry Exp Dermatol 14 176-181

207

Levine M (1986) New concepts in the biology and biochemistry of ascorbic acid N

Engl J Med 314 892-902

Lewin S (1976) Vitamin C Its Molecular Biology and Medical Potential Academic

Press New York

Liau LS Lee BL New AL Ong CN (1993) Determination of plasma ascorbic

acid by high-performance liquid chromatography with ultraviolet and electrochemical

detection J Chromatogr 612 63-70

Lin JY Selim MA Shea CR Grichnik JM Omar MM Monteiro-Riviere NA

Pinnell SR (2003) UV photoprotection by combination topical antioxidants vitamin

C and vitamin E J Am Acad Dermatol 48 866-874

Lin FH Lin JY Gupta RD Tournas JA Burch JA Selim MA Monteiro-

Riviere NA Grichnik JM Zielinski J Pinnell SR (2005) Ferulic acid

stabilizes a solution of vitamins C and E and doubles its photoprotection of skin J

Invest Dermatol 125 826-832

Lindahl T (1993) Instability and decay of the primary structure of DNA Nature 362

709-715

Litner CJ (1973) Drug Product Stability In Cooper MS Ed Quality Control in

Pharmaceutical Industry Vol 2 Academic Press New York p 185

Liu TZ Chin N Kiser MD Bigler WN (1982) Specific spectrophotometry of

ascorbic acid in serum or plasma by use of ascorbate oxidase Clin Chem 28 2225-

2228

Lu GW Flynn GL (2009) Cutaneous and transdermal delivery-processes and

systems of delivery In Florence AT Siepmann J Eds Modern Pharmaceutics-

208

Applications and Advances Vol 2 5th ed Informa Healthcare Inc New York

Chap 3

Lykkesfeldt J (2000) Determination of ascorbic acid and dehydroascorbic acid in

biological samples by high-performance liquid chromatography using subtraction

methods reliable reduction with tris [2-carboxyethyl] phosphine hydrochloride Anal

Biochem 282 89-93

Macek TJ (1960) Stability problems with some vitamins in pharmaceuticals Am J

Pharm 132 433-455

Mader WJ (1961) Vitamins In Higuchi T Brochmann-Hanssen E Eds

Pharmaceutical Analysis Interscience New York pp 689-693

Maggi N Pasqualucci CR Ballotta R Sensi P (1966) Rifampicin a new orally

active rifamycin Chemotherapy 11 285-292

Maia AM Baby AR Pinto CA Yasaka WJ Suenaga E Kaneko TM Velasco

MV (2006) Influence of sodium metabisulfite and glutathione on the stability of

vitamin C in OW emulsion and extemporaneous aqueous gel Int J Pharm 322

130-135

Mancini M Edwards AM Becker MI De Ioannes A Silva E (2000) Reactivity

of monoclonal antibodies against a tryptophan-riboflavin adduct toward irradiated

and non-irradiated bovine-eye-lens protein fractions an indicator of in vivo visible

light mediated phototransformations J Photochem Photobiol B Biol 55 9-15

Manoharan M Schwille PO (1994) Measurement of ascorbic acid in human plasma

and urine by high performance liquid chromatography Results in healthy subjects

209

and patients with idiopathic calcium urolithiasis J Chromatogr B Biomed Appl

654 134-139

Margolis SA Schmidt HL (1996) Measurement of ascorbic acid in human plasma

and serum stability intralaboratory repeatability and interlaboratory reproducibility

Clin Chem 42 1257-1262

Mason P (2007) Dietary Supplements 3rd ed Pharmaceutical Press pp 330-339

Massad WA Bertolotti S Garcia N (2004) Kinetics and mechanism of the vitamin

B2-sensitized photooxidation of isoproterenol Photochem Photobiol 79 428-433

Matsubayashi T Sakaeda T Kita T Kurimoto Y Nakamura T Nishiguchi K

Fujita T Kamiyama F Yamamoto A Okumura K (2003) Intradermal

concentration of hydroquinone after application of hydroquinone ointments is higher

than its cytotoxic concentration Biol Pharm Bull 26 1365-1367

McBride MM Moore WM (1967) The photochemistry of riboflavin II

Polarographic studies on the hydroxyethyl and formylmethyl analogues of riboflavin

Photochem Photobiol 6 103-113

McDowell LR (2000) Vitamins in Animal and Human Nutrition 2nd ed Iowa State

University Press Chap 15

Meister A (1994) Glutathione-ascorbic acid antioxidant system in animals J Biol

Chem 269 9397-9400

Meister A (1995) Mitochondrial changes associated with glutathione deficiency

Biochim Biophys Acta 1271 35-42

Min DB Boff JM (2002) Chemistry and reaction of singlet oxygen in foods

CRFSFS 1 58-72

210

Moeslinger T Brunner M Volf I Spieckermann PG (1995) Spectrophotometric

determination of ascorbic acid and dehydroascorbic acid Clin Chem 41 1177-1181

Moffat AC Osselton MD Widdop B (2004) Clarkersquos Analysis of Drugs and

Poisons 3rd ed Pharmaceutical Press London pp 649-650

Moore DE (2004) Standardization of kinetic studies of photodegradation reactions In

Tonnesen HH Ed Photostability of Drugs and Drug Formulations 2nd ed CRC

Press Boca Raton FL Chap 3

Moura T Gaudy D Jacob M Cassanas G (1994) pH influence on the stability of

ascorbic acid spray-drying solutions Pharm Acta Helv 69 77-80

Murray JC Burch JA Streilein RD Iannacchione MA Hall RP Pinnell SR

(2008) A topical antioxidant solution containing vitamins C and E stabilized by

ferulic acid provides protection for human skin against damage caused by ultraviolet

irradiation J Am Acad Dermatol 59 418-425

Mushran SP Agarwal MC (1977) Mechanistic studies on the oxidation of ascorbic

acid 5 J Sci Res (India) 36 274-283

Nerlo H Palak W Tomaszek J (1968) Stabilization of vitamin preparations II

Syrup of vitamin D3 and its stabilization Acta Polon Pharm 25 183-188

Niki E Noguchi N (1997) Protection of human low-density lipoprotein from oxidative

modification by vitamin C In Packer L Fuchs J Eds Vitamin C in Health and

Disease Marcel Dekker New York pp 183-192

Nishikimi M Fukuyama R Minoshima S Shimizu N Yagi K (1994) Cloning and

chromosomal mapping of the human nonfunctional gene for L-gulono-gamma-

211

lactone oxidase the enzyme for L-ascorbic acid biosynthesis missing in man J Biol

Chem 269 13685-13688

Njus D Wigle M Kelly PM Kipp BH Schlegel HB (2001) Mechanism of

ascorbic acid oxidation by Cytochrome B561 Biochemistry 40 11905-11911

Nusgens BV Humbert P Rougier A Colige AC Haftek M Lambert CA

Richard A Creidi P Lapiegravere CM (2001) Topically applied vitamin C enhances

the mRNA level of collagens I and III their processing enzymes and tissue inhibitor

of matrix metalloproteinase 1 in the human dermis J Invest Dermatol 116 853-859

Ogata Y Kosugi Y (1969) Solvent effect on the autooxidation of L-ascorbic acid

Tetrahedron 25 1055-1062

OrsquoKeefe T (2001) Ascorbic acid and stable ascorbate esters as sources of vitamin C in

aquaculture feeds ASA Technical Bulletin AQ48 wwwsoyaquaorg

OrsquoNeil MJ Ed (2001) The Merck Index 13th ed Merck amp Co Inc Rahway NJ

USA Electronic version

Ortega GR Delgado JN (1998) Vitamins and related compounds In Wilson and

Gisvoldrsquos Textbook of Organic Medicinal and Pharmaceutical Chemistry 10th ed

Lippincott-Raven Philadelphia Chap 27

Ortwerth BJ Monnier VM (1997) Protein glycation by the oxidation products of

ascorbic acid In Packer L Fuchs J Eds Vitamin C in Health and Disease Marcel

Dekker New York pp 123ndash142

Ozer O Muguet V Roy E Grossiord JL Seiller M (2000) Stability study of

WOW viscosified multiple emulsions Drug Dev Ind Pharm 26 1185-1189

Packer I Fuchs J (1999) Vitamin C in Health and Disease Marcel Dekker New York

212

Packer JE Slater TF Wilson RL (1979) Direct observation of a free radical

interaction between vitamin E and vitamin C Nature 278 737-738

Packer L Traber MG Kraemer K Frei B (2002) The Antioxidant Vitamins C and

E AOCS Press Illinois

Padayatty SJ Katz A Wang Y Eck P Kwon O Lee JH Chen S Corpe C

Dutta A Dutta SK Levine M (2003) Vitamin C as an antioxidant Evaluation of

its role in disease prevention J Am Coll Nutr 22 18-35

Parker CA (1953) A new sensitive chemical actinometer I Some trials with potassium

ferrioxalate Proc Roy Soc (Lond) A220 104-116

Parker JN Parker PM (2003) Antioxidants ICON Group International

Pastore P Rizzetto T Curcuruto O Cin MD Zaramella A Martin D (2001)

Characterization of dehydroascorbic acid solutions by liquid chromatography mass

spectrometry Rapid Commun Mass Spectrom 15 2051-2057

Pelletier O (1985) In Augustin J Klein BP Becker D Venugopal PB Eds

Methods of Vitamins Assay Wiley New York pp 303-347

Peyrat-Maillard MN Bonnely S Rondini L Berset C (2001) Effect of vitamin E

and vitamin C on the antioxidant activity of malt rootlets extracts Lebensmittel-

Wissenschaft Und-Technological 34 176-182

Pinnell SR (2003) Cutaneous photodamage oxidative stress and topical antioxidant

protection J Am Acad Dermatol 48 1-19

Pinnell SR Madey DL (1998) Topical vitamin C in skin care Aesthet Surg J 18

468-470

213

Pinnell SR Yang H Omar M Monteiro-Riviere N DeBuys HV Walker LC

Wang Y Levine M (2001) Topical L-ascorbic acid percutaneous absorption

studies Dermatol Surg 27 137-142

Placzek M Gaube S Kerkmann U Gilbertz KP Herzinger T Haen E Przybilla

B (2005) Ultraviolet B-induced DNA damage in human epidermis is modified by

the antioxidants ascorbic acid and D-alpha-tocopherol J Invest Dermatol 124 304-

307

Puskas F Gergely PJr Banki K Perl A (2000) Stimulation of the pentose

phosphate pathway and glutathione levels by dehydroascorbate the oxidized form of

vitamin C FASEB J 14 1352-1361

Puskas F Gergely P Niland B Banki K Perl A (2002) Differential regulation of

hydrogen peroxide and Fas-dependent apoptosis pathways by dehydroascorbate the

oxidized form of vitamin C Antioxid Redox Signal 4 357-369

Racz I (1989) Drug Formulation Wiley New York pp 121-122

Ramu A Mehta MM (2000) The riboflavin-mediated photooxidation of doxorubicin

Cancer Chemoth Pharm 46 449-458

Raschke T Koop U Duumlsing HJ Filbry A Sauermann K Jaspers S Wenck H

Wittern KP (2004) Topical activity of ascorbic acid from in vitro optimization to in

vivo efficacy Skin Pharmacol Physiol 17 200-206

Reed RA Harmon P Manas D Wasylaschuk W Galli C Biddell R Bergquist

PA Hunke W Templeton AC Ip D (2003) The role of excipients and package

components in the photostability of liquid formulations PDA J Pharm Sci Tech 57

351-368

214

Reichstein T Griissner A Oppcnuuer R (1933) Synthese der d- und l-Ascorbinsaumlure

(C-Vitamin) Helv Chim Acta 16 1019-1033

Riehl J Maupin C Layloff T (1995) On the choice of a photolysis source for the

photostability testing of pharmaceuticals Pharmacop Forum 21 1654-1663

Rivlin RS (2007) Riboflavin (vitamin B2) In Zempleni J Rucker RB McCormick

DB Suttie JW Eds Handbook of Vitamins 4th ed Taylor amp Francis CRC

Press Boca Raton FL p 241

Rogers AR Yacomeni JA (1971) The effect of pH on the aerobic degradation of

ascorbic acid solutions J Pharm Pharmacol 23 218S

Rosen MR Ed (2005) Delivery System Handbook for Personal Care and Cosmetic

Products-Technology Applications and Formulations William Andrew Publishing

Norwich NY

Rosenberg HR (1945) Chemistry and Physiology of the Vitamins Interscience New

York pp 289-338

Roth HJ Eger K Troschutz R (1991) Pharmaceutical Chemistry Vol 2 Drug

Analysis Ellis Horwood London pp 282-288

Rowe RC Sheskey PJ Quinn ME (2009) Handbook of Pharmaceutical Excipients

6th ed Pharmaceutical Press London pp 43-47 181-183 731-732

Roacutezanowska M Bober A Burke JM Sarna T (1997) The role of retinal pigment

epithelium melanin in photoinduced oxidation of ascorbate Photochem Photobiol

65 472-479

Rozman B Gasperlin M (2007) Stability of vitamins C and E in topical

microemulsions for combined antioxidant therapy Drug Deliv 14 235-245

215

Rozman B Gasperlin M Tinois-Tessoneaud E Pirot F Falson F (2009)

Simultaneous absorption of vitamins C and E from topical microemulsions using

reconstructed human epidermis as a skin model Eur J Pharm Biopharm 72 69-75

Ruumlmelin A Fauth U Halmagyi M (1999) Determination of ascorbic acid in plasma

and urine by high performance liquid chromatography with ultraviolet detection Clin

Chem Lab Med 37 533-536

Rumsey SC Levine M (2000) Vitamin C In Song WO Beecher GR

Eitenmiller RR Eds Modern Analytical Methodologies in Fat- and Water-Soluble

Vitamins Wiley New York Chap 13

Saari JC Baker EM Sauberlich HE (1967) Thin-layer chromatographic separation

of the oxidative degradation products of ascorbic acid Anal Biochem 18 173-177

Salkić M Keran H Jašić M (2007) Direct spectrophotometric determination of l-

ascorbic acid in the presence of potassium cyanide Agric Conspec Sci 72 371-375

Salvador A Chisvert A (2007) Analysis of Cosmetic Products Elsevier Amsterdam

Sattar A DeMan JM Alexander JC (1977) Light-induced degradation of vitamins

II Kinetic studies on ascorbic acid decomposition in solution Can Inst Food Sci

Technol J 10 65-68

Satoh K Sakagami H Terasaka H Ida Y Fujisawa S (2000) Interaction between

hydroquinone and ascorbic acid derivatives quenching effects of organic solvents

Antican Res 20 1577-1581

Sauberlich HE (1994a) Pharmacology of vitamin C Annu Rev Nutr 14 371-391

Sauberlich HE (1994b) Vitamin C and Cancer In Kritchevsky D Carroll K Eds

Nutrition and Disease Update Cancer AOCS Press Champaign IL pp 111-172

216

Sauermann K Jaspers S Koop U Wenck H (2004) Topically applied vitamin C

increases the density of dermal papillae in aged human skin BMC Dermatol 4 13

Sawicka J (1991) The influence of excipients and technological process on

cholecalciferol stability and its liberation from tablets Pharmazie 46 519-521

Scheuplein RJ Blank IH (1973) Mechanism of percutaneous absorption IV

Penetration of non-electrolytes (alcohols) from aqueous solutions and from pure

liquids J Invest Dermatol 60 286-296

Schick B (1943) Scurvy and pine needles Science 98 241-242

Schuman Jorns M Schollnhammer G Hemmerich P (1975) Intramolecular addition

of the riboflavin side chain Anion-catalysed neutral photochemistry Eur J

Biochem 57 35-48

Seib PA Tolbert BM (1982) Ascorbic Acid Chemistry Metabolism and Uses

Advances in Chemistry Series 200 American Chemical Society Washington DC

Sequeira F Vozone C (2000) Photostability studies of drug substances and products

Practical implications of the ICH guideline Pharm Tech 24 30-35

Shacter E (2000) Quantification and significance of protein oxidation in biological

samples Drug Metab Rev 32 307-326

Shah VP (1997) FDA Guidance for Industry Document Nonsterile Semisolid Dosage

Forms Centre for Drug Evaluation and Research Rockville MD pp 1-40

Sheraz MA Ahmed S Ahmad S (2007) Developments in the clinical and food

analysis of vitamin C J Baqai Med Univ 10 19-24

217

Shindo Y Witt E Han D Packer L (1994) Dose-response effect of acute ultraviolet

irradiation on antioxidants and molecular markers of oxidation in murine epidermis

and dermis J Invest Dermatol 102 470-475

Sibi M Batcu A Gheorghiu I (1953) Vitamin oxidation-reduction effect II Effect of

light on the oxidation-reduction reactions of lactoflavin Studii cercetari stiinf

(Romania) 4 291-305 through CA (1956) 50 14816g

Sidhu DS Sugden JK (1992) Effect of food dyes in the photostability of aqueous

solutions of L-ascorbic acid Int J Pharm 83 263-266

Silva E Quina FH (2006) Photoinduced processes in the eye lens do flavins really

play a role In Silva E Edwards AM Eds Flavins Photochemisry and

Photobiology The Royal Society of Chemistry Cambridge Chap 7

Silva E Ugarte P Andrade A Edwards AM (1994) Riboflavin-sensitized photo-

processes of tryptophan J Photochem Photobiol B Biol 23 43-48

Sim AK (1972) Ascorbic acid-A scurvy past and present Chem Indust 8 160-165

Singh S (2006) Stability testing during product development In Jain NK ED

Pharmaceutical Product Development CBS Publishers New Delhi Chap 9

Singh S Bakshi M (2000) Guidance on conduct of stress testing to determine inherent

stability of drugs Pharm Tech 24 1-14

Sinko PJ (2006) Chemical Kinetics and Stability Martinrsquos Physical Pharmacy and

Pharmaceutical Sciences 5th ed Lippincott Williams amp Wilkins Baltimore Chap

8 15 18

218

Smith JL Canham JE Kirkland WD Wells PA (1988) Effect of intralipid amino

acids container temperature and duration of storage on vitamin stability in total

parenteral nutrition admixtures J Parenter Enteral Nutr 12 478-483

Speek AJ Schrijver J Schreurs WHP (1984) Fluorimetric determination of total

vitamin C and total isovitamin C in food stuffs and beverages by high-performance

liquid chromatography with precolumn derivatization J Agric Food Chem 32 352-

355Spratley JE Hellstroumlm SO Mattsson CK Pais-Clemente M (2001)

Topical ascorbic acid reduces myringosclerosis in perforated tympanic membranes A

study in the rat Ann Otol Rhinol Laryngol 110 585-591

Spiclin P Homer M Zupancic A Gasperlin M (2003) Sodium ascorbyl phosphate

in topical microemulsions Int J Pharm 256 65-73

Stadtman ER (1991) Ascorbic acid and oxidative inactivation of proteins Am J Clin

Nutr 54 1125S-1128S

Stone I (1969) The Healing Factor Vitamin C Against Disease Grosset and Dunlap

New York

Sugden JK (1985) Photostability of cosmetic materials Int J Cosmet Sci 7 165-173

Swarbrick J Rubino JT Rubino OP (2006) Coarse Dispersions In Hendrickson

R Ed Remington The Science and Practice of Pharmacy 21st ed Lippincott

Williams amp Wilkins Baltimore Chap22

Sweetman SC Ed (2009) Martindale The Complete Drug Reference 36th ed

Pharmaceutical Press London Electronic version

219

Szent-Gyoumlrgi A (1928) Observation on function of peroxidase systems and chemistry

of adrenal cortex description of new carbohydrate derivative Biochem J 22 1387-

1389

Szymula M (2005) The influence of ascorbic acid on the rheological properties of the

microemulsion region of the SDSpentanolwater system J Cosmet Sci 56 267-

277

Taqui Khan MM Martell AE (1967) Metal ion and metal chelate catalyzed oxidation

of ascorbic acid by molecular oxygen 1 Cupric acid and ferric ion catalyzed

oxidation J Am Chem Soc 89 4176-4185

Tangney CC (1988) Analysis of vitamin C in biological samples with an emphasis on

recent chromatographic techniques Prog Clin Biol Res 259 331-362

Tatsumi K Ichikawa H Wada S (1992) Flavin-sensitized photooxidation of

substituted phenols in natural water J Contam Hydrol 9 207-219

Tessier F Birlouez-Aragon I Tjani C Guilland JC (1996) Validation of a

micromethod for determining oxidized and reduced vitamin C in plasma by HPLC-

fluorescence Int J Vitam Nutr Res 66 166-170

Thatcher SR Mansfield RK Miller RB Davis CW Baertschi SW (2001a)

Pharmaceutical photostability a technical and practical interpretation of the ICH

guideline and its application to pharmaceutical stability Part 1 Pharm Tech 25 98-

110

Thatcher SR Mansfield RK Miller RB Davis CW Baertschi SW (2001b)

Pharmaceutical photostability a technical and practical interpretation of the ICH

220

guideline and its application to pharmaceutical stability Part 2 Pharm Tech 25 50-

62

Thiele JJ Dreher F Packer L (2000) Antioxidant defense systems in skin In

Elsner P Maibach HI Eds Cosmeceuticals- Drugs vs Cosmetics Marcel

Dekker New York Chap 11

Thomas DP (1997) Sailors scurvy and science J R Soc Med 90 50-54

Thomson CO Trenerry VC (1995) A rapid method for the determination of total L-

ascorbic acid in fruits and vegetables by micellar electrokinetic capillary

chromatography Food Chem 53 43-50

Tonnesen HH (1991) Photochemical degradation of components in drug formulations

1 An approach to the standardization of degradation studies Pharmazie 46 263-265

Tonnesen HH (2002) Photodecomposition of Drugs In Swarbrick J Boylan JC

Eds Encyclopedia of Pharmaceutical Technology Vol 3 Marcel Dekker New

York pp 2197-2203

Tonnesen HH Ed (2004) The Photostability of Drugs and Drug Formulations

Culinary and Hospitality Industry Publications Services Weimex TX

Tonnesen HH Karlsen J (1997) A comment on photostability testing according to

ICH guideline calibration of light sources PharmEuropa 9 735-736

Tonnesen HH Moore DE (1993) Photochemical degradation of components in drug

formulations Pharm Technol Int 5 27-33

Tournas JA Lin FH Burch JA Selim MA Monteiro-Riviere NA Zielinski

JE Pinnell SR (2006) Ubiquinone idebenone and kinetin provide ineffective

221

photoprotection to skin when compared to a topical antioxidant combination of

vitamins C and E with ferulic acid J Invest Dermatol 126 1185-1187

Traber MG (2007) Vitamin E In Zempleni J Rucker RB McCormick DB

Suttie JW Eds Handbook of Vitamins 4th ed Taylor amp Francis CRC Press

Boca Raton FL Chap 4

Traikovich SS (1999) Use of topical ascorbic acid and its effect on photodamaged skin

topography Arch Otolaryngol Head Neck Surg 125 1091-1098

Treadwell GE Cairns WL Metzler DE (1968) Photochemical degradation of

flavins V Chromatographic studies of the products of photolysis of riboflavin J

Chromatogr 35 376-388

Truswell AS (2003) ABC of Nutrition 4th ed BMJ Publishing Group Chap 10

Tsao CS Young M (1985) Analysis of ascorbic acid derivatives by high-performance

liquid chromatography with electrochemical detection J Chromatogr 13 855-856

Underberg WJM (1978) Oxidative degradation of pharmaceutically important

phenothiazines III Kinetics and mechanism of promethazine oxidation J Pharm Sci

67 1133-1138

Urso ML Clarkson PM (2003) Oxidative stress exercise and antioxidant

supplementation Toxicology 189 41-54

Vaid FHM (1998) Photolysis and Interaction of Thiamine Hydrochloride with

Riboflavin in Aqueous Solution PhD Thesis University of Karachi Karachi

Vaid FHM Shaikh RH Ansari IA Ahmad I (2005) Spectral study of the

photolysis of aqueous thiamine hydrochloride and ascorbic acid solution in the

presence and absence of riboflavin J Chem Soc Pak 27 227-232

222

Vaid F H M Shaikh RH Ansari I A Ahmad I (2006) Chromatographic study of

the photolysis of aqueous thiamine hydrochloride and ascorbic acid solutions in the

presence and absence of riboflavin J Chem Soc Pak 28 481-464

Valvani SC (2000) Industrial stability testing in the United States and computerization

of stability data In Carstensen JT Rhodes CT Eds Drug Stability Principles

and Practices 3rd

ed Marcel Dekker New York pp 534-536

Vanderslice JT Higgs DJ (1988) Chromatographic separation of ascorbic acid

isoascorbic acid dehydroascorbic acid and dehydroisoascorbic acid and their

quantitation in food products J Micronutr Anal 4 109-118

Vanderslice JT Higgs DJ Hayes JM Block G (1990) Ascorbic acid and

dehydroascorbic acid content of foods-as-eaten J Food Comp Anal 3 105-118

Vanderslice JT Higgs DJ (1993) Quantitative determination of ascorbic acid

dehydroascorbic acid by HPLC in foods and other matrices J Nutr Biochem 4 184-

190

Varvaresou A Tsirivas E Iakovou K Gikas E Papathomas Z Vonaparti A

Panderi I (2006) Development and validation of a reversed-phase ion-pair liquid

chromatography method for the determination of magnesium ascorbyl phosphate and

melatonin in cosmetic creams Anal Chim Acta 573-574 284-290

Verma KK Jain A Verma A Chaurasia A (1991) Spectrophotometric

determination of ascorbic acid in pharmaceuticals by background correction and flow

injection Analyst (London) 116 641-645

Vimaladevi M (2005) Textbook of Cosmetics CBS Publishers New Dehli Chap 2

and 17

223

Wallwork SC Grant DJW (1977) Physical Chemistry for Students of Pharmacy and

Biology 3rd ed Longmann New York pp 502-505

Waugh WA King CG (1932) Isolation and identification of vitamin C J Biol

Chem 97 325-331

Wechtersbach L Cigic B (2007) Reduction of dehydroascorbic acid at low pH J

Biochem Biophys Methods 70 767-772

Weiner N (1996) Introduction In Liberman HA Rieger MM Banker GS Eds

Pharmaceutical Dosage Forms Disperse Systems 2nd ed vol1 Marcel Dekker

New York Chap1

Werner W (1949) The reaction of l-ascorbic and d-isoascorbic acid with nicotinic acid

and its amide J Org Chem 14 22-26

Wiechers JW (2003) Formulating for Efficacy Proceedings of the 2003 IFSCC

Conference Seoul Korea

Wiechers JW (2005) Optimizing skin delivery of active ingredients from emulsions

from theory to practice In Rosen MR Ed Delivery System Handbook for

Personal Care and Cosmetic Products-Technology Applications and Formulations

William Andrew Inc Norwich NY Chap 20

Wille JJ (2005) Thixogel-Novel topical delivery systems for hydrophobic plant actives

In Rosen MR Ed Delivery System Handbook for Personal Care and Cosmetic

Products-Technology Applications and Formulations William Andrew Inc

Norwich NY Chap 36

224

Wright JL (1986) Vitamin A monograph In Connors KA Amidon GL Stella

VJ Eds Chemical Stability of Pharmaceuticals A Handbook for Pharmacists 2nd

ed Wiley New York pp 791-803

Yao AA Wathelet B Thonart P (2009) Effect of protective compounds on the

survival electrolyte leakage and lipid degradation of freeze-dried Weissella

paramesenteroides LC11 during storage J Microbiol Biotechnol 19 810-817

Yeh SY Lach JL (1961) Stability of morphine in aqueous solutions III Kinetics of

morphine degradation in aqueous solution J Pharm Sci 50 35-42

Yoshimura A Ohno T (1988) Lumiflavin-sensitized photooxygenation of indole

Photochem Photobiol 48 561-565

Yoshioka S Stella VJ (2000) Stability of Drugs and Dosage Forms Kluwer

Academic Plenum Publishers New York Chap 2

Zaeslein C (1982) Vitamins in the Field of Medicine Hoffman La Roche Basle pp

89-96

Zeng W Martinuzzi F MacGregor A (2005) Development and application of a novel

UV method for the analysis of ascorbic acid J Pharm Biomed Anal 36 1107-1111

Zhang GF Chen HY (2000) Chemiluminescence studies of the oxidation of ascorbic

acid with copper (II) catalyzed by halide anions and its applications to the

determination of halide anions and ascorbic acid Anal Sci 16 1317-1321

Zhang L Lerner S Rustrum WV Hofmann GA (1999) Electroporation-mediated

topical delivery of vitamin C for cosmetic applications Bioelectrochem Bioenerg

48 453-461

225

Zilva SS (1932) The non-specificity of the phenolindophenol reducing capacity of

lemon juice and its fractions as a measure of their antiscorbutic activity Biochem J

26 1624-1627

226

AUTHORrsquoS PUBLICATIONS

The author obtained his B Pharm degree in 2003 and joined the post graduate

program securing an M Phil degree in Pharmaceutics in 2006 from Baqai Medical

University He is a co-author of following publications

CHAPTER IN BOOK

1 Chapter on ldquoBorate Toxicity Effect on Drug Stability and Analytical

Applicationsrdquo by Iqbal Ahmad Sofia Ahmed MA Sheraz and Faiyaz H M

Vaid In Handbook on Borates Chemistry Production and Applications (MP

Chung Ed) Nova Science Publishers Inc NY USA (in press)

PAPERS PUBLISHED

INTERNATIONAL

2 Iqbal Ahmad Sofia Ahmed MA Sheraz and Faiyaz HM Vaid ldquoEffect of Borate

Buffer on the Photolysis of Riboflavin in Aqueous Solutionrdquo Journal of

Photochemistry and Photobiology B Biology 93 82-87 (2008)

3 Iqbal Ahmad Sofia Ahmed MA Sheraz M Aminuddin and Faiyaz HM Vaid

ldquoEffect of Caffeine Complexation on the Photolysis of Riboflavin in Aqueous

Solution A Kinetic Studyrdquo Chemical and Pharmaceutical Bulletin 57 (2009)

published online September 14 2009

4 Iqbal Ahmad MA Sheraz Sofia Ahmed and Faiyaz HM Vaid ldquoAnalytical

Applications of Boratesrdquo Materials Science Research Journal (in press)

5 Iqbal Ahmad Sofia Ahmed MA Sheraz Kefi Iqbal and Faiyaz HM Vaid

ldquoPharmacological Aspects of Boratesrdquo International Journal of Medical and

Biological Frontiers (in press)

6 Iqbal Ahmad Sofia Ahmed MA Sheraz Faiyaz HM Vaid and Izhar A Ansari

ldquoEffect of Divalent Ions on Photodegradation Kinetics and Pathways of

Riboflavin in Aqueous Solutionrdquo Photochemical and Photobiological Sciences

accepted

227

NATIONAL

7 Sofia Ahmed MA Sheraz and Iqbal Ahmad ldquoAdvances in Antioxidant Activity of

Vitamin Erdquo Journal of Baqai Medical University 10 13-18 (2007)

8 MA Sheraz Sofia Ahmed and Iqbal Ahmad ldquoDevelopments in the Clinical and

Food Analysis of Vitamin Crdquo Journal of Baqai Medical University 10 19-24

(2007)

9 A Azmi SNH Naqvi M Usman MA Sheraz and Sofia Ahmed ldquoPancreatic

Glucagon in Certain Ungulates Comparative Study of Extraction and

Bioassayrdquo Pakistan Journal of Entomology 20 23-28 (2005)

10 Iqbal Ahmad Sofia Ahmed MA Sheraz Faiyaz HM Vaid and S Hasan

ldquoAdvances in Biochemical Functions and the Photochemistry of Flavins and

Flavoproteinsrdquo Pakistan Journal of Pharmaceutical Sciences in press

11 MA Sheraz Sofia Ahmed and Iqbal Ahmad ldquoEffect of Borates on the Stability of

Chemical and Pharmaceutical Compoundsrdquo Journal of Baqai Medical University

accepted

PAPERS SUBMITTED

12 Iqbal Ahmad MA Sheraz Sofia Ahmed Riaz H Shaikh and Faiyaz HM Vaid

ldquoPhotostability of Ascorbic Acid in Organic Solvents and Cream Formulationsrdquo

Chemical and Pharmaceutical Bulletin

13 Iqbal Ahmad MA Sheraz Sofia Ahmed Riaz H Shaikh and Faiyaz HM Vaid

ldquoPhotochemical Interaction of Ascorbic Acid with Riboflavin Nicotinamide and

Alpha-Tocopherol in Cream Formulationsrdquo Journal of Cosmetic Science

14 Iqbal Ahmad Kefi Iqbal Sofia Ahmed MA Sheraz ldquoApplications of Laser Flash

Photolysis Spectroscopy and Electron Microscopy in Photopolymerization and

Development of Glass Ionomer Dental Cementsrdquo Materials Science Research

Journal

15 Sofia Ahmed MA Sheraz M Aminuddin I Ahmad and Faiyaz HM Vaid ldquoA

Rapid Titrimetric Assay for Quantitation of Vitamin B1 in Neat and

Pharmaceutical Preparationsrdquo Pakistan Journal of Pharmaceutical Sciences

• 01 SZ-786
• 02 SZ-title
• 03 SZ-Certificate
• 04 SZ-Abstract
• 05 SZ-Acknowledgement
• 06 SZ-Dedication
• 07 SZ-Contents
• 08 SZ-Chapter 1
• 09 SZ-Chapter 2
• 10 SZ-Chapter 3
• 11 SZ-Object of Present Investigation
• 12 SZ-Chapter 4
• 13 SZ-Chapter 5
• 14 SZ-Chapter 6
• 15 SZ-Chapter 7
• 16 SZ-Conclusion
• 17 SZ-References
• 18 SZ-Authors Publications

vii

I feel immense pleasure to pay my sincere and special thanks to Ms Sofia

Ahmed Assistant Professor and In charge Department of Pharmaceutics who lent all

sort of cooperation and spared no effort in helping me during this work

Special thanks are due to Mr Saif-ur-Rehman Khatak Deputy Drug Controller

for his cooperation and help during this study

I acknowledge with sincere thanks the contribution of Tabros Pharmaceutical

Industry Karachi for providing me the opportunity to use their facilities for certain

measurements without which the completion of this work would not have been possible

I highly appreciate the technical services rendered by Mr Anees Mr Wajahat

and Mr Sajjad in pursuance of this study

I am very grateful to Mrs Prof Dr Iqbal Ahmad for her kindness and generous

hospitality during my innumerable visits to their residence

Last but not the least I would like to express my immense indebtedness to My

Gracious Parents Beloved Brothers and Sisters for their moral support kindness and

encouragement throughout my career

I am also thankful to all my students for their affectionate feelings

M A S

viii

To

My Beloved Parents amp

Late Prof Dr S Sabir Ali for their interest and endless support

ix

CONTENTS

Chapter Page

ABSTRACT iv

ACKNOWLEDGEMENTS vi

I INTRODUCTION 1

11 HISTORICAL BACKGROUND 2

12 PHYSICOCHEMICAL CHARACTERISTICS OF

ASCORBIC ACID

2

13 CHEMISTRY OF ASCORBIC ACID 3

131 Nomenclature and Structure 3

132 Chemical Stability 3

14 BIOCHEMICAL FUNCTIONS 7

15 ANTIOXIDANT ACTIVITY 8

16 PHOTOSTABILITY OF DRUGS 9

17 KINETIC TREATMENTS OF PHOTOCHEMICAL

REACTIONS

12

18 LITERATURE ON ASCORBIC ACID 15

II PHOTODEGRADATION REACTIONS AND ASSAY OF

ASCORBIC ACID

17

21 PHOTODEGRADATION REACTIONS 18

211 Photodegradation of Ascorbic Acid 18

212 Effect of Various Substances on Photodegradation of Ascorbic

Acid

20

213 Photosensitized Oxidation of Ascorbic Acid 22

214 Ascorbic Acid Interaction with Other Water-Soluble Vitamins 25

22 ASSAY OF ASCORBIC ACID 26

221 Spectrophotometric Methods 26

222 Fluorimetric Methods 28

x

223 Mass spectrometric Methods 28

224 Chromatographic Methods 28

225 Enzymatic Methods 29

226 Commercial Kits for Clinical Analysis 30

227 Analysis in Creams 30

III FORMULATION AND STABILITY OF CREAM

PREPARATIONS

31

31 FORMULATION OF CREAM PREPARATIONS 32

311 Choice of Emulsion Type 32

312 Choice of Oil Phase 33

313 Emulsion Consistency 33

314 Choice of Emulsifying Agent 34

315 Formulation by the HLB Method 34

316 Concept of Relative Polarity Index 35

32 FORMULATION OF ASCORBIC ACID CREAMS 37

33 STABILITY OF CREAMS 39

331 Physical Stability 39

332 Chemical Stability 39

333 Microbial Stability 40

334 Stability of Ascorbic Acid in Liquid Formulations 41

335 Stability of Ascorbic Acid in Emulsions and Creams 41

336 Stability Testing of Emulsions 45

3361 Macroscopic examination 46

3362 Globule size analysis 46

3363 Change in viscosity 46

3364 Accelerated stability tests 46

337 FDA Guidelines for Semisolid Preparations 46

xi

OBJECT OF PRESENT INVESTIGATION 48

IV MATERIALS AND METHODS 51

41 MATERIALS 52

42 METHODS 55

421 Cream Formulations 55

422 Preparation of Creams 56

423 Thin-Layer Chromatography 57

424 pH Measurements 57

425 Ultraviolet and Visible Spectrometry 58

426 Photolysis of Ascorbic Acid 59

4261 Creams 59

4262 Aqueous and organic solvents 59

4263 Storage of creams in dark 59

427 Measurement of Light Intensity 59

428 Procedure 60

4281 Calculation 62

429 Viscosity Measurements 63

4210 Assay method 65

42101 UV spectrophotometric method for the assay of creams

containing ascorbic acid alone

65

42102 Iodimetric method for the assay of ascorbic acid in creams

containing riboflavin nicotinamide and alpha-tocopherol 65

42103 Spectrophotometric method for the assay of ascorbic acid in

aqueous and organic solvents

67

V PHOTODEGRADATION OF ASCORBIC ACID IN

ORGANIC SOLVENTS AND CREAM FORMULATIONS

68

51 INTRODUCTION 69

52 PHOTOPRODUCTS OF ASCORBIC ACID 71

xii

53 SPECTRAL CHARACTERISTICS OF PHOTOLYSED

SOLUTIONS

71

54 ASSAY OF ASCORBIC ACID IN CREAMS AND

SOLUTIONS

73

55 EFFECT OF SOLVENT 74

56 EFFECT OF CONCENTRATION 80

57 EFFECT OF CARBON CHAIN LENGTH OF

EMULSIFYING AGENT

88

58 EFFECT OF VISCOSITY 94

59 EFFECT OF pH 94

510 EFFECT OF REDOX POTENTIAL 96

511 PRIMARY PHOTOCHEMICAL REACTIONS IN THE

OXIDATION OF ASCORBIC ACID

97

512 DEGRADATION OF ASCORBIC ACID IN THE DARK 98

VI PHOTOCHEMICAL INTERACTION OF ASCORBIC

ACID WITH RIBOFLAVIN NICOTINAMIDE AND

ALPHA-TOCOPHEROL IN CREAM FORMULATIONS

109

61 INTRODUCTION 110

62 ABSORPTION CHARACTERISTICS OF PHOTOLYSED

CREAMS

114

63 PHOTOPRODUCTS OF ASCORBIC ACID AND OTHER

VITAMINS

114

64 ASSAY METHOD 116

65 KINETICS OF PHOTODEGRADATION OF ASCORBIC

ACID

117

66 INTERACTION OF RIBOFLAVIN WITH ASCORBIC ACID 128

67 INTERACTION OF NICOTINAMIDE WITH ASCORBIC

ACID

129

68 INTERACTION OF ΑLPHA-TOCOPHEROL WITH

ASCORBIC ACID

130

69 EFFECT OF CARBON CHAIN LENGTH OF

EMULSIFYING AGENT

130

xiii

610 EFFECT OF VISCOSITY OF CREAMS 132

611 DEGRADATION OF ASCORBIC ACID IN THE PRESENCE

OF OTHER VITAMINS IN THE DARK

135

VII STABILIZATION OF ASCORBIC ACID WITH CITRIC

ACID TARTARIC ACID AND BORIC ACID IN CREAM

FORMULATIONS

141

71 INTRODUCTION 142

72 CREAM FORMULATIONS 142

73 PRODUCTS OF ASCORBIC ACID

PHOTODEGRADATION

145

74 SPECTRAL CHANGES IN PHOTODEGRADED CREAMS 145

75 ASSAY OF ASCORBIC ACID IN CREAMS 145

76 KINETICS OF PHOTODEGRADATION 146

77 EFFECT OF STABILIZING AGENTS 146

78 DEGRADATION OF ASCORBIC ACID IN PRESENCE OF

STABILIZING AGENTS IN THE DARK

162

79 EFFECT OF ADDITIVES ON TRANSMISSION OF

ASCORBIC ACID

163

CONCLUSIONS AND SUGGESTIONS 179

CONCLUSIONS 180

SUGGESTIONS 184

REFERENCES 185

AUTHORrsquoS PUBLICATIONS 226

CHAPTER I

INTRODUCTION

2

11 HISTORICAL BACKGROUND

The disease scurvy which now is known as a condition due to a deficiency of

ascorbic acid in the diet has considerable historical significance (Schick 1943

Carpenter 1986 Bardolph and Taylor 1997 Thomas 1997 Bors 2005) Zilva (1932)

isolated the antiscorbutic activity factor from a crude fraction of lemon and showed that

the activity was destroyed by oxidation and protected by reducing agents Waugh and

King (1932) isolated crystalline vitamin C from lemon juice and showed it to be the

antiscorbutic factor Szent-Gyorgyi (1928) had isolated the same factor from pepper in

connection with his biological oxidation-reduction studies Hirst and Zilva (1933)

identified the antiscorbutic factor as ascorbic acid Early work on the chemical

identification and elucidation of the structure of ascorbic acid has been well documented

(Carpenter 1986) The first synthesis of L-ascorbic acid was achieved almost

simultaneously by Ault et al (1933) and Reichstein et al (1933)

Plants and most animals synthesize their own vitamin C but humans lack this

ability due to the deficiency in an enzyme L-gulono-gamma-lactone oxidase that

catalyzes the terminal step in ascorbic acid biosynthesis (Nishikimi et al 1994)

Therefore humans obtain this vitamin from diet and or vitamin supplements to not only

avoid the development of scurvy but also for overall well being (Stone 1969 Lewin

1976 Davies et al 1991) The minimal daily requirement for ascorbic acid in healthy

adults is 40ndash60 mg (Truswell 2003 Mason 2007 Eitenmiller et al 2008 Elia 2009)

12 PHYSICOCHEMICAL CHARACTERISTICS OF ASCORBIC ACID

The important physicochemical characteristics of ascorbic acid (Table 1) involved

in its identification and degradation are described by many authors (Connors et al 1986

3

OrsquoNeil 2001 Moffat et al 2004 Sinko 2006 Johnston et al 2007) The most

important chemical property of ascorbic acid is the reversible oxidation to semidehydro-

L-ascorbic acid and further oxidation to dehydro-L-ascorbic acid This property is the

basis for its physiological activity In addition the proton on oxygenndash3 is acidic (pKa1 =

417) which contributes to the acidic nature of ascorbic acid (1)

13 CHEMISTRY OF ASCORBIC ACID

131 Nomenclature and Structure

The IUPAC-IUB Commission on Biochemical Nomenclature changed the name

vitamin C (2-oxo-L-theo-hexono-4-lactone-23-enediol) to ascorbic acid or L-ascorbic

acid in 1965 (Johnston et al 2007) The chemical structure of ascorbic acid (1) is

HO OH

O

OHHO

H

(1)

O

The molecule has a near planar five-membered ring with two chiral centers

which contain four stereoisomers

132 Chemical Stability

Ascorbic acid is sensitive to air and light and is kept in a well-closed container

protected from light (British Pharmacopoeia 2009) The degradation reactions of

ascorbic acid in aqueous solution depend on a number of factors such as pH temperature

presence of oxygen or metal It is not very stable in aqueous media at room

temperature and undergoes oxidative degradation to dehydroascorbic acid and

4

Table 1 Physicochemical characteristics of ascorbic acid

Empirical formula C6H8O6

Molar mass 17613

Crystalline form Monoclinic mix of platelets and needles

Melting point 190 to 192 degC

[α]25

+205deg to +215deg

pH

5 mg ml

50 mg ml

~3

~2

pKa 417 1157 (20deg)

Redox potential

(dehydroascorbic acid ascorbate)

(H+ ascorbate

ndash)

ndash174 mV

+282 mV

Solubility g ml

Water

Ethanol absolute

Ether chloroform benzene

033

002

Insoluble

UV spectrum

Absorption maximum [A(1 1 cm)]

pH 20

pH 70

245 nm [695]

265 nm [940]

Infrared spectrum

Principal peaks (Nujol mull)

1026 (CminusOH str) 1111(CminusOminusC str) 1312

(minusCminusOminus str) 1653 (C=O str) 990 (C=C str)

cmndash1

Mass spectrum

Principal ions at mz

29 41 39 42 69 116 167 168

D

5

23-diketogulonic acid The stability of ascorbic acid and dehydroascorbic acid can be

improved by lowering the pH below 2 (Wechtersbach and Cigic 2007) Above pH 7

alkali-catalyzed degradation by cleavage at Cndash1 or Cndash2 results in a number of

compounds mainly monondash dindash and tricarboxylic acids (Connors et al 1986 Bors and

Buettner 1997 Halliwell and Whiteman 1997) The oxidative degradation of ascorbic

acid and dehydroascorbic acid in parenteral nutrition mixtures is catalyzed by trace

elements particularly copper (Allwood 1984ab Allwood et al 1992 Allwood and

Kearney 1998 Kearney et al 1998 Gibbons et al 2001) Stabilized ascorbic acid

preparations in hydroalcoholic vehicle (Kaplan et al 1989) and aquaculture feeds

(OrsquoKeefe 2001) have been reported The various oxidation products of ascorbic acid are

shown in Fig 1

It is interesting to note that in addition to redox and acid-base properties ascorbic

acid can exist as a free radical (Bielski et al 1981 Bielski 1982 Halliwell 1996 Bors

and Buettner 1997) The ascorbate radical anion is an important intermediate in the

reactions involving oxidants and ascorbic acidrsquos antioxidant activity Rate constants for

the generation of ascorbate radicals are in the range of 104ndash10

8 s

ndash1 When ascorbate

radicals are generated by oxyanions the rate constants are of the order of 104ndash10

7 s

ndash1

when generated by halide radicals 106ndash10

8 s

ndash1 and when generated by tocopherols and

flavonoids radicals 106ndash10

8 s

ndash1 (Bielski 1982 Halliwell and Whiteman 1997) The

ascorbate radicals decay usually by disproportionation However a change in ionic

strength or pH can influence the rate of dismutation of ascorbic acid Certain oxyanions

such as phosphates accelerate dismutation (Bielski et al 1981) The acceleration is

attributed to the activity of various protonated forms of phosphate to donate a proton

6

Fig 1 Oxidation products of ascorbic acid

O

OHOH

H

OO

OHOH

H

OO

OHOH

H

O

Ascorbyl radical anion

(interm ediate)

Ascorbic acid

(1)

-e- -2H

+

+e- +2H

+

-e-

+e-

Dehydroascorbic acid

(2)

23-diketo-L-gulonic acid

O xalic acid

+

L-Threonic acid

L-Xylose

+

C O 2

CO 2

L-Xylonic acid

+

L-Lyxonic acid CO 2

HO OH O O-

O O

7

efficiently to the ascorbate radical particularly the dimer form of ascorbate

The unusual stability of the ascorbate radical in biological systems dictates that

accessory enzymatic systems be made available to reduce the potential transient

accumulation of ascorbate radical The excess ascorbate radicals may initiate a chain of

free-radicals reactions In plants NADHmonodehydroascorbate reductase maintains

ascorbic acid in its reduced form NADHmonodehydroascorbate reductase plays a major

role in stress related responses in plants Glutathione dehydroascorbate reductase serves

this purpose in animal tissues Such enzymes keep ascorbic acid operating at maximum

efficiency so that other enzyme systems may take advantage of the univalent redox

cycling capacity of ascorbate (Asard et al 2004 Johnston et al 2007)

The anaerobic degradation of ascorbic acid has been studied by Finholt et al

(1963) Under these conditions the molecule is dehydrated and hydrolyzed in aqueous

solution to give furfural and carbon dioxide The rate of degradation is maximum at pH

41 corresponding to the pKa of ascorbic acid This has been suggested due to the

formation of a saltndashacid complex in solution The reaction is dependent on buffer

concentration but has relatively small effect of ionic strength

14 BIOCHEMICAL FUNCTIONS

Ascorbic acid plays an essential role in the activities of several enzymes It is vital

for the growth and maintenance of healthy bones teeth gums ligaments and blood

vessels It is important for the manufacture of certain neurotransmitters and adrenal

hormones Ascorbic acid is required for the utilization of folic acid and the absorption of

iron It is also necessary for normal immune responses to infection and for wound healing

(Henry 1997)

8

Ascorbic acid deprivation and scurvy include a range of signs and symptoms that

involves defects in specific enzymatic processes (Johnston et al 2007) The

administration of ascorbic acid improves most of the signs of chemically induced

glutathione (L-γ-glutamyl-L-cysteine-glycine GSH) deficiency (Meister 1994) The

effect is very pronounced in newborn rats which do not efficiently synthesize ascorbic

acid in contrast to adult rats and guinea pigs When L-buthionine-(SR)-sulphoxime is

administered in addition to the loss in GSH there is a marked increase in

dehydroascorbic acid This has led to the hypothesis that GSH is very important to

dehydroascorbic acid reduction and as a sequence to ascorbic acid recycling (Meister

1995)

Ascorbic acid also possesses pro-oxidant properties and may cause apoptosis

lymphoid and myeloid cells It has been shown that dehydroascorbic acid also stimulates

the antioxidant defenses in some cells by preferentially importing dehydroascorbate over

ascorbate (Braun et al 1997 Banhegyi et al 1998 Puskas et al 2000 2002)

15 ANTIOXIDANT ACTIVITY

Ascorbic acid is known to readily scavenge reactive oxygen and nitrogen species

such as superoxide and hydroperoxyl radicals aqueous peroxyl radicals singlet oxygen

ozone peroxynitrite nitrogen dioxide nitroxide radicals and hypochlorous acid Excess

of such products has been associated with lipids (Niki and Noguchi 1997 Carr et al

2000 Urso and Clarkson 2003) DNA (Fraga et al 1991 1996 Lindahl 1993) and

protein oxidation (Stadtman 1991 Berlett and Stadtman 1997 Dean et al 1997

Ortwerth and Monnier 1997 Padayatty et al 2003)

9

The electron donor character of ascorbate may be responsible for many of its

known biological functions Inspite of the availability of ascorbic acid to influence the

production of hydroxyl and alkoxyl radicals it remains uncertain whether this is the

principal effect or mechanism that occurs in vivo There is a good evidence that ascorbic

acid protects lipids in biological fluids as an antioxidant (Johnston et al 2007) A

detailed account of the function of ascorbate as an antioxidant and its reactions with

reactive nitrogen species and singlet oxygen has been reported by Packer et al (2002)

and Buettner and Schafer (2004)

Ascorbic acid (Eordm ndash0115 V pH 52 Sinko 2006) has been used as an antioxidant

for the stabilization of drugs with a higher oxidation potential These drugs include

morphine (Yeh and Lach 1961) vitamin A (Wright 1986) rifampin (Maggi et al

1966) cholecalciferol (Nerlo et al 1968 Sawicka 1991) promethazine (Underberg

1978) and sulphacetamide and sulphanilamide (Ahmad and Ahmad 1983)

16 PHOTOSTABILITY OF DRUGS

Many drug substances are sensitive to light (British Pharmacopoeia 2009) and

may degrade in pharmaceutical formulations to inactive or toxic compounds This could

make a product therapeutically inactive while in use by the patients The

photodegradation (photolysis) of drug substances may occur not only during storage but

also during the use of the product It may involve several mechanisms including

oxidation reduction hydrolysis decarboxylation isomerization rearrangement and other

reactions Normal sunlight or room light may cause substantial degradation of drug

molecules The study of degradation of drug substances under the action of UVvisible

light is relevant to the process of drug development for several reasons such as

10

Exposure to light can influence the stability of a drug formulation resulting in the

loss of potency

Inappropriate exposure to light of the raw material or the final product can lead to

the formation of toxic photoproducts that are dangerous to health

Information about the stability of drug substances and formulations is needed to

predict the shelf-life of the final product (Tonnesen and Moore 1993)

The development of light-activated drugs involves activation of the compound

through photochemical reactions (Tonnesen 1991)

Adverse effects due to the formation of minor degradation products during

storage and administration have been reported (de Vries et al 1984) The drugs

substances may also cause light-induced side effects after administration to the patient by

interaction with endogenous substances The study of the photochemical properties of

drug substances and formulated products is an integral part of formulation development

to ensure the safety and efficacy of the product

The photodegradation of drug substances occurs as a result of the absorption of

radiation energy by a molecule (A) to produce an excited state species (A) (11) The

absorbed energy can be lost either by a radiative process involving fluorescence or

phosphorescence (12) or by a physical or chemical radiationless process The physical

process results in the loss of energy as heat (13) or by collisional quenching (14) The

chemical decay leads to the formation of a new species (15) The whole process is

represented as

11

A A (11)

A A + hυprime (12)

A A + heat (13)

A + A 2A (14)

A product (s) (15)

According to the Stark-Einstein law the absorption of one quantum of radiation

results in the formation of one excited molecule which may take part in several

photochemical processes [Eqs (11)ndash(15)] The quantum yield φ for any one of these

processes is defined by

Number of molecules undergoing the photochemical process φ =

Number of quanta absorbed

Considering a pure photochemical reaction the quantum yield has a value of 0ndash1

however if A is a radical that can take part in a free-radical chain reaction so that the

absorption of energy simply initiates the reaction then each quantum of energy may

result in the decomposition of molecules and φ may appear to be greater than 1 (Connors

et al 1986)

Detailed information on the photostability and photodegradation of drug

substances including vitamins alone or in solid or liquid formulations is available in the

reviews published by DeRitter (1982) Albini and Fasani (1998) Sequeira and Vozone

(2000) Tonnesen (2002 2004) Yoshioka and Stella (2002) Min and Boff (2002) Reed

et al (2003) Fasani and Albini (2005) and Sinko (2006) The photostability of cosmetic

materials has been reviewed by Sugden (1985) Important aspects dealing with the

photostability testing of drug substances have been dealt by Anderson et al (1991)

k1

k2

k3

k4

hυ

12

Tonnesen and Moore (1993) Tonnesen and Karlsen (1997) Riehl et al (1995) ICH

(1997) Singh and Bakshi (2000) Valvani (2000) Thatcher et al (2001ab) Fasani and

Albini (2005) Klick et al (2005) Singh (2006) and Ahmad and Vaid (2006)

17 KINETIC TREATMENT OF PHOTOCHEMICAL REACTIONS

The kinetic treatment of photochemical reactions with reference to the

photostability of drug substances has been considered by Moore (2004) and is presented

in this section

The photostability testing of a drug substance at the preformulation stage involves

a study of the drugrsquos rate of degradation in solution on exposure to light for a period of

time The value of the degradation rate constant depends very much on the design of the

experimental conditions (eg concentration solvent pH irradiation source oxygen

content) The factors that determine the rate of a photochemical reaction are simply the

rate at which the radiation is absorbed by the test sample (ie the number N of photons

absorbed per second) and the efficiency of the photochemical process (ie the quantum

yield of the reaction φ) For a monochromatic photon source the number of photons

absorbed depends upon the intensity of the photon source and the absorbance at that

wavelength of the absorbing species The rate of a photochemical reaction is defined as

Rate = number of molecules transformed per second = N φ (16)

In the first instance the rate can be determined for a homogeneous liquid sample

in which the only photon absorption is due to the drug molecule undergoing

transformation with the restriction that the concentration is low so that the drug does not

absorb all of the available radiation in the wavelength range corresponding to its

13

absorption spectrum The value of N can be derived at a particular wavelength λ and is

given by

Nλ = Iλ ndash It = Iλ (1 ndash 10ndashA

) (17)

where Iλ and It are the incident and transmitted radiation intensities respectively and A is

the absorbance of the sample at the wavelength of irradiation This expression can be

expanded as a power series

Nλ = 2303 Iλ (A + A22 + A

36 + hellip) (18)

When the absorbance is low (Alt 002) the second- and higher-order terms are negligible

and the expression simplifies to the first term in Eq 18 Given the Beerrsquos law relation

between absorbance and concentration N can be seen to be directly proportional to

concentration

Nλ = 2303 Iλ A = 2303 Iλ ελ b C (19)

where ελ is the molar absorptivity at wavelength λ C the molar concentration of the

absorbing species and b the optical path length of the reaction vessel Now Iλ and ελ vary

with wavelength so the expression must be integrated over the relevant wavelength range

where each has a non-zero value

N = 2303 b C int (Iλ ελ) dλ integrated from λ1 to λ2 (110)

Thus

Rate = 2303 b C φ int (Iλ ελ) dλ (111)

Now the overlap integral (int Iλ ελ dλ) is a constant for a particular combination of photon

source and absorbing substance b is determined by the reaction vessel chosen and φ is a

characteristic of the reaction Thus by grouping the constant terms into an overall

constant k1 the expression is simplified to a first-order kinetic equation

14

Rate = ndashd [Drug] dt = k1C (112)

The integrated form of Eq 112 can be expressed in exponential form (Eq 113) or

logarithmic form (Eq 114)

[Drug]t = [Drug]0 endashk1t

(113)

ln [Drug]t = ln [Drug]0 ndash k1t (114)

Verification of first-order kinetics is obtained when a plot of the logarithm of the

concentration of drug remaining is linear with slope equal to (ndashk1)

Eq 112 predicts that a photodegradation reaction studied at low concentrations in

solution will follow first-order kinetics however the rate constant derived from a study

performed in one laboratory will not be the same as that found in another The reason for

this is the inherent difficulty in reproducing exactly the experimental arrangement of

photon source and sample irradiation geometry Therefore the relative values of the rate

constants are useful in a given experimental arrangement for making comparisons of

degradation of the absorbing substance in different formulations eg those containing

ingredients designed to inhibit the photoreaction The use of rate constants is helpful for

comparative purposes when studying a number of different reaction mixtures under the

same irradiation conditions such as the effect of pH on the degradation of a drug

However the reaction order and numerical values of the rate constants are relative to the

specific conditions used

15

18 LITERATURE ON ASCORBIC ACID

A large number of reviews have been published on various aspects of ascorbic

acid A list of important reviews is given below

Chemistry biochemical functions and related aspects

Rosenberg (1945) Burns (1961) King and Burns (1975) Sim (1972) Hanck

(1982) Zaeslein (1982) Seib and Tolbert (1982) Carpenter (1986) Levine

(1986) Davies et al (1991) Halliwell and Whiteman (1997) Ortega and Delgado

(1998) Asard et al (2004) Hickey and Roberts (2004) Johnston et al (2007)

Eitenmiller (2008)

Chemical and pharmaceutical stability

Macek (1960) Garrett (1967) Carstensen (1972) Dale and Booth (1976) Hashmi

(1973) Litner (1973) DeRitter (1982) Allwood (1984ab) Allwood and Kearney

(1998) Connors et al (1986) Smith et al (1988) Racz (1989) Roth et al 1991

Ball (2006) Eitenmiller et al (2008) Sweetman (2009)

Methods of assay and chromatography

Mader (1961) Gyorgy and Pearson (1967) Bolliger and Konig (1969) Hashmi

(1973) Al-Meshal and Hassan (1982) Pelletier (1985) Lambert and deLeenheer

(1992) Halver and Felton (2001) Moffat et al (2004) Ball (2006) Eitenmiller et

al (2008)

Pharmacology and related aspects

Levine (1986) Dollery (1999) Sauberlich (1994ab) McDowell (2000)

Kaushansky and Kipps (2006) Sweetman (2009)

16

Antioxidant activity

Basu et al (1999) Shacter (2000) Thiele et al (2000) Cadenas and Packer

(2002) Packer et al (2002) Padayathy et al (2003) Parker and Parker (2003)

Burke (2006) Johnston et al (2007)

Cosmetic Preparations

Barel et al (2001) Salvador and Chisvert (2007) Rosen (2005) Bissett (2006)

Chaudhri and Jain (2009)

CHAPTER II

PHOTODEGRADATION

REACTIONS AND ASSAY

OF ASCORBIC ACID

18

21 PHOTODEGRADATION REACTIONS

211 Photodegradation of Ascorbic Acid

Aqueous ascorbic acid (1) solutions are degraded by UV light to give

dehydroascorbic acid (2) (Arcus and Zilva 1940) Ascorbic acid degradation at a

concentration of 52 and 50 mg on UV irradiation for 2 hours gave a loss of 43 and 8

respectively Dehydroascorbic acid solutions are more stable to UV light than the

ascorbic acid (Kitagawa 1968) In many natural products the vitamin is oxidized on

exposure to air and light (OrsquoNeil 2001) When solutions of multivitamin preparations are

exposed to light H2O2 as well as organic peroxides are generated and specific

byproducts that differ from dehydroascorbic acid and 23-diketogulonic acid (3) are

produced (Lavoie et al 2004)

In aqueous neutral or alkaline solution ascorbic acid (1) undergoes chemical or

photochemical oxidation to dehydroascorbic acid (2) which upon saponification of the

lactone ring under the influence of the base water produces 23-diketo-L-gulonic acid (an

α szlig- diketogulonic acid) (3) This acid undergoes further oxidation to oxalic acid (4) and

L-threonic acid (5) (Racz 1989) (Fig 2a) At room temperature oxalic acid (4) is also

formed along with threonolactone (6) by photochemical degradation of ascorbic acid (1)

in the presence of singlet oxygen (1O2) (Silva and Quina 2006) (Fig 2a) The low-

temperature photooxygenation of ascorbic acid (1) gives a mixture of unstable

hydroperoxide ketones (7) and (8) which on standing interconvert and cyclize to

hydroperoxyhemiketal (9) The hydroperoxyhemiketal breaks down on warming to

produce the oxalate esters of threonic acid (10) (Fig 2b) (Kwon and Foote 1988)

19

COOH

COOH

O

OHHO

O

HOH2C

HO2

O

O

HO

OO

O O2H

OHHO

O

HOH2C

OH

O

O

OH

O2H

OO

HO O2CCO2H

(1)hv

room temperature

(4)(6)

(1)hv

85 oC

(7)

(a)

(8)

+

cyclization

(9)

ring cleavage

(b)

(10)

(2)

OH O

OHHO

OH O O

(3)

OH OH

OH

OH O

O

OH

1O2 [O]

+

(5)

COOH

COOH

(4)

+

OH

Fig 2 Photooxidation of ascorbic acid at room and low temperature

20

An important consideration in the stability of ascorbic acid in total parenteral

nutrition (TPN) solutions is the generation of hydrogen peroxide in the presence of light

(Laborie et al 1998 1999 2000 2002 Chessex et al 2002) This may result from the

oxidation of ascorbate anion by molecular oxygen (Homann and Gaffron 1964 Taqui

Khan and Martell 1967 Mushran and Agarwal 1977 Hughes 1985 De La Rochette et

al 2000) leading to further degradation of ascorbic acid (Deutsch 1998a 1998b

1998c) The kinetics and mechanism of oxidation reactions of ascorbic acid have been

studied by several workers (Taqui Khan and Martell 1967 Ogata and Kosugi 1969

Blaugh and Hajratwala 1972 Fessenden and Verma 1978 Abe et al 1986 Kwon et al

1989 Fornaro and Coicher 1998 Njus et al 2001)

The photostability of various ascorbic acid tablets on exposure to UV light has

been studied and the influence of antioxidants and moisture on the potency loss of

ascorbic acid has been evaluated The physical characteristics of ascorbic acid tablets are

also affected on UV irradiation (Ahmad et al 1973 Jamil et al 1980ab Jamil and

Ahmad 1984)

212 Effect of Various Substances on Photodegradation of Ascorbic Acid

The oxidation-reduction reactions of ascorbic acid in the presence of riboflavin at

pH 8ndash9 under the influence of light have been studied Under these conditions ascorbic

acid is a more active H donor to riboflavin than phenolphthalein (Sibi et al 1953)

Riboflavin has been found to catalyze the photodegradation of ascorbic acid solutions

during exposure to light and air The losses of ascorbic acid are markedly increased by

the presence of Cu2+

and Fe3+

ions under light exposed and unexposed conditions (Sattar

et al 1977) A spectral study of the UV photolysis of ascorbic acid solutions in the

21

presence of riboflavin has shown that the degradation of ascorbic acid is enhanced to the

extent of about 15 (Vaid et al 2005) The influence of DL- methionine on the

photostability of ascorbic acid solutions has also been studied DL- methionine (10 mg

) enhances the photostability of ascorbic acid (40 mg ) in acetate and phosphate

buffers but not in citrate buffer at pH 45 The photoprotective action of DL-methionine

on ascorbic acid appears to be influenced by its concentration pH of the medium and the

buffer species (Asker et al 1985)

The degradation of ascorbic acid solutions on irradiation with simulated sunlight

in the presence of the food dye quinolone yellow (E 104) is enhanced However this

effect is reversed by the addition of mannitol indicating that this dye facilitates the

photogeneration of hydroxyl radicals which may cause degradation of the vitamin The

incorporation of triplet quenchers enhances the stability of substrate solutions suggesting

that the dye acts as a triplet sensitizer to facilitate the reaction (Sidhu and Sugden 1992)

The photostability of ascorbic acid solutions is enhanced by sweetening agents (mannitol

sorbitol sucrose dextrose and Canderal) at 5 wv concentration However the addition

of stoichiometric amounts of hydrogen peroxide as a source of hydroxyl radicals and 2

2rsquo-azobis (2-amidopropane) as a source of hydroperoxyl radicals results in diminished

stability of ascorbic acid solutions The diminished activity may be due to the action of

hydroperoxyl radicals in the presence of hydroxyl radical scavengers (Ho et al 1994)

Metal-complexing agents (eg disodium ethylenediaminetetraacetic acid N-

hydroxylethyl ethylenediaminetetraacetic acid 8-hydroxyquinoline) have a stabilizing

effect on the photolysis of ascorbic acid injectable solutions (Kassem et al 1969ab

22

1972) This may be due to the interaction of these agents with metal ions and other

impurities

213 Photosensitized Oxidation of Ascorbic Acid

In the presence of visible light a photosensitizer such as riboflavin can exhibit

photosensitizing properties through a mixed Type IndashType II mechanism (Yoshimura and

Ohno 1988 Foote 1991 Silva et al 1994 Silva and Quina 2006) as presented below

Type I mechanism (low oxygen concentration)

RF + hυ rarr 1RF rarr

3RF (21)

3RF + SH rarr RF

middot ndash + SH

middot + rarr RFH

middot + S

middot (22)

RFmiddot ndash

+ O2 rarr RF + O2middot ndash

(23)

2RFHmiddot rarr RF + RFH2 (24)

RFH2 + O2 rarr RF + H2O2 (25)

H2O2 + O2middot ndashrarr

ndashOH +

middotOH + O2 (26)

Smiddot and or SH

middot +

+ H2O2 O2middot ndash

rarr Soxid (27)

Type II mechanism (high oxygen concentration)

RF + hυ rarr 1RF rarr

3RF (28)

3RF + O2 rarr RF +

1O2 (29)

SH + 1O2 rarr Soxid (210)

In these reactions RF 1RF and

3RF represent RF in the ground state and in the excited

singlet and triplet states respectively RFmiddot ndash

RFHmiddot and RFH2 are the radical anion the

radical and the reduced form of RF SH is the reduced substrate and SHmiddot

+ S

middot and Soxid

23

represent the intermediate radical cation the radical and the oxidized form of the

substrate respectively

An early study of the riboflavin-sensitized photooxidation of ascorbic acid has

been carried out by flash photolysis (Heelis et al 1981) ESR spectrometry has been

used to investigate the photosensitized formation of ascorbate radicals by riboflavin (Kim

et al 1993) The photochemical behavior of a system consisting of ascorbate ion (AHndash)

and riboflavin has been studied by Mancini et al (2000) and De La Rochette et al (2000

2003) The photosensitized processes were examined as a function of oxygen pressure

and the efficiency of RF induced degradation of AHndash

at various oxygen concentrations

was compared on the basis of the respective initial photosensitization quantum yields

(Table 2)

In this reaction a Type I photosensitization mechanism (Karlsen 1996) implies a

direct electron transfer between AHndash and the RF triplet-excited state followed by the

oxidation of semioxidized ascorbyl radical (AHmiddot) by molecular oxygen or some other

reactive species On the contrary in a Type II photosensitization mechanism singlet

oxygen is produced directly by energy transfer from the RF triplet-excited state to

molecular oxygen and the singlet oxygen then oxidizes the AHndash Thus by irradiating

under increasing oxygen pressure it is possible to control the relative prevalence and

efficiency of Type I or Type II mechanisms The absence of a linear relationship between

the quantum yields of ascorbate degradation and oxygen concentration indicates that the

photosensitization mechanism involved in not exclusively Type II

24

Table 2 Initial quantum yield (φ) for ascorbate (AHndash) degradation during

photosensitization by RF (35 microM) in solutions irradiated at 365 nm and

37ordmC

O2 103 times φ (AH

ndash)a

0

5

20

14 plusmn 06

1670 plusmn 220

1940 plusmn 200

a Data are the mean plusmn SD of three independent experiments

25

In the presence of RF and O2 the quantum yields for degradation of ascorbate ion

have been found to be greater than one suggesting the participation of chain reactions

initiated by the ascorbyl radical as given by the following reactions

3RF + AH

ndash rarr RFmiddot

ndash + AHmiddot (211)

AHmiddot + O2 rarr A + HO2middot (212)

HO2middot + AHndash rarr H2O2 + AHmiddot (213)

The generation of the ascorbyl radical by the reaction between the RF excited-

triplet state and the ascorbate ion (Eq 211) is the only step that requires the absorption of

photons (to form the excited-triplet state of RF) The subsequent reactions (Eqs 212 and

213) are independent of light and lead to further degradation of the ascorbate ion In the

presence of transition metal ions such as Fe3+

in trace amounts in the buffer solution

containing RF and ascorbate ions further oxidation of ascorbate ion could also occur As

a result the reduced form of the metal ion (ie Fe2+

) can be generated by the metal

catalyzed oxidation of ascorbate ion This has been confirmed by the significant decrease

in the AHndash photooxidation quantum yield in the presence of the metal chelator EDTA

which inactivates the trace amounts of iron present in the buffer solution The quantum

yields for the photosensitized oxidation of ascorbate ion are decreased twofold at 20 O2

and fourfold at 5 O2 concentration in the presence of EDTA (Silva and Quina 2006)

Amino acids have been found to affect the photosensitized oxidation of ascorbic acid

(Jung et al 1995)

214 Ascorbic Acid Interaction with Other Water-Soluble Vitamins

The stability of ascorbic acid is reported to be enhanced in syrups containing B-

complex vitamins (Connors et al 1986) This may be due to the increased viscosity of

the syrups inhibiting the oxidation of ascorbic acid The rate of photolysis in solution

26

containing cyanocobalamin and ascorbic acid is reported to decrease with an increase in

pH (Ansari et al 2004) where as use of certain halide salts has been reported to be

beneficial in stabilizing pharmaceutical products and dietary supplements when vitamin

B12 and vitamin C are combined in solution (Ichikawa et al 2005) When a solution of

multivitamins is exposed to light it is reported that organic peroxidases are generated and

the concentration of ascorbic acid decreases (Lavoie et al 2004)

22 ASSAY OF ASCORBIC ACID

Recent accounts of the development and application of analytical methods to the

determination of ascorbic acid in pharmaceuticals biological samples and food materials

are reported in the literature (Rumsey and Levine 2000 Halver and Felton 2001 Moffat

et al 2004 Ball 2006 Sheraz et al 2007 Eitenmiller et al 2008 Salkic and Kubicek

2008) Most of these methods are based on the application of spectrophotometric

fluorimetric and chromatographic techniques to suit the requirements of a particular assay

and are summarized below

221 Spectrophotometric Methods

Spectrophotometric methods are the most widely used methods for the assay of

ascorbic acid in aqueous solution Ascorbic acid exhibits strong absorption in the

ultraviolet region (absorption maxima 243 nm at pH 2 and 265 nm at pH 4ndash10 OrsquoNeil

2001 Moffat et al 2004 British Pharmacopoeia 2009) This is the basis of

spectrophotometric methods for the determination of the vitamins in pure solutions and in

sample preparations where no interference is observed from UV absorbing impurities

The value of A (1 1 cm) at the analytical wavelength of 245 nm (pH 20) is high (695)

which makes the method very sensitive for the determination of mg quantities of the

27

vitamin Treatment of the material to be analyzed with ascorbic acid oxidase is often used

as a blank to correct for the presence of interfering substances in biological samples (Liu

et al 1982) A spectrophotometric method for the determination of ascorbic acid in

pharmaceuticals by background correction (245 nm) has been reported (Verma et al

1991) The direct determination of ascorbic acid in mixtures involves the use of 22prime-

dipyridyl as a colorimetric reagent The method is based on the reduction of Fe (III) by

ascorbic acid to Fe (II) which reacts with 2 2prime-dipyridyl to form a colored complex

(absorption maximum 510 nm) that can be used for quantitative determination (Margolis

and Schmidt 1996) A spectrophotometric method has been developed for the

determination of ascorbic acid and its oxidation product dehydroascorbic acid in

biological samples (Moeslinger et al 1995) A sensitive method has been reported for

the determination of ascorbic acid in pharmaceutical formulations and fruit juices by

interaction with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (Br-PADAP) (Ferreira

et al 1997) A novel UV method has been developed for the analysis of ascorbic acid in

methanol at 245 nm in various formulations (Zeng et al 2005)

Ascorbic acid in aqueous solutions has been assayed at 244 nm (pH ~2) (Ogata

and Kosugi 1969) 245 nm (pH 35) (Blaugh and Hajratwala 1972) 264 nm (pH 7)

(Salkic et al 2007) 265 nm (pH 7) (Hashmi 1973) 275 nm (pH 41 and 70) (Heelis et

al 1981) 265 nm (pH 7) (Al-Meshal and Hassan 1982) 245 nm (pH ~2) (Verma et al

1991) and 265 nm (pH ~7) (Erb et al 2004) Dehydroascorbic acid and 23-

diketogulonic acid do not significantly absorb in this region (Pelletier 1985 Davies et

al 1991 Rumsey and Levine 2000) and therefore do not interfere with the assay of

ascorbic acid in degraded solutions

28

222 Fluorimetric Methods

Fluorimetry is a highly sensitive technique for the determination of fluorescent

compounds or fluorescent derivatives of non-fluorescent compounds The technique has

been used for the detection of microg quantities of ascorbic acid Methods based on

fluorimetric (Kampfenkel et al 1995) and chemiluminescence detection (Zhang and

Chen 2000) provide highly sensitive methods for the determination of ascorbic acid in

plant and other materials

223 Mass Spectrometric Methods

Conventional and isotope mass spectrometric techniques have also been used for

the analysis of ascorbic acid Isotope ratio mass spectrometry is particularly useful and

sensitive when 13

C ascorbic acid is used as a standard in the analysis of complex matrices

(Gensler et al 1995)

224 Chromatographic Methods

High-performance liquid chromatographic (HPLC) methods have extensively

been employed for the determination of ascorbic acid in biological samples These

methods include ion exchange reversed phase and ion-pairing HPLC protocols

Spectrophotometric fluorimetric and electrochemical detection has been used in the

HPLC analysis of ascorbic acid The electrochemical detection is used for the

simultaneous determination of ascorbic acid dehydroascorbic acid and their isomers and

derivatives A number of HPLC methods have been developed for the detection and

determination of ascorbic acid and its oxidation products and derivatives in biological

samples and plant materials (Tsao and Young 1985 Tangney 1988 Dabrowski and

Huiterleitner 1989 Thomson and Trenerry 1995 Kimoto et al 1997 Kall and

29

Anderson 1999 Rumelin et al 1999 Lykkesfeldt 2000 Zhang et al 2000 Pastore et

al 2001 Frenich et al 2005) The limit of detection of ascorbic acid in plasma or urine

with UV detection lies in the range of 100-120 microg (Liau et al 1993 Manoharan and

Schwille 1994) Fluorescence detection of ascorbic acid and dehydroascorbic acid in

plasma and its comparison with coulometric detection has been reported (Tessier et al

1996) A liquid chromatography-diode-array detection (LCndashDAD) method has been

reported for the determination of 10 water-soluble and 10 fat-soluble vitamins including

ascorbic acid in pharmaceutical preparations with a coefficient of variation lt 65

(Konings 2006)

Liquid chromatography methods based on precolumn and o-phenylenediamine

(OPD) derivatization have been used for the determination of total vitamin C and total

isovitamin C in foods and dehydro forms of the vitamin Isoascorbic acid has been used

as an internal standard in the analysis (Speek et al 1985 Vanderslice et al 1990

Dodsun et al 1992 Vanderslice and Higgs 1988 1993 Hagg et al 1994 1995) The

limits of detection of ascorbic acid by HPLC using different detectors are in the range of

16ndash400 microgl (Capellmann and Bolt 1992 Iwase and Ono 1994 Karatepe 2004)

225 Enzymatic Methods

Enzymatic methods using ascorbate oxidase are specific and have the advantage

of selectively measuring the biological activity of ascorbic acid in serum or plasma (Liu

et al 1982) Ascorbate oxidase and OPD derivatization has been used to develop a rapid

automated method for the routine assay of ascorbic acid in serum and plasma The

method has a sample throughput of 100h (Ihara et al 2000)

30

226 Commercial Kits for Clinical Analysis

Commercial kits (eg Immunodiagnostic Germany Biovision USA) are also

used for the determination of ascorbic acid in biological samples (serum or plasma) in

clinical laboratories

227 Analysis in Creams

The general methods for the analysis of active ingredients and excipients in

cosmetic products including creams are described by Salvador and Chisvert (2007)

Ascorbic acid and derivatives in creams have been determined by liquid chromatography

(Irache et al 1993 Varvaresou et al 2006) gas chromatography-mass spectrometry

(Leveque et al 2005) and electrochemical methods (Beissenhirtz et al 2003 Guitton et

al 2007)

CHAPTER III

FORMULATION AND

STABILITY OF CREAM

PREPARATIONS

32

31 FORMULATION OF CREAM PREPARATIONS

Traditionally emulsions have been defined as dispersions of macroscopic droplets

of one liquid in another liquid with a droplet diameter approximately in the range of 05-

100 microm (Becher 1965) According to the definition of International Union of Pure and

Applied Chemistry (IUPAC) (1971) ldquoIn an emulsion liquid droplets and or liquid

crystals are dispersed in a liquidrdquo

Creams are semisolid emulsions intended for external applications They are often

composed of two phases Oil-in-water (ow) emulsions are most useful as water-washable

bases whereas water-in-oil (wo) emulsions are emollient and cleansing agents The

active ingredient is often dissolved in one or both phases thus creating a three-phase

system Patients often prefer a wo cream to an ointment because the cream spreads more

readily is less greasy and the evaporating water soothes the inflamed tissue OW creams

(vanishing creams) rub into the skin the continuous phase evaporates and increases the

concentration of a water-soluble drug in the adhering film The concentration gradient for

drug across the stratum corneum therefore increases promoting percutaneous absorption

(Barry 2002 Betageri and Prabhu 2002)

The various factors involved in the formulation of emulsions and topical products

have been discussed by Block (1996) Barry (2002) and Jain et al (2006) and are briefly

presented in the following sections

311 Choice of Emulsion Type

Oil-in-water emulsions are used for the topical application of water-soluble drugs

mainly for local effect They do not have the greasy texture associated with oily bases

and are therefore pleasant to use and easily washed from skin surfaces Moisturizing

33

creams designed to prevent moisture loss from the skin and thus inhibit drying of the

stratum corneum are more efficient if formulated as ow emulsions which produce a

coherent water-repellent film

312 Choice of Oil Phase

Many emulsions for external use contain oils that are present as carriers for the

active ingredient It must be realized that the type of oil used may also have an effect both

on the viscosity of the product and on the transport of the drug into the skin (Barry

2002) One of the most widely used oils for this type of preparation is liquid paraffin

This is one of a series of hydrocarbons which also includes hard paraffin soft paraffin

and light liquid paraffin They can be used individually or in combination with each other

to control emulsion consistency This will ensure that the product can be spread easily but

will be sufficiently viscous to form a coherent film over the skin The film-forming

capabilities of the emulsion can be further modified by the inclusion of various waxes

such as bees wax carnauba wax or higher fatty alcohols

313 Emulsion Consistency

A consideration of the texture or feel of a product intended for external use is

important A wo preparation will have a greasy texture and often exhibits a higher

apparent viscosity than ow emulsions This fact imparts a feeling of richness to many

cosmetic formulations Oil-in-water emulsions will however feel less greasy or sticky on

application to the skin will be absorbed more readily because of their lower oil content

and can be more easily washed from skin surface Ideally emulsions should exhibit the

rheological properties of plasticity pseudoplasticity and thixotropy Emulsions of high

apparent viscosity for external use (cream) are of a semisolid consistency There are

34

several methods by which the rheological properties of an emulsion can be controlled

(Billany 2002)

314 Choice of Emulsifying Agent

The choice of emulgent to be used would depend on factors such as its

emulsifying ability route of administration and toxicity Most of the non-ionic emulgents

are less irritant and less toxic than their anionic and cationic counter parts Some

emulgents such as the ionic alkali soaps often have a high pH and are thus unsuitable for

application to broken skin Even in normal intact skin with a pH of 55 the application of

such alkaline materials can cause irritation Some emulsifiers in particular wool fat can

cause sensitizing reactions in susceptible people The details of various types of

emulsifying agents are available in the literature (Betageri and Prabhu 2002 Billany

2002 Swarbrick et al 2006)

315 Formulation by the HLB Method

The physically stable emulsions are best achieved by the presence of a condensed

layer of emulgent at the oil water interface and the complex interfacial films formed by a

blend of an oil-soluble emulsifying agent with a water-soluble one produces the most

satisfactory emulsions

It is possible to calculate the relative quantities of the emulgents necessary to

produce the most physically stable emulsions for a particular formulation with water

combination This approach is called the hydrophilic-lipophilic balance (HLB) method

Each surfactant is allocated an HLB number representing the relative properties of the

lipophilic and hydrophilic parts of the molecule High numbers (up to a theoretical

number of 20) therefore indicates a surfactant exhibiting mainly hydrophilic or polar

35

properties whereas low numbers represent lipophilic or non-polar characteristics Each

type of oil requires an emulgent of a particular HLB number in order to ensure a stable

product For an ow emulsion the more polar the oil phase the more polar must be the

emulgent system (Billany 2002 Im-Emsap et al 2002 Swarbrick et al 2006)

316 Concept of Relative Polarity Index

In the ingredient selection in cosmetic formulations a new concept of relative

polarity index (RPI) has been presented (Wiechers 2005) The physicochemical

characteristics of the ingredients determine their skin delivery to a greater extent than the

formulation type The cosmetic formulation cannot change the chemistry of the active

molecule that needs to penetrate to a specific site within the skin However the

formulation type can be selected based on the polarity of the active ingredient and the

desired site of action for the active ingredient For optimum skin delivery the solubility of

the active ingredient needs to be as high as possible (to create a large concentration

gradient) and as small as possible (to create a large partition coefficient) To achieve this

it is necessary to determine the following parameters

The total amount dissolved in the formulation that is available for skin penetration

the higher this amount the more will penetrate until a solution concentration is

reached in the skin therefore a high absolute solubility in the formulation is required

The polarity of the formulation relative to that of the stratum corneum if an active

ingredient dissolves better in the stratum corneum than in the formulation then the

partition of the active ingredient will favour the stratum corneum therefore a low

(relative to that in the stratum corneum) solubility in the formulation is required

(Wiechers 2005)

36

These requirements can be met by considering the concept of RPI (Wiechers

2003 2005) In this systematic approach it is essential to consider the stratum corneum

as another solvent with its own polarity The stratum corneum appears to behave very

similarly to and in a more polar fashion than butanol with respect to its solubilizing

ability for active ingredients (Scheuplein and Blank 1973) The polarity of stratum

corneum as expressed by its octanol water partition coefficient is 63

The relative polarity index may be used to compare the polarity of an active

ingredient with both that of the skin and that of the oil phase of a cosmetic formulation

predominantly consisting of emollients It may be visualized as a vertical line with a high

polarity at the top and a high lipophilicity at the bottom The polarity is expressed as the

log10 of the octanol water coefficient For example the relative polarity index values of

glycerin and isostearyl isostearate are -176 and 2698 respectively (Wiechers 2005) In

order to use the concept of the relative polarity index three numbers (on log10 scale) are

required

The polarity of the stratum corneum is set at 08 However in reality this value will

change with the hydration state of the stratum corneum that is determined in part by

the external relative humidity (Bonwstra et al 2003)

The polarity of the active molecule

The polarity of the formulation

For multiphase or multipolarity systems like emulsions the active ingredient is dissolved

in the phase For example in an ow emulsion where a lipophilic active ingredient is

dissolved in the oil phase it is the polarity of the homogenous mixture of the lipophilic

active ingredient and internal oil For the same lipophilic active in a wo emulsion it is

37

the polarity of the homogenous mixture of the lipophilic active ingredients and external

oil For water-soluble active ingredients it is the polarity of the homogenous mixture of

the hydrophilic active ingredient and the aqueous phase regardless whether it is internal

(wo emulsions) or external (ow emulsions)

Once the active ingredient and the formulation type have been chosen it is

necessary to create the delivery system that will effectively deliver the molecule The

concept of relative polarity index allows the formulator to select the polarity of the phase

in which the active ingredient is incorporated on the basis of its own properties and those

of the stratum corneum In order to achieve maximum delivery the polarity of the active

ingredient and the stratum corneum need to be considered In order to improve the skin

delivery of active ingredients the first step involves selecting a primary emollient with a

polarity close to that of the active ingredient in which it will have a high solubility The

second step is to reduce the solubility of the active ingredient in the primary emollient via

the addition of a secondary emollient with a different polarity and therefore lower

solubility for the active ingredient This approach has shown a 3-4 fold increase in skin

penetration with out increasing the amount of active ingredients in the formulation

(Wiechers 2005)

32 FORMULATION OF ASCORBIC ACID CREAMS

Ascorbic acid is a water-soluble material and is included frequently in skin care

formulations to restore skin health It is very unstable and is easily oxidized in aqueous

solution This vitamin is known to be a reducing agent in biological systems and causes

the reduction of both oxygen- and nitrogen- based free radicals (Higdon and Frei 2002)

It can also act as a co-antioxidant with the tocopheroxyl radical to regenerate alpha-

38

tocopherol (Packer et al 1979 Buettner 1993 Peyrat-Maillard et al 2001) In this

reaction the two vitamins act synergistically Alpha-tocopherol first functions as the

primary antioxidant that reacts with an organic free radical Thereafter ascorbic acid

reacts with the free radical tocopheroxyl to general alpha-tocopherol In physiological

conditions the ascorbyl radical formed by regenerating tocopherol is then converted back

to ascorbate by the redox cycle (Davies et al 1991) The interaction of ascorbic acid

with a redox partner such as alpha-tocopherol has been found useful to slow its oxidation

and prolong its action

The instability of ascorbic acid makes this antioxidant active ingredient a

formulation challenge to deliver to the skin and retain its effective form In addition to its

use in combination with alpha-tocopherol in cream formulations the stability of ascorbic

acid may be improved by its use in the form of a fatty acid ester such as ascorbyl

palmitate Ascorbyl palmitate has been used in thixogel formulations and is typically

incorporated into the mineral oil phase Preliminary experiments have shown that it could

be slowly released from the starch-oil emulsion matrix and act as an antioxidant (Wille

2005)

Various physical and chemical factors involved in the formulation of cream

preparations have been discussed in the above sections For polar and air light sensitive

compounds such as ascorbic acid it is important to consider factors such as the choice of

formulation ingredients polar character of formulation HLB value pH viscosity etc to

achieve stability

39

33 STABILITY OF CREAMS

331 Physical Stability

The most important consideration with respect to pharmaceutical and cosmetic

emulsions (creams) is the stability of the finished product The stability of a

pharmaceutical emulsion is characterized by the absence of coalescence of the internal

phase absence of creaming and maintenance of elegance with respect to appearance

odor color and other physical properties An emulsion is a dynamic system however

any flocculation and resultant creaming represent potential steps towards complete

coalescence of the internal phase In pharmaceutical emulsions creaming results as a lack

of uniformity of drug distribution and poses a problem to the pharmaceutical

compounder Another important factor in the stabilization of emulsions is phase inversion

which involves the change of emulsion type from ow to wo or vice versa and is

considered as a case of instability The four major phenomena associated with the

physical instability of emulsions are flocculation creaming coalescence and breaking

These have been discussed by Garti and Aserin (1996) Im-Emsap et al (2002) and Sinko

(2006)

332 Chemical Stability

The instability of a drug may lead to the loss of its concentration through a

chemical reaction under normal or stress conditions This results in a reduction of the

potency and is a well-recognized cause of poor product quality The degradation of the

drug may make the product esthetically unacceptable if significant changes in color or

odor have occurred The degradation product may also be a toxic substance The various

pathways of chemical degradation of a drug depend on the structural characteristics of the

40

drug and may involve hydrolysis dehydration isomerization and racemization

decarboxylation and elimination oxidation photodegradation drug-excipients and drug-

drug interactions Factors determining the chemical stability of drug substances include

intrinsic factors such as molecular structure of the drug itself and environmental factors

such as temperature light pH buffer species ionic strength oxygen moisture additives

and excipients The application of well-established kinetic principles may throw light on

the role of each variable in altering the kinetics of degradation and to provide valuable

insight into the mechanism of degradation (Baertschi and Alsante 2005 Yoshioka and

Stella 2002 Lachman et al 1986) The chemical stability of individual components

within an emulsion system may be very different from their stability after incorporation

into other formulation types For example many unsaturated oils are prone to oxidation

and their degree of exposure to oxygen may be influenced by factors that affect the extent

of molecular dispersion (eg droplet size) This could be particularly troublesome in

emulsions because emulsification may introduce air into the product and because of the

high interfacial contact area between the phases (Barry 2002) The use of antioxidants

retards oxidation of unsaturated oils minimizes changes in color and texture and prevents

rancidity in the formulation Moreover they can retard the degradation of certain active

ingredients such as vitamin C (Vimaladevi 2005) The stability problems of dispersed

systems and the factors leading to these stability problems have been discussed by

Weiner (1996) and Lu and Flynn (2009)

333 Microbial Stability

Topical bases often contain aqueous and oily phases together with carbohydrates

and proteins and are susceptible to bacterial and fungal attack Microbial growth spoils

41

the formulation and is a potential toxic hazard Therefore topical formulations need

appropriate preservatives to prevent microbial growth and to maintain their quality and

shelf-life (Barry 2002 Arger et al 1996) Cream formulations may contain fats and oils

with high percentage of unsaturated linkages that are susceptible to oxidation degradation

and development of rancidity The addition of antioxidants retards oxidation of fats and

oils minimizes changes in color and texture and prevents rancidity in the formulation

Moreover they can retard the degradation of certain active ingredients such as vitamin C

These aspects in relation to dermatological formulations have been discussed by Barry

(1983 2002) and Vimaladevi 2005)

334 Stability of Ascorbic Acid in Liquid Formulations

Ascorbic acid is very unstable in aqueous solution Different workers have studied

the stability of ascorbic acid in liquid formulations (Connors et al 1986 Austria et al

1997) Its shelf-life can be prolonged by appropriate choice of vehicle and control of

other variables such as pH stabilizers temperature light and oxygen (Table 3)

Similarly the stability of various concentrations of ascorbic acid in solution form may

vary depending upon the type of solvent used (Table 4) (Connors et al 1986 Satoh et

al 2000 Lee et al 2004 Zeng et al 2005)

335 Stability of Ascorbic Acid in Emulsions and Creams

Ascorbic acid exerts several functions on skin such as collagen synthesis

depigmentation and antioxidant activity Ultraviolet radiation generates reactive oxygen

species (ROS) which produce some harmful effects on the skin including photocarcinoma

and photoaging In order to combat these problems ascorbic acid as an antioxidant has

42

Table 3 Effect of vehicles on the stability of ascorbic acid ( ascorbic acid remaining in

solutions after storage at room temperature) (Connors et al 1986)

Storage Time (days) No Vehicle

30 60 90 120 180 240 360

1 Corn Syrup 965 925 920 920 900 860 760

2 Sorbitol 990 990 990 970 960 925 890

3 4 Carboxymethyl

Cellulose

840 680 565 380 ndash ndash ndash

4 Glycerin 100 100 990 990 970 935 920

5 Propylene glycol 995 990 980 945 920 900 900

6 Syrup USP 100 100 980 980 930 900 880

7 Syrup 212 gL 880 810 775 745 645 590 440

8 25 Tragacanth 785 620 510 320 ndash ndash ndash

9 Saturated solution of

Dextrose

990 935 875 800 640 580 510

10 Distilled Water 900 815 745 675 405 185 ndash

11 50 Propylene glycol +

50 Glycerin

980 ndash 960 ndash 933 ndash ndash

12 25 Distilled Water +

75 Sorbo (70 solution

of Sorbitol)

955 954 ndash 942 930 ndash ndash

13 50 Glycerin + 50

Sorbo

982 984 978 ndash ndash 914 ndash

43

Table 4 Stability of various concentrations of ascorbic acid in water propylene glycol

and USP syrup at room temperature ( of ascorbic acid remaining in solution)

(Connors et al 1986)

Storage Time (days) Concentration

(mg ml)

Solvent

30 60 90 120 180 240 360

10 Water 930 840 820 670 515 410 ndash

50 Water 940 920 880 795 605 590 300

100 Water 970 930 910 835 705 680 590

10 Propylene glycol 100 985 980 975 960 920 860

50 Propylene glycol 100 970 980 980 980 965 935

100 Propylene glycol 100 100 100 100 990 100 925

10 Syrup 100 100 980 990 970 960 840

50 Syrup 100 100 100 100 990 100 960

100 Syrup 100 100 100 100 100 100 995

44

been used in various dosage forms and in different concentrations (Darr et al 1996

Gallarate et al 1999 Zhang et al 1999 Pinnell et al 2001 Lee et al 2004 Raschke

et al 2004 Elmore 2005 Farahmand et al 2006 Maia et al 2006) Ascorbic acid has

good photoprotective ability against UVA-mediated phototoxicity (Darr et al 1996) A

variety of formulations containing ascorbic acid or its derivatives have been studied in

order to evaluate their stability and delivery through the skin (Gallarate et al 1999

Zhang et al 1999 Ozer et al 2000 Pinnell et al 2001 Lee et al 2004 Raschke et al

2004 Farahmand et al 2006) Formulations containing derivatives of ascorbic acid are

found to be more stable than ascorbic acid but they do not produce the same effect as that

of the parent compound probably due to the lack of redox properties (Heber et al 2006)

Effective delivery of ascorbic acid through topical preparations is a major factor that

should be critically evaluated as it may be dependent upon the nature or type of the

formulation (Gallarate et al 1999 Pinnell et al 2001) The pH of the formulation

should be on the acidic side (~ pH 35) for effective penetration of the vitamin in the skin

(Pinnell et al 2001) and for its stabilization in the formulation (Gallarate et al 1999)

Some other antioxidants such as alpha-tocopherol ferulic acid and sodium metabisulphite

have also been used in combination with ascorbic acid for the purpose of its stabilization

in topical formulations and to produce some synergistic effects (Darr et al 1996 Lin et

al 2005 Maia et al 2006 Tournas et al 2006) Effect of some rheological properties

such as viscosity and dielectric constant on the stability of ascorbic acid in emulsions has

also been investigated (Connors et al 1986) Viscosity of the medium is an important

factor that should be considered for the purpose of ascorbic acid stability as higher

viscosity formulations have shown some degree of protection (Ozer et al 2000

45

Szymula 2005) Along with other factors formulation type also plays an important role in

the stability of ascorbic acid It is reported that ascorbic acid is more stable in emulsified

system as compared to aqueous solutions (Gallarate et al 1999 Lee et al 2004) In

multiemulsions ascorbic acid is reported to be more stable as compared to simple

emulsions (Gallarate et al 1999 Ozer et al 2000 Lee et al 2004 Farahmand et al

2006)

Ascorbic acid and its derivatives have been used in a variety of cosmetic

formulations as an antioxidant pH adjuster anti-aging and photoprotectant (Elmore

2005) The control of instability of ascorbic acid poses a significant challenge in the

development of cosmetic formulations It is also reported that certain metal ions or

enzyme systems effectively convert ascorbic acidrsquos antioxidant action to pro-oxidant

activity (Elmore 2005) Therefore utilization of an effective antioxidant system is

required to maintain the stability of vitamin C in various preparations (Zhang et al 1999

Pinnell et al 2001 Maia et al 2006) The chemical stability of ascorbic acid has been

studied in emulsions and creams by several workers (Darr et al 1996 Gallarate et al

1999 Lee et al 2004 Raschke et al 2004 Elmore 2005 Farahmand et al 2006)

however there is a lack of information on the photostability of ascorbic acid in cream

formulations

336 Stability Testing of Emulsions

The stability testing of emulsions (creams) may be carried out by performing the

following tests (Billany 2002)

46

3361 Macroscopic examination

The assessment of the physical stability of an emulsion is made by an

examination of the degree of creaming or coalescence occurring over a period of time

This involves the calculation of the ratio of the volume of the creamed or separated part

of the emulsion and the total volume A comparison of these values can be made for

different products

3362 Globule size analysis

An increase in mean globule size with time (coupled with a decrease in globule

numbers) indicates that coalescence is the cause of this behavior This can be used to

compare the rates of coalescence for a variety of emulsion formulations For this purpose

microscopic examination or electronic particle counting devices (coulter counter) or

laser diffraction sizing are widely used

3363 Change in viscosity

Many factors may influence the viscosity of emulsions A change in apparent

viscosity may result from any variation in globule size or number or in the orientation or

migration of emulsifier over a period of time

3264 Accelerated stability tests

In order to compare the relative stabilities of a range of similar products it is

necessary to speed up the processes of creaming and coalescence by storage at elevated

temperatures and then carrying out the tests described in the above sections

337 FDA guidelines for semisolid preparations

According to FDA draft guidelines to the industry (Shah 1997) semisolid

preparations (eg creams) should be evaluated for appearance clarity color

47

homogencity odour pH consistency viscosity particle size distribution (when feasible)

assay degradation products preservative and antioxidant content (if present) microbial

limits sterility and weight loss when appropriate Additionally samples from

production lot or approved products are retained for stability testing in case of product

failure in the field Retained samples can be tested along with returned samples to

ascertain if the problem was manufacturing or storage related Appropriate stability data

should be provided for products supplied in closed-end tubes to support the maximum

anticipated use period during patient use and after the seal is punctured allowing product

contact with the cap cap lever Creams in large containers including tubes should be

assayed by sampling at the surface top middle and bottom of the container In addition

tubes should be sampled near the crimp The objective of stability testing is to determine

whether the product has adequate shelf-life under market and use conditions

48

OBJECT OF PRESENT INVESTIGATION

Ascorbic acid (vitamin C) is extensively used as a single ingredient or in

combination with vitamin B complex and other vitamins in the form of drops injectables

lotions and syrups It is an ingredient of anti-aging cosmetic products alone or along with

alpha-tocopherol (vitamin E) Ascorbic acid exerts several functions on the skin as

collagen synthesis depigmentation and antioxidant activity It protects the signs of

degenerative skin conditions caused by oxy-radical damage In solutions and creams

ascorbic acid is susceptible to air and light and undergoes oxidative degradation to

dehydroascorbic acid and inactive products The degradation is influenced by

temperature viscosity and polarity of the medium and is catalysed by metal ions

particularly Cu+2

Fe+2

and Zn+2

One of the major problems faced in cream preparations is the instability of

ascorbic acid as it may be exposed to light during formulation manufacturing and

storage and the possibility of photochemical degradation can not be neglected The

behaviour of ascorbic acid in light is of particular interest since no systematic kinetic

studies have been conducted on its photodegradation in these preparations under various

conditions The study of the formulation variables such as emulsifier humectants and pH

may throw light on the photostabilization of ascorbic acid in creams

The main object of this investigation is to study the behaviour of ascorbic acid in

cream preparations on exposure to UV light in the pharmaceutically useful pH range An

important aspect of the work is to study the interaction of ascorbic acid with other

vitamins such as riboflavin nicotinamide and alpha-tocopherol and the effect of certain

stabilizers such as citric acid tartaric acid and boric acid on its photodegradation In

49

addition it is intended to study the photolysis of ascorbic acid in organic solvents to

evaluate the effect of solvent characteristics (eg dielectric constant and viscosity) on the

stability of the vitamin The study of all these aspects may provide useful information to

improve the photostability and efficacy of ascorbic acid in cream preparations

An outline of the proposed plan of work is presented as follows

1 To prepare a number of oil-in-water cream formulations based on different

emulsifying agents and humectants containing ascorbic acid alone and in

combination with other vitamins and stabilizing agents

2 To perform photodegradation studies on ascorbic acid in creams using a UV

irradiation source with emission corresponding to the absorption maximum of

ascorbic acid

3 To identify the photoproducts of ascorbic acid in creams using chromatographic

and spectrophotometric methods

4 To apply appropriate and validated analytical methods for the assay of ascorbic

acid alone and in combination with other vitamins and stabilizing agents

5 To study the effect of solvent characteristics such as dielectric constant and

viscosity on the photolysis of ascorbic acid in aqueous and organic solvents

6 To evaluate the kinetics of photodegradation of ascorbic acid and its interactions

with other vitamins (riboflavin nicotinamide and alpha-tocopherol) in creams

7 To evaluate the effect of carbon chain length of the emulsifying agent and the

viscosity of the humectant on the photodegradation of ascorbic acid

50

8 To develop relationships between the rate of photodegradation of ascorbic acid

and the concentration pH carbon chain length of emulsifier viscosity of the

creams

9 To determine the effect of compounds such as citric acid tartaric acid and boric

acid used as stabilizing agents on the rate of photodegradation and stabilization

of ascorbic acid in creams

10 To present reaction schemes for the photodegradation of ascorbic acid and its

interactions with other vitamins

CHAPTER IV

MATERIALS

AND

METHODS

52

41 MATERIALS

Vitamins and Related Compounds

L-Ascorbic Acid vitamin C (5R)-5-[(1S)-12-dihydroxyethyl]-34-dihydroxyfuran-2(5H)-

one Merck

C6H8O6 Mr 1761

Dehydroascorbic Acid L-threo-23-hexodiulosonic acid γ-lactone Sigma

C6H6O6 Mr 1741

23-Diketogulonic Acid

C6H8O7 Mr 192

It was prepared according to the method of Homann and Gaffron (1964) by the

hydrolysis of dehydroascorbic acid

Riboflavin vitamin B2 (310-dihydro-78-dimethyl-10-[(2S3S4R)-2345-

tetrahydroxypentyl] benzopteridine-24-dione) Merck

C17H20N4O6 Mr 3764

Nicotinamide vitamin B3 (pyridine-3-carboxamide) Merck

C6H6N2O Mr 1221

Alpha-Tocopherol vitamin E ((2R)-2578-tetramethyl-2-[(4R8R)-4812-

trimethyltridecyl]-34-dihydro-2H-1-benzopyran-6-ol) Merck

C29H50O2 Mr 4307

Formylmethylflavin (78-dimethyl-10-formylmethylisoalloxazine)

C14H12N4O3 Mr 2843

53

Formylmethylflavin was synthesized according to the method of Fall and Petering

(1956) by the periodic acid oxidation of riboflavin It was recrystallized from absolute

methanol dried in vacuo and stored in the dark in a refrigerator

Lumichrome (78-dimethylalloxazine) Sigma

C12H10N4O2 Mr 2423

It was stored in the dark in a desiccator

Stabilizers

Boric Acid orthoboric acid Merck

H3BO3 Mr 618

Citric Acid 2-hydroxypropane-123-tricarboxylic acid Merck

C6H8O7H2O Mr 2101

L-Tartaric acid [(2R3R)-23-dihydroxybutanedioic acid] Merck

C4H6O6 Mr 1501

Emulsifying Agents

Stearic Acid (95) octadecanoic acid Merck

C18H36O2 Mr 2845

Palmitic Acid hexadecanoic acid Merck

C16H32O2 Mr 2564

Myristic Acid tetradecanoic acid Merck

C14H28O2 Mr 2284

Cetyl alcohol hexadecan-1-ol Merck

C16H34O Mr 2424

54

Humectants

Glycerin glycerol (propane-123-triol) Merck

C3H8O3 Mr 921

Propylene glycol (RS)-propane-12-diol Merck

C3H8O2 Mr 7610

Ethylene glycol ethane-12-diol Merck

C2H6O2 Mr 6207

Potassium Ferrioxalate Actinometry

Potassium Ferrioxalate

K3Fe(C2O4)3 3H2O Mr 4912

Potassium Ferrioxalate was prepared according to the method of Hatchard and

Parker (1956) Three volumes of 15 M potassium oxalate was mixed with one volume of

15 M ferric chloride with vigorous stirring The yellow green precipitate of potassium

ferrioxalate was recrystallized twice from water dried at 45 ordmC and stored in the dark in a

desiccator

Reagents

All the reagents and solvents used were of analytical grade obtained from BDH

Merck

Water

Freshly boiled distilled water was used throughout the work

55

42 METHODS

421 Cream Formulations

On the basis of the various cream formulations reported in the literature (Block

1996 Flynn 2002 Betageri and Prabhu 2002 Vimaladevi 2005 EIRI Board Lu and

Flynn 2009) the following basic formula was used for the preparation of oil-in-water

creams containing ascorbic acid

Oil phase Percentage (ww)

Emulsifier

Myristic palmitic stearic acid

Cetyl alcohol

120

30

Aqueous phase

Humectant

Ethylene glycol propylene glycol glycerin

50

Active ingredient

Ascorbic acid

20 (0114 M)

Neutralizer

Potassium hydroxide

10

Continuous phase

Distilled water

QS

Additional ingredientsa

Vitamins

Riboflavin (Vitamin B2)

Nicotinamide (Vitamin B3)

Alpha-Tocopherol (Vitamin E)

0002ndash001 (053ndash266times10ndash4

M)

028ndash140 (0023ndash0115 M)

017ndash086 (0395ndash200times10ndash2

M)

Stabilizers

Citric acid

Tartaric acid

Boric acid

010ndash040 (0476ndash190times10ndash2

M)

010ndash040 (067ndash266times10ndash2

M)

010ndash040 (0016ndash0065 M)

a The vitamin stabilizer concentrations used were found to be effective in promotion

inhibition of photodegradation of ascorbic acid in cream formulations

56

422 Preparation of Creams

The emulsifiers were melted at 70ndash80 ordmC in a glass jar immersed in a water bath

Ascorbic acid was separately dissolved in a small portion of distilled water Potassium

hydroxide and humectant were dissolved in the remaining portion of water and mixed

with the oily phase with constant stirring until the formation of a thick white mass It was

cooled to ~40 ordmC and the ascorbic acid solution was added The thick mass was mixed

using a mechanical mixer with a glass stirrer at 1000 rpm for 5 minutes The pH of the

cream was adjusted to the desired value and the contents again mixed for 10 minutes at

500 rpm All the creams were prepared under uniform conditions to maintain their

individual physical characteristics and stored at room temperature in airtight glass

containers protected from light

In the case of other vitamins nicotinamide was dissolved along with ascorbic acid

in water and added to the cream Riboflavin or alpha-tocopherol were directly added to

the cream and mixed thoroughly according to the procedure described above

In the case of stabilizing agents (citric tartaric and boric acids) the individual

compounds were dissolved in the ascorbic acid solution and added to the cream followed

by the procedure described above

The details of the various cream formulations used in this study are given in

chapters 5ndash7

57

423 Thin-Layer Chromatography (TLC)

The following TLC systems were used for the separation and identification of

ascorbic acid and photodegradation products

Adsorbent a) Silica gel GF 254 (250-microm) precoated plates

(Merck)

Solvent systems S1 acetic acid-acetone-methanol-benzene

(552070 vv) (Ganshirt and Malzacher 1960)

S2 ethanol-10 acetic acid-water (9010 vv)

(Bolliger and Konig 1969)

S3 acetonitrile-butylnitrile-water (66332 vv)

(Saari et al 1967)

Temperature 25ndash27 ordmC

Location of spots Ascorbic acid UV light 254 nm (Uvitec lamp

UK)

Dehydroascorbic acid Spray with a 3 aqueous

phenylhydrazine hydrochloride solution

424 pH Measurements

The measurements of pH of aqueous solutions and cream formulations were

carried out using an Elmetron LCD display pH meter (modelndashCP501 sensitivity plusmn 001

pH units) (Poland) with a combination electrode The electrode was calibrated

automatically in the desired pH range (25 ordmC) using the following buffer solutions

58

Phthalate pH 4008

Phosphate pH 6865

Disodium tetraborate pH 9180

The electrode was immersed directly into the cream (British Pharmacopoeia

2009) kept for few seconds to equilibrate and the pH adjusted in the range of 40ndash70

with phosphoric acid sodium hydroxide solution

425 Ultraviolet and Visible Spectrometry

The absorbance measurements and spectral determinations were performed on

Shimadzu UVndashVisible recording spectrophotometer (model UVndash1601) using matched

silica cells of 10 mm path length The cells were employed always in the same orientation

using appropriate control solutions in the reference beam The baseline was automatically

corrected by the built-in baseline memory at the initializing period Auto-zero adjustment

was made by a one-touch operation The instrument checked the wavelength calibration

(6561 nm) using the deuterium lamp at the initializing period The absorbance scale was

periodically checked using the following calibration standard (British Pharmacopoeia

2009)

0057ndash0063 gl of potassium dichromate in 0005 M sulphuric acid

The specific absorbance [A(1 1 cm)] of the solution should match the

following values with the stated limit of tolerance

Wavelength

(nm)

Specific absorbance

A (1 1 cm)

Maximum

tolerance

235 1245 1229ndash1262

257 1445 1428ndash1462

313 486 470ndash503

350 1073 1056ndash109

430 159 157ndash161

59

426 Photolysis of Ascorbic Acid

4261 Creams

A 2 g quantity of the cream was uniformly spread on several rectangular glass

plates (5 times 15 cm) covered with a 1 cm tape on each side to give a 1 mm thick layer The

plates were irradiated in a dark chamber using a Philips 30 watt TUV tube (100

emission at 254 nm the wavelength absorbed by ascorbic acid at pH 4ndash7) fixed

horizontally at a distance of 30 cm from the centre of the plates Each plate was removed

at appropriate interval and the cream was subjected to spectrophotometric assay and

chromatographic examination

4262 Aqueous and organic solvents

A 10ndash3

M solution of ascorbic acid (50 ml) prepared in water (pH 70 005 M

phosphate buffer) or in an organic solvent in a 100 ml beaker (Pyrex) was placed in a

water bath maintained at 20 plusmn 1 ordmC The solution was irradiated with the Philips 30 watt

TUV tube in a dark chamber as stated above Samples were withdrawn at appropriate

intervals for assay and chromatography

4263 Storage of creams in dark

In order to determine the stability of various cream formulations in the dark

samples were stored at room temperature in a cupboard protected from light for a period

of three months The samples were analyzed periodically for the content of ascorbic acid

and the presence of any degradation product

427 Measurement of Light Intensity

The potassium ferrioxalate actinometry was used for the measurement of light

intensity of the radiation source employed in this work This actinometer has been

60

developed by Parker (1953) and Hatchard and Parker (1956) and is considered as the

most useful actinometer over a wide range of wavelengths (254ndash577 nm) It has been

used by Holmstrom and Oster (1961) Byrom and Turnbull (1967) McBride and Moore

(1967) Ahmad (1968) Ahmad (1978) Ahmad et al (2004a 2004b 2005 2006a

2006b 2008 2009ab) Fasihullah (1988) Vaid (1998) Ansari (2002) and Ahmad (2009)

for the measurement of light intensity

The irradiation of potassium ferrioxalate solutions in sulphuric acid results in the

reduction of ferric ion to ferrous ion according to the following reaction

2Fe [(C2O4)3]3ndash

rarr 2 Fe (C2O4) + 3 (C2O4)2ndash

+ 2CO2 (31)

The amount of Fe2+

ions formed in the reaction may be determined by

complexation with 110-phenanthroline to give a red colored complex The absorbance of

the complex is measured at 510 nm

428 Procedure

An oxygen free 0006 M solution of potassium ferrioxalate (2947 gl) in 01 N

H2SO4 was placed in the reaction vessel and irradiated with the lamp used for the

photolysis of riboflavin The irradiation was carried out under nitrogen (90ndash120

bubblesminute) which also caused stirring of the solution The temperature of the

reaction vessel was maintained at 25 plusmn 1 ordmC during the reaction

An aliquot of the photolysed solution (1ndash2 ml) was pipetted out at suitable

intervals (up to 30 minutes) into a 10 ml volumetric flask to which was then added 09

ml of N H2SO4 + 1 ml (01) 110-phenanthroline + 05 ml buffer (60 ml N CH3COONa

+ 36 ml N H2SO4 made up to 100 ml with distilled water) The flask was made up

to the mark with distilled water (final pH 35) thoroughly shaken to mix the contents and

61

Fig 3 Spectral power distribution of TUV 30 W tube (Philips)

62

allowed to stand for one hour in the dark to develop the colorndashcomplex The absorbance

of the phenanthrolinendashferrous complex was measured in a 1 cm cell at 510 nm using the

appropriate solution as blank The amount of Fe2+

ions formed was determined from the

calibration graph The calibration graph was constructed in a similar manner using

several dilutions of 1 times 10ndash6

mole ml Fe2+

in 01 N H2SO4 (freshly prepared by dilution

from standardized 01 M FeSO4 in 01 N H2SO4) (Fig 8) The experimental value of the

molar absorptivity of Fe2+

complex as determined from the slope of the calibration graph

is equal to 111 times 104 M

ndash1 cm

ndash1 and is in agreement with the value reported by Parker

(1953) Using the values of the known quantum yield for ferrioxalate actinometer at

different wavelengths (Hatchard and Parker 1956) the number of Fe2+

ions formed

during photolysis the time of exposure and the fraction of the light absorbed by the

length of the actinometer solution employed the light intensity incident just inside the

front window of the photolysis cell can be calculated In the present case total absorption

of the light has been assumed

4281 Calculation

The number of Fe2+

ions formed during photolysis (nFe

2+) is given by the

equation

6023 times 1020

V1 V3 A Σ

n Fe

2+ =

V2 1 ε (32)

where V1 is the volume of the actinometer solution irradiated (ml)

V2 is the volume of the aliquot taken for analysis (ml)

V3 is the final volume to which the aliquot V2 is diluted (ml)

1 is the path length of the spectrophotometer cell used (1 cm)

A is the measured absorbance of the final solution at 510 nm

63

ε is the molar absorptivity of the Fe2+

complex (111 times 104 M

ndash1 cm

ndash1)

The number of quanta absorbed by the actinometer nabs can then be obtained as follows

n Fe

2+

Σ nabs = ф

(33)

where ф is the quantum yield for the Fe2+

formation at the desired wavelength

The number of quanta per second per cell nabs is therefore given by

Σ nabs 6023 times 1020

V1 V3 A nabs =

t =

ф V2 1 ε t (34)

where t is the irradiation time of the actinometer in seconds

The relative spectral energy distribution of the radiation source (Fig 3) shows

100 emission at 254 nm the wavelength used for the photolysis of ascorbic acid (λmax

265 nm at pH 4ndash7) The energy emitted by the radiation source at various wavelengths

can be calculated using the equation

1197 times 105

E (KJ molndash1

) = λ nm

(35)

The quantum efficiency of ferrioxalate actinometer at the wavelength absorbed by

ascorbic acid (ie 254 nm) is high although the sensitivity drops over 450 nm The

average intensity of the TUV tube used in this study was determined as 556 plusmn 012 times

1018

quanta sndash1

429 Viscosity Measurements

The viscosity of the cream formulations was measured with a Brookfield RV

viscometer (Model DV-II + Pro USA) The instrument was calibrated using the

manufacturerrsquos viscosity standard A 200 g quantity of the cream was placed in a beaker

and the spindle (TE) was dipped into the cream It was rotated at a speed of 06 rpm for

64

00

02

04

06

08

10

12

0 2 4 6 8 10 12

Concentration of Fe++

times 105 M

Ab

sorb

an

ce a

t 51

0 n

m

Fig 4 Calibration graph for the determination of K3Fe(C2O4)3

65

one minute and the viscosity was recorded at 25plusmn1 ordmC The test was repeated three times

to account for the experimental variability and the average viscosity was noted

4210 Assay Methods

42101 UV spectrophotometric method for the assay of creams containing ascorbic

acid alone

The creams were thoroughly mixed a quantity of 2 g was accurately weighed and

the assay of ascorbic acid was carried out by the UV method of Zeng et al (2005) In the

case of photodegraded creams (2 g) the material was completely removed from the glass

plate and transferred to a volumetric flask The method involved extraction of ascorbic

acid with methanol (3 times 10 ml) adjustment of the pH of combined methanolic solutions

to 20 (with H3PO4) dilution of the final solution with acidified methanol (pH 20) to 100

ml and measurement of the absorbance at 245 nm using appropriate blank The

concentration of ascorbic acid was calculated using 560 as the value of specific

absorbance [A (1 1 cm)] at the analytical wavelength (Table 5)

The same method was used for the assay of ascorbic acid in creams stored in the

dark and in the presence of individual stabilizing agents (citric tartaric and boric acids)

42102 Iodimetric method for the assay of ascorbic acid in creams containing

riboflavin nicotinamide and alpha-tocopherol

The assay of ascorbic acid in creams in the presence of riboflavin nicotinamide

and alpha-tocopherol was carried out according to the procedure of British

Pharmacopoeia (2009) as follows

The photolysed cream (2 g) was completely scrapped from the glass plate and

transferred to a flask containing 40 ml of distilled water and 10 ml of 1 M sulphuric acid

66

Table 5 Calibration data for ascorbic acid showing linear regression analysisa

λ max 245 nm

Concentration range 01ndash10 times 10ndash4

M (0176ndash1761 mg )

Slope 9920

SE (plusmn) of slope 00114

Intercept 00012

Correlation coefficient 09996

Molar absorptivity (ε) 9920 Mndash1

cmndash1

Specific absorbance [A (1 1 cm)] 560

a Values represent a mean of five determinations

67

was added The solution was titrated with 002 M iodine solution using 1 ml of starch

solution as indicator until a persistent violet-blue color was obtained Each ml of 002 M

iodine solution is equivalent to 352 mg of C6H8O6 The same method was used for the

assay of ascorbic acid in creams stored in the dark

42103 Spectrophotometric method for the assay of ascorbic acid in aqueous and

organic solvents

A 1 ml aliquot of the photolysed solutions of ascorbic acid in water or in an

organic solvent was evaporated to dryness under nitrogen at room temperature and the

residue redissolved in a small volume of methanol The solution was transferred to a 10

ml volumetric flask made up to volume with acidified methanol (pH 20) and the

absorbance measured at 245 nm using an appropriate blank The content of ascorbic acid

in the solutions was determined using 9920 Mndash1

cmndash1

as the value of molar absorptivity at

the analytical wavelength (Table 5)

CHAPTER V

PHOTODEGRADATION OF

ASCORBIC ACID IN

ORGANIC SOLVENTS AND

CREAM FORMULATIONS

69

51 INTRODUCTION

Ascorbic acid (vitamin C) is an essential micronutrient that performs important

metabolic functions (Packer and Fuchs 1999 Davey et al 2000 Johnston et al 2007)

It is an ingredient of anti-aging cosmetic products (Darr et al 1996 Gallarate et al

1999 Traikovich 1999 Zhang et al 1999 Ozer et al 2000 Nusgens et al 2001

Pinnell et al 2001 2003 Lee et al 2004 Raschke et al 2004 Sauermann et al 2004

Elmore 2005 Jentzsch et al 2005 Lin et al 2005 Placzek et al 2005 Carlotti et al

2006 Farahmand et al 2006 Heber et al 2006 Maia et al 2006 Tournas et al 2006)

and exerts several functions on the skin as collagen synthesis depigmentation and

antioxidant activity (Nusgens et al 2001 Spiclin et al 2003) As an antioxidant it

protects skin by neutralizing reactive oxygen species generated on exposure to sunlight

(Shindo et al 1994) In biological systems it reduces both oxygenndash and nitrogenndash based

free radicals (Higdon and Frei 2002) and thus delays the aging process In view of the

instability of ascorbic acid in skin care formulations (Bissett 2006) it is often used in

combination with another redox partner such as alpha-tocopherol (vitamin E) to retard its

oxidation (Wille 2005)

The details of the cream formulations used in this study are given in Table 6 The

results obtained on the photodegradation of ascorbic acid in aqueous organic solvents

and cream formulations are discussed in the following sections

70

Table 6 Composition of cream formulations containing ascorbic acid

Ingredients Cream

No pH

SA PA MA CA AH2 GL PG EG PH DW

1 a 4 + minus minus + + + minus minus + +

b 5 + minus minus + + + minus minus + +

c 6 + minus minus + + + minus minus + +

d 7 + minus minus + + + minus minus + +

2 a 4 minus + minus + + + minus minus + +

b 5 minus + minus + + + minus minus + +

c 6 minus + minus + + + minus minus + +

d 7 minus + minus + + + minus minus + +

3 a 4 minus minus + + + + minus minus + +

b 5 minus minus + + + + minus minus + +

c 6 minus minus + + + + minus minus + +

d 7 minus minus + + + + minus minus + +

4 a 4 + minus minus + + minus + minus + +

b 5 + minus minus + + minus + minus + +

c 6 + minus minus + + minus + minus + +

d 7 + minus minus + + minus + minus + +

5 a 4 minus + minus + + minus + minus + +

b 5 minus + minus + + minus + minus + +

c 6 minus + minus + + minus + minus + +

d 7 minus + minus + + minus + minus + +

6 a 4 minus minus + + + minus + minus + +

b 5 minus minus + + + minus + minus + +

c 6 minus minus + + + minus + minus + +

d 7 minus minus + + + minus + minus + +

7 a 4 + minus minus + + minus minus + + +

b 5 + minus minus + + minus minus + + +

c 6 + minus minus + + minus minus + + +

d 7 + minus minus + + minus minus + + +

8 a 4 minus + minus + + minus minus + + +

b 5 minus + minus + + minus minus + + +

c 6 minus + minus + + minus minus + + +

d 7 minus + minus + + minus minus + + +

9 a 4 minus minus + + + minus minus + + +

b 5 minus minus + + + minus minus + + +

c 6 minus minus + + + minus minus + + +

d 7 minus minus + + + minus minus + + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PG = propylene glycol EG = ethylene glycol

PH = potassium hydroxide DW = distilled water

71

52 PHOTOPRODUCTS OF ASCORBIC ACID

The photolysis of ascorbic acid (AH2) in aqueous and organic solvents and in

cream formulations on UV irradiation leads to the formation of dehydroascorbic acid

(DHA) as detected by TLC along with the undegraded AH2 using the solvent systems A

B and C The identification of DHA was carried out by comparison of the Rf value and

spot color with those of the authentic compound The formation of DHA on

photooxidation of ascorbic acid solutions has previously been reported (Homan and

Gaffron 1964 Sattar et al 1977 Heelis et al 1981 Rozanowska et al 1997 Lavoie et

al 2004) DGA the hydrolysis product of DHA (Homan and Gaffron 1964) could not

be detected under the present experimental conditions

53 SPECTRAL CHARACTERISTICS OF PHOTOLYSED SOLUTIONS

A typical set of the UV absorption spectra of photolysed solutions of AH2 in

methanol is shown in Fig 5 There is a gradual loss of absorbance around 245 nm with

time as a result of the oxidation of the molecule to DHA (Pelletier 1985 Davies et al

1991 Rumsey and Levine 2000) which does not absorb in this region due to the loss of

conjugation Similar absorption changes are observed on the photolysis of AH2 in other

organic solvents and in the methanolic extracts of cream formulations However the

magnitude of these changes varies with the rate of photolysis in a particular solvent or

cream and appears to be a function of the polar character pH and viscosity of the

medium

72

Fig 5 UV absorption spectra of photolysed solutions of ascorbic acid in methanol at

0 40 80 120 160 220 and 300 min

73

54 ASSAY OF ASCORBIC ACID IN CREAMS AND SOLUTIONS

The assay of AH2 in creams and solutions has been carried out in acidified

methanol (pH 20) according to the UV spectrophotometric method of Zeng et al (2005)

Aqueous solutions of AH2 (~pH 2) exhibit absorption maxima at 243 nm (OrsquoNeil 2001

Moffat et al 2004 Sweetman 2009) 244 nm (Ogata and Kosugi 1969) and 245 nm

(Verma et al 1991 Johnston et al 2007) The absorption maxima of AH2 in methanol

and phosphate buffer (pH 25) occur at 245 nm (Zeng et al 2005) Since dilute solutions

of AH2 are highly susceptible to oxidation the pH was adjusted to 20 with phosphoric

acid to convert the molecule to the non-ionized form (99) to minimize degradation

during the assay AH2 in acidified methanol (pH 20) was found to exhibit the absorption

maximum at 245 nm as reported by Zeng et al (2005) The method was also used for the

assay of AH2 in aqueous and organic solvents

The validity of Beerrsquos law relation in the concentration range used was confirmed

prior to the assay The calibration data for AH2 at the analytical wavelength are presented

in Table 5 (Chapter 4) The correlation coefficient (r = 09996) indicates a good linear

relationship over the concentration range employed The values of specific absorbance

and molar absorptivity at 245 nm determined from the slope of the curve are in good

agreement with those reported by previous workers (Davies et al 1991 Johnston et al

2007) The method of Zeng et al (2005) has been found to be satisfactory for the assay of

AH2 in cream formulations and solutions and has been used to study the kinetics of

photolysis reactions The method was validated before its application to the assay of AH2

in photolysed creams The reproducibility of the method was confirmed by the analysis of

known amounts of AH2 in the concentration range likely to be found in photodegraded

74

creams The values of the recoveries of AH2 in creams by the UV spectrophotometric

method are in the range of 90ndash96 The values of RSD for the assays indicate the

precision of the method within plusmn5 (Table 7)

In order to compare the UV spectrophotometric method with the British

Pharmacopoeia iodimetric method (2009) using a dilute iodine solution (002 M) the

creams were simultaneously assayed for AH2 content by the two methods and the results

are reported in Table 8 The statistical evaluation of the accuracy and precision of the two

methods was carried out by the application of the F test and the t test respectively The F

test showed that there is no significant difference between the precision of the two

methods and the calculated value of F is lower than the critical value (F = 639 P = 005)

in each case The t test indicated that the calculated t values are lower than the tabulated t

values (t = 2776 P = 005) suggesting that at 95 confidence level the differences

between the results of the two methods are statistically non-significant Thus the accuracy

and precision of the UV spectrophotometric method is comparable to that of the official

iodimetric method for the assay of AH2 in cream formulations The results of the assays

of AH2 in aqueous organic solvents and cream formulations are reported in Table 9

55 EFFECT OF SOLVENT

The influence of solvent on the rate of degradation of drugs is of considerable

importance to the formulator since the stability of drug species in solution media may be

predicted on the basis of their chemical reactivity The reactivity of drugs in a particular

medium depends to a large extent on solvent characteristics such as the dielectric

constant and viscosity (Connors et al 1986 Yoshioka and Stella 2000 Sinko 2006)

75

Table 7 Recovery of ascorbic acid added to cream formulationsa

Cream

Formulationb

Added

(mg)

Found

(mg)

Recovery

()

RSD

()

1a 400

200

380

183

950

915

21

25

2b 400

200

371

185

928

925

15

25

3c 400

200

375

181

938

905

11

31

4d 400

200

384

189

960

945

13

21

5b 400

200

370

189

925

945

14

26

6c 400

200

369

190

923

950

10

22

7d 400

200

374

182

935

910

17

39

8c 400

200

380

188

950

940

15

33

9d 400

200

367

189

918

945

20

42

a Values expressed as a mean of three to five determinations

b The cream formulations represent combinations of each emulsifier (stearic acid

palmitic acid myristic acid) with each humectant (glycerin propylene glycol ethylene

glycol) to observe the efficiency of methanol to extract AH2 from different creams

(Table 6)

76

Table 8 Assay of ascorbic acid in creams using UV spectrophotometric and iodimetric

methods

Ascorbic acid (mg) Cream

Formulationb Added UV method

a

Iodimetric

methoda

Fcalc tcalc

1a 40

20

380 plusmn 081

183 plusmn 046

375 plusmn 095

185 plusmn 071

138

238

245

104

2b 40

20

371 plusmn 056

185 plusmn 047

373 plusmn 064

193 plusmn 038

130

065

181

200

3c 40

20

375 plusmn 040

181 plusmn 056

374 plusmn 046

183 plusmn 071

132

160

101

223

4d 40

20

384 plusmn 051

189 plusmn 039

381 plusmn 066

190 plusmn 052

167

178

176

231

5b 40

20

370 plusmn 052

189 plusmn 050

372 plusmn 042

185 plusmn 067

065

179

162

125

6c 40

20

369 plusmn 037

190 plusmn 042

371 plusmn 058

188 plusmn 056

245

177

122

197

7d 40

20

374 plusmn 062

182 plusmn 072

370 plusmn 070

184 plusmn 082

127

129

144

168

8c 40

20

380 plusmn 058

188 plusmn 062

375 plusmn 075

192 plusmn 060

167

094

123

162

9d 40

20

367 plusmn 072

189 plusmn 080

365 plusmn 082

187 plusmn 075

149

092

130

203

Theoretical values (P = 005) for F is 639 and for t is 2776

a Mean plusmn SD (n = 5)

b Table 6

77

Table 9 Photodegradation of ascorbic acid in cream formulations

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) pHa 40 50 60 70

0 383 382 384 383

60 374 369 366 361

120 361 354 346 325

180 351 345 325 305

240 345 327 301 284

1

300 336 316 287 264

0 380 383 382 379

60 371 376 362 346

120 359 357 342 320

180 352 345 322 301

240 341 335 299 283

2

300 336 321 291 261

0 384 376 381 385

60 377 367 360 358

120 366 348 334 324

180 356 337 317 305

240 343 320 301 282

3

300 335 307 273 253

78

Table 9 continued

0 377 378 386 372

60 365 361 371 355

120 353 345 347 322

180 344 327 325 298

240 332 320 306 279

4

300 317 303 284 252

0 381 367 372 373

60 372 358 358 353

120 360 337 336 321

180 352 325 320 302

240 341 313 300 284

5

300 327 302 278 256

0 376 386 380 377

60 366 372 350 350

120 353 347 323 316

180 337 334 308 298

240 329 320 291 274

6

300 313 306 267 245

79

Table 9 continued

0 380 372 378 380

60 373 362 350 354

120 358 340 329 321

180 344 328 304 300

240 332 315 292 283

7

300 319 302 272 252

0 380 381 378 361

60 368 364 361 335

120 355 354 340 313

180 342 340 315 281

240 337 331 303 269

8

300 323 314 281 248

0 378 382 370 375

60 370 369 349 342

120 356 347 326 321

180 339 333 298 291

240 326 314 277 271

9

300 313 302 265 242

a The values at pH 40ndash70 represent the formulations a to d of each cream respectively

80

In order to observe the effect of solvent dielectric constant the apparent first-

order rate constants (kobs) for the photolysis of AH2 in alcoholic solvents (Table 10) were

plotted against the dielectric constants of the solvents A linear relationship indicated the

dependence of the rates of photolysis on solvent dielectric constant (Fig 6) This implies

the involvement of a polar intermediate in the reaction to facilitate the formation of the

degradation products as suggested by Ahmad and Tollin (1981) in the case of flavin

electron transfer reactions The effect of solvent polarity has been observed on the

autooxidation of AH2 in organic solvents (Ogata and Kosugi 1969)

Another solvent parameter affecting the rate of a chemical reaction is viscosity

which can greatly influence the stability of oxidisable substances (Wallwork and Grant

1977 Laidler 1987 Fung 1990) A plot of kobs for the photolysis of AH2 against the

reciprocal of solvent viscosity (Table 10) is linear showing that an increase in solvent

viscosity results in a decrease in the rate of photolysis (Fig 7) The viscosity of the liquid

affects the rate at which molecules can diffuse through the solution This in turn may

affect the rate at which a compound can suffer oxidation at the liquid surface This

applies to AH2 and an increase in the viscosity of the medium makes access to air at the

surface more difficult to prevent oxidation (Wallwork and Grant 1977)

56 EFFECT OF CONCENTRATION

In order to observe the effect of concentration (Table 11) on the photostability of

AH2 in a cream using stearic palmitic and myristic acids as emulsifying agents and

glycerin as humectant plots of log concentration versus time were constructed (Fig 8)

and the apparent first-order rate constants were determined (Table 12) A graph of kobs

against concentration of AH2 (Fig 9) exhibited an apparent linear relationship between

81

Table 10 Apparent first-order rate constants (kobs) for the photolysis of ascorbic acid in

water and organic solvents

Solvent Dielectric

Constant (25 ordmC)

Viscosity

(mPas) ndash1

kobs times104

(minndash1

)

Water 785 1000 404

Methanol 326 1838 324

Ethanol 243 0931 316

1-Propanol 201 0514 302

1-Butanol 178 0393 295

82

00

20

40

60

80

0 10 20 30 40 50 60 70 80

Dielectric constant

k (

min

ndash1)

Fig 6 A plot of kobs for photolysis of ascorbic acid against solvent dielectric constant

(times) Water () methanol () ethanol (diams) 1-propanol () 1-butanol

83

00

10

20

30

40

50

00 05 10 15 20

Viscosity (mPas)ndash1

k times

10

4 (m

inndash1)

Fig 7 A plot of kobs for photolysis of ascorbic acid against reciprocal of solvent

viscosity Symbols are as in Fig 6

84

Table 11 Effect of concentration on the photodegradation of ascorbic acid in cream

formulations at pH 60

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) 05 10 15 20 25

0 95 191 290 379 471

60 90 182 277 358 453

120 82 167 260 339 431

180 77 158 239 311 401

240 70 144 225 298 382

1

300 64 134 210 282 363

0 92 186 287 380 472

60 88 175 272 369 453

120 82 160 251 342 429

180 75 152 238 326 405

240 71 144 225 309 392

2

300 65 134 215 289 366

0 94 182 286 376 470

60 87 171 265 352 454

120 78 152 251 337 426

180 69 143 227 315 404

240 62 129 215 290 378

3

300 58 119 195 271 353

85

05

10

15

20

25

06

08

10

12

14

16

18

log

co

nce

ntr

ati

on

(m

g)

a

05

10

15

20

25

06

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

b

05

10

15

20

25

06

08

10

12

14

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

c

Fig 8 Log concentration versus time plots for the photodegradation of ascorbic acid at

various concentrations in creams at pH 60 a) stearic acid b) palmitic acid

c) myristic acid

86

Table 12 Apparent first-order rate constants (kobs) for the photodegradation of various

ascorbic acid concentrations in cream formulations at pH 60

kobs times 103 (min

ndash1)a Cream

Formulationb 05 10 15 20 25

1 133

(0994)

120

(0993)

111

(0995)

101

(0994)

090

(0994)

2 118

(0992)

108

(0994)

098

(0993)

093

(0992)

084

(0994)

3 169

(0994)

144

(0995)

126

(0994)

109

(0993)

097

(0992)

a The values in parenthesis are correlation coefficients

b Table 6

87

Stearic acid

Palmitic acid

Myristic acid

00

05

10

15

20

25

00 05 10 15 20 25

Ascorbic acid concentration ()

kob

s (min

ndash1)

Fig 9 A plot of kobs for photodegradation against ascorbic acid concentrations in cream

formulations

88

the two values Thus the rate of degradation of AH2 is faster at a lower concentration on

exposure to the same intensity of light This may be due to a relatively greater number of

photons available for excitation of the molecule at lower concentration compared to that

at a higher concentration The AH2 concentrations of creams used in this study are within

the range (1ndash15) reported by previous workers for topical applications to skin (Kaplan

et al 1989 Traikovich et al 1999 Nusgens et al 2001 Matsubayashi et al 2003

Espinal-Perez et al 2004 Sauermann et al 2004 Lin et al 2005 Heber et al 2006)

57 EFFECT OF CARBON CHAIN LENGTH OF EMULSIFYING AGENT

The values of kobs for the photodegradation of AH2 (2) in various cream

formulations are reported in Table 13 The first-order plots for the photodegradation of

AH2 at pH 4ndash7 in various cream formulations are shown in Fig 10ndash12 The plots of kobs

against carbon chain length of the emulsifying agents are shown in Fig 13 They indicate

that the photodegradation of AH2 is affected by the emulsifying agent in the order

myristic acid gt stearic acid gt palmitic acid

These acids possess a polar character (Yao et al 2009) and the carbon chain of the acid

may play a part in the photostability of AH2 However the results indicate that in the

presence of palmitic acid AH2 exhibits greater stability as indicated by the plots of kobs

versus the carbon chain length of the emulsifying agents (Fig 13) This could be

predominantly due to the interaction of AH2 with palmitic acid in the cream to impart it

greater stability Ascorbic acid-6-palmitate is known to be an antioxidant in cosmetic

preparations (Lee et al 2009) and food products (Doores 2002)

In view of the above observations it may be suggested that the photodegradation

of AH2 could involve a polar semiquinone intermediate (Johnston et al 2007) which

89

Table 13 First-order rate constants (kobs) for the photodegradation of ascorbic acid in

cream formulations

kobs times 103 (min

ndash1)abc

Cream

Formulation pH 40 50 60 70

1 044

(0992)

064

(0994)

100

(0995)

126

(0995)

2 042

(0992)

060

(0991)

095

(0992)

120

(0995)

3 047

(0993)

069

(0993)

107

(0991)

137

(0995)

4 056

(0993)

072

(0992)

104

(0994)

131

(0993)

5 050

(0991)

067

(0992)

097

(0991)

124

(0992)

6 061

(0992)

079

(0993)

113

(0992)

140

(0994)

7 060

(0992)

071

(0993)

108

(0994)

133

(0992)

8 053

(0991)

062

(0992)

099

(0994)

126

(0993)

9 065

(0991)

081

(0996)

117

(0993)

142

(0995)

a The rate constants at pH 40ndash70 represent the values for formulations a to d of each

cream respectively

b The values in parenthesis are correlation coefficients

c The values of rate constants are relative and depend on specific experimental conditions

including light intensity

The estimated error is plusmn5

90

12

13

14

15

16

17

log

co

nce

ntr

ati

on

(m

g)

1

12

13

14

15

16

17

log

co

nce

ntr

ati

on

(m

g)

2

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

3

Fig 10 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (1ndash3) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

91

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

4

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

5

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

6

Fig 11 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (4ndash6) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

92

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

7

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

8

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

9

Fig 12 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (7ndash9) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

93

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

ko

bs times

10

3 (m

inndash

1)

1-3

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

ko

bs times

10

3 (

min

ndash1)

4-6

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

Carbon chain length

ko

bs times

10

3 (

min

ndash1)

7-9

Fig 13 Plots of kobs for photodegradation of ascorbic acid in creams (1ndash9) against carbon

chain length of emulsifier () Stearic acid () palmitic acid () myristic acid

Humectant used glycerin (1ndash3) propylene glycol (4ndash6) ethylene glycol (7ndash9)

94

depending on the polar character of the medium undergoes oxidation with varying rates

This is similar to the behavior of the photolysis of riboflavin analogs which is dependent

on the polar character of the medium (Ahmad and Tollin 1981) The effect of carbon

chain length on the transdermal delivery of an active ingredient has been discussed (Lu

and Flynn 2009)

58 EFFECT OF VISCOSITY

The plots of rates of AH2 degradation in cream formulations (Table 13) as a

function of carbon chain length (Fig 13) indicate that the rates vary with the humectant

and hence the viscosity of the medium in the order

ethylene glycol gt propylene glycol gt glycerin

This is in agreement with the rate of photolysis of AH2 in organic solvents that

higher the viscosity of the medium lower the rate of photolysis Thus apart from the

carbon chain length of the emulsifier viscosity of the humectant added to the cream

formulation appears to play an important part in the stability of AH2 The stabilizing

effect of viscosity imparting substances on AH2 solutions has been reported (Stone 1969

Kassem et al 1969ab)

59 EFFECT OF pH

The kobsndashpH profiles for the photodegradation of AH2 in various creams (1ndash9) at

pH 4ndash7 (Fig 14) represent a sigmoid type curve indicating the oxidation of the ionized

form (AHndash) of AH2 (pKa 41) (OrsquoNeil 2001) with pH The AH

ndash species appears to be

more susceptible to photooxidation than the non-ionized form (AH2) The behavior of

AH2 on photooxidation in the pH range 4ndash7 is similar to that observed for the chemical

oxidation of AH2 by molecular oxygen (DeRitter 1982) and involves the interaction of

95

Stearic Acid (1)

Palmitic Acid (2)

Myristic Acid (3)

04

06

08

10

12

14

kob

s times

10

3 (m

inndash

1)

Stearic Acid (4)

Palmitic Acid (5)

Myristic Acid (6)

04

06

08

10

12

14

ko

bs times

10

3 (

min

ndash1)

Stearic Acid (7)

Palmitic Acid (8)

Myristic Acid (9)

04

06

08

10

12

14

30 40 50 60 70

pH

ko

bs

times 1

03

(min

ndash1)

Fig 14 The kobsndashpH profiles for the photodegradation of ascorbic acid in creams (1ndash9)

Glycerin

Propylene glycol

Ethylene glycol

96

AH2 with singlet oxygen on UV irradiation (Silva and Quina 2006) The AHndash species

(predominant in the pH range 42ndash70 557ndash999) is more reactive towards singlet

oxygen than its protonated form the AH2 molecule as suggested by Bisby et al (1999)

and therefore the rate of photooxidation is higher in the pH range above 41

corresponding to the pKa1 of AH2 The major goal of a ratendashpH profile is to determine

the optimum pH range for a particular formulation Several workers have studied the

ratendashpH profiles of the chemical oxidation of AH2 in the pH range 2ndash7 (Garrett 1967

Taqui Khan and Martell 1967 Rogers and Yacomeni 1971 Blaugh and Hajratwala

1972 DeRitter 1982 Moura et al 1994) however the kinetics of photooxidation of

AH2 in cream formulations has so far not been reported

510 EFFECT OF REDOX POTENTIAL

The photooxidation of AH2 is also influenced by its redox potential which varies

with pH The greater photostability of AH2 at pH 5ndash6 compared to that at pH 7 and above

is due to its lower rate of oxidationndashreduction in this range (Eordm pH 50 = +0127 V)

(OrsquoNeil 2001) The increase in the rate of photooxidation with pH is due to a

corresponding increase in the redox potential (Eordm pH 70 = +0058 V) (Fasman 1976) of

AH2 and is similar to the photolysis behavior of riboflavin at pH 5ndash6 (Eordm pH 50 = ndash0117

V) (Sinko 2006) compared to that at pH 70 (Eordm pH 70 = ndash 0207 V) (Ahmad et al

2004a Sinko 2006) Since the ionization as well as the redox potentials of AH2 are a

function of pH the rate of photooxidation depends upon the specific species present and

its redox behavior at a particular pH

97

511 PRIMARY PHOTOCHEMICAL REACTIONS IN THE OXIDATION OF

ASCORBIC ACID

A reaction scheme based on general photochemical principles for the important

reactions involved in the photooxidation of ascorbic acid is presented below

0AH2 hv k1

1AH2 (51)

1AH2 k2 Products (52)

1AH2 isc k3

3AH2 (53)

3AH2 k4 Products (54)

0AH

ndash hv k5

1AH

ndash (55)

1AH

ndash k6 Products (56)

1AH

ndash k7

3AH

ndash (57)

3AH

ndash k8 Products (58)

3AH

ndash +

0AH2 k9 AH٠

ndash + AH٠ (59)

2 AH٠ k10 A + AH2 (510)

3AH2 +

3O2 k11

0AH2 +

1O2 (511)

AHndash +

1O2 k12

3AH

ndash +

3O2 (512)

AH٠ + 1O2 k13 AHOO٠ (513)

AHOO٠ k14 A + HO2٠ (514)

AHOO٠ + 0AH2 k15 AH٠ + AHOOH (515)

AHOOH k16 secondary reaction

A + H2O2 (516)

According to this reaction scheme the ground state ascorbic acid species (0AH2

0AH

ndash) each is excited to the lowest singlet state (

1AH2

1AH

ndash) by the absorption of a

quantum of UV light (51 55) These excited states may directly be converted to

98

photoproducts (52 56) or may undergo intersystem crossing (isc) to form the excited

triplet states (53 57) The excited triplet states may then degrade to the photoproducts

(54 58) The monoascorbate triplet (3AH

ndash) may react with the ground state ascorbic

acid to form a monoascorbate radical anion (AH٠ndash) and a monoascorbate radical (AH٠)

(59) Two AH٠ radical species may lead to the formation of an oxidized (A) and a

reduced ascorbic acid molecule (AH2) (510) Ascorbic acid triplet (3AH2) may react with

molecular oxygen (3O2) to yield singlet oxygen (

1O2) (511) which may then react with

monoascorbate anion (AHndash) to form the excited triplet state (

3AH

ndash) (512) or with

monoascorbate radical to form a peroxyl radical (AHOO٠) (513) The peroxyl radical

may yield dehydroascorbic acid (A) (514) or react with ground state ascorbic acid to

give monoascorbate radical and a reduced species AHOOH (515) The reduced species

may give rise to dehydroascorbic acid and hydrogen peroxide (516)

512 DEGRADATION OF ASCORBIC ACID IN THE DARK

In view of the instability of AH2 and to observe its degradation in the dark the

creams were stored in airtight containers at room temperature in a cupboard for a period

of about 3 months and assayed for AH2 content at appropriate intervals The analytical

data (Table 14) were subjected to kinetic treatment (Fig 15ndash17) and the apparent first-

order rate constants for the degradation of AH2 were determined (Table 15) The values

of the rate constants indicate that the degradation of AH2 in the dark is about 70 times

slower than those of the creams exposed to UV irradiation (Table 13) The degradation of

AH2 in creams in the dark is due to chemical oxidation (Section 132) and occurs in the

order of emulsifying agents (Fig 18)

myristic acid gt stearic acid gt palmitic acid

99

Table 14 Degradation of ascorbic acid in the dark in cream formulations

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(days) pHa 40 50 60 70

0 383 382 384 383

10 354 340 313 278

20 309 306 279 245

40 244 209 183 161

60 172 166 131 105

1

80 145 114 81 61

0 380 383 382 379

10 360 343 350 335

20 322 310 301 294

40 266 250 211 186

60 233 211 168 142

2

80 182 153 114 89

0 384 376 381 385

10 368 350 340 318

20 318 273 273 266

40 223 199 172 155

60 174 132 117 84

3

80 122 97 66 54

100

Table 14 continued

0 377 378 386 372

10 350 334 334 318

20 314 268 256 244

40 238 208 182 136

60 179 155 107 94

4

80 128 101 79 59

0 381 367 372 373

10 350 293 300 320

20 299 266 270 263

40 220 191 192 184

60 183 153 139 129

5

80 149 115 87 76

0 376 386 380 377

10 312 320 314 251

20 255 282 226 199

40 175 194 159 131

60 139 128 99 74

6

80 102 81 55 41

101

Table 14 continued

0 380 372 378 380

10 323 330 333 323

20 288 273 276 224

40 212 174 182 146

60 152 133 108 83

7

80 100 82 66 56

0 380 381 378 361

10 333 320 310 310

20 281 266 260 257

40 230 189 171 177

60 156 148 128 111

8

80 123 96 78 66

0 378 382 370 375

10 313 295 281 300

20 256 247 257 203

40 194 178 151 133

60 119 114 88 74

9

80 88 68 49 39

a The values at pH 40ndash70 represent the formulations a to d of each cream respectively

102

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

1

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

2

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

3

Fig 15 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (1ndash3) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

103

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

4

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

5

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

6

Fig 16 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (4ndash6) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

104

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

7

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

8

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

9

Fig 17 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (7ndash9) at pH 40 (times) 50 () 60 () and 70 ()

Palmitic acid

Myristic acid

Stearic acid

105

Table 15 First-order rate constants (kobs) for the degradation of ascorbic acid in cream

formulations in the dark

kobs times 102 (day

ndash1)abc

Cream

Formulation pH 40 50 60 70

1 128

(0991)

152

(0994)

191

(0995)

220

(0994)

2 091

(0992)

110

(0991)

152

(0993)

182

(0992)

3 148

(0991)

176

(0995)

220

(0993)

254

(0995)

4 137

(0992)

161

(0993)

205

(0994)

236

(0995)

5 121

(0991)

141

(0994)

175

(0993)

195

(0993)

6 162

(0992)

194

(0995)

237

(0994)

265

(0994)

7 164

(0994)

189

(0994)

222

(0993)

246

(0996)

8 143

(0994)

167

(0995)

193

(0996)

212

(0993)

9 184

(0995)

208

(0994)

251

(0992)

280

(0996)

a The rate constants at pH 40ndash70 represent the values for formulations a to d of each

cream respectively

b The values in parenthesis are correlation coefficients

c The values of rate constants are relative and depend on specific experimental

conditions

The estimated error is plusmn5

106

pH 4

pH 5

pH 6

pH 7

00

10

20

30

k times

10

2 (d

ayndash

1)

1-3

pH 4

pH 5

pH 6

pH 7

00

10

20

30

k times

10

2 (

da

yndash1)

4-6

pH 4

pH 5

pH 6

pH 7

00

10

20

30

12 14 16 18

Carbon chain length

k times

10

2 (

da

yndash1)

7-9

Fig 18 Plots of kobs for degradation of ascorbic acid in the dark in creams (1ndash9) against

carbon chain length of emulsifier () Stearic acid () palmitic acid

() myristic acid Humectant used glycerin (1ndash3) propylene glycol (4ndash6)

ethylene glycol (7ndash9)

107

Although it is logical to expect a linear relationship between the rate of degradation and

the carbon chain length of the emulsifier due to its polar character (Yao et al 2009) it

has not been observed in the present case The reason for the slowest rate of degradation

of AH2 in the presence of palmitic acid appears to be due to the interaction of AH2 with

palmitic acid (Lee et al 2009) as explained in Section 57

The degradation of AH2 also appears to be affected by the viscosity of the cream

in the order of humectant (Fig 19)

ethylene glycol gt propylene glycol gt glycerin

Thus the presence of glycerin imparts the most stabilizing effect on the degradation of

AH2 This is the same order as observed in the case of photodegradation of AH2 in the

creams The airtight containers used for the storage of creams make the access of air to

the creams difficult to cause chemical oxidation of AH2 However it has been observed

that the degradation of AH2 is highest in the upper layer of the creams compared to that

of the middle and the bottom layers Therefore the creams were thoroughly mixed before

sampling for the assay of AH2 However the scattering in kinetic plots (Fig 15ndash17) is

due to non-uniform distribution of AH2 in degraded creams

The effect of pH on the degradation of AH2 in the creams (Fig 19) shows that the

degradation increases with an increase in pH as observed in the case of photodegradation

of AH2 in the creams This is due to an increase in the ionization and redox potential of

AH2 with pH causing greater oxidation of the molecule and has been discussed in

Sections 59 and 510

108

Stearic Acid (1)

Palmitic Acid (2)

Myristic Acid (3)

00

10

20

30

k times

10

2 (d

ayndash

1)

Glycerin

Stearic Acid (4)

Palmitic Acid (5)

Myristic Acid (6)

00

10

20

30

k times

10

2 (

da

yndash1)

Propylene glycol

Stearic Acid (7)

Palmitic Acid (8)

Myristic Acid (9)

00

10

20

30

30 40 50 60 70

pH

k times

10

2 (d

ayndash

1)

Ethylene glycol

Fig 19 The kobsndashpH profiles for the degradation of ascorbic acid in the dark in creams

(1ndash9)

CHAPTER VI

PHOTOCHEMICAL INTERACTION

OF ASCORBIC ACID WITH

RIBOFLAVIN NICOTINAMIDE

AND ALPHA-TOCOPHEROL IN

CREAM FORMULATIONS

110

61 INTRODUCTION

It is now medically recognized that sagging skin and other signs of degenerative

skin conditions such as wrinkles and age spots are caused primarily by oxy-radical

damage Ascorbic acid can accelerate wound healing protect fatty tissues from oxidative

damage and play an integral role collagen synthesis (Zhang et al 1999) It is used in

cosmetic preparations for its anti-aging depigmentation and antioxidant properties

(Spiclin 2003 Ehrlich et al 2006) It is also used in combination with other vitamins

such as alpha-tocopherol as a co-antioxidant to stabilize cosmetic preparations (Eberlein-

Koumlnig and Ring 2005 Bissett 2006 Murray 2008) Ascorbic acid in the presence of air

or light is known to interact with alpha-tocopherol (Packer et al 2002 Johnston et al

2007) riboflavin (Homann and Gaffron 1964 Sattar et al 1977 Heelis et al 1981

Kim et al 1993 Jung et al 1995 De La Rochette et al 2000 2003 Lavoie et al

2004 Vaid et al 2005 Ahmad and Vaid 2006 Silva and Quina 2006) and

nicotinamide (Bailey et al 1945 Werner et al 1949 Guttman and Brooke 1963

DeRitter 1982) The present work involves a study of the effect of alpha-tocopherol

riboflavin and nicotinamide on the photostability of ascorbic acid in cream formulations

to observe whether the interaction in these formulations leads to the stabilization of

ascorbic acid The chemical structures of nicotinamide (NA) alpha-tocopherol (TP)

riboflavin (RF) formylmethylflavin (FMF) and lumichrome (LC) are shown in Fig 20

The details of the cream formulations used in this study are given in Table 16

The results obtained on the photodegradation of ascorbic acid in cream formulations are

discussed in the following sections

111

Riboflavin

N

N

NH

N

CH2

CH

C OHH

CH OH

CH2OH

N

N

NH

N

CH2

CHO

Formylmethylflavin

N

N

NH

HN

Lumichrome

OH

N

NH2

O

Nicotinamide

O CH3

CH3

CH3

HO

H3C

CH3 CH3 CH3

CH3

Alpha-Tocopherol

O

O

H3C

H3C

H3C

H3C

O

O

H3C

H3C

O

O

Fig 20 Chemical structures of alpha-tocopherol nicotinamide riboflavin

formylmethylflavin and lumichrome

112

Table 16 Composition of cream formulations containing ascorbic acid (2) and other

vitamins

Ingredients Cream

No SA PA MA CA GL AH2 RFa NA

b TP

c PH DW

10 a + minus minus + + + a minus minus + +

b + minus minus + + + b minus minus + +

c + minus minus + + + c minus minus + +

d + minus minus + + + d minus minus + +

e + minus minus + + + e minus minus + +

11 a minus + minus + + + a minus minus + +

b minus + minus + + + b minus minus + +

c minus + minus + + + c minus minus + +

d minus + minus + + + d minus minus + +

e minus + minus + + + e minus minus + +

12 a minus minus + + + + a minus minus + +

b minus minus + + + + b minus minus + +

c minus minus + + + + c minus minus + +

d minus minus + + + + d minus minus + +

e minus minus + + + + e minus minus + +

13 a + minus minus + + + minus a minus + +

b + minus minus + + + minus b minus + +

c + minus minus + + + minus c minus + +

d + minus minus + + + minus d minus + +

e + minus minus + + + minus e minus + +

14 a minus + minus + + + minus a minus + +

b minus + minus + + + minus b minus + +

c minus + minus + + + minus c minus + +

d minus + minus + + + minus d minus + +

e minus + minus + + + minus e minus + +

113

Table 16 continued

15 a minus minus + + + + minus a minus + +

b minus minus + + + + minus b minus + +

c minus minus + + + + minus c minus + +

d minus minus + + + + minus d minus + +

e minus minus + + + + minus e minus + +

16 a + minus minus + + + minus minus a + +

b + minus minus + + + minus minus b + +

c + minus minus + + + minus minus c + +

d + minus minus + + + minus minus d + +

e + minus minus + + + minus minus e + +

17 a minus + minus + + + minus minus a + +

b minus + minus + + + minus minus b + +

c minus + minus + + + minus minus c + +

d minus + minus + + + minus minus d + +

e minus + minus + + + minus minus e + +

18 a minus minus + + + + minus minus a + +

b minus minus + + + + minus minus b + +

c minus minus + + + + minus minus c + +

d minus minus + + + + minus minus d + +

e minus minus + + + + minus minus e + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PH = potassium hydroxide DW = distilled water

RF = riboflavin NA = nicotinamide TP = alpha-tocopherol

a RF(g ) a = 0002 b = 0004 c = 0006 d = 0008 e = 0010

b NA (g ) a = 028 b = 056 c = 084 d = 112 e = 140

c TP (g ) a = 017 b = 034 c = 052 d = 069 e = 086

The molar concentrations of these vitamins are given in Section 421

114

62 ABSORPTION CHARACTERISTICS OF PHOTOLYSED CREAMS

A typical set of the absorption spectra of the methanolic extracts (pH 20) of the

freshly prepared and photolysed creams containing AH2 and TP is shown in Fig 21 AH2

in acidified methanol exhibits absorption maximum at 245 nm (Zeng et al 2005) as

observed in Fig 21 The absorption due to TP at 284 nm (Moffat et al 2004) was

cancelled by using an appropriate blank containing an equivalent concentration of TP

The gradual decrease in absorption at around 245 nm during UV irradiation indicates the

transformation of AH2 to DHA which does not absorb in this region (Davies et al 1991)

as a result of the loss of C3=C2 chromophore Similar spectral changes around 245 nm are

observed in the presence of RF and NA which also strongly absorb in this region A

decrease in the absorption of AH2 around 266 nm in aqueous solution (pH 60) in the

presence of RF has been reported (Vaid et al 2005) The spectral changes and loss of

absorbance in methanolic extracts of creams depends on the rate of photolysis of AH2 in

the presence of these vitamins

63 PHOTOPRODUCTS OF ASCORBIC ACID AND OTHER VITAMINS

The UV irradiation of AH2 in cream formulations (pH 60) in the presence of RF

NA and TP results in the degradation of AH2 and RF and the following photoproducts

have been identified on comparison of their RF values and spot color fluorescence with

those of the authentic compounds

AH2 DHA

RF FMF LC CMF

In the TLC systems used NA and TP did not show the formation of any

degradation product in creams

115

Fig 21 UV absorption spectra of methanolic extracts of photodegraded ascorbic acid in

cream at 0 60 120 180 300 and 480 min

116

The formation of DHA in the photooxidation of AH2 has previously been reported by

many workers (Homann and Gaffron 1964 Sattar et al 1977 Heelis et al 1981

Rozanowska et al 1997 Lavoie et al 2004 Vaid et al 2006) RF is sensitive to light in

aqueous solutions (DeRitter 1982 British Pharmacopoeia 2009 Sweetman 2009) and is

known to form a number of products under aerobic conditions (Treadwell et al 1968

Cairns and Metzler 1971 Schuman Jorns et al 1975 Ahmad and Rapson 1990 Ahmad

and Vaid 2006 Ahmad et al 2004ab 2005 2008 Vaid et al 2006) It has been found

to degrade on UV irradiation in cream formulations to give FMF LC and CMF and these

products have been reported in the photolysis of RF by the workers cited above The

formation of these products may be affected by the interaction of AH2 and RF in creams

(Section 66) NA and TP individually did not appear to form any photoproduct of their

own directly or on interaction with AH2 in creams and may influence the degradation of

AH2 on UV irradiation

64 ASSAY METHOD

In view of the presence of RF (absorption maxima 223 267 373 and 444 nm)

(British Pharmacopoeia 2009) NA (absorption maximum 261 nm) (Moffat et al 2004)

and TP (absorption maximum 284 nm) (Moffat et al 2004) in the cream formulations

and the interference of these vitamins with the absorption of AH2 (absorption maximum

265 nm) (Davies et al 1991) the direct spectrophotometric method cannot be applied for

the determination of AH2 Therefore the iodimetric method (British Pharmacopoeia

2009) was used to determine AH2 in cream formulations The method was validated in

the presence of RF NA and TP before its application to the determination of AH2 in

photodegraded creams The reproducibility of the method has been confirmed by the

117

assay of known concentrations of AH2 in the range present in photodegraded creams The

recovery of AH2 in the creams has been found to be in the range 90ndash96 The values of

RSD indicate that the precision of the method is within plusmn5 (Table 17) and it can be

applied to study the kinetics of AH2 photolysis in cream formulations The assay data on

AH2 in various cream formulations are given in Table 18

65 KINETICS OF PHOTODEGRADATION OF ASCORBIC ACID

Several chemical and physical factors play a role in the photodegradation of AH2

in the presence of other vitamins (RF NA TP) and affect the rate of its degradation in

cream formulations The present work involves the study of photodegradation of AH2 in

cream formulations containing glycerin as humectant as AH2 has been found to be most

stable in these creams (Chapter 5) The apparent first-order rate constants (kobs) for the

photodegradation of AH2 in the presence of other vitamins in cream formulations

derived from the kinetic plots (Fig 22ndash24) are reported in Table 19 The second-order

rate constants (correlation coefficients 0991ndash0996) determined from the slopes of the

graphs of kobs versus vitamin concentration for the individual vitamins (Fig 25) and the

values of k0 determined from the intercept on the vertical axis at zero concentration are

reported in Table 20 The values of k0 give an indication of the effect of other vitamins on

the rate of degradation of AH2 These values are about 13 times lower than the values of

kobs obtained at the highest concentrations of RF and NA indicating that RF and NA both

accelerate the photodegradation of AH2 in creams RF is known to act as a

photosensitizer for the degradation of AH2 (Section 66) and therefore its presence in

creams would accelerate the degradation of AH2 The increase in the rate of

photodegradation of AH2 in the presence of NA has not previously been reported NA

118

Table 17 Recovery of ascorbic acid in cream formulations in the presence of other

vitamins by iodimetric methoda

Cream

Formulationb

Added

(mg )

Found

(mg )

Recovery

()

RSD

()

10e (RF) 400

200

373

187

933

935

29

22

11e (RF) 400

200

379

187

948

935

25

31

12e (RF) 400

200

375

188

938

940

29

28

13e (NA) 400

200

382

191

955

955

23

27

14e (NA) 400

200

380

185

950

925

19

26

15e (NA) 400

200

379

191

948

955

21

17

16e (TP) 400

200

368

183

920

915

29

44

17e (TP) 400

200

391

195

978

975

11

13

18e (TP) 400

200

377

182

943

910

32

37

a Values expressed as a mean of three to five determinations

b The cream formulations represent all the emulsifiers (stearic acid palmitic acid

myristic acid) to observe the efficiency of iodimetric method for the recovery of

ascorbic acid in presence of the highest concentration of vitamins (Table 16)

119

Table 18 Photodegradation of ascorbic acid in cream formulations in the presence of

other vitamins

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) a b C d e

0 373 372 374 372 375

60 362 354 354 360 359

150 342 336 336 332 334

240 315 314 308 310 302

10 (RF)

330 301 291 288 281 282

0 380 379 376 374 374

60 370 366 362 362 361

150 343 337 340 332 328

240 329 323 320 313 310

11 (RF)

330 307 301 294 288 282

0 379 380 375 372 376

60 362 366 361 351 342

150 341 335 319 307 312

240 310 306 295 284 282

12 (RF)

330 285 278 263 254 243

120

Table 18 continued

0 372 370 371 368 365

60 361 358 348 350 349

120 342 343 329 326 330

180 327 325 319 312 308

240 317 309 299 289 285

13 (NA)

300 299 291 283 278 273

0 386 380 375 378 370

60 371 362 365 362 355

120 359 351 343 339 336

200 341 332 325 316 311

14 (NA)

300 313 303 296 294 280

0 375 371 374 370 366

60 362 356 352 352 345

120 343 332 336 326 314

200 323 315 311 295 293

15 (NA)

300 293 283 275 270 259

121

Table 18 continued

0 380 378 380 377 377

60 362 365 369 369 371

120 351 352 360 360 364

180 340 346 349 353 355

240 331 334 343 343 346

16 (TP)

300 320 323 330 332 337

0 383 380 378 380 377

60 372 371 372 373 370

120 363 360 361 366 365

180 348 348 350 356 355

240 341 343 343 348 348

17 (TP)

300 330 332 336 339 341

0 380 383 377 375 373

60 364 370 366 367 366

120 352 356 351 352 351

180 334 338 339 343 342

240 324 328 324 332 330

18 (TP)

300 307 315 317 318 322

122

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

10

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

11

ab

c

de

13

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

12

Fig 22 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing riboflavin (a) 0002 (b) 0004 (c) 0006 (d) 0008

(e) 0010

Stearic acid

Palmitic acid

Myristic acid

123

abcde

13

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

13

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

14

abcde

13

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

15

Fig 23 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing nicotinamide (a) 028 (b) 056 (c) 084 (d) 112 (e) 140

Stearic acid

Palmitic acid

Myristic acid

124

abcde

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

16

abcde

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

17

abcde

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

18

Fig 24 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing alpha-tocopherol (a) 017 (b) 034 (c) 052 (d) 069

(e) 086

Stearic acid

Myristic acid

Palmitic acid

125

Table 19 First-order rate constants (kobs) for the photodegradation of ascorbic acid in the

presence of other vitamins in cream formulations

kobs times 103 (min

ndash1)ab

Cream

formulationd

Other

vitaminc

a b C d e

10 RF 068

(0991)

073

(0996)

079

(0995)

085

(0992)

089

(0995)

11 RF 065

(0992)

070

(0992)

073

(0994)

080

(0995)

086

(0993)

12 RF 087

(0993)

096

(0995)

109

(0993)

116

(0994)

127

(0992)

13 NA 073

(0993)

081

(0992)

088

(0994)

096

(0994)

101

(0993)

14 NA 069

(0992)

074

(0992)

080

(0991)

086

(0995)

094

(0995)

15 NA 083

(0994)

090

(0993)

101

(0993)

109

(0994)

115

(0995)

16 TP 055

(0991)

051

(0994)

046

(0994)

042

(0993)

038

(0991)

17 TP 050

(0995)

045

(0993)

041

(0992)

038

(0995)

034

(0994)

18 TP 070

(0996)

066

(0996)

060

(0994)

055

(0993)

051

(0993)

a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental conditions

including the light intensity

c Vitamin concentrations (andashe) are as given in Table 16

d All the creams contain glycerin as humectant

The estimated error is plusmn5

126

00

05

10

15

00 10 20 30

Riboflavin concentration (M times 104)

kob

s times

10

3 (

min

ndash1)

10-12

00

05

10

15

00 20 40 60 80 100 120

Nicotinamide concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

13-15

00

02

04

06

08

00 04 08 12 16 20

Alpha-Tocopherol concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

16-18

Fig 25 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against individual vitamin concentration in cream formulations (10ndash18)

127

Table 20 First-order rate constants (k0)a for the photodegradation of ascorbic acid in the

absence of other vitamins and second-order rate constants (k) for the

photochemical interaction of ascorbic acid with RF NA and TP

Cream

formulation

Other

vitamin

k0 times 103

(minndash1

)

k

(Mndash1

minndash1

)

Correlation

coefficient

10 RF 062 102 0994

11 RF 059 097 0992

12 RF 077 189 0995

13 NA 066 032 times 10ndash2

0995

14 NA 062 027 times 10ndash2

0993

15 NA 074 037 times 10ndash2

0994

16 TP 059 110 times 10ndash2b

0996

17 TP 053 096 times 10ndash2b

0992

18 TP 075 123 times 10ndash2b

0994

a

The variations in the values of k0 are due to the degradation of AH2 in the presence of

different emulsifying agents in cream formulations

b Values for the inhibition of photodegradation of AH2

128

forms a complex with AH2 (Section 67) and may also act as a photosensitizer for AH2 by

energy transfer in the excited state on UV irradiation The absorption maximum of NA

(261 nm) (Moffat et al 2004) is very close to that of AH2 (265 nm) (Davies et al 1991)

and the possibility of energy transfer in the excited state (Moore 2004) is greater leading

to the photodegradation of AH2

The value of k0 is about 13 times greater than the values of kobs obtained for the

degradation of AH2 in the presence of the highest concentrations of TP in the creams

This indicates that TP has a stabilising effect on the photodegradation of AH2 in the

cream formulations This is in agreement with the view that the TP acts as a redox partner

with AH2 to retard its oxidation (Wille 2005) Thus among the three vitamins studied

only TP appears to have a stabilising effect on photodegradation of AH2 The

photochemical interaction of individual vitamins with AH2 is discussed below

66 INTERACTION OF RIBOFLAVIN WITH ASCORBIC ACID

The interaction of RF with the ascorbate ion (AHndash) may be represented by the

following reactions proposed by Silva and Quina (2006)

RF rarr 1RF (61)

1RF rarr

3RF (62)

3RF + AH

ndash rarr RF

ndashmiddot + AHmiddot (63)

AHmiddot + O2 rarr A + HO2middot (64)

HO2middot + AHndash rarr H2O2 + AHmiddot (65)

RF on the absorption of a quantum of light is promoted to the excited singlet state (1RF)

(61) 1RF may undergo intersystem crossing (isc) to form the excited triplet state (

3RF)

(62) The excited triplet state may react with the ascorbate ion to generate the ascorbyl

hv

isc

129

radical (AH) (63) (Kim et al 1993) The ascorbyl radical reacts with oxygen to give

dehydroascorbic acid (A) and peroxyl radical (HO2) (64) This radical may interact with

ascorbate ion to generate further ascorbyl radicals (65) These radicals may again take

part in the sequence of reactions to form A The role of RF in this reaction is to act as a

photosensitiser in the oxidation of ascorbic acid to A Ascorbic acid is reported to protect

riboflavin in milk under the influence of light by reacting with singlet oxygen (Hall et al

2009) (Section 511)

67 INTERACTION OF NICOTINAMIDE WITH ASCORBIC ACID

NA is known to form a 11 complex with ascorbic acid (Guttman and Brooke

1963 OrsquoNeil 2001 Doores 2002) The complexation of NA and AH2 may result from

the donor-acceptor interaction between AH2 (donor) and NA (acceptor) as observed in

the case of tryptophan and NA (Florence and Attwood 2006) In the presence of light the

interaction may cause reduction of NA (NAH) to form the ascorbyl radical (AH) ((66)-

(68)) which is oxidized to dehydroascorbic acid (A) (69) The NAH may be oxidized to

NA and H2O2 (610)

NA rarr 1NA (66)

1NA rarr

3NA (67)

3NA + AH2 rarr NAH + AHmiddot (68)

2 AH٠ rarr A + AH2 (69)

NAH + O2 rarr NA + H2O2 (610)

The proposed reactions suggest that on photochemical interaction AH2 undergoes

photosensitised oxidation in the presence of NA indicating that the photostability of

ascorbic acid is affected by NA

isc

130

68 INTERACTION OF ΑLPHA-TOCOPHEROL WITH ASCORBIC ACID

TP is an unstable compound and its oxidation by air results in the formation of an

epoxide which than produces a quinone that is inactive (Connors et al 1986) TP is

destroyed by sun light and artificial light containing the wavelengths in the UV region

(Ball 2006) It interacts with other antioxidants such as ascorbic acid (AH2) according to

the following reactions

TPndashO + AH2 rarr TP + AHmiddot (611)

2 AHmiddot rarr A + AH2 (612)

TP + AHmiddot rarr TPndashO + AH2 (613)

The tocopheroxyl radical (TPndashO) reacts with AH2 to regenerate TP and form the

ascorbyl radical (AHmiddot) (611) This radical undergoes further reactions as described in

equations (64) and (65) (Traber 2007) It may also disproportionate back to A and AH2

(612) TP reacts with AHmiddot to produce again the TPndashO radical and AH2 Thus in the

presence of light TP leads to the oxidation of AH2 which is ultimately regenerated in the

reaction (Davies et al 1991) Ascorbic acid has the ability to act as a co-antioxidant with

the TPndashO to regenerate TP (Packer et al 1979 2002) In this manner AH2 and TP act

synergistically to function in a redox cycle and AH2 is stabilized in the cream

formulations and microemulsions (Rozman and Gasperlin 2007 Rozman et al 2009)

69 EFFECT OF CARBON CHAIN LENGTH OF EMULSIFYING AGENT

The graphs of kobs for the photodegradation of AH2 in the presence of RF NA and

TP versus the carbon chain length of emulsifying agents are shown in Fig 26 It appears

that the photodegradation of AH2 in the presence of all the three vitamins in the creams

lies in the order

131

Fig 26 Plots of k for photodegradation of ascorbic acid in creams (10ndash18) against

carbon chain length of emulsifier () Stearic acid () palmitic acid

() myristic acid

00

05

10

15

20

25

k

(Mndash

1 m

inndash

1)

00

05

10

15

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash

1 m

inndash

1)

132

myristic acid gt stearic acid gt palmitic acid

The same order of emulsifying agents has been observed in the absence of the

added vitamins (Section 57) The polar character of these acids (Yao et al 2009) on the

basis of their carbon chain length may play a part in the photostability of AH2 The

greater stability of AH2 in creams in the presence of palmitic acid (Fig 26) may be due to

the interaction of AH2 with palmitic acid as discussed in Section 57 Ascorbic acid-6-

palmitate is known to be an antioxidant in cosmetic preparations (Lee et al 2009) and

food products (Doores 2002)

610 EFFECT OF VISCOSITY OF CREAMS

The plots of kobs for the degradation of AH2 in the presence of the highest

concentration of vitamins versus reciprocal of the viscosity of creams (Table 21) are

linear (Fig 27) and indicate that the increase in cream viscosity leads to a decrease in the

rate of degradation of AH2 The slopes of the plots indicate the effect of viscosity on the

interaction of AH2 with other vitamins in the order

riboflavin gt nicotinamide gt alpha-tocopherol

The relatively slow rate of degradation of AH2 in creams containing palmitic acid may be

due to the interaction of AH2 with the vitamins as well as palmitic acid (Lee et al 2009)

Thus viscosity is an important factor in the stability of AH2 in cream formulations and

may affect its rate of interaction with other vitamins It has been suggested that an

increase in the viscosity of the medium makes access to air at the surface more difficult to

prevent the oxidation of a drug (Wallwork and Grant 1977) This is in agreement with

the photolysis of AH2 in aqueous and organic solvents cream formulations (Chapter 5)

and aerobic oxidation of Ah2 in syrups (Blaug and Hajratwala 1972)

133

Table 21 Average viscosity of cream formulations containing different emulsifying

agents and glycerin as humectant (25 plusmn 1 ordmC) and the photodegradation rate

constants of AH2

Cream No Emulsifying

agent

Viscosityab

(mPa s)

kobs times 103c

10 (RF)

13 (NA)

16 (TP)

Stearic acid 9000 089

101

038

11 (RF)

14 (NA)

17 (TP)

Palmitic acid 8600 086

094

034

12 (RF)

15 (NA)

18 (TP)

Myristic acid 7200 127

115

051

a plusmn10

b Average viscosity of creams containing the individual vitamins (RF NA TP)

c The values have been obtained in the presence of highest concentration of the

vitamins

134

00

05

10

15

20

25

30

100 110 120 130 140

Viscosity (mPa s)ndash1

times 103

kob

s (m

inndash1)

Fig 27 Plots of kobs in the presence of highest concentration of vitamins versus

reciprocal of the viscosity of creams () riboflavin

( ) nicotinamide (- - -- - -) alpha-tocopherol

135

611 DEGRADATION OF ASCORBIC ACID IN THE PRESENCE OF OTHER

VITAMINS IN THE DARK

In order to observe the effect of riboflavin nicotinamide and alpha-tocopherol on

the degradation of AH2 in the creams stored in the dark the AH2 contents of the creams

were assayed at appropriate intervals (Table 22) The apparent first-order rate constants

determined from the kinetic plots (Fig 28) for the degradation of AH2 in the presence of

the highest concentrations of the individual vitamins in cream formulations (10ndash18) are

reported in Table 23 These rate constants indicate that the overall degradation of AH2 in

the presence of the highest concentration of the individual vitamins (RF NA and TP) is

about 70 times slower than that obtained on the exposure of creams to UV irradiation

This decrease in the rate of degradation of AH2 in the creams is the same as observed in

the case of AH2 alone In the absence of light the degradation of AH2 occurs due to

chemical oxidation (Section 132) and does not appear to be affected by the presence of

riboflavin and nicotinamide as indicated by the comparisons of the values of kobs in the

presence and absence of these vitamins (Table 15 and 23) In the presence of alpha-

tocopherol the degradation is slower than that in the presence of riboflavin and

nicotinamide This may be due to some interaction of AH2 and alpha-tocopherol causing

stabilisation of AH2 in the creams

As observed in the case of AH2 degradation alone in creams in the dark the AH2

degradation in the presence of the highest concentrations of other vitamins also occurs in

the same order of emulsifying agents (Fig 29)

myristic acid gt stearic acid gt palmitic acid

136

Table 22 Degradation of ascorbic acid in cream formulations in the dark in presence of

highest concentration of other vitamins

Concentration of ascorbic acid (mg 2g) Cream

No Time (days) 0 10 20 40 60 80

10e (RF) 375 285 233 171 110 69

11e (RF) 374 341 281 221 148 113

12e (RF) 372 259 203 130 89 59

13e (NA) 365 330 255 187 126 81

14e (NA) 370 321 289 219 159 109

15e (NA) 366 289 249 159 110 63

16e (TP) 377 359 321 261 211 159

17e (TP) 377 366 333 275 228 191

18e (TP) 373 361 304 252 200 167

137

02

07

12

17lo

g c

on

cen

tra

tio

n (

mg

)

10-12Riboflavin

02

07

12

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

13-15Nicotinamide

10

12

14

16

18

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

16-18Alpha-Tocopherol

Fig 28 First-order plots for the degradation of ascorbic acid in the dark in presence of

other vitamins using the emulsifying agents (minusminusminusminus) Stearic acid

(minus minusminus minus) palmitic acid (----) myristic acid

138

Table 23 First-order rate constants (kobs) for the degradation of ascorbic acid in presence

of other vitamins in cream formulations in the dark

Cream

formulation

Other

vitaminc

kobs times 102

(dayndash1

)ab

10e RF 204

(0995)

11e RF 156

(0992)

12e RF 222

(0992)

13e NA 189

(0995)

14e NA 151

(0993)

15e NA 214

(0995)

16e TP 100

(0994)

17e TP 088

(0995)

18e TP 105

(0993)

a The values in parenthesis are correlation coefficients and range from 0991ndash0996 due to

some variations in AH2 distribution in the creams

b The values of rate constants are relative and depend on specific experimental

conditions

c Vitamin concentrations (andashe) are as given in Table 16

The estimated error is plusmn5

139

Riboflavin

Nicotinamide

Alpha-Tocopherol

00

10

20

30

12 14 16 18Carbon chain length

ko

bs times

10

2 (

da

yndash1)

Fig 29 Plots of kobs for degradation of ascorbic acid in the dark in creams (10ndash18)

against carbon chain length of the emulsifier () Stearic acid () palmitic acid

() myristic acid

140

This indicates that the rate of degradation of AH2 is slowest in the creams containing

palmitic acid as the emulsifying agent The reason for AH2 degradation in the dark in this

order has already been explained in section 512

CHAPTER VII

STABILIZATION OF

ASCORBIC ACID WITH

CITRIC ACID TARTARIC

ACID AND BORIC ACID IN

CREAM FORMULATIONS

142

71 INTRODUCTION

Ascorbic acid is an ingredient of cosmetic preparations (Section 51) and is

sensitive to light (Rowe et al 2009 Sweetman 2009 British Pharmacopoeia 2009)

degrading to dehydroascorbic acid on UV irradiation by photooxidation (Kitagawa

1968) The photosensitivity of ascorbic acid makes it unstable in pharmaceutical and

cosmetic preparations (DeRitter 1982) The present work is an attempt to study the

photodegradation of ascorbic acid in cream formulations in the presence of certain

compounds (eg citric acid tartaric acid and boric acid) to investigate their role in the

stabilization of the vitamin on exposure to light and in the dark Citric acid and tartaric

acid are used as an antioxidant synergist (Rowe et al 2009) and boric acid is a

complexing agent for hydroxy compounds (Ahmad et al 2009cd)

72 CREAM FORMULATIONS

The details of the various cream formulations used in this study are given in Table

24 and the results obtained on the photodegradation of ascorbic acid in the presence of

stabilizing agents in these formulations are discussed in the following sections

143

Table 24 Composition of cream formulations containing ascorbic acid (2) and

stabilizers

Ingredients Cream

No SA PA MA CA GL PG EG AH2 CTa TA

b BA

c PH DW

19 a + minus minus + + minus minus + a minus minus + +

b + minus minus + + minus minus + b minus minus + +

c + minus minus + + minus minus + c minus minus + +

20 a minus + minus + + minus minus + a minus minus + +

b minus + minus + + minus minus + b minus minus + +

c minus + minus + + minus minus + c minus minus + +

21 a minus minus + + + minus minus + a minus minus + +

b minus minus + + + minus minus + b minus minus + +

c minus minus + + + minus minus + c minus minus + +

22 a + minus minus + + minus minus + minus a minus + +

b + minus minus + + minus minus + minus b minus + +

c + minus minus + + minus minus + minus c minus + +

23 a minus + minus + + minus minus + minus a minus + +

b minus + minus + + minus minus + minus b minus + +

c minus + minus + + minus minus + minus c minus + +

24 a minus minus + + + minus minus + minus a minus + +

b minus minus + + + minus minus + minus b minus + +

c minus minus + + + minus minus + minus c minus + +

25 a + minus minus + + minus minus + minus minus a + +

b + minus minus + + minus minus + minus minus b + +

c + minus minus + + minus minus + minus minus c + +

26 a minus + minus + + minus minus + minus minus a + +

b minus + minus + + minus minus + minus minus b + +

c minus + minus + + minus minus + minus minus c + +

27 a minus minus + + + minus minus + minus minus a + +

b minus minus + + + minus minus + minus minus b + +

c minus minus + + + minus minus + minus minus c + +

144

Table 24 continued

28 a + minus minus + minus + minus + minus minus a + +

b + minus minus + minus + minus + minus minus b + +

c + minus minus + minus + minus + minus minus c + +

29 a minus + minus + minus + minus + minus minus a + +

b minus + minus + minus + minus + minus minus b + +

c minus + minus + minus + minus + minus minus c + +

30 a minus minus + + minus + minus + minus minus a + +

b minus minus + + minus + minus + minus minus b + +

c minus minus + + minus + minus + minus minus c + +

31 a + minus minus + minus minus + + minus minus a + +

b + minus minus + minus minus + + minus minus b + +

c + minus minus + minus minus + + minus minus c + +

32 a minus + minus + minus minus + + minus minus a + +

b minus + minus + minus minus + + minus minus b + +

c minus + minus + minus minus + + minus minus c + +

33 a minus minus + + minus minus + + minus minus a + +

b minus minus + + minus minus + + minus minus b + +

c minus minus + + minus minus + + minus minus c + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PG = propylene glycol EG = ethylene glycol

PH = potassium hydroxide DW = distilled water CT = citric acid TA = tartaric acid

BA = boric acid

a CT (g ) a = 01 b = 02 c = 04

b TA (g ) a = 01 b = 02 c = 04

c BA (g ) a = 01 b = 02 c = 04

145

73 PRODUCTS OF ASCORBIC ACID PHOTODEGRADATION

The photodegradation of AH2 in cream formulations leads to the formation of

DHA as detected by TLC and reported earlier in the photolysis of AH2 in aqueous

solutions (Vaid et al 2006) and cream formulations (Sections 52 and 63) AH2 and

DHA in the methanolic extracts of the degraded creams were identified by comparison of

their Rf and color of the spots with those of the reference standards DHA is also

biologically active (Gardner 1972 Doores 2002) but its further degradation to 23-

diketo-gulonic acid (DGA) results in the loss of vitamin activity (Section 132)

However this product has not been detected in the present cream formulations

Therefore the creams may still possess their biological efficacy

74 SPECTRAL CHANGES IN PHOTODEGRADED CREAMS

In order to observe the spectral changes in photodegraded creams in the presence

of stabilizing agents the absorption spectra of the methanolic extracts of a degraded

cream were determined The spectra show a gradual loss of absorbance around 245 nm

due to the oxidation of AH2 to DHA on UV irradiation and similar to that shown for the

photodegradation of AH2 alone in Fig 5 DHA has negligible absorbance around 245 nm

(Davies et al 1991) and therefore it does not interfere with the absorbance of AH2 in

methanolic solutions The spectral changes and loss of absorbance around 245 nm in

methanolic solution depend on the extent of photooxidation of AH2 in a particular cream

75 ASSAY OF ASCORBIC ACID IN CREAMS

The UV spectrophotometric method (Zeng et al 2005) has previously been

applied to the determination of AH2 in cream formulations (Section 54) The absorbance

of the methanolic extracts of creams containing AH2 during photodegradation was used

146

to determine the concentration of AH2 The method was validated in the presence of citric

acid (CT) tartaric acid (TA) and boric acid (BA) before its application to the evaluation

of the kinetics of AH2 degradation in cream formulations The recovery of AH2 in creams

has been found to be in the range of 90ndash96 and is similar to that reported in Table 7

The reproducibility of the method lies within plusmn5 The assay data on the degradation of

AH2 in various creams in the presence of the stabilizing agents are reported in Table 25

76 KINETICS OF PHOTODEGRADATION

The effect of CT TA and BA as stabilizing agents on the photodegradation of

AH2 was studied by adding 01ndash04 of each compound to the cream formulations (19ndash

33) at pH 60 This concentration range is normally used for the stabilization of drugs in

pharmaceutical preparations (Im-Emsap et al 2002) The apparent first-order rate

constants (kobs) determined from the plots of log concentration versus time (Fig 30ndash34)

are reported in Table 26 The second-order rate constants (k) determined from the plots

of kobs versus concentration of the individual compounds (Fig 35ndash36) are given in Table

27 The values of k indicate the rate of inhibition of photodegradation of AH2 by each

compound

77 EFFECT OF STABILIZING AGENTS

In order to compare the effectiveness of CT TA and BA as stabilizing agents for

AH2 plots of k versus carbon chain length of the emulsifying agents were constructed

(Fig 37) The k values for the interaction of these compounds with AH2 are in the order

citric acid gt tartaric acid gt boric acid

The curves indicate that the highest interaction of these compounds with AH2 is in the

order

147

Table 25 Photodegradation of ascorbic acid in cream formulations in the presence of

stabilizers

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) a b c

0 374 378 379

60 362 362 372

120 349 355 367

210 333 335 349

19 (CT)

300 319 322 336

400 296 309 324

0 381 378 380

60 368 370 369

120 355 363 364

210 344 345 355

20 (CT)

300 328 335 341

400 312 319 331

21 (CT) 0 368 370 374

60 355 356 360

120 340 344 343

210 321 322 333

300 296 299 315

400 272 285 299

148

Table 25 continued

0 375 374 378

60 363 363 368

120 352 354 362

210 329 335 345

22 (TA)

300 307 314 333

400 292 299 313

0 370 377 374

60 364 365 368

120 352 357 357

210 332 344 349

23 (TA)

300 317 330 335

400 301 310 322

24 (TA) 0 376 379 377

60 367 369 368

120 351 348 352

210 325 330 344

300 306 317 326

400 284 294 310

149

Table 25 continued

0 370 375 380

60 356 362 359

120 331 339 344

210 311 318 330

25 (BA)

300 279 288 305

400 260 269 283

0 377 375 370

60 364 363 361

120 351 353 351

210 331 332 337

26 (BA)

300 323 324 325

400 301 307 313

27 (BA) 0 380 377 375

60 369 368 366

120 333 338 341

210 305 313 318

300 292 294 304

400 262 266 281

150

Table 25 continued

0 373 376 378

60 348 349 360

120 329 336 339

210 315 312 323

28 (BA)

300 282 283 299

400 249 264 280

0 370 373 380

60 358 355 367

120 343 346 356

210 325 329 347

29 (BA)

300 307 312 325

400 287 295 315

30 (BA) 0 369 375 372

60 353 358 362

120 321 330 335

210 283 294 303

300 265 281 293

400 242 254 270

151

Table 25 continued

0 374 376 379

60 348 366 352

120 324 340 337

210 303 319 322

31 (BA)

300 275 289 293

400 243 260 275

0 370 374 375

60 355 354 366

120 339 344 345

210 313 319 330

32 (BA)

300 288 297 308

400 261 271 290

33 (BA) 0 377 380 377

60 357 361 367

120 324 335 339

210 288 294 307

300 270 280 293

400 233 248 265

Creams 19ndash27 contain glycerin 28ndash30 contain propylene glycol and 31ndash33 contain

ethylene glycol as humectants

152

a

b

c

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

19

ab

c

14

15

16

log

co

nce

ntr

ati

on

(m

g)

20

a

b

c

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

21

Fig 30 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and citric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

153

a

b

c

14

15

16lo

g c

on

cen

tra

tio

n (

mg

)

22

a

b

c

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

23

a

b

c

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

24

Fig 31 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and tartaric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

154

ab

c

13

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

25

abc

13

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

26

ab

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

27

Fig 32 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and boric acid (a) 01 (b) 02 (c) 04

Palmitic acid

Stearic acid

Myristic acid

155

a

b

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

28

ab

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

29

a

b

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

30

Fig 33 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing propylene glycol and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

156

a

b

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

31

ab

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

32

a

b

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

33

Fig 34 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing ethylene glycol and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

157

Table 26 Apparent first-order rate constants (kobs) for the photodegradation of ascorbic

acid in presence of different stabilizers in cream formulations

kobs times 103 (min

ndash1)ab

Cream

formulation Stabilizer

c

a b c

19 CT 057

(0995)

050

(0992)

041

(0991)

20 CT 049

(0996)

043

(0995)

034

(0993)

21 CT 076

(0995)

067

(0995)

055

(0992)

22 TA 065

(0995)

058

(0995)

046

(0991)

23 TA 054

(0994)

047

(0993)

038

(0994)

24 TA 072

(0996)

063

(0992)

049

(0991)

25 BA 091

(0994)

086

(0995)

071

(0993)

26 BA 055

(0994)

050

(0993)

042

(0993)

27 BA 095

(0995)

089

(0992)

074

(0996)

28 BA 097

(0995)

088

(0992)

075

(0993)

29 BA 064

(0994)

057

(0991)

047

(0993)

30 BA 110

(0994)

100

(0996)

084

(0992)

31 BA 105

(0995)

094

(0994)

078

(0992)

32 BA 088

(0994)

079

(0993)

066

(0993)

33 BA 120

(0995)

108

(0993)

091

(0993) a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental conditions

including the light intensity

c Stabilizer concentrations (andashc) are as given in Table 24

The estimated error is plusmn5

158

00

04

08

12

00 04 08 12 16 20

Citric acid concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

19-21

00

04

08

12

00 05 10 15 20 25

Tartaric acid concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

22-24

Fig 35 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against citric acid (19ndash21) and tartaric acid concentrations (22ndash24) in cream

formulations

159

00

04

08

12k

ob

s times

10

3 (

min

ndash1)

25-27

00

04

08

12

00 20 40 60

ko

bs times

10

3 (

min

ndash1)

28-30

00

04

08

12

00 20 40 60

Boric acid concentration (M times 102)

kob

s times

10

3 (

min

ndash1)

30-33

Fig 36 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against boric acid concentrations in cream formulations (25ndash33)

Propylene glycol

Glycerin

Ethylene glycol

160

Table 27 First-order rate constants (k0)a for the photodegradation of ascorbic acid in the

absence of stabilizers and second-order rate constants (k) for the interaction of

ascorbic acid with CT TA and BA

Cream

formulation Stabilizers

k0 times 103

(minndash1

)

k times 102

(Mndash1

minndash1

)

Correlation

coefficient

19 CT 062 111 0991

20 CT 053 103 0994

21 CT 082 145 0995

22 TA 071 092 0995

23 TA 059 080 0993

24 TA 080 118 0996

25 BA 098 041 0994

26 BA 059 026 0994

27 BA 102 044 0995

28 BA 104 046 0992

29 BA 069 033 0995

30 BA 118 054 0994

31 BA 113 053 0995

32 BA 095 045 0995

33 BA 129 060 0993

a The variations in the values of k0 are due to the presence of different emulsifying agents

in cream formulations

161

00

04

08

12

16

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash1

min

ndash1)

18-33

a

b

e

cd

Fig 37 Plots of k for photodegradation of ascorbic acid in creams (19ndash33) against

carbon chain length of emulsifier () Stearic acid () palmitic acid ()

myristic acid (a) CT (b) TA (c) BA with glycerin (d) BA with propylene

glycol (e) BA with ethylene glycol

162

myristic acid gt stearic acid gt palmitic acid

In the case of myristic acid and stearic acid it may be explained on the basis of the

decreasing polarity (Yao et al 2009) It is interesting to observe the lowest rates of

interaction of these compounds in the creams containing palmitic acid This could be due

to the interaction of AH2 with palmitic acid to form a palmitate derivative in addition to

its interaction with the individual stabilizing agents CT and TA are known to act as

antioxidant synergists (Rowe et al 2009 Sweetman 2009) and in this capacity may

inhibit the photooxidation of AH2 as indicated by the values of the degradation rate

constants in the presence of these compounds The addition of CT to nutritional

supplements is known to inhibit the oxidation of AH2 (Doores 2002) Boric acid forms a

complex with AH2 (Rivlin 2007) and there by may inhibit its degradation Boric acid

may also interact with glycerin added to the creams as a humectant and form a complex

(Rowe et al 2009) This may influence its interaction and stabilizing effect on AH2 in

creams as indicated by the lower k values compared to those in the presence of CT and

TA It has further been observed that the k values for BA are greater in propylene glycol

and ethylene glycol compared to those in glycerin (Table 27) Again this may be due to

greater interaction of BA with glycerin compared to other humectants in the creams

78 DEGRADATION OF ASCORBIC ACID IN PRESENCE OF STABILIZING

AGENTS IN THE DARK

An important factor in the formulation of cosmetic preparations is to ensure the

chemical and photostability of the active ingredient by the use of appropriate stabilizing

agents The choice of these agents would largely depend on the nature and

physicochemical characteristics of the active ingredient AH2 possesses a redox system

163

and can be easily oxidized by air or light In order to observe the effect of CT TA and

BA on the stability of AH2 the cream formulations containing the individual compounds

were stored in the dark for a period of about three months and the rate of degradation of

AH2 was determined The assay data are reported in Table 28 and the kinetic plots are

shown in Fig 38ndash42 The values of apparent first-order rate constants for the degradation

of AH2 in the presence of the stabilizing agents are reported in Table 29 The second

order-rate constants for the interaction of CT TA and BA with AH2 are reported in Table

30 (Fig 43ndash44) The plots of k against the carbon chain length of the emulsifiers are

shown in Fig 45 The kinetic data indicate the same pattern of rates of degradation and

interaction of AH2 with these compounds as observed in the presence of light except that

the rates are much slower in the dark Thus the stabilizing agents are equally effective in

inhibiting the rate of degradation of AH2 in the dark The effect of emulsifying agents and

the humectants on the rate of degradation of AH2 in the presence of the stabilizers has

been discussed in the above Section 77

79 EFFECT OF ADDITIVES ON TRANSMISSION OF ASCORBIC ACID

In order to observe the effect of additives (citric tartaric and boric acids) on the

transmission characteristics of ascorbic acid (0002 mg100 ml) in methanol containing

the highest concentration of the additives (004) used in this study the transmission

spectra were measured It has been found that these additives produce a hypsochromic

shift in the absorption maximum of ascorbic acid This may result in the reduction of the

fraction of light absorbed by ascorbic acid to the extent of about 10 and thus influence

the rate of photodegradation reactions However since all the additives produce similar

effects the rate constants can be considered on a comparative basis

164

Table 28 Degradation of ascorbic acid in cream formulations in the presence of

stabilizers in the dark

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(days) a b c

0 374 378 379

10 355 346 362

20 326 328 342

40 293 297 322

19 (CT)

60 264 269 295

80 241 245 262

0 381 378 380

10 361 364 372

20 339 350 348

40 309 312 330

20 (CT)

60 279 286 301

80 260 266 282

21 (CT) 0 368 370 374

10 342 346 364

20 310 321 348

40 278 282 313

60 249 251 278

80 217 228 249

165

Table 28 continued

0 375 374 378

10 339 344 351

20 317 326 336

40 282 288 306

22 (TA)

60 251 258 280

80 222 235 252

0 370 377 374

10 340 354 355

20 332 336 343

40 297 303 310

23 (TA)

60 266 282 294

80 238 248 267

24 (TA) 0 376 379 377

10 341 339 350

20 306 319 323

40 263 284 279

60 223 241 249

80 196 202 223

166

Table 28 continued

0 370 375 380

10 331 341 334

20 287 289 301

40 225 247 245

25 (BA)

60 189 185 214

80 141 154 170

0 377 375 370

10 355 357 349

20 326 314 324

40 264 267 286

26 (BA)

60 232 238 254

80 189 199 211

27 (BA) 0 380 377 375

10 346 339 337

20 309 288 301

40 233 241 260

60 192 196 211

80 140 147 163

167

Table 28 continued

0 373 376 378

10 314 322 333

20 267 281 305

40 217 233 253

28 (BA)

60 167 177 204

80 122 135 151

0 370 373 380

10 336 329 343

20 283 277 306

40 233 243 267

29 (BA)

60 189 190 217

80 144 154 173

30 (BA) 0 369 375 372

10 308 319 329

20 255 275 310

40 210 226 244

60 158 163 191

80 113 131 147

168

Table 28 continued

0 374 376 379

10 303 311 329

20 266 260 289

40 211 219 239

31 (BA)

60 155 158 178

80 112 121 149

0 370 374 375

10 314 323 339

20 276 280 305

40 222 233 258

32 (BA)

60 172 187 193

80 126 136 162

33 (BA) 0 377 380 377

10 308 306 320

20 254 265 280

40 205 214 237

60 144 155 175

80 107 118 138

169

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

19

abc

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

20

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

21

Fig 38 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing citric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

170

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

22

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

23

ab

c

12

13

14

15

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

24

Fig 39 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing tartaric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

171

a

b

c

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

25

abc

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

26

ab

c

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

27

Fig 40 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing glycerin and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

172

a

b

c

10

12

14

16

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

28

ab

c

10

12

14

16

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

29

a

b

c

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

30

Fig 41 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing propylene glycol and boric acid (a) 01 (b) 02 (c)

04

Palmitic acid

Stearic acid

Myristic acid

173

ab

c

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

31

ab

c

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

32

a

b

c

08

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

33

Fig 42 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing ethylene glycol and boric acid (a) 01 (b) 02 (c)

04

Myristic acid

Palmitic acid

Stearic acid

174

Table 29 Apparent first-order rate constants (kobs) for the degradation of ascorbic acid in

presence of different stabilizers in cream formulations in the dark

kobs times 102 (day

ndash1)ab

Cream

formulation Stabilizer

c

a b c

19 CT 055

(0994)

052

(0992)

044

(0991)

20 CT 048

(0995)

046

(0995)

038

(0992)

21 CT 064

(0994)

061

(0995)

052

(0994)

22 TA 063

(0994)

058

(0995)

049

(0996)

23 TA 054

(0995)

050

(0995)

041

(0994)

24 TA 081

(0995)

075

(0993)

066

(0995)

25 BA 118

(0996)

113

(0994)

097

(0994)

26 BA 087

(0995)

079

(0993)

068

(0994)

27 BA 124

(0995)

114

(0994)

101

(0993)

28 BA 134

(0995)

124

(0996)

110

(0992)

29 BA 116

(0996)

108

(0992)

096

(0995)

30 BA 142

(0993)

131

(0995)

115

(0995)

31 BA 145

(0995)

137

(0992)

117

(0995)

32 BA 130

(0996)

120

(0993)

107

(0994)

33 BA 153

(0995)

141

(0994)

122

(0994) a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental

conditions

c Stabilizer concentrations (andashc) are as given in Table 24

The estimated error is plusmn5

175

176

Table 30 First-order rate constants (k0)a for the degradation of ascorbic acid in the

absence of stabilizers and second-order rate constants (k) for the chemical

interaction of ascorbic acid with CT TA and BA in the dark

Cream

formulation Stabilizers

k0 times 102

(dayndash1

)

k times 102

(Mndash1

dayndash1

)

Correlation

coefficient

19 CT 060 797 0996

20 CT 052 723 0995

21 CT 069 850 0994

22 TA 068 710 0996

23 TA 058 636 0994

24 TA 086 758 0994

25 BA 126 444 0993

26 BA 092 375 0992

27 BA 131 480 0991

28 BA 141 488 0993

29 BA 122 418 0994

30 BA 149 531 0991

31 BA 155 578 0996

32 BA 137 472 0994

33 BA 163 627 0996

a The variations in the values of k0 are due to the presence of different emulsifying agents

in cream formulations

177

00

04

08

12

00 04 08 12 16 20

Citric acid concentration (M times 102)

kob

s times

10

2 (

da

yndash1)

19-21

00

04

08

12

00 05 10 15 20 25

Tartaric acid concentration (M times 102)

kob

s times

10

2 (

da

yndash1)

22-24

Fig 35 Plots of first-order rate constants (kobs) for the degradation of ascorbic acid in the

dark against citric acid (19ndash21) and tartaric acid (22ndash24) concentrations in

cream formulations

178

00

10

20

00 20 40 60

kob

s times

10

3 (

min

ndash1)

25-27

00

10

20

00 20 40 60

kob

s times

10

3 (

min

ndash1)

28-30

00

10

20

00 20 40 60

Boric acid concentration (M times 102)

kob

s times

10

3 (

min

ndash1)

30-33

Fig 44 Plots of first-order rate constants (kobs) for the degradation of ascorbic acid in the

dark against boric acid concentrations in cream formulations (25ndash33)

Glycerin

Propylene glycol

Ethylene glycol

179

00

04

08

12

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash

1 d

ayndash

1)

18-33

b

a

e

dc

Fig 45 Plots of k for degradation of ascorbic acid in the dark in creams (19ndash33) against

carbon chain length of emulsifier () Stearic acid () palmitic acid ()

myristic acid (a) CT (b) TA (c) BA with glycerin (d) BA with propylene

glycol (e) BA with ethylene glycol

CONCLUSIONS

AND

SUGGESTIONS

180

CONCLUSIONS

The main conclusions of the present study on the photodegradation of the ascorbic

acid in organic solvents and cream formulations are as follows

1 Identification of Photodegradation Products

The photodegradation of ascorbic acid in aqueous organic solvents and

laboratory prepared oil-in-water cream preparations on UV irradiation leads to the

formation of dehydroascorbic acid No further degradation products of dehydroascorbic

acid have been detected under the present experimental conditions The product was

identified by comparison of its Rf value and color of the spot with those of the authentic

compound by thin-layer chromatography and spectral changes

2 Assay of Ascorbic Acid

Ascorbic acid in aqueous organic solvents and cream preparations was assayed

in acidified methanolic solutions (pH 20) at 245 nm using a UV spectrophotometric

method Ascorbic acid in combination with other vitamins (riboflavin nicotinamide and

alpha-tocopherol) was assayed by the official iodimetric method due to interference by

these vitamins at the analytical wavelength Both analytical methods were validated

under the experimental conditions employed before their application to the assay of

ascorbic acid The recoveries of ascorbic acid in cream preparations are in the range of

90ndash96 and the reproducibility of both methods are within plusmn5 The F test and the t test

show that there is no significant difference between the precision of the two methods and

therefore these methods can be applied to the assay of ascorbic acid in cream

preparations with comparable results

181

3 Kinetics of Photodegradation

a) Photodegradation of ascorbic acid in organic solvents

Ascorbic acid degradation follows apparent first-order kinetics in aqueous

organic solvents A plot of the first-order rate constants (log kobs) versus solvent dielectric

constant is linear with positive slope indicating an increase in the rate with dielectric

constant On the contrary a plot of kobs verses reciprocal of solvent viscosity is linear with

a positive slope showing a decrease in the rate with solvent viscosity Thus the rate of

photodegradation of ascorbic acid (an oxidizable drug) depends on the solvent

characteristics

b) Photodegradation of ascorbic acid in cream preparations

Ascorbic acid has been found to follow apparent first-order kinetics in cream

preparations and the rate of degradation is affected by the following factors

i Effect of concentration

An apparent linear relationship has been observed between log kobs and

concentration (05ndash25) of ascorbic acid in a cream preparation Thus the rate of

degradation of ascorbic acid appears to be faster at a lower concentration

compared to that of a higher concentration on exposure to the same intensity of

light

ii Effect of carbon chain length of the emulsifying agent

The plots of kobs verses carbon chain length of the emulsifying agent show that the

photodegradation of ascorbic acid is affected in the order myristic acid gt stearic

acid gt palmitic acid This is predominantly due to the interaction of ascorbic acid

with palmitic acid and the carbon chain length (measure of relative polar

182

character) of the emulsifying acid probably does not play a part in the

photodegradation kinetics of ascorbic acid in creams This is evident from the

non-linear relationship between the rate constants for ascorbic acid degradation

and the carbon chain length of the emulsifying acids

iii Effect of viscosity

The values of kobs for the photodegradation of ascorbic acid in cream preparations

are in the order of humectant ethylene glycol gt propylene glycol gt glycerin

showing that the rates of degradation are influenced by the viscosity of the

humectant and decrease with an increase in the viscosity as observed in the case

of organic solvents

iv Effect of pH

The log kndashpH profiles for the photodegradation of ascorbic acid in creams

represent sigmoid type curves indicating an increase in the rate of oxidation of the

molecule with ionization (pH 42ndash70 557ndash999) The AHndash species appears to

be more susceptible to oxidation than the non-ionized molecule in the pH range

studied

v Effect of redox potential

The values of kobs show that the rate of photooxidation of ascorbic acid is

influenced by its redox potential which varies with pH The greater photostability

of ascorbic acid at pH 5ndash6 compared to that at pH 7 and above is due to its lower

rate of oxidation-reduction in the lower range The increase in the rate of

photooxidation with pH is due to a corresponding increase in the redox potential

of ascorbic acid

183

c) Photodegradation of ascorbic acid in the presence of other vitamins (riboflavin

nicotinamide alpha-tocopherol) in cream preparations

The photodegradation of ascorbic acid is affected by the presence of other

vitamins in creams The kinetic data on the photochemical interactions indicate that

riboflavin and nicotinamide act as photosensitizers in the degradation of ascorbic acid

and have an adverse effect on the photostability of the vitamin in creams Whereas

alpha-tocopherol exerts an inhibitory effect on the degradation of ascorbic acid by acting

as a redox partner in the creams Thus a combination of ascorbic acid and alpha-

tocopherol has a synergistic effect on the stabilization of ascorbic acid in creams These

vitamins do not appear to influence the rate of degradation of ascorbic acid in the dark

d) Photodegradation of ascorbic acid in the presence of citric acid tartaric acid and

boric acid in cream preparations

The rate of photodegradation of ascorbic acid in creams has been found to be

inhibited by the addition of compounds such as citric acid tartaric acid and boric acid in

creams These compounds show a stabilizing effect on the photodegradation of ascorbic

acid in the order citric acid gt tartaric acid gt boric acid The lower effect of boric acid

may be due to its interaction with the emulsifying agents and humectants Boric acid

exerts this effect by complex formation with ascorbic acid Citric acid and tartaric acid

are antioxidant synergists and in combination with ascorbic acid may exert a stabilizing

effect on its degradation

184

Salient Features of the Work

In the present work an attempt has been made to study the effects of solvent

characteristics formulation factors particularly the emulsifying agents in terms of the

carbon chain length and humectants in terms of viscosity medium pH drug

concentration redox potential and interactions with other vitamins and stabilizers on the

kinetics of photodegradation of ascorbic acid in cream preparations The study may

provide useful information to improve the photostability and efficacy of ascorbic acid in

cream preparations

SUGGESTIONS

The present work may provide guidelines for a systematic study of the stability of

drug substances in cream ointment preparations and the evaluation of the influence of

formulation variables such as emulsifying agents and humectants concentration pH

polarity viscosity redox potential on the rate of degradation and stabilization of drug

substances This may enable the formulator in the judicious design of formulations that

have improved stability and efficacy for therapeutic use The kinetic parameters may

throw light on the comparative stability of the preparations and help in the choice of

appropriate formulation ingredients

REFERENCES

186

Abe Y Horii H Taniguchi S (1986) A theoretical study of the oxidation mechanism

of triose reductone in reference to L-ascorbic acid Can J Chem 64 360-365

Ahmad I (1968) A Study of the Degradation of Riboflavin and Related Compounds

PhD Thesis University of London London

Ahmad I Rapson HDC (1990) Multicomponent spectrophotometric assay of

riboflavin and photoproducts J Pharm Biomed Anal 8 217-223

Ahmad I Vaid FHM (2006) Photochemistry of flavins in aqueous and organic

solvents In Silva E Edwards AM Eds Flavins Photochemistry and

Photobiology Royal Society of Chemistry Cambridge Chap 2

Ahmad I Tollin G (1981) Solvent effect on flavin electron transfer reactions

Biochemistry 20 5925-5928

Ahmad I Jamil N Zoha SMS (1973) Statistical evaluation of antioxidants used in

the manufacture of ascorbic acid tablets J Sci Univ Kar 2 269-287

Ahmad I Fasihullah Q Vaid FHM (2004b) A study of simultaneous photolysis and

photoaddition reactions of riboflavin in aqueous solution J Photochem Photobiol B

Biol 75 13-20

Ahmad I Fasihullah Q Vaid FHM (2005) Effect of phosphate buffer on

photodegradation reactions of riboflavin in aqueous solution J Photochem

Photobiol B Biol 78 229-234

Ahmad I Fasihullah Q Vaid FHM (2006a) Effect of light intensity and wavelength

on photodegradation reactions of riboflavin in aqueous solution J Photochem

Photobiol B Biol 82 21-27

187

Ahmad I Fasihullah Q Vaid F H M (2006b) Photolysis of formylmethylflavin in

aqueous and organic solvents Photochem Photobiol Sci 5 680-685

Ahmad I Ahmed S Sheraz MA Vaid FHM (2008) Effect of borate buffer on the

photolysis of riboflavin in aqueous solution J Photochem Photobiol B Biol 93 82-

87

Ahmad I Ahmed S Sheraz MA Vaid FHM (2009c) Borate toxicity effect on

drug stability and analytical applications In Chung MP Ed Handbook on

Borates Chemistry Production and Applications Nova Science Publishers Inc

New York in press

Ahmad I Sheraz MA Ahmed S Vaid FHM (2009d) Analytical applications of

borates Mater Sci Res J in press

Ahmad I Fasihullah Q Noor A Ansari IA Ali QNM (2004a) Photolysis of

riboflavin in aqueous solution a kinetic study Int J Pharm 280 199-208

Ahmad I Ahmed S Sheraz MA Aminuddin M Vaid FHM (2009a) Effect of

caffeine complexation on the photolysis of riboflavin in aqueous solution a kinetic

study Chem Pharm Bull in press

Ahmad I Ahmed S Sheraz MA Vaid FHM Ansari IA (2009b) Effect of

divalent ions on photodegradation kinetics and pathways of riboflavin in aqueous

solution Photochem Photobiol Sci accepted

Ahmed S (2009) Effect of Complexing Agents on the Photodegradation of Riboflavin

in Aqueous Solution PhD Thesis Baqai Medical University Karachi

Ahmad T (1978) Studies on the Stability of Sulphacetamide and Degradation Products

PhD Thesis University of Karachi Karachi

188

Ahmad T Ahmad I (1983) The effect of antioxidants on the photolysis of aqueous

sulfacetamide and sulfanilamide solutions Pol J Pharmacol Pharm 35 69-75

Albini A Fasani E Eds (1998) Drugs Photochemistry and Photostability The Royal

Society of Chemistry Cambridge

Allwood MC (1984a) Factors influencing the stability of ascorbic acid in total

parenteral nutrition infusions J Clin Hosp Pharm 9 75-85

Allwood MC (1984b) Compatibility and stability of TPN mixtures in big bags J Clin

Pharm Ther 9 181-198

Allwood MC Kearney MCJ (1998) Compatibility and stability of additives in

parenteral nutrition admixtures Nutrition 14 697-706

Al-Meshal IA Hassan MMA (1982) Ascorbic Acid In Florey K Ed Analytical

Profiles of Drug Substances Vol 11 Academic Press New York pp 45-78

Anderson NH Johnston D McLelland MA Munden P (1991) Photostability

testing of drug substances and drug products in UK pharmaceutical laboratories J

Pharm Biomed Anal 9 443-449

Ansari IA (2002) Photolysis and Interactions of Cyanocobalamin with some B-

Vitamins and Ascorbic Acid in Parenteral Solutions PhD Thesis University of

Karachi Karachi

Ansari IA Vaid Vaid HM Ahmad I (2004) Spectral Study of Photolysis of

Aqueous Cyanocobalamin Solutions in Presence of Vitamins B and C Pak J Pharm

Sci 17 83-93

Arcus CL Zilva SS (1940) The photochemical decomposition of L-ascorbic acid II

Biochem J 34 61-66

189

Arger CB Rupp D Lo P (1996) Preservation of dispersed systems In Liberman

HA Rieger MM Banker GS Eds Pharmaceutical Dosage Forms Disperse

Systems 2nd ed vol1 Marcel Dekker New York Chap9

Asard H May J Smirnoff N Eds (2004) Vitamin C Function and Biochemistry in

Animals and Plants BIOS Scientific Publishers New York

Asker AF Canady D Cobb C (1985) Influence of DL-methionine on the

photostability of ascorbic acid solutions Drug Dev Ind Pharm 11 2109-2125

Ault RG Baird DK Carrington HC Haworth WN Herbert R Hirst EL

Percival EGV Smith F Stacey M (1933) Synthesis of d- and of l-ascorbic acid

and of analogous substances J Chem Soc 1419-1423

Austria R Semenzato A Bettero A (1997) Stability of vitamin C derivatives in

solution and topical formulations J Pharm Biomed Anal 15 795-801

Baertschi SW Alsante KM (2005) Stress testing The chemistry of drug degradation

In Baertschi SW Ed Pharmaceutical Stress testing Predicting Drug Degradation

Taylor amp Francis London Chap 3

Bailey CW Bright JR Jasper JJ (1945) A study of the binary system nicotinamide-

ascorbic acid J Am Chem Soc 67 1184-1186

Ball GFM (2006) Vitamins in Foods Analysis Bioavailability and Stability CRC

Press Boca Raton FL Chap 15

Banhegyi G Marcolongo P Puskas F Fulceri R Mandl J Benedetti A (1998)

Dehydroascorbate and ascorbate transport in rat liver microsomal vesicles J Biol

Chem 273 2758-2762

190

Bardolph EM Taylor RH (1997) Sailors scurvy science and authority J R Soc

Med 90 238

Barel AO Paye M Maibach HI Eds (2001) Handbook of Cosmetic Science and

Technology Marcel Dekker New York

Barry BW (1983) Dermatological Formulations Percutaneous Absorption Marcel

Dekker New York

Barry BW (2002) Transdermal drug delivery In Aulton ME Ed Pharmaceutics

The Science of Dosage Form Design Churchill Livingstone London Chap 33

Basu TK Temple NJ Garg ML (1999) Antioxidants in Human Health and

Disease CABI Publishing Oxford

Becher P (1965) Emulsions Theory and Practice Reinhold New York p 1

Beissenhirtz M Scheller F Lisdat F (2003) Immobilized cytochrome c sensor in

organicaqueous media for the characterization of hydrophilic and hydrophobic

antioxidants Electroanalysis 15 1425-1435

Berlett BS Stadtman ER (1997) Protein oxidation in aging disease and oxidative

stress J Biol Chem 272 20313-20316

Betageri G Prabhu S (2002) Semisolid preparations In Swarbrick J Boylan JC

Eds Encyclopedia of Pharmaceutical Technology 2nd ed Marcel Dekker New

York pp 2436-2457

Bielski BHJ (1982) Chemistry of ascorbic acid radicals In Seib PA Tolbert BM

Eds Ascorbic Acid Chemistry Metabolism and Uses Advances in Chemistry

Series American Chemical Society Washington DC pp 81-100

191

Bielski BHJ Allen AO Schwartz HA (1981) Mechanism of disproportionation of

ascorbate radicals J Am Chem Soc 103 3516-3518

Billany M (2002) Suspensions and emulsions In Aulton ME Ed Pharmaceutics

The Science of Dosage Form Design 2nd ed Churchill Livingstone London Chap

23

Bisby RH Morgan CG Hamblett I Gorman AA (1999) Quenching of singlet

oxygen by trolox C ascorbate and amino acids effect of pH and temperature J Phys

Chem 103 7454-7459

Bissett DL (2006) Anti-aging skin care formulations In Draelos ZD Thaman LA

Eds Cosmetic Formulation of Skin Care Products Taylor amp Francis Group New

York Chap 11

Blaugh SM Hajratwala B (1972) Kinetics of aerobic oxidation of ascorbic acid J

Pharm Sci 61 556-562

Block LH (1996) Pharmaceutical emulsions and microemulsions In Lieberman HA

Rieger MM Banker GS Eds Pharmaceutical Dosage Forms Disperse Systems

Vol 2 2nd ed Chap 2

Bolliger HR Konig A (1969) Water-soluble vitamins In Stahl E Ed Thin-layer

Chromatography Springer-Verlag Berlin pp 304-306

Bonwstra JA De Graaff A Gooris GS Nijsse J Wiechers JW Van Aelst AC

(2003) Water distribution and related morphology in human stratum corneum at

different hydration levels J Invest Dermatol 120 750-758

Bors SR (2005) Scurvy Thomas Dunne Books St Martinrsquos Griffen New York

192

Bors W Buettner GR (1997) The vitamin C radical and its reactions In Packer L

Fuchs J Eds Vitamin C in Health and Disease Marcel Dekker New York Chap

4

Braun L Puskas F Csala M Meszaros G Mandl J Banhegyi G (1997)

Ascorbate as a substrate for glycolysis or gluconeogenesis evidence for an interorgan

ascorbate cycle Free Radic Biol Med 23 804-808

British Pharmacopoeia (2009) Her Majestyrsquos Stationary Office London Electronic

version

Buettner G (1993) The pecking order of free radicals and antioxidants lipid

peroxidation α-tocopherol and ascorbate Arch Biochem Biphys 300 535-543

Buettner GR Schafer FQ (2004) Ascorbate as an antioxidant In Asard H May

JM Smirnoff N Eds Vitamin C Function and Biochemistry in Animals and

Plants BIOS Scientific Publishers Oxford Chap 10

Burke KE (2006) Topical nutritional antioxidants In Draelos ZD Thaman LA

Eds Cosmetic Formulation of Skin Care Products Taylor amp Francis Group New

York Chap 21

Burns JJ Ed (1961) Vitamin C Ann NY Acad Sci 92 1

Byrom P Turnbull JH (1967) Excited states of flavin coenzymes II Anaerobic

oxidation of amino acids by excited riboflavin derivatives Photochem Photobiol 6

125-131

Cadenas E Packer L (2002) Handbook of Antioxidants 2nd ed Marcel Dekker New

York Chap 7-9

193

Capellmann M Bolt HM (1992) Simultaneous determination of ascorbic acid and

dehydroascorbic acid by HPLC with postcolumn derivatisation and fluorometric

detection Fresenius J Anal Chem 342 462-466

Cairns WL Metzler DE (1971) Photochemical degradation of flavins VI A new

photoproduct and its use in studying photolytic mechanism J Am Chem Soc 93

2772-2777

Carlotti ME Ugazio E Sapino S Peira E Gallarate M (2006) Photodegradation

of retinol and anti-aging effectiveness of two commercial emulsions J Cosmet Sci

57 261-277

Carpenter K (1986) The History of Scurvy and Vitamin C Cambridge University Press

Cambridge

Carr AC Zhu BZ Frei B (2000) Potential antiatherogenic mechanisms of ascorbate

(vitamin C) and alpha-tocopherol (vitamin E) Circ Res 87 349-354

Carstensen JT (1972) Theory of Pharmaceutical Systems Academic Press New York

pp 211-214

Chaudhri SK Jain NK (2009) History of cosmetics Asian J Pharm 3 164-167

Chessex P Lovoic JC Roulean T Brochu P St Louis P Levy E Al Varez F

(2002) Photooxidation of parenteral multivitamins induced hepatic steatosis in a

neonatal guinea pig model of intravenous infusion Pediatr Res 52 958-963

Connors KA Amidon GL Stella VJ (1986) Chemical Stability of Pharmaceuticals

A Handbook for Pharmacists 2nd ed Wiley New York pp 105-106 142 208-220

194

Dabrowski K Huiterleitner S (1989) Application of a simultaneous assay of ascorbic

acid dehydroascorbic acid and ascorbic sulfate in biological matter Analyst

(London) 114 83-87

Dale JK Booth RE (1976) In Hoover JE Ed Dispensing of Medication 8th ed

Mack Easton Pennsylvania pp 479-481

Darr D Dunston S Faust H Pinnell S (1996) Effectiveness of antioxidants (vitamin

C and E) with and without sunscreens as topical photoprotectants Acta Derm

Venereol 76 264-268

Davey MW Montagu MV Inze D Sanmartin M Kanellis A Smirnoff N

Benzie IJJ Strain JJ Favell D Fletcher J (2000) Plant L-ascorbic acid

chemistry function metabolism bioavailability and effect of processing J Sci Food

Agr 80 825-860

Davies MB Austin J Partridge DA (1991) Vitamin C Its Chemistry and

Biochemistry The Royal Society of Chemistry Cambridge

De La Rochette A Birlouez-Aragon I Silva E Morliere P (2003) Advanced

glycation endproducts as UVA photosensitizers of tryptophan and ascorbic acid

consequences for the lens Biochem Biophys Acta 1621 235-241

De La Rochette A Silva E Birlouez-Aragon I Mancini M Edwards AM

Morliere P (2000) Riboflavin photodegradation and photosensitization effects are

highly dependent on oxygen and ascorbate concentration Photochem Photobiol 72

815-820

195

De Vries H Beijersbergen Van Henegouwen GMJ Huff FA (1984) Photochemical

decomposition of chloramphenicol in a 025 eye drop and in a therapeutic

intraocular concentration Int J Pharm 20 265-271

Dean RT Fu S Stocker R Davies MJ (1997) Biochemistry and pathology of

radicalmediated protein oxidation Biochem J 324 1-18

DeRitter E (1982) Vitamins in pharmaceutical formulations J Pharm Sci 71 1073-

1096

Deutsch JC (1998a) Ascorbic acid oxidation by hydrogen peroxide Anal Biochem

255 1-7

Deutsch JC (1998b)Spontaneous hydrolysis and dehydration of dehydroascorbic acid

in aqueous solution Anal Biochem 260 223-229

Deutsch JC (1998c)oxygen-accepting antioxidants which arise during ascorbate

oxidation Anal Biochem 265 238-245

Dodsun KY Young ER Soliman AGM (1992) Determination of total vitamin C

in various food matrices by liquid chromatography and fluorescence detection J

AOAC Int 75 887-891

Dollery C Ed (1999) Therapeutic Drugs 2nd ed Vol 1 Churchill Livingstone

London pp A213-A216

Doores S (2002) pH control agents and acidulants In Branen LA Davidson PM

Salminen S Thorngate JH Eds Food Additives Marcel Dekker New York p

637

Eberlein-Koumlnig B Ring J (2005) Relevance of vitamins C and E in cutaneous

photoprotection J Cosmet Dermatol 4 4-9

196

Ehrlich M Rao J Pabby A Goldman MP (2006) Improvement in the appearance

of wrinkles with topical transforming growth factor beta(1) and l-ascorbic acid

Dermatol Surg 32 618-625

EIRI Board (2007) Profitable Small Scale Manufacture of Cosmetics Engineers India

Research Institute Nai Sarak Delhi

Eitenmiller RR Ye L Landen WOJr (2008) Vitamin Analysis for the Health and

Food Sciences 2nd ed CRC Press Boca Raton FL Chap 5

Elia S (2009) Nutrition In Kumar P Clark M Eds Kumar and Clarkrsquos Clinical

Medicine 7th ed Elsevier Limited Chap 5

Elmore AR (2005) Final report of the safety assessment of L-ascorbic acid calcium

ascorbate magnesium ascorbate magnesium ascorbyl phosphate sodium ascorbate

and sodium ascorbyl phosphate as used in cosmetics Int J Toxicol 24 51-111

Erb C Nau-Staudt K Flammer J Nau W (2004) Ascorbic acid as a free radical

scavenger in porcine and bovine aqueous humour Ophthal Res 36 38-42

Espinal-Perez LE Moncada B Castanedo-Cazares JP (2004) A double-blind

randomized trial of 5 ascorbic acid vs 4 hydroquinone in melasma Int J

Dermatol 43 604-607

Fall HH Petering HG (1956) Metabolic inhibitors 1 67-Dimethyl-9-

formylmethylisoalloxazine67-dimethyl-9-(2-hydroxyethyl)-isoalloxazine and

derivatives J Am Chem Soc 78 377-381

Farahmand S Tajerzadeh H Farboud ES (2006) Formulation and evaluation of a

vitamin C multiple emulsion Pharm Dev Technol 11 255-261

197

Fasani E Albini A (2005) Photostability Stress Testing Predicting Drug Degradation

Taylor amp Francis London Chap 10

Fasihullah Q (1988) Photolysis of Flavins in Aqueous and Organic Solvents PhD

Thesis University of Karachi Karachi

Fasman GD Ed (1976) CRC Handbook of Biochemistry and Molecular Biology 3rd

ed Physical Chemical Data Vol 1 CRC Press Cleveland Ohio pp 122-130

Ferreira SLC Bandeira MLSF Lemos VA Dos Santos HC Spinola Costa

AC De Jesus DS (1997) Sensitive spectrophotometric determination of ascorbic

acid in fruit juices and pharmaceutical formulations using 2-(5-bromo-2-pyridylazo)-

5-diethylaminophenol (Br-PADAP) Anal Bioanal Chem 357 1174-1178

Fessenden RW Verma NC (1978) A time-resolved electron spin resonance study of

the oxidation of ascorbic acid by hydroxyl radical Biophys J 24 413-430

Finholt P Paulssen RB Higuchi T (1963) Rate of anaerobic degradation of ascorbic

acid in aqueous solution J Pharm Sci 52 948-954

Florence AT Attwood D (2006) Drug Stability Physicochemical Principles of

Pharmacy 4th ed Pharmaceutical Press London p 411

Flynn GL (2002) Cutaneous and transdermal delivery-processes and systems of

delivery In Banker GS and Rhodes CT Eds Modern Pharmaceutics Marcel

Dekker New York Chap 8

Foote CS (1991) Definition of Type I and Type II Photosensitized oxidation

Photochem Photobiol 54 659

Fornaro A Coicher N (1998) L-ascorbic acid complexation and redox reactions with

some transition metal ions Quni Nova 21 642-650

198

Fraga CG Motchink PA Wyrobek AJ Rempel DM Ames BN (1996)

Smoking and low antioxidant levels increase oxidative damage to sperm DNA

Mutat Res 351 199-203

Fraga CG Motchnik PA Shigenaga MK Helbock HJ Jacob RA Ames BN

(1991) Ascorbic acid protects against endogenous oxidative DNA damage in human

sperm Proc Natl Acad Sci USA 88 11003-11006

Frenich AG Torres ME Vega AB Vidal JL Bolanos PP (2005) Determination

of ascorbic acid and carotenoids in food commodities by liquid chromatography with

mass spectrometry detection J Agric Food Chem 53 7371-7376

Fung HL (1990) Drug stability In Banker GS Rhodes CT Eds Modern

Pharmaceutics 2nd ed Marcel Dekker New York Chap 6

Gallarate M Carlotti ME Trotta M Bovo S (1999) On the stability of ascorbic

acid in emulsified systems for topical and cosmetic use Int J Pharm 188 233-241

Ganshirt H Malzacher A (1960) Separation of several vitamins of the B group and C

by chromatography Naturwiss 47 279-280

Gardner WH (1972) Acidulants in food processing In Furia TE Ed Handbook of

Food Additives 2nd ed Vol 1 CRC Press Cleveland OH pp 225ndash270

Garrett ER (1967) Kinetics and mechanisms in stability of drugs In Bean HS

Beckett AH Carless JE Eds Advances in Pharmaceutical Sciences Vol 2

Academic Press London Chap 1

Garti N Aserin A (1996) Pharmaceutical emulsions double emulsions and

microemulsions In Benita S Ed Microencapsulation Methods and Industrial

Applications Marcel Dekker New York pp 411-534

199

Gensler M Rossmann A Schmidt HL (1995) Detection of added L-ascorbic acid in

fruit juice by isotope ratio mass spectrometry J Agric Food Chem 43 2662-2666

Gibbons E Allwood MC Neal T Hardy G (2001) Degradation of dehydroascorbic

acid in parenteral nutrition mixtures J Pharm Biomed Anal 25 605-611

Guitton C Gros P Comtat M (2007) Direct electrochemical measurements in dermo-

cosmetic creams In Alegret Merkoci Eds Comprehensive Analytical Chemistry

Elsevier Amsterdam 49 e59-e68

Guttman DE Brooke D (1963) Solution phase interaction of nicotinamide with

ascorbic acid J Pharm Sci 52 941-945

Gyorgy P Pearson WN Eds (1967) Vitamin Methods Vol VII Academic Press

New York pp 27-49

Hagg M Ylikoski S Kumpulainen J (1994) Vitamin C and α- and β-carotene in

vegetables consumed in Finland during 1988-1989 and 1992-1993 J Food Compos

Anal 7 252-259

Hagg M Ylikoski S Kumpulainen A (1995) Vitamin C content in fruits and berries

consumed in Finland J Food Compos Anal 8 12-20

Hall NK Chapman TM Kim HJ Min DB (2009) Antioxidant mechanisms of

Trolox and ascorbic acid on the oxidation of riboflavin in milk under light Food

Chem in press doi 101016jfoodchem200905017

Halliwell B (1996) Vitamin C antioxidant or pro-oxidant in vivo Free Radic Res 25

439-454

200

Halliwell B Whiteman M (1997) Antioxidant and prooxidant properties of vitamin C

In Packer L Fuchs J Eds Vitamin C in Health and Disease Marcel Dekker New

York pp 25-94

Halver JE Felton SP (2001) Analytical enigmas in assaying for vitamers C In

Dabrowski K Ed Ascorbic Acid in Aquatic Organisms CRC Press Boca Raton

Chap 3

Hanck A Ed (1982) Vitamin C New Clinical Applications in Immunology Lipid

Metabolism and Cancer Hans Huber Bern

Hashmi MH (1973) Assay of Vitamins in Pharmaceutical Preparations John Wiley

New York Chap 13

Hatchard CG Parker CA (1956) A new sensitive chemical actinometer II Potassium

ferrioxalate as a standard chemical actinometer Proc R Soc London Ser A A235

518-536

Heber GK Markovic B Hayes A (2006) An immunohistological study of anhydrous

topical ascorbic acid compositions on ex vivo human skin J Cosmet Dermatol 5

150-156

Heelis PF Parsons BJ Phillips GO McKellar JF (1981) The flavin sensitized

photooxidation of ascorbic acid a continuous and flash photolysis study Photochem

Photobiol 37 7-13

Henry JA Ed (1997) The New Guide to Medicines and Drugs The British Medical

Association 4th ed Dorling Kindersley London p 434

Hickey S Roberts H (2004) Ascorbate-The Science of Vitamin C Electronic Version

(ISBN 1-4116-0724-4)

201

Higdon JV Frei B (2002) The Antioxidant Vitamin C and E AOAC Press Arlington

VA pp 1-16

Hirst EL Zilva SS (1933) Ascorbic acid as the anti-scorbutic factor Biochem J 27

1271-1278

Ho AHL Puri A Sugden JK (1994) Effect of sweetening agents on the light

stability of aqueous solutions of L-ascorbic acid Int J Pharm 107 199-203

Holmstrom B Oster G (1961) Riboflavin as an electron donor in photochemical

reactions J Am Chem Soc 83 1867-1871

Homann P Gaffron H (1964) Photochemistry and metal catalyzes studies in a flavin

sensitized oxidation of ascorbic acid Photochem Photobiol 3 499-515

Hughes DE (1985) Irreversible reaction kinetics of the aerobic oxidation of ascorbic

acid Anal Chem 57 555-558

Humbert PG Haftek M Creidi P Lapiegravere C Nusgens B Richard A Schmitt D

Rougier A Zahouani H (2003) Topical ascorbic acid on photoaged skin Clinical

topographical and ultrastructural evaluation double-blind study vs placebo Exp

Dermatol 12 237-244

ICH Harmonized Tripartite Guideline (1997) QIB Photostability Testing of New Drug

Substances and Products Federal Register 62 27115-27122

Ichikawa M Ide N Shiraishi S Ono K (2005) Effect of various halide salts on the

incompatibility of cyanocobalamin and ascorbic acid in aqueous solution Chem

Pharm Bull 53 688-690

202

Ihara H Shino Y Aoki Y Hashizume N Minegishi N (2000) A simple and rapid

method for the routine assay of total ascorbic acid in serum and plasma using

ascorbate oxidase and o-phenylenediamine J Nutr Sci Vitaminol 46 321-324

Im-Emsap W Paeratakul O Siepmann J (2002) Disperse Systems In Banker GS

Rhodes CT Ed Modern Pharmaceutics Maecel Dekker New York Chap 9

Irache JM Ezpeleta I Vega FA (1993) HPLC determination of antioxidant

synergists and ascorbic acid in some fatty pharmaceuticals cosmetics and food

Chromatographia 35 232-23

Iwase H Ono I (1994) Determination of ascorbic acid in human plasma by high-

performance liquid chromatography with electrochemical detection using a

hydroxyapatite cartridge for precolumn deproteinization J Chromatogr B Biomed

Appl 655 195-200

Jain S Tiwari AK Jain NK (2006) Topical products In Jain NK Ed

Pharmaceutical Product Development CBS Publishers New Delhi Chap 7

Jamil N Ahmad I (1984) Moisture content of ascorbic acid tablets stored at various

temperatures and humidities Pak J Sci Ind Res 27 252

Jamil N Ahmad I Beg AE (1980a) The effect of UV and tungsten light on physical

characteristics of ascorbic acid tablets Laboratory prepared samples IV Kar Univ

J Sci 8 77-87

Jamil N Ahmad I Beg AE (1980b) The effect of UV and tungsten light on physical

characteristics of ascorbic acid tablets Commercial samples III Kar Univ J Sci 8

117-126

203

Jentzsch A Aikens P (2005) RetiSTARTM

for cosmetic formulations-stabilized retinol

In Rosen MR Ed Delivery System Handbook for Personal Care and Cosmetic

Products Technology Applications and Formulations William Andrew Publishing

Norwich NY pp 862-866

Johnston CS Steinberg FM Rucker RB (2007) Ascorbic Acid In Zempleni J

Rucker RB McCormick DB Suttie JW (Eds) Handbook of Vitamins 4th ed

CRC Press Boca Raton FL Chap 15

Jung MY Kim SM Kim SY (1995) Riboflavin-sensitized photooxidation of

ascorbic acid kinetics and amino acid effects Food Chem 53 397-403

Kall MA Anderson CJ (1999) Improved method for simultaneous determination of

ascorbic acid and dehydroascorbic acid isoascorbic acid and dehydroisoascorbic acid

in food and biological samples J Chromatogr B Biomed Sci Appl 730 101-111

Kampfenkel K Van Montagu M Inze D (1995) Extraction and determination of

ascorbate and dehydroascorbate from plant tissue Anal Biochem 225 165-167

Kaplan DL Moloney SJ Troy WR Dickens MS Pinnell SR (1989) A new

stabilized ascorbic acid solution percutaneous absorption and effect on relative

collagen synthesis J Cutan Aging Cosmet Derm I 115-121

Karatepe M (2004) Simultaneous determination of ascorbic acid and free

malondialdehyde in human serum by HPLC-UV LC GC (North America) 22 362-

365

Karlsen J (1996) Light-activated drugs and drug formulations in drug targeting In

Tonneson HH Ed The Photostability of Drugs and Formulations Taylor amp

Francis London Chap 8

204

Kassem MA Kassem AA Ammar HO (1969a) Studies on the stability of

injectable L-ascorbic acid solutions I Effect of pH solvent light and container

Pharm Acta Helv 44 611-623

Kassem MA Kassem AA Ammar HO (1969b) Studies on the stability of

injectable L-ascorbic acid solutions II Effect of metal ions and oxygen content of

solvent water Pharm Acta Helv 44 667-675

Kassem MA Kassem AA Ammar HO (1972) Studies on the stability of injectable

L-ascorbic acid solutions 3 Effect of metal-complexing agents Pharm Acta Helv

47 89-97

Kaushansky K Kipps TJ (2006) Hematopoietic agents growth factors minerals and

vitamins In Brunton L Ed Goodman amp Gilmans The Pharmacologic Basis of

Therapeutics 11th ed digital ed McGraw-Hill Companies USA Chap 53

Kearney MCJ Allwood MC Martin H Neal T Hardy G (1998) The influence

of amino acid source on the stability of ascorbic acid in TPN mixtures Nutrition 14

173-178

Kim H Kirshenbaum LJ Rosenithol I Riesz P (1993) Photosensitized formation

of ascorbate radicals by riboflavin an ESR study Photochem Photobiol 57 777-

784

Kimoto K Terada S Yamaguchi T (1997) Analysis of ascorbic acid

dehydroascorbic acid and transformation products by ion-pairing high-performance

liquid chromatography with multi-wavelength ultraviolet and electrochemical

detection Methods Enzymol 279 3-12

205

King CG Burns JJ Ed (1975) Second Conference on Vitamin C Ann N Y Acad

Sci 258

Kitagawa Y (1968) The effects of ultraviolet rays on vitamin C Eiyo To Shokuryo 20

416-421 through CA (1968) 68 111560f

Klick S Muijselaar PG Waterval J EichingerT Korn C Gerding TK Debets

AJ de Griend CS van den Beld C Somsen GW De Jong GJ (2005)Toward

a generic approach for stress testing of drug substances and drug products Pharm

Tech 29 48-66

Konings EJ (2006) Water-soluble vitamins J AOAC Int 89 285-288

Kwon BM Foote CS (1988) Chemistry of singlet oxygen 50 Hydroperoxide

intermediates in the photooxygenation of ascorbic acid J Am Chem Soc 110

6582-6583

Kwon BM Foote CS Khan SI (1989) Photooxygenation of ascorbic acid

derivatives and model compounds J Am Chem Soc 111 1854-1860

Laborie S Lavoie JC Chessex P (1998) Paradoxical role of ascorbic acid and

riboflavin in solutions of total parenteral nutrition implication in photoinduced

peroxide generation Pediatr Res 43 601-601

Laborie S Lavoie JC Pineault M Chessex P (1999) Protecting solutions of

parenteral nutrition from peroxidation JPEN 23 104-108

Laborie S Lavoie JC Pineault M Chessex P (2000) Contribution of multivitamin

air and light in the generation of peroxides in adult and neonatal parenteral nutrition

solutions Ann Pharmacother 34440-445

206

Laborie S Lavoie JC Rouleau T Chessex P (2002) Multivitamin solutions for

enteral supplementation a source of peroxides Nutrition 18 470-473s

Lachman L DeLuca P Akers MJ (1986) Kinetic principles and stability testing In

Lachman L Liberman HA Kanig JL Eds The Theory and Practice of

Industrial Pharmacy 3rd ed Lea amp Febiger Philadelphia Chap 26

Laidler KJ (1987) Chemical Kinetics 3rd ed Harper and Row New York pp 183-

185

Lambert WE de Leenheer A (1992) In Nollet LML Ed Food Analysis by HPLC

Marcel Dekker New York Chap 10

Lavoie JC Chessex P Rouleau T Migneault D Comte B (2004) Light-induced

byproducts of vitamin C in multivitamin solutions Clin Chem 50 135-140

Lee JS Kim JW Han SH Chang IS Kang HH Lee OS Oh SG Suh KD

(2004) The stabilization of L-ascorbic acid in aqueous solution and water-in-oil-in-

water double emulsion by controlling pH and electrolyte concentration J Cosmet

Sci 55 1-12

Lee KM Kwon JY Lee KW Lee HJ (2009) Ascorbic acid 6-palmitate

suppresses gap-junctional intercellular communication through phosphorylation of

connexin 43 via activation of the MEK-ERK pathway Mutat Res 660 51-56

Leacutevecircque N Mac-Mary S Muret P Makki S Aubin F Kantelip JP Heusegravele C

SS Humbert P (2005) Evaluation of a sunscreen photoprotective effect by ascorbic

acid assessment in human dermis using microdialysis and gas chromatography mass

spectrometry Exp Dermatol 14 176-181

207

Levine M (1986) New concepts in the biology and biochemistry of ascorbic acid N

Engl J Med 314 892-902

Lewin S (1976) Vitamin C Its Molecular Biology and Medical Potential Academic

Press New York

Liau LS Lee BL New AL Ong CN (1993) Determination of plasma ascorbic

acid by high-performance liquid chromatography with ultraviolet and electrochemical

detection J Chromatogr 612 63-70

Lin JY Selim MA Shea CR Grichnik JM Omar MM Monteiro-Riviere NA

Pinnell SR (2003) UV photoprotection by combination topical antioxidants vitamin

C and vitamin E J Am Acad Dermatol 48 866-874

Lin FH Lin JY Gupta RD Tournas JA Burch JA Selim MA Monteiro-

Riviere NA Grichnik JM Zielinski J Pinnell SR (2005) Ferulic acid

stabilizes a solution of vitamins C and E and doubles its photoprotection of skin J

Invest Dermatol 125 826-832

Lindahl T (1993) Instability and decay of the primary structure of DNA Nature 362

709-715

Litner CJ (1973) Drug Product Stability In Cooper MS Ed Quality Control in

Pharmaceutical Industry Vol 2 Academic Press New York p 185

Liu TZ Chin N Kiser MD Bigler WN (1982) Specific spectrophotometry of

ascorbic acid in serum or plasma by use of ascorbate oxidase Clin Chem 28 2225-

2228

Lu GW Flynn GL (2009) Cutaneous and transdermal delivery-processes and

systems of delivery In Florence AT Siepmann J Eds Modern Pharmaceutics-

208

Applications and Advances Vol 2 5th ed Informa Healthcare Inc New York

Chap 3

Lykkesfeldt J (2000) Determination of ascorbic acid and dehydroascorbic acid in

biological samples by high-performance liquid chromatography using subtraction

methods reliable reduction with tris [2-carboxyethyl] phosphine hydrochloride Anal

Biochem 282 89-93

Macek TJ (1960) Stability problems with some vitamins in pharmaceuticals Am J

Pharm 132 433-455

Mader WJ (1961) Vitamins In Higuchi T Brochmann-Hanssen E Eds

Pharmaceutical Analysis Interscience New York pp 689-693

Maggi N Pasqualucci CR Ballotta R Sensi P (1966) Rifampicin a new orally

active rifamycin Chemotherapy 11 285-292

Maia AM Baby AR Pinto CA Yasaka WJ Suenaga E Kaneko TM Velasco

MV (2006) Influence of sodium metabisulfite and glutathione on the stability of

vitamin C in OW emulsion and extemporaneous aqueous gel Int J Pharm 322

130-135

Mancini M Edwards AM Becker MI De Ioannes A Silva E (2000) Reactivity

of monoclonal antibodies against a tryptophan-riboflavin adduct toward irradiated

and non-irradiated bovine-eye-lens protein fractions an indicator of in vivo visible

light mediated phototransformations J Photochem Photobiol B Biol 55 9-15

Manoharan M Schwille PO (1994) Measurement of ascorbic acid in human plasma

and urine by high performance liquid chromatography Results in healthy subjects

209

and patients with idiopathic calcium urolithiasis J Chromatogr B Biomed Appl

654 134-139

Margolis SA Schmidt HL (1996) Measurement of ascorbic acid in human plasma

and serum stability intralaboratory repeatability and interlaboratory reproducibility

Clin Chem 42 1257-1262

Mason P (2007) Dietary Supplements 3rd ed Pharmaceutical Press pp 330-339

Massad WA Bertolotti S Garcia N (2004) Kinetics and mechanism of the vitamin

B2-sensitized photooxidation of isoproterenol Photochem Photobiol 79 428-433

Matsubayashi T Sakaeda T Kita T Kurimoto Y Nakamura T Nishiguchi K

Fujita T Kamiyama F Yamamoto A Okumura K (2003) Intradermal

concentration of hydroquinone after application of hydroquinone ointments is higher

than its cytotoxic concentration Biol Pharm Bull 26 1365-1367

McBride MM Moore WM (1967) The photochemistry of riboflavin II

Polarographic studies on the hydroxyethyl and formylmethyl analogues of riboflavin

Photochem Photobiol 6 103-113

McDowell LR (2000) Vitamins in Animal and Human Nutrition 2nd ed Iowa State

University Press Chap 15

Meister A (1994) Glutathione-ascorbic acid antioxidant system in animals J Biol

Chem 269 9397-9400

Meister A (1995) Mitochondrial changes associated with glutathione deficiency

Biochim Biophys Acta 1271 35-42

Min DB Boff JM (2002) Chemistry and reaction of singlet oxygen in foods

CRFSFS 1 58-72

210

Moeslinger T Brunner M Volf I Spieckermann PG (1995) Spectrophotometric

determination of ascorbic acid and dehydroascorbic acid Clin Chem 41 1177-1181

Moffat AC Osselton MD Widdop B (2004) Clarkersquos Analysis of Drugs and

Poisons 3rd ed Pharmaceutical Press London pp 649-650

Moore DE (2004) Standardization of kinetic studies of photodegradation reactions In

Tonnesen HH Ed Photostability of Drugs and Drug Formulations 2nd ed CRC

Press Boca Raton FL Chap 3

Moura T Gaudy D Jacob M Cassanas G (1994) pH influence on the stability of

ascorbic acid spray-drying solutions Pharm Acta Helv 69 77-80

Murray JC Burch JA Streilein RD Iannacchione MA Hall RP Pinnell SR

(2008) A topical antioxidant solution containing vitamins C and E stabilized by

ferulic acid provides protection for human skin against damage caused by ultraviolet

irradiation J Am Acad Dermatol 59 418-425

Mushran SP Agarwal MC (1977) Mechanistic studies on the oxidation of ascorbic

acid 5 J Sci Res (India) 36 274-283

Nerlo H Palak W Tomaszek J (1968) Stabilization of vitamin preparations II

Syrup of vitamin D3 and its stabilization Acta Polon Pharm 25 183-188

Niki E Noguchi N (1997) Protection of human low-density lipoprotein from oxidative

modification by vitamin C In Packer L Fuchs J Eds Vitamin C in Health and

Disease Marcel Dekker New York pp 183-192

Nishikimi M Fukuyama R Minoshima S Shimizu N Yagi K (1994) Cloning and

chromosomal mapping of the human nonfunctional gene for L-gulono-gamma-

211

lactone oxidase the enzyme for L-ascorbic acid biosynthesis missing in man J Biol

Chem 269 13685-13688

Njus D Wigle M Kelly PM Kipp BH Schlegel HB (2001) Mechanism of

ascorbic acid oxidation by Cytochrome B561 Biochemistry 40 11905-11911

Nusgens BV Humbert P Rougier A Colige AC Haftek M Lambert CA

Richard A Creidi P Lapiegravere CM (2001) Topically applied vitamin C enhances

the mRNA level of collagens I and III their processing enzymes and tissue inhibitor

of matrix metalloproteinase 1 in the human dermis J Invest Dermatol 116 853-859

Ogata Y Kosugi Y (1969) Solvent effect on the autooxidation of L-ascorbic acid

Tetrahedron 25 1055-1062

OrsquoKeefe T (2001) Ascorbic acid and stable ascorbate esters as sources of vitamin C in

aquaculture feeds ASA Technical Bulletin AQ48 wwwsoyaquaorg

OrsquoNeil MJ Ed (2001) The Merck Index 13th ed Merck amp Co Inc Rahway NJ

USA Electronic version

Ortega GR Delgado JN (1998) Vitamins and related compounds In Wilson and

Gisvoldrsquos Textbook of Organic Medicinal and Pharmaceutical Chemistry 10th ed

Lippincott-Raven Philadelphia Chap 27

Ortwerth BJ Monnier VM (1997) Protein glycation by the oxidation products of

ascorbic acid In Packer L Fuchs J Eds Vitamin C in Health and Disease Marcel

Dekker New York pp 123ndash142

Ozer O Muguet V Roy E Grossiord JL Seiller M (2000) Stability study of

WOW viscosified multiple emulsions Drug Dev Ind Pharm 26 1185-1189

Packer I Fuchs J (1999) Vitamin C in Health and Disease Marcel Dekker New York

212

Packer JE Slater TF Wilson RL (1979) Direct observation of a free radical

interaction between vitamin E and vitamin C Nature 278 737-738

Packer L Traber MG Kraemer K Frei B (2002) The Antioxidant Vitamins C and

E AOCS Press Illinois

Padayatty SJ Katz A Wang Y Eck P Kwon O Lee JH Chen S Corpe C

Dutta A Dutta SK Levine M (2003) Vitamin C as an antioxidant Evaluation of

its role in disease prevention J Am Coll Nutr 22 18-35

Parker CA (1953) A new sensitive chemical actinometer I Some trials with potassium

ferrioxalate Proc Roy Soc (Lond) A220 104-116

Parker JN Parker PM (2003) Antioxidants ICON Group International

Pastore P Rizzetto T Curcuruto O Cin MD Zaramella A Martin D (2001)

Characterization of dehydroascorbic acid solutions by liquid chromatography mass

spectrometry Rapid Commun Mass Spectrom 15 2051-2057

Pelletier O (1985) In Augustin J Klein BP Becker D Venugopal PB Eds

Methods of Vitamins Assay Wiley New York pp 303-347

Peyrat-Maillard MN Bonnely S Rondini L Berset C (2001) Effect of vitamin E

and vitamin C on the antioxidant activity of malt rootlets extracts Lebensmittel-

Wissenschaft Und-Technological 34 176-182

Pinnell SR (2003) Cutaneous photodamage oxidative stress and topical antioxidant

protection J Am Acad Dermatol 48 1-19

Pinnell SR Madey DL (1998) Topical vitamin C in skin care Aesthet Surg J 18

468-470

213

Pinnell SR Yang H Omar M Monteiro-Riviere N DeBuys HV Walker LC

Wang Y Levine M (2001) Topical L-ascorbic acid percutaneous absorption

studies Dermatol Surg 27 137-142

Placzek M Gaube S Kerkmann U Gilbertz KP Herzinger T Haen E Przybilla

B (2005) Ultraviolet B-induced DNA damage in human epidermis is modified by

the antioxidants ascorbic acid and D-alpha-tocopherol J Invest Dermatol 124 304-

307

Puskas F Gergely PJr Banki K Perl A (2000) Stimulation of the pentose

phosphate pathway and glutathione levels by dehydroascorbate the oxidized form of

vitamin C FASEB J 14 1352-1361

Puskas F Gergely P Niland B Banki K Perl A (2002) Differential regulation of

hydrogen peroxide and Fas-dependent apoptosis pathways by dehydroascorbate the

oxidized form of vitamin C Antioxid Redox Signal 4 357-369

Racz I (1989) Drug Formulation Wiley New York pp 121-122

Ramu A Mehta MM (2000) The riboflavin-mediated photooxidation of doxorubicin

Cancer Chemoth Pharm 46 449-458

Raschke T Koop U Duumlsing HJ Filbry A Sauermann K Jaspers S Wenck H

Wittern KP (2004) Topical activity of ascorbic acid from in vitro optimization to in

vivo efficacy Skin Pharmacol Physiol 17 200-206

Reed RA Harmon P Manas D Wasylaschuk W Galli C Biddell R Bergquist

PA Hunke W Templeton AC Ip D (2003) The role of excipients and package

components in the photostability of liquid formulations PDA J Pharm Sci Tech 57

351-368

214

Reichstein T Griissner A Oppcnuuer R (1933) Synthese der d- und l-Ascorbinsaumlure

(C-Vitamin) Helv Chim Acta 16 1019-1033

Riehl J Maupin C Layloff T (1995) On the choice of a photolysis source for the

photostability testing of pharmaceuticals Pharmacop Forum 21 1654-1663

Rivlin RS (2007) Riboflavin (vitamin B2) In Zempleni J Rucker RB McCormick

DB Suttie JW Eds Handbook of Vitamins 4th ed Taylor amp Francis CRC

Press Boca Raton FL p 241

Rogers AR Yacomeni JA (1971) The effect of pH on the aerobic degradation of

ascorbic acid solutions J Pharm Pharmacol 23 218S

Rosen MR Ed (2005) Delivery System Handbook for Personal Care and Cosmetic

Products-Technology Applications and Formulations William Andrew Publishing

Norwich NY

Rosenberg HR (1945) Chemistry and Physiology of the Vitamins Interscience New

York pp 289-338

Roth HJ Eger K Troschutz R (1991) Pharmaceutical Chemistry Vol 2 Drug

Analysis Ellis Horwood London pp 282-288

Rowe RC Sheskey PJ Quinn ME (2009) Handbook of Pharmaceutical Excipients

6th ed Pharmaceutical Press London pp 43-47 181-183 731-732

Roacutezanowska M Bober A Burke JM Sarna T (1997) The role of retinal pigment

epithelium melanin in photoinduced oxidation of ascorbate Photochem Photobiol

65 472-479

Rozman B Gasperlin M (2007) Stability of vitamins C and E in topical

microemulsions for combined antioxidant therapy Drug Deliv 14 235-245

215

Rozman B Gasperlin M Tinois-Tessoneaud E Pirot F Falson F (2009)

Simultaneous absorption of vitamins C and E from topical microemulsions using

reconstructed human epidermis as a skin model Eur J Pharm Biopharm 72 69-75

Ruumlmelin A Fauth U Halmagyi M (1999) Determination of ascorbic acid in plasma

and urine by high performance liquid chromatography with ultraviolet detection Clin

Chem Lab Med 37 533-536

Rumsey SC Levine M (2000) Vitamin C In Song WO Beecher GR

Eitenmiller RR Eds Modern Analytical Methodologies in Fat- and Water-Soluble

Vitamins Wiley New York Chap 13

Saari JC Baker EM Sauberlich HE (1967) Thin-layer chromatographic separation

of the oxidative degradation products of ascorbic acid Anal Biochem 18 173-177

Salkić M Keran H Jašić M (2007) Direct spectrophotometric determination of l-

ascorbic acid in the presence of potassium cyanide Agric Conspec Sci 72 371-375

Salvador A Chisvert A (2007) Analysis of Cosmetic Products Elsevier Amsterdam

Sattar A DeMan JM Alexander JC (1977) Light-induced degradation of vitamins

II Kinetic studies on ascorbic acid decomposition in solution Can Inst Food Sci

Technol J 10 65-68

Satoh K Sakagami H Terasaka H Ida Y Fujisawa S (2000) Interaction between

hydroquinone and ascorbic acid derivatives quenching effects of organic solvents

Antican Res 20 1577-1581

Sauberlich HE (1994a) Pharmacology of vitamin C Annu Rev Nutr 14 371-391

Sauberlich HE (1994b) Vitamin C and Cancer In Kritchevsky D Carroll K Eds

Nutrition and Disease Update Cancer AOCS Press Champaign IL pp 111-172

216

Sauermann K Jaspers S Koop U Wenck H (2004) Topically applied vitamin C

increases the density of dermal papillae in aged human skin BMC Dermatol 4 13

Sawicka J (1991) The influence of excipients and technological process on

cholecalciferol stability and its liberation from tablets Pharmazie 46 519-521

Scheuplein RJ Blank IH (1973) Mechanism of percutaneous absorption IV

Penetration of non-electrolytes (alcohols) from aqueous solutions and from pure

liquids J Invest Dermatol 60 286-296

Schick B (1943) Scurvy and pine needles Science 98 241-242

Schuman Jorns M Schollnhammer G Hemmerich P (1975) Intramolecular addition

of the riboflavin side chain Anion-catalysed neutral photochemistry Eur J

Biochem 57 35-48

Seib PA Tolbert BM (1982) Ascorbic Acid Chemistry Metabolism and Uses

Advances in Chemistry Series 200 American Chemical Society Washington DC

Sequeira F Vozone C (2000) Photostability studies of drug substances and products

Practical implications of the ICH guideline Pharm Tech 24 30-35

Shacter E (2000) Quantification and significance of protein oxidation in biological

samples Drug Metab Rev 32 307-326

Shah VP (1997) FDA Guidance for Industry Document Nonsterile Semisolid Dosage

Forms Centre for Drug Evaluation and Research Rockville MD pp 1-40

Sheraz MA Ahmed S Ahmad S (2007) Developments in the clinical and food

analysis of vitamin C J Baqai Med Univ 10 19-24

217

Shindo Y Witt E Han D Packer L (1994) Dose-response effect of acute ultraviolet

irradiation on antioxidants and molecular markers of oxidation in murine epidermis

and dermis J Invest Dermatol 102 470-475

Sibi M Batcu A Gheorghiu I (1953) Vitamin oxidation-reduction effect II Effect of

light on the oxidation-reduction reactions of lactoflavin Studii cercetari stiinf

(Romania) 4 291-305 through CA (1956) 50 14816g

Sidhu DS Sugden JK (1992) Effect of food dyes in the photostability of aqueous

solutions of L-ascorbic acid Int J Pharm 83 263-266

Silva E Quina FH (2006) Photoinduced processes in the eye lens do flavins really

play a role In Silva E Edwards AM Eds Flavins Photochemisry and

Photobiology The Royal Society of Chemistry Cambridge Chap 7

Silva E Ugarte P Andrade A Edwards AM (1994) Riboflavin-sensitized photo-

processes of tryptophan J Photochem Photobiol B Biol 23 43-48

Sim AK (1972) Ascorbic acid-A scurvy past and present Chem Indust 8 160-165

Singh S (2006) Stability testing during product development In Jain NK ED

Pharmaceutical Product Development CBS Publishers New Delhi Chap 9

Singh S Bakshi M (2000) Guidance on conduct of stress testing to determine inherent

stability of drugs Pharm Tech 24 1-14

Sinko PJ (2006) Chemical Kinetics and Stability Martinrsquos Physical Pharmacy and

Pharmaceutical Sciences 5th ed Lippincott Williams amp Wilkins Baltimore Chap

8 15 18

218

Smith JL Canham JE Kirkland WD Wells PA (1988) Effect of intralipid amino

acids container temperature and duration of storage on vitamin stability in total

parenteral nutrition admixtures J Parenter Enteral Nutr 12 478-483

Speek AJ Schrijver J Schreurs WHP (1984) Fluorimetric determination of total

vitamin C and total isovitamin C in food stuffs and beverages by high-performance

liquid chromatography with precolumn derivatization J Agric Food Chem 32 352-

355Spratley JE Hellstroumlm SO Mattsson CK Pais-Clemente M (2001)

Topical ascorbic acid reduces myringosclerosis in perforated tympanic membranes A

study in the rat Ann Otol Rhinol Laryngol 110 585-591

Spiclin P Homer M Zupancic A Gasperlin M (2003) Sodium ascorbyl phosphate

in topical microemulsions Int J Pharm 256 65-73

Stadtman ER (1991) Ascorbic acid and oxidative inactivation of proteins Am J Clin

Nutr 54 1125S-1128S

Stone I (1969) The Healing Factor Vitamin C Against Disease Grosset and Dunlap

New York

Sugden JK (1985) Photostability of cosmetic materials Int J Cosmet Sci 7 165-173

Swarbrick J Rubino JT Rubino OP (2006) Coarse Dispersions In Hendrickson

R Ed Remington The Science and Practice of Pharmacy 21st ed Lippincott

Williams amp Wilkins Baltimore Chap22

Sweetman SC Ed (2009) Martindale The Complete Drug Reference 36th ed

Pharmaceutical Press London Electronic version

219

Szent-Gyoumlrgi A (1928) Observation on function of peroxidase systems and chemistry

of adrenal cortex description of new carbohydrate derivative Biochem J 22 1387-

1389

Szymula M (2005) The influence of ascorbic acid on the rheological properties of the

microemulsion region of the SDSpentanolwater system J Cosmet Sci 56 267-

277

Taqui Khan MM Martell AE (1967) Metal ion and metal chelate catalyzed oxidation

of ascorbic acid by molecular oxygen 1 Cupric acid and ferric ion catalyzed

oxidation J Am Chem Soc 89 4176-4185

Tangney CC (1988) Analysis of vitamin C in biological samples with an emphasis on

recent chromatographic techniques Prog Clin Biol Res 259 331-362

Tatsumi K Ichikawa H Wada S (1992) Flavin-sensitized photooxidation of

substituted phenols in natural water J Contam Hydrol 9 207-219

Tessier F Birlouez-Aragon I Tjani C Guilland JC (1996) Validation of a

micromethod for determining oxidized and reduced vitamin C in plasma by HPLC-

fluorescence Int J Vitam Nutr Res 66 166-170

Thatcher SR Mansfield RK Miller RB Davis CW Baertschi SW (2001a)

Pharmaceutical photostability a technical and practical interpretation of the ICH

guideline and its application to pharmaceutical stability Part 1 Pharm Tech 25 98-

110

Thatcher SR Mansfield RK Miller RB Davis CW Baertschi SW (2001b)

Pharmaceutical photostability a technical and practical interpretation of the ICH

220

guideline and its application to pharmaceutical stability Part 2 Pharm Tech 25 50-

62

Thiele JJ Dreher F Packer L (2000) Antioxidant defense systems in skin In

Elsner P Maibach HI Eds Cosmeceuticals- Drugs vs Cosmetics Marcel

Dekker New York Chap 11

Thomas DP (1997) Sailors scurvy and science J R Soc Med 90 50-54

Thomson CO Trenerry VC (1995) A rapid method for the determination of total L-

ascorbic acid in fruits and vegetables by micellar electrokinetic capillary

chromatography Food Chem 53 43-50

Tonnesen HH (1991) Photochemical degradation of components in drug formulations

1 An approach to the standardization of degradation studies Pharmazie 46 263-265

Tonnesen HH (2002) Photodecomposition of Drugs In Swarbrick J Boylan JC

Eds Encyclopedia of Pharmaceutical Technology Vol 3 Marcel Dekker New

York pp 2197-2203

Tonnesen HH Ed (2004) The Photostability of Drugs and Drug Formulations

Culinary and Hospitality Industry Publications Services Weimex TX

Tonnesen HH Karlsen J (1997) A comment on photostability testing according to

ICH guideline calibration of light sources PharmEuropa 9 735-736

Tonnesen HH Moore DE (1993) Photochemical degradation of components in drug

formulations Pharm Technol Int 5 27-33

Tournas JA Lin FH Burch JA Selim MA Monteiro-Riviere NA Zielinski

JE Pinnell SR (2006) Ubiquinone idebenone and kinetin provide ineffective

221

photoprotection to skin when compared to a topical antioxidant combination of

vitamins C and E with ferulic acid J Invest Dermatol 126 1185-1187

Traber MG (2007) Vitamin E In Zempleni J Rucker RB McCormick DB

Suttie JW Eds Handbook of Vitamins 4th ed Taylor amp Francis CRC Press

Boca Raton FL Chap 4

Traikovich SS (1999) Use of topical ascorbic acid and its effect on photodamaged skin

topography Arch Otolaryngol Head Neck Surg 125 1091-1098

Treadwell GE Cairns WL Metzler DE (1968) Photochemical degradation of

flavins V Chromatographic studies of the products of photolysis of riboflavin J

Chromatogr 35 376-388

Truswell AS (2003) ABC of Nutrition 4th ed BMJ Publishing Group Chap 10

Tsao CS Young M (1985) Analysis of ascorbic acid derivatives by high-performance

liquid chromatography with electrochemical detection J Chromatogr 13 855-856

Underberg WJM (1978) Oxidative degradation of pharmaceutically important

phenothiazines III Kinetics and mechanism of promethazine oxidation J Pharm Sci

67 1133-1138

Urso ML Clarkson PM (2003) Oxidative stress exercise and antioxidant

supplementation Toxicology 189 41-54

Vaid FHM (1998) Photolysis and Interaction of Thiamine Hydrochloride with

Riboflavin in Aqueous Solution PhD Thesis University of Karachi Karachi

Vaid FHM Shaikh RH Ansari IA Ahmad I (2005) Spectral study of the

photolysis of aqueous thiamine hydrochloride and ascorbic acid solution in the

presence and absence of riboflavin J Chem Soc Pak 27 227-232

222

Vaid F H M Shaikh RH Ansari I A Ahmad I (2006) Chromatographic study of

the photolysis of aqueous thiamine hydrochloride and ascorbic acid solutions in the

presence and absence of riboflavin J Chem Soc Pak 28 481-464

Valvani SC (2000) Industrial stability testing in the United States and computerization

of stability data In Carstensen JT Rhodes CT Eds Drug Stability Principles

and Practices 3rd

ed Marcel Dekker New York pp 534-536

Vanderslice JT Higgs DJ (1988) Chromatographic separation of ascorbic acid

isoascorbic acid dehydroascorbic acid and dehydroisoascorbic acid and their

quantitation in food products J Micronutr Anal 4 109-118

Vanderslice JT Higgs DJ Hayes JM Block G (1990) Ascorbic acid and

dehydroascorbic acid content of foods-as-eaten J Food Comp Anal 3 105-118

Vanderslice JT Higgs DJ (1993) Quantitative determination of ascorbic acid

dehydroascorbic acid by HPLC in foods and other matrices J Nutr Biochem 4 184-

190

Varvaresou A Tsirivas E Iakovou K Gikas E Papathomas Z Vonaparti A

Panderi I (2006) Development and validation of a reversed-phase ion-pair liquid

chromatography method for the determination of magnesium ascorbyl phosphate and

melatonin in cosmetic creams Anal Chim Acta 573-574 284-290

Verma KK Jain A Verma A Chaurasia A (1991) Spectrophotometric

determination of ascorbic acid in pharmaceuticals by background correction and flow

injection Analyst (London) 116 641-645

Vimaladevi M (2005) Textbook of Cosmetics CBS Publishers New Dehli Chap 2

and 17

223

Wallwork SC Grant DJW (1977) Physical Chemistry for Students of Pharmacy and

Biology 3rd ed Longmann New York pp 502-505

Waugh WA King CG (1932) Isolation and identification of vitamin C J Biol

Chem 97 325-331

Wechtersbach L Cigic B (2007) Reduction of dehydroascorbic acid at low pH J

Biochem Biophys Methods 70 767-772

Weiner N (1996) Introduction In Liberman HA Rieger MM Banker GS Eds

Pharmaceutical Dosage Forms Disperse Systems 2nd ed vol1 Marcel Dekker

New York Chap1

Werner W (1949) The reaction of l-ascorbic and d-isoascorbic acid with nicotinic acid

and its amide J Org Chem 14 22-26

Wiechers JW (2003) Formulating for Efficacy Proceedings of the 2003 IFSCC

Conference Seoul Korea

Wiechers JW (2005) Optimizing skin delivery of active ingredients from emulsions

from theory to practice In Rosen MR Ed Delivery System Handbook for

Personal Care and Cosmetic Products-Technology Applications and Formulations

William Andrew Inc Norwich NY Chap 20

Wille JJ (2005) Thixogel-Novel topical delivery systems for hydrophobic plant actives

In Rosen MR Ed Delivery System Handbook for Personal Care and Cosmetic

Products-Technology Applications and Formulations William Andrew Inc

Norwich NY Chap 36

224

Wright JL (1986) Vitamin A monograph In Connors KA Amidon GL Stella

VJ Eds Chemical Stability of Pharmaceuticals A Handbook for Pharmacists 2nd

ed Wiley New York pp 791-803

Yao AA Wathelet B Thonart P (2009) Effect of protective compounds on the

survival electrolyte leakage and lipid degradation of freeze-dried Weissella

paramesenteroides LC11 during storage J Microbiol Biotechnol 19 810-817

Yeh SY Lach JL (1961) Stability of morphine in aqueous solutions III Kinetics of

morphine degradation in aqueous solution J Pharm Sci 50 35-42

Yoshimura A Ohno T (1988) Lumiflavin-sensitized photooxygenation of indole

Photochem Photobiol 48 561-565

Yoshioka S Stella VJ (2000) Stability of Drugs and Dosage Forms Kluwer

Academic Plenum Publishers New York Chap 2

Zaeslein C (1982) Vitamins in the Field of Medicine Hoffman La Roche Basle pp

89-96

Zeng W Martinuzzi F MacGregor A (2005) Development and application of a novel

UV method for the analysis of ascorbic acid J Pharm Biomed Anal 36 1107-1111

Zhang GF Chen HY (2000) Chemiluminescence studies of the oxidation of ascorbic

acid with copper (II) catalyzed by halide anions and its applications to the

determination of halide anions and ascorbic acid Anal Sci 16 1317-1321

Zhang L Lerner S Rustrum WV Hofmann GA (1999) Electroporation-mediated

topical delivery of vitamin C for cosmetic applications Bioelectrochem Bioenerg

48 453-461

225

Zilva SS (1932) The non-specificity of the phenolindophenol reducing capacity of

lemon juice and its fractions as a measure of their antiscorbutic activity Biochem J

26 1624-1627

226

AUTHORrsquoS PUBLICATIONS

The author obtained his B Pharm degree in 2003 and joined the post graduate

program securing an M Phil degree in Pharmaceutics in 2006 from Baqai Medical

University He is a co-author of following publications

CHAPTER IN BOOK

1 Chapter on ldquoBorate Toxicity Effect on Drug Stability and Analytical

Applicationsrdquo by Iqbal Ahmad Sofia Ahmed MA Sheraz and Faiyaz H M

Vaid In Handbook on Borates Chemistry Production and Applications (MP

Chung Ed) Nova Science Publishers Inc NY USA (in press)

PAPERS PUBLISHED

INTERNATIONAL

2 Iqbal Ahmad Sofia Ahmed MA Sheraz and Faiyaz HM Vaid ldquoEffect of Borate

Buffer on the Photolysis of Riboflavin in Aqueous Solutionrdquo Journal of

Photochemistry and Photobiology B Biology 93 82-87 (2008)

3 Iqbal Ahmad Sofia Ahmed MA Sheraz M Aminuddin and Faiyaz HM Vaid

ldquoEffect of Caffeine Complexation on the Photolysis of Riboflavin in Aqueous

Solution A Kinetic Studyrdquo Chemical and Pharmaceutical Bulletin 57 (2009)

published online September 14 2009

4 Iqbal Ahmad MA Sheraz Sofia Ahmed and Faiyaz HM Vaid ldquoAnalytical

Applications of Boratesrdquo Materials Science Research Journal (in press)

5 Iqbal Ahmad Sofia Ahmed MA Sheraz Kefi Iqbal and Faiyaz HM Vaid

ldquoPharmacological Aspects of Boratesrdquo International Journal of Medical and

Biological Frontiers (in press)

6 Iqbal Ahmad Sofia Ahmed MA Sheraz Faiyaz HM Vaid and Izhar A Ansari

ldquoEffect of Divalent Ions on Photodegradation Kinetics and Pathways of

Riboflavin in Aqueous Solutionrdquo Photochemical and Photobiological Sciences

accepted

227

NATIONAL

7 Sofia Ahmed MA Sheraz and Iqbal Ahmad ldquoAdvances in Antioxidant Activity of

Vitamin Erdquo Journal of Baqai Medical University 10 13-18 (2007)

8 MA Sheraz Sofia Ahmed and Iqbal Ahmad ldquoDevelopments in the Clinical and

Food Analysis of Vitamin Crdquo Journal of Baqai Medical University 10 19-24

(2007)

9 A Azmi SNH Naqvi M Usman MA Sheraz and Sofia Ahmed ldquoPancreatic

Glucagon in Certain Ungulates Comparative Study of Extraction and

Bioassayrdquo Pakistan Journal of Entomology 20 23-28 (2005)

10 Iqbal Ahmad Sofia Ahmed MA Sheraz Faiyaz HM Vaid and S Hasan

ldquoAdvances in Biochemical Functions and the Photochemistry of Flavins and

Flavoproteinsrdquo Pakistan Journal of Pharmaceutical Sciences in press

11 MA Sheraz Sofia Ahmed and Iqbal Ahmad ldquoEffect of Borates on the Stability of

Chemical and Pharmaceutical Compoundsrdquo Journal of Baqai Medical University

accepted

PAPERS SUBMITTED

12 Iqbal Ahmad MA Sheraz Sofia Ahmed Riaz H Shaikh and Faiyaz HM Vaid

ldquoPhotostability of Ascorbic Acid in Organic Solvents and Cream Formulationsrdquo

Chemical and Pharmaceutical Bulletin

13 Iqbal Ahmad MA Sheraz Sofia Ahmed Riaz H Shaikh and Faiyaz HM Vaid

ldquoPhotochemical Interaction of Ascorbic Acid with Riboflavin Nicotinamide and

Alpha-Tocopherol in Cream Formulationsrdquo Journal of Cosmetic Science

14 Iqbal Ahmad Kefi Iqbal Sofia Ahmed MA Sheraz ldquoApplications of Laser Flash

Photolysis Spectroscopy and Electron Microscopy in Photopolymerization and

Development of Glass Ionomer Dental Cementsrdquo Materials Science Research

Journal

15 Sofia Ahmed MA Sheraz M Aminuddin I Ahmad and Faiyaz HM Vaid ldquoA

Rapid Titrimetric Assay for Quantitation of Vitamin B1 in Neat and

Pharmaceutical Preparationsrdquo Pakistan Journal of Pharmaceutical Sciences

• 01 SZ-786
• 02 SZ-title
• 03 SZ-Certificate
• 04 SZ-Abstract
• 05 SZ-Acknowledgement
• 06 SZ-Dedication
• 07 SZ-Contents
• 08 SZ-Chapter 1
• 09 SZ-Chapter 2
• 10 SZ-Chapter 3
• 11 SZ-Object of Present Investigation
• 12 SZ-Chapter 4
• 13 SZ-Chapter 5
• 14 SZ-Chapter 6
• 15 SZ-Chapter 7
• 16 SZ-Conclusion
• 17 SZ-References
• 18 SZ-Authors Publications

viii

To

My Beloved Parents amp

Late Prof Dr S Sabir Ali for their interest and endless support

ix

CONTENTS

Chapter Page

ABSTRACT iv

ACKNOWLEDGEMENTS vi

I INTRODUCTION 1

11 HISTORICAL BACKGROUND 2

12 PHYSICOCHEMICAL CHARACTERISTICS OF

ASCORBIC ACID

2

13 CHEMISTRY OF ASCORBIC ACID 3

131 Nomenclature and Structure 3

132 Chemical Stability 3

14 BIOCHEMICAL FUNCTIONS 7

15 ANTIOXIDANT ACTIVITY 8

16 PHOTOSTABILITY OF DRUGS 9

17 KINETIC TREATMENTS OF PHOTOCHEMICAL

REACTIONS

12

18 LITERATURE ON ASCORBIC ACID 15

II PHOTODEGRADATION REACTIONS AND ASSAY OF

ASCORBIC ACID

17

21 PHOTODEGRADATION REACTIONS 18

211 Photodegradation of Ascorbic Acid 18

212 Effect of Various Substances on Photodegradation of Ascorbic

Acid

20

213 Photosensitized Oxidation of Ascorbic Acid 22

214 Ascorbic Acid Interaction with Other Water-Soluble Vitamins 25

22 ASSAY OF ASCORBIC ACID 26

221 Spectrophotometric Methods 26

222 Fluorimetric Methods 28

x

223 Mass spectrometric Methods 28

224 Chromatographic Methods 28

225 Enzymatic Methods 29

226 Commercial Kits for Clinical Analysis 30

227 Analysis in Creams 30

III FORMULATION AND STABILITY OF CREAM

PREPARATIONS

31

31 FORMULATION OF CREAM PREPARATIONS 32

311 Choice of Emulsion Type 32

312 Choice of Oil Phase 33

313 Emulsion Consistency 33

314 Choice of Emulsifying Agent 34

315 Formulation by the HLB Method 34

316 Concept of Relative Polarity Index 35

32 FORMULATION OF ASCORBIC ACID CREAMS 37

33 STABILITY OF CREAMS 39

331 Physical Stability 39

332 Chemical Stability 39

333 Microbial Stability 40

334 Stability of Ascorbic Acid in Liquid Formulations 41

335 Stability of Ascorbic Acid in Emulsions and Creams 41

336 Stability Testing of Emulsions 45

3361 Macroscopic examination 46

3362 Globule size analysis 46

3363 Change in viscosity 46

3364 Accelerated stability tests 46

337 FDA Guidelines for Semisolid Preparations 46

xi

OBJECT OF PRESENT INVESTIGATION 48

IV MATERIALS AND METHODS 51

41 MATERIALS 52

42 METHODS 55

421 Cream Formulations 55

422 Preparation of Creams 56

423 Thin-Layer Chromatography 57

424 pH Measurements 57

425 Ultraviolet and Visible Spectrometry 58

426 Photolysis of Ascorbic Acid 59

4261 Creams 59

4262 Aqueous and organic solvents 59

4263 Storage of creams in dark 59

427 Measurement of Light Intensity 59

428 Procedure 60

4281 Calculation 62

429 Viscosity Measurements 63

4210 Assay method 65

42101 UV spectrophotometric method for the assay of creams

containing ascorbic acid alone

65

42102 Iodimetric method for the assay of ascorbic acid in creams

containing riboflavin nicotinamide and alpha-tocopherol 65

42103 Spectrophotometric method for the assay of ascorbic acid in

aqueous and organic solvents

67

V PHOTODEGRADATION OF ASCORBIC ACID IN

ORGANIC SOLVENTS AND CREAM FORMULATIONS

68

51 INTRODUCTION 69

52 PHOTOPRODUCTS OF ASCORBIC ACID 71

xii

53 SPECTRAL CHARACTERISTICS OF PHOTOLYSED

SOLUTIONS

71

54 ASSAY OF ASCORBIC ACID IN CREAMS AND

SOLUTIONS

73

55 EFFECT OF SOLVENT 74

56 EFFECT OF CONCENTRATION 80

57 EFFECT OF CARBON CHAIN LENGTH OF

EMULSIFYING AGENT

88

58 EFFECT OF VISCOSITY 94

59 EFFECT OF pH 94

510 EFFECT OF REDOX POTENTIAL 96

511 PRIMARY PHOTOCHEMICAL REACTIONS IN THE

OXIDATION OF ASCORBIC ACID

97

512 DEGRADATION OF ASCORBIC ACID IN THE DARK 98

VI PHOTOCHEMICAL INTERACTION OF ASCORBIC

ACID WITH RIBOFLAVIN NICOTINAMIDE AND

ALPHA-TOCOPHEROL IN CREAM FORMULATIONS

109

61 INTRODUCTION 110

62 ABSORPTION CHARACTERISTICS OF PHOTOLYSED

CREAMS

114

63 PHOTOPRODUCTS OF ASCORBIC ACID AND OTHER

VITAMINS

114

64 ASSAY METHOD 116

65 KINETICS OF PHOTODEGRADATION OF ASCORBIC

ACID

117

66 INTERACTION OF RIBOFLAVIN WITH ASCORBIC ACID 128

67 INTERACTION OF NICOTINAMIDE WITH ASCORBIC

ACID

129

68 INTERACTION OF ΑLPHA-TOCOPHEROL WITH

ASCORBIC ACID

130

69 EFFECT OF CARBON CHAIN LENGTH OF

EMULSIFYING AGENT

130

xiii

610 EFFECT OF VISCOSITY OF CREAMS 132

611 DEGRADATION OF ASCORBIC ACID IN THE PRESENCE

OF OTHER VITAMINS IN THE DARK

135

VII STABILIZATION OF ASCORBIC ACID WITH CITRIC

ACID TARTARIC ACID AND BORIC ACID IN CREAM

FORMULATIONS

141

71 INTRODUCTION 142

72 CREAM FORMULATIONS 142

73 PRODUCTS OF ASCORBIC ACID

PHOTODEGRADATION

145

74 SPECTRAL CHANGES IN PHOTODEGRADED CREAMS 145

75 ASSAY OF ASCORBIC ACID IN CREAMS 145

76 KINETICS OF PHOTODEGRADATION 146

77 EFFECT OF STABILIZING AGENTS 146

78 DEGRADATION OF ASCORBIC ACID IN PRESENCE OF

STABILIZING AGENTS IN THE DARK

162

79 EFFECT OF ADDITIVES ON TRANSMISSION OF

ASCORBIC ACID

163

CONCLUSIONS AND SUGGESTIONS 179

CONCLUSIONS 180

SUGGESTIONS 184

REFERENCES 185

AUTHORrsquoS PUBLICATIONS 226

CHAPTER I

INTRODUCTION

2

11 HISTORICAL BACKGROUND

The disease scurvy which now is known as a condition due to a deficiency of

ascorbic acid in the diet has considerable historical significance (Schick 1943

Carpenter 1986 Bardolph and Taylor 1997 Thomas 1997 Bors 2005) Zilva (1932)

isolated the antiscorbutic activity factor from a crude fraction of lemon and showed that

the activity was destroyed by oxidation and protected by reducing agents Waugh and

King (1932) isolated crystalline vitamin C from lemon juice and showed it to be the

antiscorbutic factor Szent-Gyorgyi (1928) had isolated the same factor from pepper in

connection with his biological oxidation-reduction studies Hirst and Zilva (1933)

identified the antiscorbutic factor as ascorbic acid Early work on the chemical

identification and elucidation of the structure of ascorbic acid has been well documented

(Carpenter 1986) The first synthesis of L-ascorbic acid was achieved almost

simultaneously by Ault et al (1933) and Reichstein et al (1933)

Plants and most animals synthesize their own vitamin C but humans lack this

ability due to the deficiency in an enzyme L-gulono-gamma-lactone oxidase that

catalyzes the terminal step in ascorbic acid biosynthesis (Nishikimi et al 1994)

Therefore humans obtain this vitamin from diet and or vitamin supplements to not only

avoid the development of scurvy but also for overall well being (Stone 1969 Lewin

1976 Davies et al 1991) The minimal daily requirement for ascorbic acid in healthy

adults is 40ndash60 mg (Truswell 2003 Mason 2007 Eitenmiller et al 2008 Elia 2009)

12 PHYSICOCHEMICAL CHARACTERISTICS OF ASCORBIC ACID

The important physicochemical characteristics of ascorbic acid (Table 1) involved

in its identification and degradation are described by many authors (Connors et al 1986

3

OrsquoNeil 2001 Moffat et al 2004 Sinko 2006 Johnston et al 2007) The most

important chemical property of ascorbic acid is the reversible oxidation to semidehydro-

L-ascorbic acid and further oxidation to dehydro-L-ascorbic acid This property is the

basis for its physiological activity In addition the proton on oxygenndash3 is acidic (pKa1 =

417) which contributes to the acidic nature of ascorbic acid (1)

13 CHEMISTRY OF ASCORBIC ACID

131 Nomenclature and Structure

The IUPAC-IUB Commission on Biochemical Nomenclature changed the name

vitamin C (2-oxo-L-theo-hexono-4-lactone-23-enediol) to ascorbic acid or L-ascorbic

acid in 1965 (Johnston et al 2007) The chemical structure of ascorbic acid (1) is

HO OH

O

OHHO

H

(1)

O

The molecule has a near planar five-membered ring with two chiral centers

which contain four stereoisomers

132 Chemical Stability

Ascorbic acid is sensitive to air and light and is kept in a well-closed container

protected from light (British Pharmacopoeia 2009) The degradation reactions of

ascorbic acid in aqueous solution depend on a number of factors such as pH temperature

presence of oxygen or metal It is not very stable in aqueous media at room

temperature and undergoes oxidative degradation to dehydroascorbic acid and

4

Table 1 Physicochemical characteristics of ascorbic acid

Empirical formula C6H8O6

Molar mass 17613

Crystalline form Monoclinic mix of platelets and needles

Melting point 190 to 192 degC

[α]25

+205deg to +215deg

pH

5 mg ml

50 mg ml

~3

~2

pKa 417 1157 (20deg)

Redox potential

(dehydroascorbic acid ascorbate)

(H+ ascorbate

ndash)

ndash174 mV

+282 mV

Solubility g ml

Water

Ethanol absolute

Ether chloroform benzene

033

002

Insoluble

UV spectrum

Absorption maximum [A(1 1 cm)]

pH 20

pH 70

245 nm [695]

265 nm [940]

Infrared spectrum

Principal peaks (Nujol mull)

1026 (CminusOH str) 1111(CminusOminusC str) 1312

(minusCminusOminus str) 1653 (C=O str) 990 (C=C str)

cmndash1

Mass spectrum

Principal ions at mz

29 41 39 42 69 116 167 168

D

5

23-diketogulonic acid The stability of ascorbic acid and dehydroascorbic acid can be

improved by lowering the pH below 2 (Wechtersbach and Cigic 2007) Above pH 7

alkali-catalyzed degradation by cleavage at Cndash1 or Cndash2 results in a number of

compounds mainly monondash dindash and tricarboxylic acids (Connors et al 1986 Bors and

Buettner 1997 Halliwell and Whiteman 1997) The oxidative degradation of ascorbic

acid and dehydroascorbic acid in parenteral nutrition mixtures is catalyzed by trace

elements particularly copper (Allwood 1984ab Allwood et al 1992 Allwood and

Kearney 1998 Kearney et al 1998 Gibbons et al 2001) Stabilized ascorbic acid

preparations in hydroalcoholic vehicle (Kaplan et al 1989) and aquaculture feeds

(OrsquoKeefe 2001) have been reported The various oxidation products of ascorbic acid are

shown in Fig 1

It is interesting to note that in addition to redox and acid-base properties ascorbic

acid can exist as a free radical (Bielski et al 1981 Bielski 1982 Halliwell 1996 Bors

and Buettner 1997) The ascorbate radical anion is an important intermediate in the

reactions involving oxidants and ascorbic acidrsquos antioxidant activity Rate constants for

the generation of ascorbate radicals are in the range of 104ndash10

8 s

ndash1 When ascorbate

radicals are generated by oxyanions the rate constants are of the order of 104ndash10

7 s

ndash1

when generated by halide radicals 106ndash10

8 s

ndash1 and when generated by tocopherols and

flavonoids radicals 106ndash10

8 s

ndash1 (Bielski 1982 Halliwell and Whiteman 1997) The

ascorbate radicals decay usually by disproportionation However a change in ionic

strength or pH can influence the rate of dismutation of ascorbic acid Certain oxyanions

such as phosphates accelerate dismutation (Bielski et al 1981) The acceleration is

attributed to the activity of various protonated forms of phosphate to donate a proton

6

Fig 1 Oxidation products of ascorbic acid

O

OHOH

H

OO

OHOH

H

OO

OHOH

H

O

Ascorbyl radical anion

(interm ediate)

Ascorbic acid

(1)

-e- -2H

+

+e- +2H

+

-e-

+e-

Dehydroascorbic acid

(2)

23-diketo-L-gulonic acid

O xalic acid

+

L-Threonic acid

L-Xylose

+

C O 2

CO 2

L-Xylonic acid

+

L-Lyxonic acid CO 2

HO OH O O-

O O

7

efficiently to the ascorbate radical particularly the dimer form of ascorbate

The unusual stability of the ascorbate radical in biological systems dictates that

accessory enzymatic systems be made available to reduce the potential transient

accumulation of ascorbate radical The excess ascorbate radicals may initiate a chain of

free-radicals reactions In plants NADHmonodehydroascorbate reductase maintains

ascorbic acid in its reduced form NADHmonodehydroascorbate reductase plays a major

role in stress related responses in plants Glutathione dehydroascorbate reductase serves

this purpose in animal tissues Such enzymes keep ascorbic acid operating at maximum

efficiency so that other enzyme systems may take advantage of the univalent redox

cycling capacity of ascorbate (Asard et al 2004 Johnston et al 2007)

The anaerobic degradation of ascorbic acid has been studied by Finholt et al

(1963) Under these conditions the molecule is dehydrated and hydrolyzed in aqueous

solution to give furfural and carbon dioxide The rate of degradation is maximum at pH

41 corresponding to the pKa of ascorbic acid This has been suggested due to the

formation of a saltndashacid complex in solution The reaction is dependent on buffer

concentration but has relatively small effect of ionic strength

14 BIOCHEMICAL FUNCTIONS

Ascorbic acid plays an essential role in the activities of several enzymes It is vital

for the growth and maintenance of healthy bones teeth gums ligaments and blood

vessels It is important for the manufacture of certain neurotransmitters and adrenal

hormones Ascorbic acid is required for the utilization of folic acid and the absorption of

iron It is also necessary for normal immune responses to infection and for wound healing

(Henry 1997)

8

Ascorbic acid deprivation and scurvy include a range of signs and symptoms that

involves defects in specific enzymatic processes (Johnston et al 2007) The

administration of ascorbic acid improves most of the signs of chemically induced

glutathione (L-γ-glutamyl-L-cysteine-glycine GSH) deficiency (Meister 1994) The

effect is very pronounced in newborn rats which do not efficiently synthesize ascorbic

acid in contrast to adult rats and guinea pigs When L-buthionine-(SR)-sulphoxime is

administered in addition to the loss in GSH there is a marked increase in

dehydroascorbic acid This has led to the hypothesis that GSH is very important to

dehydroascorbic acid reduction and as a sequence to ascorbic acid recycling (Meister

1995)

Ascorbic acid also possesses pro-oxidant properties and may cause apoptosis

lymphoid and myeloid cells It has been shown that dehydroascorbic acid also stimulates

the antioxidant defenses in some cells by preferentially importing dehydroascorbate over

ascorbate (Braun et al 1997 Banhegyi et al 1998 Puskas et al 2000 2002)

15 ANTIOXIDANT ACTIVITY

Ascorbic acid is known to readily scavenge reactive oxygen and nitrogen species

such as superoxide and hydroperoxyl radicals aqueous peroxyl radicals singlet oxygen

ozone peroxynitrite nitrogen dioxide nitroxide radicals and hypochlorous acid Excess

of such products has been associated with lipids (Niki and Noguchi 1997 Carr et al

2000 Urso and Clarkson 2003) DNA (Fraga et al 1991 1996 Lindahl 1993) and

protein oxidation (Stadtman 1991 Berlett and Stadtman 1997 Dean et al 1997

Ortwerth and Monnier 1997 Padayatty et al 2003)

9

The electron donor character of ascorbate may be responsible for many of its

known biological functions Inspite of the availability of ascorbic acid to influence the

production of hydroxyl and alkoxyl radicals it remains uncertain whether this is the

principal effect or mechanism that occurs in vivo There is a good evidence that ascorbic

acid protects lipids in biological fluids as an antioxidant (Johnston et al 2007) A

detailed account of the function of ascorbate as an antioxidant and its reactions with

reactive nitrogen species and singlet oxygen has been reported by Packer et al (2002)

and Buettner and Schafer (2004)

Ascorbic acid (Eordm ndash0115 V pH 52 Sinko 2006) has been used as an antioxidant

for the stabilization of drugs with a higher oxidation potential These drugs include

morphine (Yeh and Lach 1961) vitamin A (Wright 1986) rifampin (Maggi et al

1966) cholecalciferol (Nerlo et al 1968 Sawicka 1991) promethazine (Underberg

1978) and sulphacetamide and sulphanilamide (Ahmad and Ahmad 1983)

16 PHOTOSTABILITY OF DRUGS

Many drug substances are sensitive to light (British Pharmacopoeia 2009) and

may degrade in pharmaceutical formulations to inactive or toxic compounds This could

make a product therapeutically inactive while in use by the patients The

photodegradation (photolysis) of drug substances may occur not only during storage but

also during the use of the product It may involve several mechanisms including

oxidation reduction hydrolysis decarboxylation isomerization rearrangement and other

reactions Normal sunlight or room light may cause substantial degradation of drug

molecules The study of degradation of drug substances under the action of UVvisible

light is relevant to the process of drug development for several reasons such as

10

Exposure to light can influence the stability of a drug formulation resulting in the

loss of potency

Inappropriate exposure to light of the raw material or the final product can lead to

the formation of toxic photoproducts that are dangerous to health

Information about the stability of drug substances and formulations is needed to

predict the shelf-life of the final product (Tonnesen and Moore 1993)

The development of light-activated drugs involves activation of the compound

through photochemical reactions (Tonnesen 1991)

Adverse effects due to the formation of minor degradation products during

storage and administration have been reported (de Vries et al 1984) The drugs

substances may also cause light-induced side effects after administration to the patient by

interaction with endogenous substances The study of the photochemical properties of

drug substances and formulated products is an integral part of formulation development

to ensure the safety and efficacy of the product

The photodegradation of drug substances occurs as a result of the absorption of

radiation energy by a molecule (A) to produce an excited state species (A) (11) The

absorbed energy can be lost either by a radiative process involving fluorescence or

phosphorescence (12) or by a physical or chemical radiationless process The physical

process results in the loss of energy as heat (13) or by collisional quenching (14) The

chemical decay leads to the formation of a new species (15) The whole process is

represented as

11

A A (11)

A A + hυprime (12)

A A + heat (13)

A + A 2A (14)

A product (s) (15)

According to the Stark-Einstein law the absorption of one quantum of radiation

results in the formation of one excited molecule which may take part in several

photochemical processes [Eqs (11)ndash(15)] The quantum yield φ for any one of these

processes is defined by

Number of molecules undergoing the photochemical process φ =

Number of quanta absorbed

Considering a pure photochemical reaction the quantum yield has a value of 0ndash1

however if A is a radical that can take part in a free-radical chain reaction so that the

absorption of energy simply initiates the reaction then each quantum of energy may

result in the decomposition of molecules and φ may appear to be greater than 1 (Connors

et al 1986)

Detailed information on the photostability and photodegradation of drug

substances including vitamins alone or in solid or liquid formulations is available in the

reviews published by DeRitter (1982) Albini and Fasani (1998) Sequeira and Vozone

(2000) Tonnesen (2002 2004) Yoshioka and Stella (2002) Min and Boff (2002) Reed

et al (2003) Fasani and Albini (2005) and Sinko (2006) The photostability of cosmetic

materials has been reviewed by Sugden (1985) Important aspects dealing with the

photostability testing of drug substances have been dealt by Anderson et al (1991)

k1

k2

k3

k4

hυ

12

Tonnesen and Moore (1993) Tonnesen and Karlsen (1997) Riehl et al (1995) ICH

(1997) Singh and Bakshi (2000) Valvani (2000) Thatcher et al (2001ab) Fasani and

Albini (2005) Klick et al (2005) Singh (2006) and Ahmad and Vaid (2006)

17 KINETIC TREATMENT OF PHOTOCHEMICAL REACTIONS

The kinetic treatment of photochemical reactions with reference to the

photostability of drug substances has been considered by Moore (2004) and is presented

in this section

The photostability testing of a drug substance at the preformulation stage involves

a study of the drugrsquos rate of degradation in solution on exposure to light for a period of

time The value of the degradation rate constant depends very much on the design of the

experimental conditions (eg concentration solvent pH irradiation source oxygen

content) The factors that determine the rate of a photochemical reaction are simply the

rate at which the radiation is absorbed by the test sample (ie the number N of photons

absorbed per second) and the efficiency of the photochemical process (ie the quantum

yield of the reaction φ) For a monochromatic photon source the number of photons

absorbed depends upon the intensity of the photon source and the absorbance at that

wavelength of the absorbing species The rate of a photochemical reaction is defined as

Rate = number of molecules transformed per second = N φ (16)

In the first instance the rate can be determined for a homogeneous liquid sample

in which the only photon absorption is due to the drug molecule undergoing

transformation with the restriction that the concentration is low so that the drug does not

absorb all of the available radiation in the wavelength range corresponding to its

13

absorption spectrum The value of N can be derived at a particular wavelength λ and is

given by

Nλ = Iλ ndash It = Iλ (1 ndash 10ndashA

) (17)

where Iλ and It are the incident and transmitted radiation intensities respectively and A is

the absorbance of the sample at the wavelength of irradiation This expression can be

expanded as a power series

Nλ = 2303 Iλ (A + A22 + A

36 + hellip) (18)

When the absorbance is low (Alt 002) the second- and higher-order terms are negligible

and the expression simplifies to the first term in Eq 18 Given the Beerrsquos law relation

between absorbance and concentration N can be seen to be directly proportional to

concentration

Nλ = 2303 Iλ A = 2303 Iλ ελ b C (19)

where ελ is the molar absorptivity at wavelength λ C the molar concentration of the

absorbing species and b the optical path length of the reaction vessel Now Iλ and ελ vary

with wavelength so the expression must be integrated over the relevant wavelength range

where each has a non-zero value

N = 2303 b C int (Iλ ελ) dλ integrated from λ1 to λ2 (110)

Thus

Rate = 2303 b C φ int (Iλ ελ) dλ (111)

Now the overlap integral (int Iλ ελ dλ) is a constant for a particular combination of photon

source and absorbing substance b is determined by the reaction vessel chosen and φ is a

characteristic of the reaction Thus by grouping the constant terms into an overall

constant k1 the expression is simplified to a first-order kinetic equation

14

Rate = ndashd [Drug] dt = k1C (112)

The integrated form of Eq 112 can be expressed in exponential form (Eq 113) or

logarithmic form (Eq 114)

[Drug]t = [Drug]0 endashk1t

(113)

ln [Drug]t = ln [Drug]0 ndash k1t (114)

Verification of first-order kinetics is obtained when a plot of the logarithm of the

concentration of drug remaining is linear with slope equal to (ndashk1)

Eq 112 predicts that a photodegradation reaction studied at low concentrations in

solution will follow first-order kinetics however the rate constant derived from a study

performed in one laboratory will not be the same as that found in another The reason for

this is the inherent difficulty in reproducing exactly the experimental arrangement of

photon source and sample irradiation geometry Therefore the relative values of the rate

constants are useful in a given experimental arrangement for making comparisons of

degradation of the absorbing substance in different formulations eg those containing

ingredients designed to inhibit the photoreaction The use of rate constants is helpful for

comparative purposes when studying a number of different reaction mixtures under the

same irradiation conditions such as the effect of pH on the degradation of a drug

However the reaction order and numerical values of the rate constants are relative to the

specific conditions used

15

18 LITERATURE ON ASCORBIC ACID

A large number of reviews have been published on various aspects of ascorbic

acid A list of important reviews is given below

Chemistry biochemical functions and related aspects

Rosenberg (1945) Burns (1961) King and Burns (1975) Sim (1972) Hanck

(1982) Zaeslein (1982) Seib and Tolbert (1982) Carpenter (1986) Levine

(1986) Davies et al (1991) Halliwell and Whiteman (1997) Ortega and Delgado

(1998) Asard et al (2004) Hickey and Roberts (2004) Johnston et al (2007)

Eitenmiller (2008)

Chemical and pharmaceutical stability

Macek (1960) Garrett (1967) Carstensen (1972) Dale and Booth (1976) Hashmi

(1973) Litner (1973) DeRitter (1982) Allwood (1984ab) Allwood and Kearney

(1998) Connors et al (1986) Smith et al (1988) Racz (1989) Roth et al 1991

Ball (2006) Eitenmiller et al (2008) Sweetman (2009)

Methods of assay and chromatography

Mader (1961) Gyorgy and Pearson (1967) Bolliger and Konig (1969) Hashmi

(1973) Al-Meshal and Hassan (1982) Pelletier (1985) Lambert and deLeenheer

(1992) Halver and Felton (2001) Moffat et al (2004) Ball (2006) Eitenmiller et

al (2008)

Pharmacology and related aspects

Levine (1986) Dollery (1999) Sauberlich (1994ab) McDowell (2000)

Kaushansky and Kipps (2006) Sweetman (2009)

16

Antioxidant activity

Basu et al (1999) Shacter (2000) Thiele et al (2000) Cadenas and Packer

(2002) Packer et al (2002) Padayathy et al (2003) Parker and Parker (2003)

Burke (2006) Johnston et al (2007)

Cosmetic Preparations

Barel et al (2001) Salvador and Chisvert (2007) Rosen (2005) Bissett (2006)

Chaudhri and Jain (2009)

CHAPTER II

PHOTODEGRADATION

REACTIONS AND ASSAY

OF ASCORBIC ACID

18

21 PHOTODEGRADATION REACTIONS

211 Photodegradation of Ascorbic Acid

Aqueous ascorbic acid (1) solutions are degraded by UV light to give

dehydroascorbic acid (2) (Arcus and Zilva 1940) Ascorbic acid degradation at a

concentration of 52 and 50 mg on UV irradiation for 2 hours gave a loss of 43 and 8

respectively Dehydroascorbic acid solutions are more stable to UV light than the

ascorbic acid (Kitagawa 1968) In many natural products the vitamin is oxidized on

exposure to air and light (OrsquoNeil 2001) When solutions of multivitamin preparations are

exposed to light H2O2 as well as organic peroxides are generated and specific

byproducts that differ from dehydroascorbic acid and 23-diketogulonic acid (3) are

produced (Lavoie et al 2004)

In aqueous neutral or alkaline solution ascorbic acid (1) undergoes chemical or

photochemical oxidation to dehydroascorbic acid (2) which upon saponification of the

lactone ring under the influence of the base water produces 23-diketo-L-gulonic acid (an

α szlig- diketogulonic acid) (3) This acid undergoes further oxidation to oxalic acid (4) and

L-threonic acid (5) (Racz 1989) (Fig 2a) At room temperature oxalic acid (4) is also

formed along with threonolactone (6) by photochemical degradation of ascorbic acid (1)

in the presence of singlet oxygen (1O2) (Silva and Quina 2006) (Fig 2a) The low-

temperature photooxygenation of ascorbic acid (1) gives a mixture of unstable

hydroperoxide ketones (7) and (8) which on standing interconvert and cyclize to

hydroperoxyhemiketal (9) The hydroperoxyhemiketal breaks down on warming to

produce the oxalate esters of threonic acid (10) (Fig 2b) (Kwon and Foote 1988)

19

COOH

COOH

O

OHHO

O

HOH2C

HO2

O

O

HO

OO

O O2H

OHHO

O

HOH2C

OH

O

O

OH

O2H

OO

HO O2CCO2H

(1)hv

room temperature

(4)(6)

(1)hv

85 oC

(7)

(a)

(8)

+

cyclization

(9)

ring cleavage

(b)

(10)

(2)

OH O

OHHO

OH O O

(3)

OH OH

OH

OH O

O

OH

1O2 [O]

+

(5)

COOH

COOH

(4)

+

OH

Fig 2 Photooxidation of ascorbic acid at room and low temperature

20

An important consideration in the stability of ascorbic acid in total parenteral

nutrition (TPN) solutions is the generation of hydrogen peroxide in the presence of light

(Laborie et al 1998 1999 2000 2002 Chessex et al 2002) This may result from the

oxidation of ascorbate anion by molecular oxygen (Homann and Gaffron 1964 Taqui

Khan and Martell 1967 Mushran and Agarwal 1977 Hughes 1985 De La Rochette et

al 2000) leading to further degradation of ascorbic acid (Deutsch 1998a 1998b

1998c) The kinetics and mechanism of oxidation reactions of ascorbic acid have been

studied by several workers (Taqui Khan and Martell 1967 Ogata and Kosugi 1969

Blaugh and Hajratwala 1972 Fessenden and Verma 1978 Abe et al 1986 Kwon et al

1989 Fornaro and Coicher 1998 Njus et al 2001)

The photostability of various ascorbic acid tablets on exposure to UV light has

been studied and the influence of antioxidants and moisture on the potency loss of

ascorbic acid has been evaluated The physical characteristics of ascorbic acid tablets are

also affected on UV irradiation (Ahmad et al 1973 Jamil et al 1980ab Jamil and

Ahmad 1984)

212 Effect of Various Substances on Photodegradation of Ascorbic Acid

The oxidation-reduction reactions of ascorbic acid in the presence of riboflavin at

pH 8ndash9 under the influence of light have been studied Under these conditions ascorbic

acid is a more active H donor to riboflavin than phenolphthalein (Sibi et al 1953)

Riboflavin has been found to catalyze the photodegradation of ascorbic acid solutions

during exposure to light and air The losses of ascorbic acid are markedly increased by

the presence of Cu2+

and Fe3+

ions under light exposed and unexposed conditions (Sattar

et al 1977) A spectral study of the UV photolysis of ascorbic acid solutions in the

21

presence of riboflavin has shown that the degradation of ascorbic acid is enhanced to the

extent of about 15 (Vaid et al 2005) The influence of DL- methionine on the

photostability of ascorbic acid solutions has also been studied DL- methionine (10 mg

) enhances the photostability of ascorbic acid (40 mg ) in acetate and phosphate

buffers but not in citrate buffer at pH 45 The photoprotective action of DL-methionine

on ascorbic acid appears to be influenced by its concentration pH of the medium and the

buffer species (Asker et al 1985)

The degradation of ascorbic acid solutions on irradiation with simulated sunlight

in the presence of the food dye quinolone yellow (E 104) is enhanced However this

effect is reversed by the addition of mannitol indicating that this dye facilitates the

photogeneration of hydroxyl radicals which may cause degradation of the vitamin The

incorporation of triplet quenchers enhances the stability of substrate solutions suggesting

that the dye acts as a triplet sensitizer to facilitate the reaction (Sidhu and Sugden 1992)

The photostability of ascorbic acid solutions is enhanced by sweetening agents (mannitol

sorbitol sucrose dextrose and Canderal) at 5 wv concentration However the addition

of stoichiometric amounts of hydrogen peroxide as a source of hydroxyl radicals and 2

2rsquo-azobis (2-amidopropane) as a source of hydroperoxyl radicals results in diminished

stability of ascorbic acid solutions The diminished activity may be due to the action of

hydroperoxyl radicals in the presence of hydroxyl radical scavengers (Ho et al 1994)

Metal-complexing agents (eg disodium ethylenediaminetetraacetic acid N-

hydroxylethyl ethylenediaminetetraacetic acid 8-hydroxyquinoline) have a stabilizing

effect on the photolysis of ascorbic acid injectable solutions (Kassem et al 1969ab

22

1972) This may be due to the interaction of these agents with metal ions and other

impurities

213 Photosensitized Oxidation of Ascorbic Acid

In the presence of visible light a photosensitizer such as riboflavin can exhibit

photosensitizing properties through a mixed Type IndashType II mechanism (Yoshimura and

Ohno 1988 Foote 1991 Silva et al 1994 Silva and Quina 2006) as presented below

Type I mechanism (low oxygen concentration)

RF + hυ rarr 1RF rarr

3RF (21)

3RF + SH rarr RF

middot ndash + SH

middot + rarr RFH

middot + S

middot (22)

RFmiddot ndash

+ O2 rarr RF + O2middot ndash

(23)

2RFHmiddot rarr RF + RFH2 (24)

RFH2 + O2 rarr RF + H2O2 (25)

H2O2 + O2middot ndashrarr

ndashOH +

middotOH + O2 (26)

Smiddot and or SH

middot +

+ H2O2 O2middot ndash

rarr Soxid (27)

Type II mechanism (high oxygen concentration)

RF + hυ rarr 1RF rarr

3RF (28)

3RF + O2 rarr RF +

1O2 (29)

SH + 1O2 rarr Soxid (210)

In these reactions RF 1RF and

3RF represent RF in the ground state and in the excited

singlet and triplet states respectively RFmiddot ndash

RFHmiddot and RFH2 are the radical anion the

radical and the reduced form of RF SH is the reduced substrate and SHmiddot

+ S

middot and Soxid

23

represent the intermediate radical cation the radical and the oxidized form of the

substrate respectively

An early study of the riboflavin-sensitized photooxidation of ascorbic acid has

been carried out by flash photolysis (Heelis et al 1981) ESR spectrometry has been

used to investigate the photosensitized formation of ascorbate radicals by riboflavin (Kim

et al 1993) The photochemical behavior of a system consisting of ascorbate ion (AHndash)

and riboflavin has been studied by Mancini et al (2000) and De La Rochette et al (2000

2003) The photosensitized processes were examined as a function of oxygen pressure

and the efficiency of RF induced degradation of AHndash

at various oxygen concentrations

was compared on the basis of the respective initial photosensitization quantum yields

(Table 2)

In this reaction a Type I photosensitization mechanism (Karlsen 1996) implies a

direct electron transfer between AHndash and the RF triplet-excited state followed by the

oxidation of semioxidized ascorbyl radical (AHmiddot) by molecular oxygen or some other

reactive species On the contrary in a Type II photosensitization mechanism singlet

oxygen is produced directly by energy transfer from the RF triplet-excited state to

molecular oxygen and the singlet oxygen then oxidizes the AHndash Thus by irradiating

under increasing oxygen pressure it is possible to control the relative prevalence and

efficiency of Type I or Type II mechanisms The absence of a linear relationship between

the quantum yields of ascorbate degradation and oxygen concentration indicates that the

photosensitization mechanism involved in not exclusively Type II

24

Table 2 Initial quantum yield (φ) for ascorbate (AHndash) degradation during

photosensitization by RF (35 microM) in solutions irradiated at 365 nm and

37ordmC

O2 103 times φ (AH

ndash)a

0

5

20

14 plusmn 06

1670 plusmn 220

1940 plusmn 200

a Data are the mean plusmn SD of three independent experiments

25

In the presence of RF and O2 the quantum yields for degradation of ascorbate ion

have been found to be greater than one suggesting the participation of chain reactions

initiated by the ascorbyl radical as given by the following reactions

3RF + AH

ndash rarr RFmiddot

ndash + AHmiddot (211)

AHmiddot + O2 rarr A + HO2middot (212)

HO2middot + AHndash rarr H2O2 + AHmiddot (213)

The generation of the ascorbyl radical by the reaction between the RF excited-

triplet state and the ascorbate ion (Eq 211) is the only step that requires the absorption of

photons (to form the excited-triplet state of RF) The subsequent reactions (Eqs 212 and

213) are independent of light and lead to further degradation of the ascorbate ion In the

presence of transition metal ions such as Fe3+

in trace amounts in the buffer solution

containing RF and ascorbate ions further oxidation of ascorbate ion could also occur As

a result the reduced form of the metal ion (ie Fe2+

) can be generated by the metal

catalyzed oxidation of ascorbate ion This has been confirmed by the significant decrease

in the AHndash photooxidation quantum yield in the presence of the metal chelator EDTA

which inactivates the trace amounts of iron present in the buffer solution The quantum

yields for the photosensitized oxidation of ascorbate ion are decreased twofold at 20 O2

and fourfold at 5 O2 concentration in the presence of EDTA (Silva and Quina 2006)

Amino acids have been found to affect the photosensitized oxidation of ascorbic acid

(Jung et al 1995)

214 Ascorbic Acid Interaction with Other Water-Soluble Vitamins

The stability of ascorbic acid is reported to be enhanced in syrups containing B-

complex vitamins (Connors et al 1986) This may be due to the increased viscosity of

the syrups inhibiting the oxidation of ascorbic acid The rate of photolysis in solution

26

containing cyanocobalamin and ascorbic acid is reported to decrease with an increase in

pH (Ansari et al 2004) where as use of certain halide salts has been reported to be

beneficial in stabilizing pharmaceutical products and dietary supplements when vitamin

B12 and vitamin C are combined in solution (Ichikawa et al 2005) When a solution of

multivitamins is exposed to light it is reported that organic peroxidases are generated and

the concentration of ascorbic acid decreases (Lavoie et al 2004)

22 ASSAY OF ASCORBIC ACID

Recent accounts of the development and application of analytical methods to the

determination of ascorbic acid in pharmaceuticals biological samples and food materials

are reported in the literature (Rumsey and Levine 2000 Halver and Felton 2001 Moffat

et al 2004 Ball 2006 Sheraz et al 2007 Eitenmiller et al 2008 Salkic and Kubicek

2008) Most of these methods are based on the application of spectrophotometric

fluorimetric and chromatographic techniques to suit the requirements of a particular assay

and are summarized below

221 Spectrophotometric Methods

Spectrophotometric methods are the most widely used methods for the assay of

ascorbic acid in aqueous solution Ascorbic acid exhibits strong absorption in the

ultraviolet region (absorption maxima 243 nm at pH 2 and 265 nm at pH 4ndash10 OrsquoNeil

2001 Moffat et al 2004 British Pharmacopoeia 2009) This is the basis of

spectrophotometric methods for the determination of the vitamins in pure solutions and in

sample preparations where no interference is observed from UV absorbing impurities

The value of A (1 1 cm) at the analytical wavelength of 245 nm (pH 20) is high (695)

which makes the method very sensitive for the determination of mg quantities of the

27

vitamin Treatment of the material to be analyzed with ascorbic acid oxidase is often used

as a blank to correct for the presence of interfering substances in biological samples (Liu

et al 1982) A spectrophotometric method for the determination of ascorbic acid in

pharmaceuticals by background correction (245 nm) has been reported (Verma et al

1991) The direct determination of ascorbic acid in mixtures involves the use of 22prime-

dipyridyl as a colorimetric reagent The method is based on the reduction of Fe (III) by

ascorbic acid to Fe (II) which reacts with 2 2prime-dipyridyl to form a colored complex

(absorption maximum 510 nm) that can be used for quantitative determination (Margolis

and Schmidt 1996) A spectrophotometric method has been developed for the

determination of ascorbic acid and its oxidation product dehydroascorbic acid in

biological samples (Moeslinger et al 1995) A sensitive method has been reported for

the determination of ascorbic acid in pharmaceutical formulations and fruit juices by

interaction with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (Br-PADAP) (Ferreira

et al 1997) A novel UV method has been developed for the analysis of ascorbic acid in

methanol at 245 nm in various formulations (Zeng et al 2005)

Ascorbic acid in aqueous solutions has been assayed at 244 nm (pH ~2) (Ogata

and Kosugi 1969) 245 nm (pH 35) (Blaugh and Hajratwala 1972) 264 nm (pH 7)

(Salkic et al 2007) 265 nm (pH 7) (Hashmi 1973) 275 nm (pH 41 and 70) (Heelis et

al 1981) 265 nm (pH 7) (Al-Meshal and Hassan 1982) 245 nm (pH ~2) (Verma et al

1991) and 265 nm (pH ~7) (Erb et al 2004) Dehydroascorbic acid and 23-

diketogulonic acid do not significantly absorb in this region (Pelletier 1985 Davies et

al 1991 Rumsey and Levine 2000) and therefore do not interfere with the assay of

ascorbic acid in degraded solutions

28

222 Fluorimetric Methods

Fluorimetry is a highly sensitive technique for the determination of fluorescent

compounds or fluorescent derivatives of non-fluorescent compounds The technique has

been used for the detection of microg quantities of ascorbic acid Methods based on

fluorimetric (Kampfenkel et al 1995) and chemiluminescence detection (Zhang and

Chen 2000) provide highly sensitive methods for the determination of ascorbic acid in

plant and other materials

223 Mass Spectrometric Methods

Conventional and isotope mass spectrometric techniques have also been used for

the analysis of ascorbic acid Isotope ratio mass spectrometry is particularly useful and

sensitive when 13

C ascorbic acid is used as a standard in the analysis of complex matrices

(Gensler et al 1995)

224 Chromatographic Methods

High-performance liquid chromatographic (HPLC) methods have extensively

been employed for the determination of ascorbic acid in biological samples These

methods include ion exchange reversed phase and ion-pairing HPLC protocols

Spectrophotometric fluorimetric and electrochemical detection has been used in the

HPLC analysis of ascorbic acid The electrochemical detection is used for the

simultaneous determination of ascorbic acid dehydroascorbic acid and their isomers and

derivatives A number of HPLC methods have been developed for the detection and

determination of ascorbic acid and its oxidation products and derivatives in biological

samples and plant materials (Tsao and Young 1985 Tangney 1988 Dabrowski and

Huiterleitner 1989 Thomson and Trenerry 1995 Kimoto et al 1997 Kall and

29

Anderson 1999 Rumelin et al 1999 Lykkesfeldt 2000 Zhang et al 2000 Pastore et

al 2001 Frenich et al 2005) The limit of detection of ascorbic acid in plasma or urine

with UV detection lies in the range of 100-120 microg (Liau et al 1993 Manoharan and

Schwille 1994) Fluorescence detection of ascorbic acid and dehydroascorbic acid in

plasma and its comparison with coulometric detection has been reported (Tessier et al

1996) A liquid chromatography-diode-array detection (LCndashDAD) method has been

reported for the determination of 10 water-soluble and 10 fat-soluble vitamins including

ascorbic acid in pharmaceutical preparations with a coefficient of variation lt 65

(Konings 2006)

Liquid chromatography methods based on precolumn and o-phenylenediamine

(OPD) derivatization have been used for the determination of total vitamin C and total

isovitamin C in foods and dehydro forms of the vitamin Isoascorbic acid has been used

as an internal standard in the analysis (Speek et al 1985 Vanderslice et al 1990

Dodsun et al 1992 Vanderslice and Higgs 1988 1993 Hagg et al 1994 1995) The

limits of detection of ascorbic acid by HPLC using different detectors are in the range of

16ndash400 microgl (Capellmann and Bolt 1992 Iwase and Ono 1994 Karatepe 2004)

225 Enzymatic Methods

Enzymatic methods using ascorbate oxidase are specific and have the advantage

of selectively measuring the biological activity of ascorbic acid in serum or plasma (Liu

et al 1982) Ascorbate oxidase and OPD derivatization has been used to develop a rapid

automated method for the routine assay of ascorbic acid in serum and plasma The

method has a sample throughput of 100h (Ihara et al 2000)

30

226 Commercial Kits for Clinical Analysis

Commercial kits (eg Immunodiagnostic Germany Biovision USA) are also

used for the determination of ascorbic acid in biological samples (serum or plasma) in

clinical laboratories

227 Analysis in Creams

The general methods for the analysis of active ingredients and excipients in

cosmetic products including creams are described by Salvador and Chisvert (2007)

Ascorbic acid and derivatives in creams have been determined by liquid chromatography

(Irache et al 1993 Varvaresou et al 2006) gas chromatography-mass spectrometry

(Leveque et al 2005) and electrochemical methods (Beissenhirtz et al 2003 Guitton et

al 2007)

CHAPTER III

FORMULATION AND

STABILITY OF CREAM

PREPARATIONS

32

31 FORMULATION OF CREAM PREPARATIONS

Traditionally emulsions have been defined as dispersions of macroscopic droplets

of one liquid in another liquid with a droplet diameter approximately in the range of 05-

100 microm (Becher 1965) According to the definition of International Union of Pure and

Applied Chemistry (IUPAC) (1971) ldquoIn an emulsion liquid droplets and or liquid

crystals are dispersed in a liquidrdquo

Creams are semisolid emulsions intended for external applications They are often

composed of two phases Oil-in-water (ow) emulsions are most useful as water-washable

bases whereas water-in-oil (wo) emulsions are emollient and cleansing agents The

active ingredient is often dissolved in one or both phases thus creating a three-phase

system Patients often prefer a wo cream to an ointment because the cream spreads more

readily is less greasy and the evaporating water soothes the inflamed tissue OW creams

(vanishing creams) rub into the skin the continuous phase evaporates and increases the

concentration of a water-soluble drug in the adhering film The concentration gradient for

drug across the stratum corneum therefore increases promoting percutaneous absorption

(Barry 2002 Betageri and Prabhu 2002)

The various factors involved in the formulation of emulsions and topical products

have been discussed by Block (1996) Barry (2002) and Jain et al (2006) and are briefly

presented in the following sections

311 Choice of Emulsion Type

Oil-in-water emulsions are used for the topical application of water-soluble drugs

mainly for local effect They do not have the greasy texture associated with oily bases

and are therefore pleasant to use and easily washed from skin surfaces Moisturizing

33

creams designed to prevent moisture loss from the skin and thus inhibit drying of the

stratum corneum are more efficient if formulated as ow emulsions which produce a

coherent water-repellent film

312 Choice of Oil Phase

Many emulsions for external use contain oils that are present as carriers for the

active ingredient It must be realized that the type of oil used may also have an effect both

on the viscosity of the product and on the transport of the drug into the skin (Barry

2002) One of the most widely used oils for this type of preparation is liquid paraffin

This is one of a series of hydrocarbons which also includes hard paraffin soft paraffin

and light liquid paraffin They can be used individually or in combination with each other

to control emulsion consistency This will ensure that the product can be spread easily but

will be sufficiently viscous to form a coherent film over the skin The film-forming

capabilities of the emulsion can be further modified by the inclusion of various waxes

such as bees wax carnauba wax or higher fatty alcohols

313 Emulsion Consistency

A consideration of the texture or feel of a product intended for external use is

important A wo preparation will have a greasy texture and often exhibits a higher

apparent viscosity than ow emulsions This fact imparts a feeling of richness to many

cosmetic formulations Oil-in-water emulsions will however feel less greasy or sticky on

application to the skin will be absorbed more readily because of their lower oil content

and can be more easily washed from skin surface Ideally emulsions should exhibit the

rheological properties of plasticity pseudoplasticity and thixotropy Emulsions of high

apparent viscosity for external use (cream) are of a semisolid consistency There are

34

several methods by which the rheological properties of an emulsion can be controlled

(Billany 2002)

314 Choice of Emulsifying Agent

The choice of emulgent to be used would depend on factors such as its

emulsifying ability route of administration and toxicity Most of the non-ionic emulgents

are less irritant and less toxic than their anionic and cationic counter parts Some

emulgents such as the ionic alkali soaps often have a high pH and are thus unsuitable for

application to broken skin Even in normal intact skin with a pH of 55 the application of

such alkaline materials can cause irritation Some emulsifiers in particular wool fat can

cause sensitizing reactions in susceptible people The details of various types of

emulsifying agents are available in the literature (Betageri and Prabhu 2002 Billany

2002 Swarbrick et al 2006)

315 Formulation by the HLB Method

The physically stable emulsions are best achieved by the presence of a condensed

layer of emulgent at the oil water interface and the complex interfacial films formed by a

blend of an oil-soluble emulsifying agent with a water-soluble one produces the most

satisfactory emulsions

It is possible to calculate the relative quantities of the emulgents necessary to

produce the most physically stable emulsions for a particular formulation with water

combination This approach is called the hydrophilic-lipophilic balance (HLB) method

Each surfactant is allocated an HLB number representing the relative properties of the

lipophilic and hydrophilic parts of the molecule High numbers (up to a theoretical

number of 20) therefore indicates a surfactant exhibiting mainly hydrophilic or polar

35

properties whereas low numbers represent lipophilic or non-polar characteristics Each

type of oil requires an emulgent of a particular HLB number in order to ensure a stable

product For an ow emulsion the more polar the oil phase the more polar must be the

emulgent system (Billany 2002 Im-Emsap et al 2002 Swarbrick et al 2006)

316 Concept of Relative Polarity Index

In the ingredient selection in cosmetic formulations a new concept of relative

polarity index (RPI) has been presented (Wiechers 2005) The physicochemical

characteristics of the ingredients determine their skin delivery to a greater extent than the

formulation type The cosmetic formulation cannot change the chemistry of the active

molecule that needs to penetrate to a specific site within the skin However the

formulation type can be selected based on the polarity of the active ingredient and the

desired site of action for the active ingredient For optimum skin delivery the solubility of

the active ingredient needs to be as high as possible (to create a large concentration

gradient) and as small as possible (to create a large partition coefficient) To achieve this

it is necessary to determine the following parameters

The total amount dissolved in the formulation that is available for skin penetration

the higher this amount the more will penetrate until a solution concentration is

reached in the skin therefore a high absolute solubility in the formulation is required

The polarity of the formulation relative to that of the stratum corneum if an active

ingredient dissolves better in the stratum corneum than in the formulation then the

partition of the active ingredient will favour the stratum corneum therefore a low

(relative to that in the stratum corneum) solubility in the formulation is required

(Wiechers 2005)

36

These requirements can be met by considering the concept of RPI (Wiechers

2003 2005) In this systematic approach it is essential to consider the stratum corneum

as another solvent with its own polarity The stratum corneum appears to behave very

similarly to and in a more polar fashion than butanol with respect to its solubilizing

ability for active ingredients (Scheuplein and Blank 1973) The polarity of stratum

corneum as expressed by its octanol water partition coefficient is 63

The relative polarity index may be used to compare the polarity of an active

ingredient with both that of the skin and that of the oil phase of a cosmetic formulation

predominantly consisting of emollients It may be visualized as a vertical line with a high

polarity at the top and a high lipophilicity at the bottom The polarity is expressed as the

log10 of the octanol water coefficient For example the relative polarity index values of

glycerin and isostearyl isostearate are -176 and 2698 respectively (Wiechers 2005) In

order to use the concept of the relative polarity index three numbers (on log10 scale) are

required

The polarity of the stratum corneum is set at 08 However in reality this value will

change with the hydration state of the stratum corneum that is determined in part by

the external relative humidity (Bonwstra et al 2003)

The polarity of the active molecule

The polarity of the formulation

For multiphase or multipolarity systems like emulsions the active ingredient is dissolved

in the phase For example in an ow emulsion where a lipophilic active ingredient is

dissolved in the oil phase it is the polarity of the homogenous mixture of the lipophilic

active ingredient and internal oil For the same lipophilic active in a wo emulsion it is

37

the polarity of the homogenous mixture of the lipophilic active ingredients and external

oil For water-soluble active ingredients it is the polarity of the homogenous mixture of

the hydrophilic active ingredient and the aqueous phase regardless whether it is internal

(wo emulsions) or external (ow emulsions)

Once the active ingredient and the formulation type have been chosen it is

necessary to create the delivery system that will effectively deliver the molecule The

concept of relative polarity index allows the formulator to select the polarity of the phase

in which the active ingredient is incorporated on the basis of its own properties and those

of the stratum corneum In order to achieve maximum delivery the polarity of the active

ingredient and the stratum corneum need to be considered In order to improve the skin

delivery of active ingredients the first step involves selecting a primary emollient with a

polarity close to that of the active ingredient in which it will have a high solubility The

second step is to reduce the solubility of the active ingredient in the primary emollient via

the addition of a secondary emollient with a different polarity and therefore lower

solubility for the active ingredient This approach has shown a 3-4 fold increase in skin

penetration with out increasing the amount of active ingredients in the formulation

(Wiechers 2005)

32 FORMULATION OF ASCORBIC ACID CREAMS

Ascorbic acid is a water-soluble material and is included frequently in skin care

formulations to restore skin health It is very unstable and is easily oxidized in aqueous

solution This vitamin is known to be a reducing agent in biological systems and causes

the reduction of both oxygen- and nitrogen- based free radicals (Higdon and Frei 2002)

It can also act as a co-antioxidant with the tocopheroxyl radical to regenerate alpha-

38

tocopherol (Packer et al 1979 Buettner 1993 Peyrat-Maillard et al 2001) In this

reaction the two vitamins act synergistically Alpha-tocopherol first functions as the

primary antioxidant that reacts with an organic free radical Thereafter ascorbic acid

reacts with the free radical tocopheroxyl to general alpha-tocopherol In physiological

conditions the ascorbyl radical formed by regenerating tocopherol is then converted back

to ascorbate by the redox cycle (Davies et al 1991) The interaction of ascorbic acid

with a redox partner such as alpha-tocopherol has been found useful to slow its oxidation

and prolong its action

The instability of ascorbic acid makes this antioxidant active ingredient a

formulation challenge to deliver to the skin and retain its effective form In addition to its

use in combination with alpha-tocopherol in cream formulations the stability of ascorbic

acid may be improved by its use in the form of a fatty acid ester such as ascorbyl

palmitate Ascorbyl palmitate has been used in thixogel formulations and is typically

incorporated into the mineral oil phase Preliminary experiments have shown that it could

be slowly released from the starch-oil emulsion matrix and act as an antioxidant (Wille

2005)

Various physical and chemical factors involved in the formulation of cream

preparations have been discussed in the above sections For polar and air light sensitive

compounds such as ascorbic acid it is important to consider factors such as the choice of

formulation ingredients polar character of formulation HLB value pH viscosity etc to

achieve stability

39

33 STABILITY OF CREAMS

331 Physical Stability

The most important consideration with respect to pharmaceutical and cosmetic

emulsions (creams) is the stability of the finished product The stability of a

pharmaceutical emulsion is characterized by the absence of coalescence of the internal

phase absence of creaming and maintenance of elegance with respect to appearance

odor color and other physical properties An emulsion is a dynamic system however

any flocculation and resultant creaming represent potential steps towards complete

coalescence of the internal phase In pharmaceutical emulsions creaming results as a lack

of uniformity of drug distribution and poses a problem to the pharmaceutical

compounder Another important factor in the stabilization of emulsions is phase inversion

which involves the change of emulsion type from ow to wo or vice versa and is

considered as a case of instability The four major phenomena associated with the

physical instability of emulsions are flocculation creaming coalescence and breaking

These have been discussed by Garti and Aserin (1996) Im-Emsap et al (2002) and Sinko

(2006)

332 Chemical Stability

The instability of a drug may lead to the loss of its concentration through a

chemical reaction under normal or stress conditions This results in a reduction of the

potency and is a well-recognized cause of poor product quality The degradation of the

drug may make the product esthetically unacceptable if significant changes in color or

odor have occurred The degradation product may also be a toxic substance The various

pathways of chemical degradation of a drug depend on the structural characteristics of the

40

drug and may involve hydrolysis dehydration isomerization and racemization

decarboxylation and elimination oxidation photodegradation drug-excipients and drug-

drug interactions Factors determining the chemical stability of drug substances include

intrinsic factors such as molecular structure of the drug itself and environmental factors

such as temperature light pH buffer species ionic strength oxygen moisture additives

and excipients The application of well-established kinetic principles may throw light on

the role of each variable in altering the kinetics of degradation and to provide valuable

insight into the mechanism of degradation (Baertschi and Alsante 2005 Yoshioka and

Stella 2002 Lachman et al 1986) The chemical stability of individual components

within an emulsion system may be very different from their stability after incorporation

into other formulation types For example many unsaturated oils are prone to oxidation

and their degree of exposure to oxygen may be influenced by factors that affect the extent

of molecular dispersion (eg droplet size) This could be particularly troublesome in

emulsions because emulsification may introduce air into the product and because of the

high interfacial contact area between the phases (Barry 2002) The use of antioxidants

retards oxidation of unsaturated oils minimizes changes in color and texture and prevents

rancidity in the formulation Moreover they can retard the degradation of certain active

ingredients such as vitamin C (Vimaladevi 2005) The stability problems of dispersed

systems and the factors leading to these stability problems have been discussed by

Weiner (1996) and Lu and Flynn (2009)

333 Microbial Stability

Topical bases often contain aqueous and oily phases together with carbohydrates

and proteins and are susceptible to bacterial and fungal attack Microbial growth spoils

41

the formulation and is a potential toxic hazard Therefore topical formulations need

appropriate preservatives to prevent microbial growth and to maintain their quality and

shelf-life (Barry 2002 Arger et al 1996) Cream formulations may contain fats and oils

with high percentage of unsaturated linkages that are susceptible to oxidation degradation

and development of rancidity The addition of antioxidants retards oxidation of fats and

oils minimizes changes in color and texture and prevents rancidity in the formulation

Moreover they can retard the degradation of certain active ingredients such as vitamin C

These aspects in relation to dermatological formulations have been discussed by Barry

(1983 2002) and Vimaladevi 2005)

334 Stability of Ascorbic Acid in Liquid Formulations

Ascorbic acid is very unstable in aqueous solution Different workers have studied

the stability of ascorbic acid in liquid formulations (Connors et al 1986 Austria et al

1997) Its shelf-life can be prolonged by appropriate choice of vehicle and control of

other variables such as pH stabilizers temperature light and oxygen (Table 3)

Similarly the stability of various concentrations of ascorbic acid in solution form may

vary depending upon the type of solvent used (Table 4) (Connors et al 1986 Satoh et

al 2000 Lee et al 2004 Zeng et al 2005)

335 Stability of Ascorbic Acid in Emulsions and Creams

Ascorbic acid exerts several functions on skin such as collagen synthesis

depigmentation and antioxidant activity Ultraviolet radiation generates reactive oxygen

species (ROS) which produce some harmful effects on the skin including photocarcinoma

and photoaging In order to combat these problems ascorbic acid as an antioxidant has

42

Table 3 Effect of vehicles on the stability of ascorbic acid ( ascorbic acid remaining in

solutions after storage at room temperature) (Connors et al 1986)

Storage Time (days) No Vehicle

30 60 90 120 180 240 360

1 Corn Syrup 965 925 920 920 900 860 760

2 Sorbitol 990 990 990 970 960 925 890

3 4 Carboxymethyl

Cellulose

840 680 565 380 ndash ndash ndash

4 Glycerin 100 100 990 990 970 935 920

5 Propylene glycol 995 990 980 945 920 900 900

6 Syrup USP 100 100 980 980 930 900 880

7 Syrup 212 gL 880 810 775 745 645 590 440

8 25 Tragacanth 785 620 510 320 ndash ndash ndash

9 Saturated solution of

Dextrose

990 935 875 800 640 580 510

10 Distilled Water 900 815 745 675 405 185 ndash

11 50 Propylene glycol +

50 Glycerin

980 ndash 960 ndash 933 ndash ndash

12 25 Distilled Water +

75 Sorbo (70 solution

of Sorbitol)

955 954 ndash 942 930 ndash ndash

13 50 Glycerin + 50

Sorbo

982 984 978 ndash ndash 914 ndash

43

Table 4 Stability of various concentrations of ascorbic acid in water propylene glycol

and USP syrup at room temperature ( of ascorbic acid remaining in solution)

(Connors et al 1986)

Storage Time (days) Concentration

(mg ml)

Solvent

30 60 90 120 180 240 360

10 Water 930 840 820 670 515 410 ndash

50 Water 940 920 880 795 605 590 300

100 Water 970 930 910 835 705 680 590

10 Propylene glycol 100 985 980 975 960 920 860

50 Propylene glycol 100 970 980 980 980 965 935

100 Propylene glycol 100 100 100 100 990 100 925

10 Syrup 100 100 980 990 970 960 840

50 Syrup 100 100 100 100 990 100 960

100 Syrup 100 100 100 100 100 100 995

44

been used in various dosage forms and in different concentrations (Darr et al 1996

Gallarate et al 1999 Zhang et al 1999 Pinnell et al 2001 Lee et al 2004 Raschke

et al 2004 Elmore 2005 Farahmand et al 2006 Maia et al 2006) Ascorbic acid has

good photoprotective ability against UVA-mediated phototoxicity (Darr et al 1996) A

variety of formulations containing ascorbic acid or its derivatives have been studied in

order to evaluate their stability and delivery through the skin (Gallarate et al 1999

Zhang et al 1999 Ozer et al 2000 Pinnell et al 2001 Lee et al 2004 Raschke et al

2004 Farahmand et al 2006) Formulations containing derivatives of ascorbic acid are

found to be more stable than ascorbic acid but they do not produce the same effect as that

of the parent compound probably due to the lack of redox properties (Heber et al 2006)

Effective delivery of ascorbic acid through topical preparations is a major factor that

should be critically evaluated as it may be dependent upon the nature or type of the

formulation (Gallarate et al 1999 Pinnell et al 2001) The pH of the formulation

should be on the acidic side (~ pH 35) for effective penetration of the vitamin in the skin

(Pinnell et al 2001) and for its stabilization in the formulation (Gallarate et al 1999)

Some other antioxidants such as alpha-tocopherol ferulic acid and sodium metabisulphite

have also been used in combination with ascorbic acid for the purpose of its stabilization

in topical formulations and to produce some synergistic effects (Darr et al 1996 Lin et

al 2005 Maia et al 2006 Tournas et al 2006) Effect of some rheological properties

such as viscosity and dielectric constant on the stability of ascorbic acid in emulsions has

also been investigated (Connors et al 1986) Viscosity of the medium is an important

factor that should be considered for the purpose of ascorbic acid stability as higher

viscosity formulations have shown some degree of protection (Ozer et al 2000

45

Szymula 2005) Along with other factors formulation type also plays an important role in

the stability of ascorbic acid It is reported that ascorbic acid is more stable in emulsified

system as compared to aqueous solutions (Gallarate et al 1999 Lee et al 2004) In

multiemulsions ascorbic acid is reported to be more stable as compared to simple

emulsions (Gallarate et al 1999 Ozer et al 2000 Lee et al 2004 Farahmand et al

2006)

Ascorbic acid and its derivatives have been used in a variety of cosmetic

formulations as an antioxidant pH adjuster anti-aging and photoprotectant (Elmore

2005) The control of instability of ascorbic acid poses a significant challenge in the

development of cosmetic formulations It is also reported that certain metal ions or

enzyme systems effectively convert ascorbic acidrsquos antioxidant action to pro-oxidant

activity (Elmore 2005) Therefore utilization of an effective antioxidant system is

required to maintain the stability of vitamin C in various preparations (Zhang et al 1999

Pinnell et al 2001 Maia et al 2006) The chemical stability of ascorbic acid has been

studied in emulsions and creams by several workers (Darr et al 1996 Gallarate et al

1999 Lee et al 2004 Raschke et al 2004 Elmore 2005 Farahmand et al 2006)

however there is a lack of information on the photostability of ascorbic acid in cream

formulations

336 Stability Testing of Emulsions

The stability testing of emulsions (creams) may be carried out by performing the

following tests (Billany 2002)

46

3361 Macroscopic examination

The assessment of the physical stability of an emulsion is made by an

examination of the degree of creaming or coalescence occurring over a period of time

This involves the calculation of the ratio of the volume of the creamed or separated part

of the emulsion and the total volume A comparison of these values can be made for

different products

3362 Globule size analysis

An increase in mean globule size with time (coupled with a decrease in globule

numbers) indicates that coalescence is the cause of this behavior This can be used to

compare the rates of coalescence for a variety of emulsion formulations For this purpose

microscopic examination or electronic particle counting devices (coulter counter) or

laser diffraction sizing are widely used

3363 Change in viscosity

Many factors may influence the viscosity of emulsions A change in apparent

viscosity may result from any variation in globule size or number or in the orientation or

migration of emulsifier over a period of time

3264 Accelerated stability tests

In order to compare the relative stabilities of a range of similar products it is

necessary to speed up the processes of creaming and coalescence by storage at elevated

temperatures and then carrying out the tests described in the above sections

337 FDA guidelines for semisolid preparations

According to FDA draft guidelines to the industry (Shah 1997) semisolid

preparations (eg creams) should be evaluated for appearance clarity color

47

homogencity odour pH consistency viscosity particle size distribution (when feasible)

assay degradation products preservative and antioxidant content (if present) microbial

limits sterility and weight loss when appropriate Additionally samples from

production lot or approved products are retained for stability testing in case of product

failure in the field Retained samples can be tested along with returned samples to

ascertain if the problem was manufacturing or storage related Appropriate stability data

should be provided for products supplied in closed-end tubes to support the maximum

anticipated use period during patient use and after the seal is punctured allowing product

contact with the cap cap lever Creams in large containers including tubes should be

assayed by sampling at the surface top middle and bottom of the container In addition

tubes should be sampled near the crimp The objective of stability testing is to determine

whether the product has adequate shelf-life under market and use conditions

48

OBJECT OF PRESENT INVESTIGATION

Ascorbic acid (vitamin C) is extensively used as a single ingredient or in

combination with vitamin B complex and other vitamins in the form of drops injectables

lotions and syrups It is an ingredient of anti-aging cosmetic products alone or along with

alpha-tocopherol (vitamin E) Ascorbic acid exerts several functions on the skin as

collagen synthesis depigmentation and antioxidant activity It protects the signs of

degenerative skin conditions caused by oxy-radical damage In solutions and creams

ascorbic acid is susceptible to air and light and undergoes oxidative degradation to

dehydroascorbic acid and inactive products The degradation is influenced by

temperature viscosity and polarity of the medium and is catalysed by metal ions

particularly Cu+2

Fe+2

and Zn+2

One of the major problems faced in cream preparations is the instability of

ascorbic acid as it may be exposed to light during formulation manufacturing and

storage and the possibility of photochemical degradation can not be neglected The

behaviour of ascorbic acid in light is of particular interest since no systematic kinetic

studies have been conducted on its photodegradation in these preparations under various

conditions The study of the formulation variables such as emulsifier humectants and pH

may throw light on the photostabilization of ascorbic acid in creams

The main object of this investigation is to study the behaviour of ascorbic acid in

cream preparations on exposure to UV light in the pharmaceutically useful pH range An

important aspect of the work is to study the interaction of ascorbic acid with other

vitamins such as riboflavin nicotinamide and alpha-tocopherol and the effect of certain

stabilizers such as citric acid tartaric acid and boric acid on its photodegradation In

49

addition it is intended to study the photolysis of ascorbic acid in organic solvents to

evaluate the effect of solvent characteristics (eg dielectric constant and viscosity) on the

stability of the vitamin The study of all these aspects may provide useful information to

improve the photostability and efficacy of ascorbic acid in cream preparations

An outline of the proposed plan of work is presented as follows

1 To prepare a number of oil-in-water cream formulations based on different

emulsifying agents and humectants containing ascorbic acid alone and in

combination with other vitamins and stabilizing agents

2 To perform photodegradation studies on ascorbic acid in creams using a UV

irradiation source with emission corresponding to the absorption maximum of

ascorbic acid

3 To identify the photoproducts of ascorbic acid in creams using chromatographic

and spectrophotometric methods

4 To apply appropriate and validated analytical methods for the assay of ascorbic

acid alone and in combination with other vitamins and stabilizing agents

5 To study the effect of solvent characteristics such as dielectric constant and

viscosity on the photolysis of ascorbic acid in aqueous and organic solvents

6 To evaluate the kinetics of photodegradation of ascorbic acid and its interactions

with other vitamins (riboflavin nicotinamide and alpha-tocopherol) in creams

7 To evaluate the effect of carbon chain length of the emulsifying agent and the

viscosity of the humectant on the photodegradation of ascorbic acid

50

8 To develop relationships between the rate of photodegradation of ascorbic acid

and the concentration pH carbon chain length of emulsifier viscosity of the

creams

9 To determine the effect of compounds such as citric acid tartaric acid and boric

acid used as stabilizing agents on the rate of photodegradation and stabilization

of ascorbic acid in creams

10 To present reaction schemes for the photodegradation of ascorbic acid and its

interactions with other vitamins

CHAPTER IV

MATERIALS

AND

METHODS

52

41 MATERIALS

Vitamins and Related Compounds

L-Ascorbic Acid vitamin C (5R)-5-[(1S)-12-dihydroxyethyl]-34-dihydroxyfuran-2(5H)-

one Merck

C6H8O6 Mr 1761

Dehydroascorbic Acid L-threo-23-hexodiulosonic acid γ-lactone Sigma

C6H6O6 Mr 1741

23-Diketogulonic Acid

C6H8O7 Mr 192

It was prepared according to the method of Homann and Gaffron (1964) by the

hydrolysis of dehydroascorbic acid

Riboflavin vitamin B2 (310-dihydro-78-dimethyl-10-[(2S3S4R)-2345-

tetrahydroxypentyl] benzopteridine-24-dione) Merck

C17H20N4O6 Mr 3764

Nicotinamide vitamin B3 (pyridine-3-carboxamide) Merck

C6H6N2O Mr 1221

Alpha-Tocopherol vitamin E ((2R)-2578-tetramethyl-2-[(4R8R)-4812-

trimethyltridecyl]-34-dihydro-2H-1-benzopyran-6-ol) Merck

C29H50O2 Mr 4307

Formylmethylflavin (78-dimethyl-10-formylmethylisoalloxazine)

C14H12N4O3 Mr 2843

53

Formylmethylflavin was synthesized according to the method of Fall and Petering

(1956) by the periodic acid oxidation of riboflavin It was recrystallized from absolute

methanol dried in vacuo and stored in the dark in a refrigerator

Lumichrome (78-dimethylalloxazine) Sigma

C12H10N4O2 Mr 2423

It was stored in the dark in a desiccator

Stabilizers

Boric Acid orthoboric acid Merck

H3BO3 Mr 618

Citric Acid 2-hydroxypropane-123-tricarboxylic acid Merck

C6H8O7H2O Mr 2101

L-Tartaric acid [(2R3R)-23-dihydroxybutanedioic acid] Merck

C4H6O6 Mr 1501

Emulsifying Agents

Stearic Acid (95) octadecanoic acid Merck

C18H36O2 Mr 2845

Palmitic Acid hexadecanoic acid Merck

C16H32O2 Mr 2564

Myristic Acid tetradecanoic acid Merck

C14H28O2 Mr 2284

Cetyl alcohol hexadecan-1-ol Merck

C16H34O Mr 2424

54

Humectants

Glycerin glycerol (propane-123-triol) Merck

C3H8O3 Mr 921

Propylene glycol (RS)-propane-12-diol Merck

C3H8O2 Mr 7610

Ethylene glycol ethane-12-diol Merck

C2H6O2 Mr 6207

Potassium Ferrioxalate Actinometry

Potassium Ferrioxalate

K3Fe(C2O4)3 3H2O Mr 4912

Potassium Ferrioxalate was prepared according to the method of Hatchard and

Parker (1956) Three volumes of 15 M potassium oxalate was mixed with one volume of

15 M ferric chloride with vigorous stirring The yellow green precipitate of potassium

ferrioxalate was recrystallized twice from water dried at 45 ordmC and stored in the dark in a

desiccator

Reagents

All the reagents and solvents used were of analytical grade obtained from BDH

Merck

Water

Freshly boiled distilled water was used throughout the work

55

42 METHODS

421 Cream Formulations

On the basis of the various cream formulations reported in the literature (Block

1996 Flynn 2002 Betageri and Prabhu 2002 Vimaladevi 2005 EIRI Board Lu and

Flynn 2009) the following basic formula was used for the preparation of oil-in-water

creams containing ascorbic acid

Oil phase Percentage (ww)

Emulsifier

Myristic palmitic stearic acid

Cetyl alcohol

120

30

Aqueous phase

Humectant

Ethylene glycol propylene glycol glycerin

50

Active ingredient

Ascorbic acid

20 (0114 M)

Neutralizer

Potassium hydroxide

10

Continuous phase

Distilled water

QS

Additional ingredientsa

Vitamins

Riboflavin (Vitamin B2)

Nicotinamide (Vitamin B3)

Alpha-Tocopherol (Vitamin E)

0002ndash001 (053ndash266times10ndash4

M)

028ndash140 (0023ndash0115 M)

017ndash086 (0395ndash200times10ndash2

M)

Stabilizers

Citric acid

Tartaric acid

Boric acid

010ndash040 (0476ndash190times10ndash2

M)

010ndash040 (067ndash266times10ndash2

M)

010ndash040 (0016ndash0065 M)

a The vitamin stabilizer concentrations used were found to be effective in promotion

inhibition of photodegradation of ascorbic acid in cream formulations

56

422 Preparation of Creams

The emulsifiers were melted at 70ndash80 ordmC in a glass jar immersed in a water bath

Ascorbic acid was separately dissolved in a small portion of distilled water Potassium

hydroxide and humectant were dissolved in the remaining portion of water and mixed

with the oily phase with constant stirring until the formation of a thick white mass It was

cooled to ~40 ordmC and the ascorbic acid solution was added The thick mass was mixed

using a mechanical mixer with a glass stirrer at 1000 rpm for 5 minutes The pH of the

cream was adjusted to the desired value and the contents again mixed for 10 minutes at

500 rpm All the creams were prepared under uniform conditions to maintain their

individual physical characteristics and stored at room temperature in airtight glass

containers protected from light

In the case of other vitamins nicotinamide was dissolved along with ascorbic acid

in water and added to the cream Riboflavin or alpha-tocopherol were directly added to

the cream and mixed thoroughly according to the procedure described above

In the case of stabilizing agents (citric tartaric and boric acids) the individual

compounds were dissolved in the ascorbic acid solution and added to the cream followed

by the procedure described above

The details of the various cream formulations used in this study are given in

chapters 5ndash7

57

423 Thin-Layer Chromatography (TLC)

The following TLC systems were used for the separation and identification of

ascorbic acid and photodegradation products

Adsorbent a) Silica gel GF 254 (250-microm) precoated plates

(Merck)

Solvent systems S1 acetic acid-acetone-methanol-benzene

(552070 vv) (Ganshirt and Malzacher 1960)

S2 ethanol-10 acetic acid-water (9010 vv)

(Bolliger and Konig 1969)

S3 acetonitrile-butylnitrile-water (66332 vv)

(Saari et al 1967)

Temperature 25ndash27 ordmC

Location of spots Ascorbic acid UV light 254 nm (Uvitec lamp

UK)

Dehydroascorbic acid Spray with a 3 aqueous

phenylhydrazine hydrochloride solution

424 pH Measurements

The measurements of pH of aqueous solutions and cream formulations were

carried out using an Elmetron LCD display pH meter (modelndashCP501 sensitivity plusmn 001

pH units) (Poland) with a combination electrode The electrode was calibrated

automatically in the desired pH range (25 ordmC) using the following buffer solutions

58

Phthalate pH 4008

Phosphate pH 6865

Disodium tetraborate pH 9180

The electrode was immersed directly into the cream (British Pharmacopoeia

2009) kept for few seconds to equilibrate and the pH adjusted in the range of 40ndash70

with phosphoric acid sodium hydroxide solution

425 Ultraviolet and Visible Spectrometry

The absorbance measurements and spectral determinations were performed on

Shimadzu UVndashVisible recording spectrophotometer (model UVndash1601) using matched

silica cells of 10 mm path length The cells were employed always in the same orientation

using appropriate control solutions in the reference beam The baseline was automatically

corrected by the built-in baseline memory at the initializing period Auto-zero adjustment

was made by a one-touch operation The instrument checked the wavelength calibration

(6561 nm) using the deuterium lamp at the initializing period The absorbance scale was

periodically checked using the following calibration standard (British Pharmacopoeia

2009)

0057ndash0063 gl of potassium dichromate in 0005 M sulphuric acid

The specific absorbance [A(1 1 cm)] of the solution should match the

following values with the stated limit of tolerance

Wavelength

(nm)

Specific absorbance

A (1 1 cm)

Maximum

tolerance

235 1245 1229ndash1262

257 1445 1428ndash1462

313 486 470ndash503

350 1073 1056ndash109

430 159 157ndash161

59

426 Photolysis of Ascorbic Acid

4261 Creams

A 2 g quantity of the cream was uniformly spread on several rectangular glass

plates (5 times 15 cm) covered with a 1 cm tape on each side to give a 1 mm thick layer The

plates were irradiated in a dark chamber using a Philips 30 watt TUV tube (100

emission at 254 nm the wavelength absorbed by ascorbic acid at pH 4ndash7) fixed

horizontally at a distance of 30 cm from the centre of the plates Each plate was removed

at appropriate interval and the cream was subjected to spectrophotometric assay and

chromatographic examination

4262 Aqueous and organic solvents

A 10ndash3

M solution of ascorbic acid (50 ml) prepared in water (pH 70 005 M

phosphate buffer) or in an organic solvent in a 100 ml beaker (Pyrex) was placed in a

water bath maintained at 20 plusmn 1 ordmC The solution was irradiated with the Philips 30 watt

TUV tube in a dark chamber as stated above Samples were withdrawn at appropriate

intervals for assay and chromatography

4263 Storage of creams in dark

In order to determine the stability of various cream formulations in the dark

samples were stored at room temperature in a cupboard protected from light for a period

of three months The samples were analyzed periodically for the content of ascorbic acid

and the presence of any degradation product

427 Measurement of Light Intensity

The potassium ferrioxalate actinometry was used for the measurement of light

intensity of the radiation source employed in this work This actinometer has been

60

developed by Parker (1953) and Hatchard and Parker (1956) and is considered as the

most useful actinometer over a wide range of wavelengths (254ndash577 nm) It has been

used by Holmstrom and Oster (1961) Byrom and Turnbull (1967) McBride and Moore

(1967) Ahmad (1968) Ahmad (1978) Ahmad et al (2004a 2004b 2005 2006a

2006b 2008 2009ab) Fasihullah (1988) Vaid (1998) Ansari (2002) and Ahmad (2009)

for the measurement of light intensity

The irradiation of potassium ferrioxalate solutions in sulphuric acid results in the

reduction of ferric ion to ferrous ion according to the following reaction

2Fe [(C2O4)3]3ndash

rarr 2 Fe (C2O4) + 3 (C2O4)2ndash

+ 2CO2 (31)

The amount of Fe2+

ions formed in the reaction may be determined by

complexation with 110-phenanthroline to give a red colored complex The absorbance of

the complex is measured at 510 nm

428 Procedure

An oxygen free 0006 M solution of potassium ferrioxalate (2947 gl) in 01 N

H2SO4 was placed in the reaction vessel and irradiated with the lamp used for the

photolysis of riboflavin The irradiation was carried out under nitrogen (90ndash120

bubblesminute) which also caused stirring of the solution The temperature of the

reaction vessel was maintained at 25 plusmn 1 ordmC during the reaction

An aliquot of the photolysed solution (1ndash2 ml) was pipetted out at suitable

intervals (up to 30 minutes) into a 10 ml volumetric flask to which was then added 09

ml of N H2SO4 + 1 ml (01) 110-phenanthroline + 05 ml buffer (60 ml N CH3COONa

+ 36 ml N H2SO4 made up to 100 ml with distilled water) The flask was made up

to the mark with distilled water (final pH 35) thoroughly shaken to mix the contents and

61

Fig 3 Spectral power distribution of TUV 30 W tube (Philips)

62

allowed to stand for one hour in the dark to develop the colorndashcomplex The absorbance

of the phenanthrolinendashferrous complex was measured in a 1 cm cell at 510 nm using the

appropriate solution as blank The amount of Fe2+

ions formed was determined from the

calibration graph The calibration graph was constructed in a similar manner using

several dilutions of 1 times 10ndash6

mole ml Fe2+

in 01 N H2SO4 (freshly prepared by dilution

from standardized 01 M FeSO4 in 01 N H2SO4) (Fig 8) The experimental value of the

molar absorptivity of Fe2+

complex as determined from the slope of the calibration graph

is equal to 111 times 104 M

ndash1 cm

ndash1 and is in agreement with the value reported by Parker

(1953) Using the values of the known quantum yield for ferrioxalate actinometer at

different wavelengths (Hatchard and Parker 1956) the number of Fe2+

ions formed

during photolysis the time of exposure and the fraction of the light absorbed by the

length of the actinometer solution employed the light intensity incident just inside the

front window of the photolysis cell can be calculated In the present case total absorption

of the light has been assumed

4281 Calculation

The number of Fe2+

ions formed during photolysis (nFe

2+) is given by the

equation

6023 times 1020

V1 V3 A Σ

n Fe

2+ =

V2 1 ε (32)

where V1 is the volume of the actinometer solution irradiated (ml)

V2 is the volume of the aliquot taken for analysis (ml)

V3 is the final volume to which the aliquot V2 is diluted (ml)

1 is the path length of the spectrophotometer cell used (1 cm)

A is the measured absorbance of the final solution at 510 nm

63

ε is the molar absorptivity of the Fe2+

complex (111 times 104 M

ndash1 cm

ndash1)

The number of quanta absorbed by the actinometer nabs can then be obtained as follows

n Fe

2+

Σ nabs = ф

(33)

where ф is the quantum yield for the Fe2+

formation at the desired wavelength

The number of quanta per second per cell nabs is therefore given by

Σ nabs 6023 times 1020

V1 V3 A nabs =

t =

ф V2 1 ε t (34)

where t is the irradiation time of the actinometer in seconds

The relative spectral energy distribution of the radiation source (Fig 3) shows

100 emission at 254 nm the wavelength used for the photolysis of ascorbic acid (λmax

265 nm at pH 4ndash7) The energy emitted by the radiation source at various wavelengths

can be calculated using the equation

1197 times 105

E (KJ molndash1

) = λ nm

(35)

The quantum efficiency of ferrioxalate actinometer at the wavelength absorbed by

ascorbic acid (ie 254 nm) is high although the sensitivity drops over 450 nm The

average intensity of the TUV tube used in this study was determined as 556 plusmn 012 times

1018

quanta sndash1

429 Viscosity Measurements

The viscosity of the cream formulations was measured with a Brookfield RV

viscometer (Model DV-II + Pro USA) The instrument was calibrated using the

manufacturerrsquos viscosity standard A 200 g quantity of the cream was placed in a beaker

and the spindle (TE) was dipped into the cream It was rotated at a speed of 06 rpm for

64

00

02

04

06

08

10

12

0 2 4 6 8 10 12

Concentration of Fe++

times 105 M

Ab

sorb

an

ce a

t 51

0 n

m

Fig 4 Calibration graph for the determination of K3Fe(C2O4)3

65

one minute and the viscosity was recorded at 25plusmn1 ordmC The test was repeated three times

to account for the experimental variability and the average viscosity was noted

4210 Assay Methods

42101 UV spectrophotometric method for the assay of creams containing ascorbic

acid alone

The creams were thoroughly mixed a quantity of 2 g was accurately weighed and

the assay of ascorbic acid was carried out by the UV method of Zeng et al (2005) In the

case of photodegraded creams (2 g) the material was completely removed from the glass

plate and transferred to a volumetric flask The method involved extraction of ascorbic

acid with methanol (3 times 10 ml) adjustment of the pH of combined methanolic solutions

to 20 (with H3PO4) dilution of the final solution with acidified methanol (pH 20) to 100

ml and measurement of the absorbance at 245 nm using appropriate blank The

concentration of ascorbic acid was calculated using 560 as the value of specific

absorbance [A (1 1 cm)] at the analytical wavelength (Table 5)

The same method was used for the assay of ascorbic acid in creams stored in the

dark and in the presence of individual stabilizing agents (citric tartaric and boric acids)

42102 Iodimetric method for the assay of ascorbic acid in creams containing

riboflavin nicotinamide and alpha-tocopherol

The assay of ascorbic acid in creams in the presence of riboflavin nicotinamide

and alpha-tocopherol was carried out according to the procedure of British

Pharmacopoeia (2009) as follows

The photolysed cream (2 g) was completely scrapped from the glass plate and

transferred to a flask containing 40 ml of distilled water and 10 ml of 1 M sulphuric acid

66

Table 5 Calibration data for ascorbic acid showing linear regression analysisa

λ max 245 nm

Concentration range 01ndash10 times 10ndash4

M (0176ndash1761 mg )

Slope 9920

SE (plusmn) of slope 00114

Intercept 00012

Correlation coefficient 09996

Molar absorptivity (ε) 9920 Mndash1

cmndash1

Specific absorbance [A (1 1 cm)] 560

a Values represent a mean of five determinations

67

was added The solution was titrated with 002 M iodine solution using 1 ml of starch

solution as indicator until a persistent violet-blue color was obtained Each ml of 002 M

iodine solution is equivalent to 352 mg of C6H8O6 The same method was used for the

assay of ascorbic acid in creams stored in the dark

42103 Spectrophotometric method for the assay of ascorbic acid in aqueous and

organic solvents

A 1 ml aliquot of the photolysed solutions of ascorbic acid in water or in an

organic solvent was evaporated to dryness under nitrogen at room temperature and the

residue redissolved in a small volume of methanol The solution was transferred to a 10

ml volumetric flask made up to volume with acidified methanol (pH 20) and the

absorbance measured at 245 nm using an appropriate blank The content of ascorbic acid

in the solutions was determined using 9920 Mndash1

cmndash1

as the value of molar absorptivity at

the analytical wavelength (Table 5)

CHAPTER V

PHOTODEGRADATION OF

ASCORBIC ACID IN

ORGANIC SOLVENTS AND

CREAM FORMULATIONS

69

51 INTRODUCTION

Ascorbic acid (vitamin C) is an essential micronutrient that performs important

metabolic functions (Packer and Fuchs 1999 Davey et al 2000 Johnston et al 2007)

It is an ingredient of anti-aging cosmetic products (Darr et al 1996 Gallarate et al

1999 Traikovich 1999 Zhang et al 1999 Ozer et al 2000 Nusgens et al 2001

Pinnell et al 2001 2003 Lee et al 2004 Raschke et al 2004 Sauermann et al 2004

Elmore 2005 Jentzsch et al 2005 Lin et al 2005 Placzek et al 2005 Carlotti et al

2006 Farahmand et al 2006 Heber et al 2006 Maia et al 2006 Tournas et al 2006)

and exerts several functions on the skin as collagen synthesis depigmentation and

antioxidant activity (Nusgens et al 2001 Spiclin et al 2003) As an antioxidant it

protects skin by neutralizing reactive oxygen species generated on exposure to sunlight

(Shindo et al 1994) In biological systems it reduces both oxygenndash and nitrogenndash based

free radicals (Higdon and Frei 2002) and thus delays the aging process In view of the

instability of ascorbic acid in skin care formulations (Bissett 2006) it is often used in

combination with another redox partner such as alpha-tocopherol (vitamin E) to retard its

oxidation (Wille 2005)

The details of the cream formulations used in this study are given in Table 6 The

results obtained on the photodegradation of ascorbic acid in aqueous organic solvents

and cream formulations are discussed in the following sections

70

Table 6 Composition of cream formulations containing ascorbic acid

Ingredients Cream

No pH

SA PA MA CA AH2 GL PG EG PH DW

1 a 4 + minus minus + + + minus minus + +

b 5 + minus minus + + + minus minus + +

c 6 + minus minus + + + minus minus + +

d 7 + minus minus + + + minus minus + +

2 a 4 minus + minus + + + minus minus + +

b 5 minus + minus + + + minus minus + +

c 6 minus + minus + + + minus minus + +

d 7 minus + minus + + + minus minus + +

3 a 4 minus minus + + + + minus minus + +

b 5 minus minus + + + + minus minus + +

c 6 minus minus + + + + minus minus + +

d 7 minus minus + + + + minus minus + +

4 a 4 + minus minus + + minus + minus + +

b 5 + minus minus + + minus + minus + +

c 6 + minus minus + + minus + minus + +

d 7 + minus minus + + minus + minus + +

5 a 4 minus + minus + + minus + minus + +

b 5 minus + minus + + minus + minus + +

c 6 minus + minus + + minus + minus + +

d 7 minus + minus + + minus + minus + +

6 a 4 minus minus + + + minus + minus + +

b 5 minus minus + + + minus + minus + +

c 6 minus minus + + + minus + minus + +

d 7 minus minus + + + minus + minus + +

7 a 4 + minus minus + + minus minus + + +

b 5 + minus minus + + minus minus + + +

c 6 + minus minus + + minus minus + + +

d 7 + minus minus + + minus minus + + +

8 a 4 minus + minus + + minus minus + + +

b 5 minus + minus + + minus minus + + +

c 6 minus + minus + + minus minus + + +

d 7 minus + minus + + minus minus + + +

9 a 4 minus minus + + + minus minus + + +

b 5 minus minus + + + minus minus + + +

c 6 minus minus + + + minus minus + + +

d 7 minus minus + + + minus minus + + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PG = propylene glycol EG = ethylene glycol

PH = potassium hydroxide DW = distilled water

71

52 PHOTOPRODUCTS OF ASCORBIC ACID

The photolysis of ascorbic acid (AH2) in aqueous and organic solvents and in

cream formulations on UV irradiation leads to the formation of dehydroascorbic acid

(DHA) as detected by TLC along with the undegraded AH2 using the solvent systems A

B and C The identification of DHA was carried out by comparison of the Rf value and

spot color with those of the authentic compound The formation of DHA on

photooxidation of ascorbic acid solutions has previously been reported (Homan and

Gaffron 1964 Sattar et al 1977 Heelis et al 1981 Rozanowska et al 1997 Lavoie et

al 2004) DGA the hydrolysis product of DHA (Homan and Gaffron 1964) could not

be detected under the present experimental conditions

53 SPECTRAL CHARACTERISTICS OF PHOTOLYSED SOLUTIONS

A typical set of the UV absorption spectra of photolysed solutions of AH2 in

methanol is shown in Fig 5 There is a gradual loss of absorbance around 245 nm with

time as a result of the oxidation of the molecule to DHA (Pelletier 1985 Davies et al

1991 Rumsey and Levine 2000) which does not absorb in this region due to the loss of

conjugation Similar absorption changes are observed on the photolysis of AH2 in other

organic solvents and in the methanolic extracts of cream formulations However the

magnitude of these changes varies with the rate of photolysis in a particular solvent or

cream and appears to be a function of the polar character pH and viscosity of the

medium

72

Fig 5 UV absorption spectra of photolysed solutions of ascorbic acid in methanol at

0 40 80 120 160 220 and 300 min

73

54 ASSAY OF ASCORBIC ACID IN CREAMS AND SOLUTIONS

The assay of AH2 in creams and solutions has been carried out in acidified

methanol (pH 20) according to the UV spectrophotometric method of Zeng et al (2005)

Aqueous solutions of AH2 (~pH 2) exhibit absorption maxima at 243 nm (OrsquoNeil 2001

Moffat et al 2004 Sweetman 2009) 244 nm (Ogata and Kosugi 1969) and 245 nm

(Verma et al 1991 Johnston et al 2007) The absorption maxima of AH2 in methanol

and phosphate buffer (pH 25) occur at 245 nm (Zeng et al 2005) Since dilute solutions

of AH2 are highly susceptible to oxidation the pH was adjusted to 20 with phosphoric

acid to convert the molecule to the non-ionized form (99) to minimize degradation

during the assay AH2 in acidified methanol (pH 20) was found to exhibit the absorption

maximum at 245 nm as reported by Zeng et al (2005) The method was also used for the

assay of AH2 in aqueous and organic solvents

The validity of Beerrsquos law relation in the concentration range used was confirmed

prior to the assay The calibration data for AH2 at the analytical wavelength are presented

in Table 5 (Chapter 4) The correlation coefficient (r = 09996) indicates a good linear

relationship over the concentration range employed The values of specific absorbance

and molar absorptivity at 245 nm determined from the slope of the curve are in good

agreement with those reported by previous workers (Davies et al 1991 Johnston et al

2007) The method of Zeng et al (2005) has been found to be satisfactory for the assay of

AH2 in cream formulations and solutions and has been used to study the kinetics of

photolysis reactions The method was validated before its application to the assay of AH2

in photolysed creams The reproducibility of the method was confirmed by the analysis of

known amounts of AH2 in the concentration range likely to be found in photodegraded

74

creams The values of the recoveries of AH2 in creams by the UV spectrophotometric

method are in the range of 90ndash96 The values of RSD for the assays indicate the

precision of the method within plusmn5 (Table 7)

In order to compare the UV spectrophotometric method with the British

Pharmacopoeia iodimetric method (2009) using a dilute iodine solution (002 M) the

creams were simultaneously assayed for AH2 content by the two methods and the results

are reported in Table 8 The statistical evaluation of the accuracy and precision of the two

methods was carried out by the application of the F test and the t test respectively The F

test showed that there is no significant difference between the precision of the two

methods and the calculated value of F is lower than the critical value (F = 639 P = 005)

in each case The t test indicated that the calculated t values are lower than the tabulated t

values (t = 2776 P = 005) suggesting that at 95 confidence level the differences

between the results of the two methods are statistically non-significant Thus the accuracy

and precision of the UV spectrophotometric method is comparable to that of the official

iodimetric method for the assay of AH2 in cream formulations The results of the assays

of AH2 in aqueous organic solvents and cream formulations are reported in Table 9

55 EFFECT OF SOLVENT

The influence of solvent on the rate of degradation of drugs is of considerable

importance to the formulator since the stability of drug species in solution media may be

predicted on the basis of their chemical reactivity The reactivity of drugs in a particular

medium depends to a large extent on solvent characteristics such as the dielectric

constant and viscosity (Connors et al 1986 Yoshioka and Stella 2000 Sinko 2006)

75

Table 7 Recovery of ascorbic acid added to cream formulationsa

Cream

Formulationb

Added

(mg)

Found

(mg)

Recovery

()

RSD

()

1a 400

200

380

183

950

915

21

25

2b 400

200

371

185

928

925

15

25

3c 400

200

375

181

938

905

11

31

4d 400

200

384

189

960

945

13

21

5b 400

200

370

189

925

945

14

26

6c 400

200

369

190

923

950

10

22

7d 400

200

374

182

935

910

17

39

8c 400

200

380

188

950

940

15

33

9d 400

200

367

189

918

945

20

42

a Values expressed as a mean of three to five determinations

b The cream formulations represent combinations of each emulsifier (stearic acid

palmitic acid myristic acid) with each humectant (glycerin propylene glycol ethylene

glycol) to observe the efficiency of methanol to extract AH2 from different creams

(Table 6)

76

Table 8 Assay of ascorbic acid in creams using UV spectrophotometric and iodimetric

methods

Ascorbic acid (mg) Cream

Formulationb Added UV method

a

Iodimetric

methoda

Fcalc tcalc

1a 40

20

380 plusmn 081

183 plusmn 046

375 plusmn 095

185 plusmn 071

138

238

245

104

2b 40

20

371 plusmn 056

185 plusmn 047

373 plusmn 064

193 plusmn 038

130

065

181

200

3c 40

20

375 plusmn 040

181 plusmn 056

374 plusmn 046

183 plusmn 071

132

160

101

223

4d 40

20

384 plusmn 051

189 plusmn 039

381 plusmn 066

190 plusmn 052

167

178

176

231

5b 40

20

370 plusmn 052

189 plusmn 050

372 plusmn 042

185 plusmn 067

065

179

162

125

6c 40

20

369 plusmn 037

190 plusmn 042

371 plusmn 058

188 plusmn 056

245

177

122

197

7d 40

20

374 plusmn 062

182 plusmn 072

370 plusmn 070

184 plusmn 082

127

129

144

168

8c 40

20

380 plusmn 058

188 plusmn 062

375 plusmn 075

192 plusmn 060

167

094

123

162

9d 40

20

367 plusmn 072

189 plusmn 080

365 plusmn 082

187 plusmn 075

149

092

130

203

Theoretical values (P = 005) for F is 639 and for t is 2776

a Mean plusmn SD (n = 5)

b Table 6

77

Table 9 Photodegradation of ascorbic acid in cream formulations

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) pHa 40 50 60 70

0 383 382 384 383

60 374 369 366 361

120 361 354 346 325

180 351 345 325 305

240 345 327 301 284

1

300 336 316 287 264

0 380 383 382 379

60 371 376 362 346

120 359 357 342 320

180 352 345 322 301

240 341 335 299 283

2

300 336 321 291 261

0 384 376 381 385

60 377 367 360 358

120 366 348 334 324

180 356 337 317 305

240 343 320 301 282

3

300 335 307 273 253

78

Table 9 continued

0 377 378 386 372

60 365 361 371 355

120 353 345 347 322

180 344 327 325 298

240 332 320 306 279

4

300 317 303 284 252

0 381 367 372 373

60 372 358 358 353

120 360 337 336 321

180 352 325 320 302

240 341 313 300 284

5

300 327 302 278 256

0 376 386 380 377

60 366 372 350 350

120 353 347 323 316

180 337 334 308 298

240 329 320 291 274

6

300 313 306 267 245

79

Table 9 continued

0 380 372 378 380

60 373 362 350 354

120 358 340 329 321

180 344 328 304 300

240 332 315 292 283

7

300 319 302 272 252

0 380 381 378 361

60 368 364 361 335

120 355 354 340 313

180 342 340 315 281

240 337 331 303 269

8

300 323 314 281 248

0 378 382 370 375

60 370 369 349 342

120 356 347 326 321

180 339 333 298 291

240 326 314 277 271

9

300 313 302 265 242

a The values at pH 40ndash70 represent the formulations a to d of each cream respectively

80

In order to observe the effect of solvent dielectric constant the apparent first-

order rate constants (kobs) for the photolysis of AH2 in alcoholic solvents (Table 10) were

plotted against the dielectric constants of the solvents A linear relationship indicated the

dependence of the rates of photolysis on solvent dielectric constant (Fig 6) This implies

the involvement of a polar intermediate in the reaction to facilitate the formation of the

degradation products as suggested by Ahmad and Tollin (1981) in the case of flavin

electron transfer reactions The effect of solvent polarity has been observed on the

autooxidation of AH2 in organic solvents (Ogata and Kosugi 1969)

Another solvent parameter affecting the rate of a chemical reaction is viscosity

which can greatly influence the stability of oxidisable substances (Wallwork and Grant

1977 Laidler 1987 Fung 1990) A plot of kobs for the photolysis of AH2 against the

reciprocal of solvent viscosity (Table 10) is linear showing that an increase in solvent

viscosity results in a decrease in the rate of photolysis (Fig 7) The viscosity of the liquid

affects the rate at which molecules can diffuse through the solution This in turn may

affect the rate at which a compound can suffer oxidation at the liquid surface This

applies to AH2 and an increase in the viscosity of the medium makes access to air at the

surface more difficult to prevent oxidation (Wallwork and Grant 1977)

56 EFFECT OF CONCENTRATION

In order to observe the effect of concentration (Table 11) on the photostability of

AH2 in a cream using stearic palmitic and myristic acids as emulsifying agents and

glycerin as humectant plots of log concentration versus time were constructed (Fig 8)

and the apparent first-order rate constants were determined (Table 12) A graph of kobs

against concentration of AH2 (Fig 9) exhibited an apparent linear relationship between

81

Table 10 Apparent first-order rate constants (kobs) for the photolysis of ascorbic acid in

water and organic solvents

Solvent Dielectric

Constant (25 ordmC)

Viscosity

(mPas) ndash1

kobs times104

(minndash1

)

Water 785 1000 404

Methanol 326 1838 324

Ethanol 243 0931 316

1-Propanol 201 0514 302

1-Butanol 178 0393 295

82

00

20

40

60

80

0 10 20 30 40 50 60 70 80

Dielectric constant

k (

min

ndash1)

Fig 6 A plot of kobs for photolysis of ascorbic acid against solvent dielectric constant

(times) Water () methanol () ethanol (diams) 1-propanol () 1-butanol

83

00

10

20

30

40

50

00 05 10 15 20

Viscosity (mPas)ndash1

k times

10

4 (m

inndash1)

Fig 7 A plot of kobs for photolysis of ascorbic acid against reciprocal of solvent

viscosity Symbols are as in Fig 6

84

Table 11 Effect of concentration on the photodegradation of ascorbic acid in cream

formulations at pH 60

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) 05 10 15 20 25

0 95 191 290 379 471

60 90 182 277 358 453

120 82 167 260 339 431

180 77 158 239 311 401

240 70 144 225 298 382

1

300 64 134 210 282 363

0 92 186 287 380 472

60 88 175 272 369 453

120 82 160 251 342 429

180 75 152 238 326 405

240 71 144 225 309 392

2

300 65 134 215 289 366

0 94 182 286 376 470

60 87 171 265 352 454

120 78 152 251 337 426

180 69 143 227 315 404

240 62 129 215 290 378

3

300 58 119 195 271 353

85

05

10

15

20

25

06

08

10

12

14

16

18

log

co

nce

ntr

ati

on

(m

g)

a

05

10

15

20

25

06

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

b

05

10

15

20

25

06

08

10

12

14

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

c

Fig 8 Log concentration versus time plots for the photodegradation of ascorbic acid at

various concentrations in creams at pH 60 a) stearic acid b) palmitic acid

c) myristic acid

86

Table 12 Apparent first-order rate constants (kobs) for the photodegradation of various

ascorbic acid concentrations in cream formulations at pH 60

kobs times 103 (min

ndash1)a Cream

Formulationb 05 10 15 20 25

1 133

(0994)

120

(0993)

111

(0995)

101

(0994)

090

(0994)

2 118

(0992)

108

(0994)

098

(0993)

093

(0992)

084

(0994)

3 169

(0994)

144

(0995)

126

(0994)

109

(0993)

097

(0992)

a The values in parenthesis are correlation coefficients

b Table 6

87

Stearic acid

Palmitic acid

Myristic acid

00

05

10

15

20

25

00 05 10 15 20 25

Ascorbic acid concentration ()

kob

s (min

ndash1)

Fig 9 A plot of kobs for photodegradation against ascorbic acid concentrations in cream

formulations

88

the two values Thus the rate of degradation of AH2 is faster at a lower concentration on

exposure to the same intensity of light This may be due to a relatively greater number of

photons available for excitation of the molecule at lower concentration compared to that

at a higher concentration The AH2 concentrations of creams used in this study are within

the range (1ndash15) reported by previous workers for topical applications to skin (Kaplan

et al 1989 Traikovich et al 1999 Nusgens et al 2001 Matsubayashi et al 2003

Espinal-Perez et al 2004 Sauermann et al 2004 Lin et al 2005 Heber et al 2006)

57 EFFECT OF CARBON CHAIN LENGTH OF EMULSIFYING AGENT

The values of kobs for the photodegradation of AH2 (2) in various cream

formulations are reported in Table 13 The first-order plots for the photodegradation of

AH2 at pH 4ndash7 in various cream formulations are shown in Fig 10ndash12 The plots of kobs

against carbon chain length of the emulsifying agents are shown in Fig 13 They indicate

that the photodegradation of AH2 is affected by the emulsifying agent in the order

myristic acid gt stearic acid gt palmitic acid

These acids possess a polar character (Yao et al 2009) and the carbon chain of the acid

may play a part in the photostability of AH2 However the results indicate that in the

presence of palmitic acid AH2 exhibits greater stability as indicated by the plots of kobs

versus the carbon chain length of the emulsifying agents (Fig 13) This could be

predominantly due to the interaction of AH2 with palmitic acid in the cream to impart it

greater stability Ascorbic acid-6-palmitate is known to be an antioxidant in cosmetic

preparations (Lee et al 2009) and food products (Doores 2002)

In view of the above observations it may be suggested that the photodegradation

of AH2 could involve a polar semiquinone intermediate (Johnston et al 2007) which

89

Table 13 First-order rate constants (kobs) for the photodegradation of ascorbic acid in

cream formulations

kobs times 103 (min

ndash1)abc

Cream

Formulation pH 40 50 60 70

1 044

(0992)

064

(0994)

100

(0995)

126

(0995)

2 042

(0992)

060

(0991)

095

(0992)

120

(0995)

3 047

(0993)

069

(0993)

107

(0991)

137

(0995)

4 056

(0993)

072

(0992)

104

(0994)

131

(0993)

5 050

(0991)

067

(0992)

097

(0991)

124

(0992)

6 061

(0992)

079

(0993)

113

(0992)

140

(0994)

7 060

(0992)

071

(0993)

108

(0994)

133

(0992)

8 053

(0991)

062

(0992)

099

(0994)

126

(0993)

9 065

(0991)

081

(0996)

117

(0993)

142

(0995)

a The rate constants at pH 40ndash70 represent the values for formulations a to d of each

cream respectively

b The values in parenthesis are correlation coefficients

c The values of rate constants are relative and depend on specific experimental conditions

including light intensity

The estimated error is plusmn5

90

12

13

14

15

16

17

log

co

nce

ntr

ati

on

(m

g)

1

12

13

14

15

16

17

log

co

nce

ntr

ati

on

(m

g)

2

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

3

Fig 10 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (1ndash3) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

91

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

4

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

5

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

6

Fig 11 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (4ndash6) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

92

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

7

12

13

14

15

16

17

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

8

12

13

14

15

16

17

0 60 120 180 240 300

Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

9

Fig 12 First-order plots for the photodegradation of ascorbic acid in various cream

formulations (7ndash9) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

93

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

ko

bs times

10

3 (m

inndash

1)

1-3

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

ko

bs times

10

3 (

min

ndash1)

4-6

pH 4

pH 5

pH 6

pH 7

00

05

10

15

12 14 16 18

Carbon chain length

ko

bs times

10

3 (

min

ndash1)

7-9

Fig 13 Plots of kobs for photodegradation of ascorbic acid in creams (1ndash9) against carbon

chain length of emulsifier () Stearic acid () palmitic acid () myristic acid

Humectant used glycerin (1ndash3) propylene glycol (4ndash6) ethylene glycol (7ndash9)

94

depending on the polar character of the medium undergoes oxidation with varying rates

This is similar to the behavior of the photolysis of riboflavin analogs which is dependent

on the polar character of the medium (Ahmad and Tollin 1981) The effect of carbon

chain length on the transdermal delivery of an active ingredient has been discussed (Lu

and Flynn 2009)

58 EFFECT OF VISCOSITY

The plots of rates of AH2 degradation in cream formulations (Table 13) as a

function of carbon chain length (Fig 13) indicate that the rates vary with the humectant

and hence the viscosity of the medium in the order

ethylene glycol gt propylene glycol gt glycerin

This is in agreement with the rate of photolysis of AH2 in organic solvents that

higher the viscosity of the medium lower the rate of photolysis Thus apart from the

carbon chain length of the emulsifier viscosity of the humectant added to the cream

formulation appears to play an important part in the stability of AH2 The stabilizing

effect of viscosity imparting substances on AH2 solutions has been reported (Stone 1969

Kassem et al 1969ab)

59 EFFECT OF pH

The kobsndashpH profiles for the photodegradation of AH2 in various creams (1ndash9) at

pH 4ndash7 (Fig 14) represent a sigmoid type curve indicating the oxidation of the ionized

form (AHndash) of AH2 (pKa 41) (OrsquoNeil 2001) with pH The AH

ndash species appears to be

more susceptible to photooxidation than the non-ionized form (AH2) The behavior of

AH2 on photooxidation in the pH range 4ndash7 is similar to that observed for the chemical

oxidation of AH2 by molecular oxygen (DeRitter 1982) and involves the interaction of

95

Stearic Acid (1)

Palmitic Acid (2)

Myristic Acid (3)

04

06

08

10

12

14

kob

s times

10

3 (m

inndash

1)

Stearic Acid (4)

Palmitic Acid (5)

Myristic Acid (6)

04

06

08

10

12

14

ko

bs times

10

3 (

min

ndash1)

Stearic Acid (7)

Palmitic Acid (8)

Myristic Acid (9)

04

06

08

10

12

14

30 40 50 60 70

pH

ko

bs

times 1

03

(min

ndash1)

Fig 14 The kobsndashpH profiles for the photodegradation of ascorbic acid in creams (1ndash9)

Glycerin

Propylene glycol

Ethylene glycol

96

AH2 with singlet oxygen on UV irradiation (Silva and Quina 2006) The AHndash species

(predominant in the pH range 42ndash70 557ndash999) is more reactive towards singlet

oxygen than its protonated form the AH2 molecule as suggested by Bisby et al (1999)

and therefore the rate of photooxidation is higher in the pH range above 41

corresponding to the pKa1 of AH2 The major goal of a ratendashpH profile is to determine

the optimum pH range for a particular formulation Several workers have studied the

ratendashpH profiles of the chemical oxidation of AH2 in the pH range 2ndash7 (Garrett 1967

Taqui Khan and Martell 1967 Rogers and Yacomeni 1971 Blaugh and Hajratwala

1972 DeRitter 1982 Moura et al 1994) however the kinetics of photooxidation of

AH2 in cream formulations has so far not been reported

510 EFFECT OF REDOX POTENTIAL

The photooxidation of AH2 is also influenced by its redox potential which varies

with pH The greater photostability of AH2 at pH 5ndash6 compared to that at pH 7 and above

is due to its lower rate of oxidationndashreduction in this range (Eordm pH 50 = +0127 V)

(OrsquoNeil 2001) The increase in the rate of photooxidation with pH is due to a

corresponding increase in the redox potential (Eordm pH 70 = +0058 V) (Fasman 1976) of

AH2 and is similar to the photolysis behavior of riboflavin at pH 5ndash6 (Eordm pH 50 = ndash0117

V) (Sinko 2006) compared to that at pH 70 (Eordm pH 70 = ndash 0207 V) (Ahmad et al

2004a Sinko 2006) Since the ionization as well as the redox potentials of AH2 are a

function of pH the rate of photooxidation depends upon the specific species present and

its redox behavior at a particular pH

97

511 PRIMARY PHOTOCHEMICAL REACTIONS IN THE OXIDATION OF

ASCORBIC ACID

A reaction scheme based on general photochemical principles for the important

reactions involved in the photooxidation of ascorbic acid is presented below

0AH2 hv k1

1AH2 (51)

1AH2 k2 Products (52)

1AH2 isc k3

3AH2 (53)

3AH2 k4 Products (54)

0AH

ndash hv k5

1AH

ndash (55)

1AH

ndash k6 Products (56)

1AH

ndash k7

3AH

ndash (57)

3AH

ndash k8 Products (58)

3AH

ndash +

0AH2 k9 AH٠

ndash + AH٠ (59)

2 AH٠ k10 A + AH2 (510)

3AH2 +

3O2 k11

0AH2 +

1O2 (511)

AHndash +

1O2 k12

3AH

ndash +

3O2 (512)

AH٠ + 1O2 k13 AHOO٠ (513)

AHOO٠ k14 A + HO2٠ (514)

AHOO٠ + 0AH2 k15 AH٠ + AHOOH (515)

AHOOH k16 secondary reaction

A + H2O2 (516)

According to this reaction scheme the ground state ascorbic acid species (0AH2

0AH

ndash) each is excited to the lowest singlet state (

1AH2

1AH

ndash) by the absorption of a

quantum of UV light (51 55) These excited states may directly be converted to

98

photoproducts (52 56) or may undergo intersystem crossing (isc) to form the excited

triplet states (53 57) The excited triplet states may then degrade to the photoproducts

(54 58) The monoascorbate triplet (3AH

ndash) may react with the ground state ascorbic

acid to form a monoascorbate radical anion (AH٠ndash) and a monoascorbate radical (AH٠)

(59) Two AH٠ radical species may lead to the formation of an oxidized (A) and a

reduced ascorbic acid molecule (AH2) (510) Ascorbic acid triplet (3AH2) may react with

molecular oxygen (3O2) to yield singlet oxygen (

1O2) (511) which may then react with

monoascorbate anion (AHndash) to form the excited triplet state (

3AH

ndash) (512) or with

monoascorbate radical to form a peroxyl radical (AHOO٠) (513) The peroxyl radical

may yield dehydroascorbic acid (A) (514) or react with ground state ascorbic acid to

give monoascorbate radical and a reduced species AHOOH (515) The reduced species

may give rise to dehydroascorbic acid and hydrogen peroxide (516)

512 DEGRADATION OF ASCORBIC ACID IN THE DARK

In view of the instability of AH2 and to observe its degradation in the dark the

creams were stored in airtight containers at room temperature in a cupboard for a period

of about 3 months and assayed for AH2 content at appropriate intervals The analytical

data (Table 14) were subjected to kinetic treatment (Fig 15ndash17) and the apparent first-

order rate constants for the degradation of AH2 were determined (Table 15) The values

of the rate constants indicate that the degradation of AH2 in the dark is about 70 times

slower than those of the creams exposed to UV irradiation (Table 13) The degradation of

AH2 in creams in the dark is due to chemical oxidation (Section 132) and occurs in the

order of emulsifying agents (Fig 18)

myristic acid gt stearic acid gt palmitic acid

99

Table 14 Degradation of ascorbic acid in the dark in cream formulations

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(days) pHa 40 50 60 70

0 383 382 384 383

10 354 340 313 278

20 309 306 279 245

40 244 209 183 161

60 172 166 131 105

1

80 145 114 81 61

0 380 383 382 379

10 360 343 350 335

20 322 310 301 294

40 266 250 211 186

60 233 211 168 142

2

80 182 153 114 89

0 384 376 381 385

10 368 350 340 318

20 318 273 273 266

40 223 199 172 155

60 174 132 117 84

3

80 122 97 66 54

100

Table 14 continued

0 377 378 386 372

10 350 334 334 318

20 314 268 256 244

40 238 208 182 136

60 179 155 107 94

4

80 128 101 79 59

0 381 367 372 373

10 350 293 300 320

20 299 266 270 263

40 220 191 192 184

60 183 153 139 129

5

80 149 115 87 76

0 376 386 380 377

10 312 320 314 251

20 255 282 226 199

40 175 194 159 131

60 139 128 99 74

6

80 102 81 55 41

101

Table 14 continued

0 380 372 378 380

10 323 330 333 323

20 288 273 276 224

40 212 174 182 146

60 152 133 108 83

7

80 100 82 66 56

0 380 381 378 361

10 333 320 310 310

20 281 266 260 257

40 230 189 171 177

60 156 148 128 111

8

80 123 96 78 66

0 378 382 370 375

10 313 295 281 300

20 256 247 257 203

40 194 178 151 133

60 119 114 88 74

9

80 88 68 49 39

a The values at pH 40ndash70 represent the formulations a to d of each cream respectively

102

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

1

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

2

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

3

Fig 15 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (1ndash3) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

103

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

4

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

5

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

6

Fig 16 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (4ndash6) at pH 40 (times) 50 () 60 () and 70 ()

Stearic acid

Palmitic acid

Myristic acid

104

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

7

05

07

09

11

13

15

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

8

05

07

09

11

13

15

17

0 20 40 60 80

Time (days)

log

co

nce

ntr

ati

on

(m

g)

9

Fig 17 First-order plots for the degradation of ascorbic acid in the dark in various cream

formulations (7ndash9) at pH 40 (times) 50 () 60 () and 70 ()

Palmitic acid

Myristic acid

Stearic acid

105

Table 15 First-order rate constants (kobs) for the degradation of ascorbic acid in cream

formulations in the dark

kobs times 102 (day

ndash1)abc

Cream

Formulation pH 40 50 60 70

1 128

(0991)

152

(0994)

191

(0995)

220

(0994)

2 091

(0992)

110

(0991)

152

(0993)

182

(0992)

3 148

(0991)

176

(0995)

220

(0993)

254

(0995)

4 137

(0992)

161

(0993)

205

(0994)

236

(0995)

5 121

(0991)

141

(0994)

175

(0993)

195

(0993)

6 162

(0992)

194

(0995)

237

(0994)

265

(0994)

7 164

(0994)

189

(0994)

222

(0993)

246

(0996)

8 143

(0994)

167

(0995)

193

(0996)

212

(0993)

9 184

(0995)

208

(0994)

251

(0992)

280

(0996)

a The rate constants at pH 40ndash70 represent the values for formulations a to d of each

cream respectively

b The values in parenthesis are correlation coefficients

c The values of rate constants are relative and depend on specific experimental

conditions

The estimated error is plusmn5

106

pH 4

pH 5

pH 6

pH 7

00

10

20

30

k times

10

2 (d

ayndash

1)

1-3

pH 4

pH 5

pH 6

pH 7

00

10

20

30

k times

10

2 (

da

yndash1)

4-6

pH 4

pH 5

pH 6

pH 7

00

10

20

30

12 14 16 18

Carbon chain length

k times

10

2 (

da

yndash1)

7-9

Fig 18 Plots of kobs for degradation of ascorbic acid in the dark in creams (1ndash9) against

carbon chain length of emulsifier () Stearic acid () palmitic acid

() myristic acid Humectant used glycerin (1ndash3) propylene glycol (4ndash6)

ethylene glycol (7ndash9)

107

Although it is logical to expect a linear relationship between the rate of degradation and

the carbon chain length of the emulsifier due to its polar character (Yao et al 2009) it

has not been observed in the present case The reason for the slowest rate of degradation

of AH2 in the presence of palmitic acid appears to be due to the interaction of AH2 with

palmitic acid (Lee et al 2009) as explained in Section 57

The degradation of AH2 also appears to be affected by the viscosity of the cream

in the order of humectant (Fig 19)

ethylene glycol gt propylene glycol gt glycerin

Thus the presence of glycerin imparts the most stabilizing effect on the degradation of

AH2 This is the same order as observed in the case of photodegradation of AH2 in the

creams The airtight containers used for the storage of creams make the access of air to

the creams difficult to cause chemical oxidation of AH2 However it has been observed

that the degradation of AH2 is highest in the upper layer of the creams compared to that

of the middle and the bottom layers Therefore the creams were thoroughly mixed before

sampling for the assay of AH2 However the scattering in kinetic plots (Fig 15ndash17) is

due to non-uniform distribution of AH2 in degraded creams

The effect of pH on the degradation of AH2 in the creams (Fig 19) shows that the

degradation increases with an increase in pH as observed in the case of photodegradation

of AH2 in the creams This is due to an increase in the ionization and redox potential of

AH2 with pH causing greater oxidation of the molecule and has been discussed in

Sections 59 and 510

108

Stearic Acid (1)

Palmitic Acid (2)

Myristic Acid (3)

00

10

20

30

k times

10

2 (d

ayndash

1)

Glycerin

Stearic Acid (4)

Palmitic Acid (5)

Myristic Acid (6)

00

10

20

30

k times

10

2 (

da

yndash1)

Propylene glycol

Stearic Acid (7)

Palmitic Acid (8)

Myristic Acid (9)

00

10

20

30

30 40 50 60 70

pH

k times

10

2 (d

ayndash

1)

Ethylene glycol

Fig 19 The kobsndashpH profiles for the degradation of ascorbic acid in the dark in creams

(1ndash9)

CHAPTER VI

PHOTOCHEMICAL INTERACTION

OF ASCORBIC ACID WITH

RIBOFLAVIN NICOTINAMIDE

AND ALPHA-TOCOPHEROL IN

CREAM FORMULATIONS

110

61 INTRODUCTION

It is now medically recognized that sagging skin and other signs of degenerative

skin conditions such as wrinkles and age spots are caused primarily by oxy-radical

damage Ascorbic acid can accelerate wound healing protect fatty tissues from oxidative

damage and play an integral role collagen synthesis (Zhang et al 1999) It is used in

cosmetic preparations for its anti-aging depigmentation and antioxidant properties

(Spiclin 2003 Ehrlich et al 2006) It is also used in combination with other vitamins

such as alpha-tocopherol as a co-antioxidant to stabilize cosmetic preparations (Eberlein-

Koumlnig and Ring 2005 Bissett 2006 Murray 2008) Ascorbic acid in the presence of air

or light is known to interact with alpha-tocopherol (Packer et al 2002 Johnston et al

2007) riboflavin (Homann and Gaffron 1964 Sattar et al 1977 Heelis et al 1981

Kim et al 1993 Jung et al 1995 De La Rochette et al 2000 2003 Lavoie et al

2004 Vaid et al 2005 Ahmad and Vaid 2006 Silva and Quina 2006) and

nicotinamide (Bailey et al 1945 Werner et al 1949 Guttman and Brooke 1963

DeRitter 1982) The present work involves a study of the effect of alpha-tocopherol

riboflavin and nicotinamide on the photostability of ascorbic acid in cream formulations

to observe whether the interaction in these formulations leads to the stabilization of

ascorbic acid The chemical structures of nicotinamide (NA) alpha-tocopherol (TP)

riboflavin (RF) formylmethylflavin (FMF) and lumichrome (LC) are shown in Fig 20

The details of the cream formulations used in this study are given in Table 16

The results obtained on the photodegradation of ascorbic acid in cream formulations are

discussed in the following sections

111

Riboflavin

N

N

NH

N

CH2

CH

C OHH

CH OH

CH2OH

N

N

NH

N

CH2

CHO

Formylmethylflavin

N

N

NH

HN

Lumichrome

OH

N

NH2

O

Nicotinamide

O CH3

CH3

CH3

HO

H3C

CH3 CH3 CH3

CH3

Alpha-Tocopherol

O

O

H3C

H3C

H3C

H3C

O

O

H3C

H3C

O

O

Fig 20 Chemical structures of alpha-tocopherol nicotinamide riboflavin

formylmethylflavin and lumichrome

112

Table 16 Composition of cream formulations containing ascorbic acid (2) and other

vitamins

Ingredients Cream

No SA PA MA CA GL AH2 RFa NA

b TP

c PH DW

10 a + minus minus + + + a minus minus + +

b + minus minus + + + b minus minus + +

c + minus minus + + + c minus minus + +

d + minus minus + + + d minus minus + +

e + minus minus + + + e minus minus + +

11 a minus + minus + + + a minus minus + +

b minus + minus + + + b minus minus + +

c minus + minus + + + c minus minus + +

d minus + minus + + + d minus minus + +

e minus + minus + + + e minus minus + +

12 a minus minus + + + + a minus minus + +

b minus minus + + + + b minus minus + +

c minus minus + + + + c minus minus + +

d minus minus + + + + d minus minus + +

e minus minus + + + + e minus minus + +

13 a + minus minus + + + minus a minus + +

b + minus minus + + + minus b minus + +

c + minus minus + + + minus c minus + +

d + minus minus + + + minus d minus + +

e + minus minus + + + minus e minus + +

14 a minus + minus + + + minus a minus + +

b minus + minus + + + minus b minus + +

c minus + minus + + + minus c minus + +

d minus + minus + + + minus d minus + +

e minus + minus + + + minus e minus + +

113

Table 16 continued

15 a minus minus + + + + minus a minus + +

b minus minus + + + + minus b minus + +

c minus minus + + + + minus c minus + +

d minus minus + + + + minus d minus + +

e minus minus + + + + minus e minus + +

16 a + minus minus + + + minus minus a + +

b + minus minus + + + minus minus b + +

c + minus minus + + + minus minus c + +

d + minus minus + + + minus minus d + +

e + minus minus + + + minus minus e + +

17 a minus + minus + + + minus minus a + +

b minus + minus + + + minus minus b + +

c minus + minus + + + minus minus c + +

d minus + minus + + + minus minus d + +

e minus + minus + + + minus minus e + +

18 a minus minus + + + + minus minus a + +

b minus minus + + + + minus minus b + +

c minus minus + + + + minus minus c + +

d minus minus + + + + minus minus d + +

e minus minus + + + + minus minus e + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PH = potassium hydroxide DW = distilled water

RF = riboflavin NA = nicotinamide TP = alpha-tocopherol

a RF(g ) a = 0002 b = 0004 c = 0006 d = 0008 e = 0010

b NA (g ) a = 028 b = 056 c = 084 d = 112 e = 140

c TP (g ) a = 017 b = 034 c = 052 d = 069 e = 086

The molar concentrations of these vitamins are given in Section 421

114

62 ABSORPTION CHARACTERISTICS OF PHOTOLYSED CREAMS

A typical set of the absorption spectra of the methanolic extracts (pH 20) of the

freshly prepared and photolysed creams containing AH2 and TP is shown in Fig 21 AH2

in acidified methanol exhibits absorption maximum at 245 nm (Zeng et al 2005) as

observed in Fig 21 The absorption due to TP at 284 nm (Moffat et al 2004) was

cancelled by using an appropriate blank containing an equivalent concentration of TP

The gradual decrease in absorption at around 245 nm during UV irradiation indicates the

transformation of AH2 to DHA which does not absorb in this region (Davies et al 1991)

as a result of the loss of C3=C2 chromophore Similar spectral changes around 245 nm are

observed in the presence of RF and NA which also strongly absorb in this region A

decrease in the absorption of AH2 around 266 nm in aqueous solution (pH 60) in the

presence of RF has been reported (Vaid et al 2005) The spectral changes and loss of

absorbance in methanolic extracts of creams depends on the rate of photolysis of AH2 in

the presence of these vitamins

63 PHOTOPRODUCTS OF ASCORBIC ACID AND OTHER VITAMINS

The UV irradiation of AH2 in cream formulations (pH 60) in the presence of RF

NA and TP results in the degradation of AH2 and RF and the following photoproducts

have been identified on comparison of their RF values and spot color fluorescence with

those of the authentic compounds

AH2 DHA

RF FMF LC CMF

In the TLC systems used NA and TP did not show the formation of any

degradation product in creams

115

Fig 21 UV absorption spectra of methanolic extracts of photodegraded ascorbic acid in

cream at 0 60 120 180 300 and 480 min

116

The formation of DHA in the photooxidation of AH2 has previously been reported by

many workers (Homann and Gaffron 1964 Sattar et al 1977 Heelis et al 1981

Rozanowska et al 1997 Lavoie et al 2004 Vaid et al 2006) RF is sensitive to light in

aqueous solutions (DeRitter 1982 British Pharmacopoeia 2009 Sweetman 2009) and is

known to form a number of products under aerobic conditions (Treadwell et al 1968

Cairns and Metzler 1971 Schuman Jorns et al 1975 Ahmad and Rapson 1990 Ahmad

and Vaid 2006 Ahmad et al 2004ab 2005 2008 Vaid et al 2006) It has been found

to degrade on UV irradiation in cream formulations to give FMF LC and CMF and these

products have been reported in the photolysis of RF by the workers cited above The

formation of these products may be affected by the interaction of AH2 and RF in creams

(Section 66) NA and TP individually did not appear to form any photoproduct of their

own directly or on interaction with AH2 in creams and may influence the degradation of

AH2 on UV irradiation

64 ASSAY METHOD

In view of the presence of RF (absorption maxima 223 267 373 and 444 nm)

(British Pharmacopoeia 2009) NA (absorption maximum 261 nm) (Moffat et al 2004)

and TP (absorption maximum 284 nm) (Moffat et al 2004) in the cream formulations

and the interference of these vitamins with the absorption of AH2 (absorption maximum

265 nm) (Davies et al 1991) the direct spectrophotometric method cannot be applied for

the determination of AH2 Therefore the iodimetric method (British Pharmacopoeia

2009) was used to determine AH2 in cream formulations The method was validated in

the presence of RF NA and TP before its application to the determination of AH2 in

photodegraded creams The reproducibility of the method has been confirmed by the

117

assay of known concentrations of AH2 in the range present in photodegraded creams The

recovery of AH2 in the creams has been found to be in the range 90ndash96 The values of

RSD indicate that the precision of the method is within plusmn5 (Table 17) and it can be

applied to study the kinetics of AH2 photolysis in cream formulations The assay data on

AH2 in various cream formulations are given in Table 18

65 KINETICS OF PHOTODEGRADATION OF ASCORBIC ACID

Several chemical and physical factors play a role in the photodegradation of AH2

in the presence of other vitamins (RF NA TP) and affect the rate of its degradation in

cream formulations The present work involves the study of photodegradation of AH2 in

cream formulations containing glycerin as humectant as AH2 has been found to be most

stable in these creams (Chapter 5) The apparent first-order rate constants (kobs) for the

photodegradation of AH2 in the presence of other vitamins in cream formulations

derived from the kinetic plots (Fig 22ndash24) are reported in Table 19 The second-order

rate constants (correlation coefficients 0991ndash0996) determined from the slopes of the

graphs of kobs versus vitamin concentration for the individual vitamins (Fig 25) and the

values of k0 determined from the intercept on the vertical axis at zero concentration are

reported in Table 20 The values of k0 give an indication of the effect of other vitamins on

the rate of degradation of AH2 These values are about 13 times lower than the values of

kobs obtained at the highest concentrations of RF and NA indicating that RF and NA both

accelerate the photodegradation of AH2 in creams RF is known to act as a

photosensitizer for the degradation of AH2 (Section 66) and therefore its presence in

creams would accelerate the degradation of AH2 The increase in the rate of

photodegradation of AH2 in the presence of NA has not previously been reported NA

118

Table 17 Recovery of ascorbic acid in cream formulations in the presence of other

vitamins by iodimetric methoda

Cream

Formulationb

Added

(mg )

Found

(mg )

Recovery

()

RSD

()

10e (RF) 400

200

373

187

933

935

29

22

11e (RF) 400

200

379

187

948

935

25

31

12e (RF) 400

200

375

188

938

940

29

28

13e (NA) 400

200

382

191

955

955

23

27

14e (NA) 400

200

380

185

950

925

19

26

15e (NA) 400

200

379

191

948

955

21

17

16e (TP) 400

200

368

183

920

915

29

44

17e (TP) 400

200

391

195

978

975

11

13

18e (TP) 400

200

377

182

943

910

32

37

a Values expressed as a mean of three to five determinations

b The cream formulations represent all the emulsifiers (stearic acid palmitic acid

myristic acid) to observe the efficiency of iodimetric method for the recovery of

ascorbic acid in presence of the highest concentration of vitamins (Table 16)

119

Table 18 Photodegradation of ascorbic acid in cream formulations in the presence of

other vitamins

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) a b C d e

0 373 372 374 372 375

60 362 354 354 360 359

150 342 336 336 332 334

240 315 314 308 310 302

10 (RF)

330 301 291 288 281 282

0 380 379 376 374 374

60 370 366 362 362 361

150 343 337 340 332 328

240 329 323 320 313 310

11 (RF)

330 307 301 294 288 282

0 379 380 375 372 376

60 362 366 361 351 342

150 341 335 319 307 312

240 310 306 295 284 282

12 (RF)

330 285 278 263 254 243

120

Table 18 continued

0 372 370 371 368 365

60 361 358 348 350 349

120 342 343 329 326 330

180 327 325 319 312 308

240 317 309 299 289 285

13 (NA)

300 299 291 283 278 273

0 386 380 375 378 370

60 371 362 365 362 355

120 359 351 343 339 336

200 341 332 325 316 311

14 (NA)

300 313 303 296 294 280

0 375 371 374 370 366

60 362 356 352 352 345

120 343 332 336 326 314

200 323 315 311 295 293

15 (NA)

300 293 283 275 270 259

121

Table 18 continued

0 380 378 380 377 377

60 362 365 369 369 371

120 351 352 360 360 364

180 340 346 349 353 355

240 331 334 343 343 346

16 (TP)

300 320 323 330 332 337

0 383 380 378 380 377

60 372 371 372 373 370

120 363 360 361 366 365

180 348 348 350 356 355

240 341 343 343 348 348

17 (TP)

300 330 332 336 339 341

0 380 383 377 375 373

60 364 370 366 367 366

120 352 356 351 352 351

180 334 338 339 343 342

240 324 328 324 332 330

18 (TP)

300 307 315 317 318 322

122

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

10

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

11

ab

c

de

13

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

12

Fig 22 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing riboflavin (a) 0002 (b) 0004 (c) 0006 (d) 0008

(e) 0010

Stearic acid

Palmitic acid

Myristic acid

123

abcde

13

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

13

abcde

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

14

abcde

13

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

15

Fig 23 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing nicotinamide (a) 028 (b) 056 (c) 084 (d) 112 (e) 140

Stearic acid

Palmitic acid

Myristic acid

124

abcde

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

16

abcde

14

15

16

0 60 120 180 240 300

log

co

nce

ntr

ati

on

(m

g)

17

abcde

14

15

16

0 60 120 180 240 300Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

18

Fig 24 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing alpha-tocopherol (a) 017 (b) 034 (c) 052 (d) 069

(e) 086

Stearic acid

Myristic acid

Palmitic acid

125

Table 19 First-order rate constants (kobs) for the photodegradation of ascorbic acid in the

presence of other vitamins in cream formulations

kobs times 103 (min

ndash1)ab

Cream

formulationd

Other

vitaminc

a b C d e

10 RF 068

(0991)

073

(0996)

079

(0995)

085

(0992)

089

(0995)

11 RF 065

(0992)

070

(0992)

073

(0994)

080

(0995)

086

(0993)

12 RF 087

(0993)

096

(0995)

109

(0993)

116

(0994)

127

(0992)

13 NA 073

(0993)

081

(0992)

088

(0994)

096

(0994)

101

(0993)

14 NA 069

(0992)

074

(0992)

080

(0991)

086

(0995)

094

(0995)

15 NA 083

(0994)

090

(0993)

101

(0993)

109

(0994)

115

(0995)

16 TP 055

(0991)

051

(0994)

046

(0994)

042

(0993)

038

(0991)

17 TP 050

(0995)

045

(0993)

041

(0992)

038

(0995)

034

(0994)

18 TP 070

(0996)

066

(0996)

060

(0994)

055

(0993)

051

(0993)

a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental conditions

including the light intensity

c Vitamin concentrations (andashe) are as given in Table 16

d All the creams contain glycerin as humectant

The estimated error is plusmn5

126

00

05

10

15

00 10 20 30

Riboflavin concentration (M times 104)

kob

s times

10

3 (

min

ndash1)

10-12

00

05

10

15

00 20 40 60 80 100 120

Nicotinamide concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

13-15

00

02

04

06

08

00 04 08 12 16 20

Alpha-Tocopherol concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

16-18

Fig 25 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against individual vitamin concentration in cream formulations (10ndash18)

127

Table 20 First-order rate constants (k0)a for the photodegradation of ascorbic acid in the

absence of other vitamins and second-order rate constants (k) for the

photochemical interaction of ascorbic acid with RF NA and TP

Cream

formulation

Other

vitamin

k0 times 103

(minndash1

)

k

(Mndash1

minndash1

)

Correlation

coefficient

10 RF 062 102 0994

11 RF 059 097 0992

12 RF 077 189 0995

13 NA 066 032 times 10ndash2

0995

14 NA 062 027 times 10ndash2

0993

15 NA 074 037 times 10ndash2

0994

16 TP 059 110 times 10ndash2b

0996

17 TP 053 096 times 10ndash2b

0992

18 TP 075 123 times 10ndash2b

0994

a

The variations in the values of k0 are due to the degradation of AH2 in the presence of

different emulsifying agents in cream formulations

b Values for the inhibition of photodegradation of AH2

128

forms a complex with AH2 (Section 67) and may also act as a photosensitizer for AH2 by

energy transfer in the excited state on UV irradiation The absorption maximum of NA

(261 nm) (Moffat et al 2004) is very close to that of AH2 (265 nm) (Davies et al 1991)

and the possibility of energy transfer in the excited state (Moore 2004) is greater leading

to the photodegradation of AH2

The value of k0 is about 13 times greater than the values of kobs obtained for the

degradation of AH2 in the presence of the highest concentrations of TP in the creams

This indicates that TP has a stabilising effect on the photodegradation of AH2 in the

cream formulations This is in agreement with the view that the TP acts as a redox partner

with AH2 to retard its oxidation (Wille 2005) Thus among the three vitamins studied

only TP appears to have a stabilising effect on photodegradation of AH2 The

photochemical interaction of individual vitamins with AH2 is discussed below

66 INTERACTION OF RIBOFLAVIN WITH ASCORBIC ACID

The interaction of RF with the ascorbate ion (AHndash) may be represented by the

following reactions proposed by Silva and Quina (2006)

RF rarr 1RF (61)

1RF rarr

3RF (62)

3RF + AH

ndash rarr RF

ndashmiddot + AHmiddot (63)

AHmiddot + O2 rarr A + HO2middot (64)

HO2middot + AHndash rarr H2O2 + AHmiddot (65)

RF on the absorption of a quantum of light is promoted to the excited singlet state (1RF)

(61) 1RF may undergo intersystem crossing (isc) to form the excited triplet state (

3RF)

(62) The excited triplet state may react with the ascorbate ion to generate the ascorbyl

hv

isc

129

radical (AH) (63) (Kim et al 1993) The ascorbyl radical reacts with oxygen to give

dehydroascorbic acid (A) and peroxyl radical (HO2) (64) This radical may interact with

ascorbate ion to generate further ascorbyl radicals (65) These radicals may again take

part in the sequence of reactions to form A The role of RF in this reaction is to act as a

photosensitiser in the oxidation of ascorbic acid to A Ascorbic acid is reported to protect

riboflavin in milk under the influence of light by reacting with singlet oxygen (Hall et al

2009) (Section 511)

67 INTERACTION OF NICOTINAMIDE WITH ASCORBIC ACID

NA is known to form a 11 complex with ascorbic acid (Guttman and Brooke

1963 OrsquoNeil 2001 Doores 2002) The complexation of NA and AH2 may result from

the donor-acceptor interaction between AH2 (donor) and NA (acceptor) as observed in

the case of tryptophan and NA (Florence and Attwood 2006) In the presence of light the

interaction may cause reduction of NA (NAH) to form the ascorbyl radical (AH) ((66)-

(68)) which is oxidized to dehydroascorbic acid (A) (69) The NAH may be oxidized to

NA and H2O2 (610)

NA rarr 1NA (66)

1NA rarr

3NA (67)

3NA + AH2 rarr NAH + AHmiddot (68)

2 AH٠ rarr A + AH2 (69)

NAH + O2 rarr NA + H2O2 (610)

The proposed reactions suggest that on photochemical interaction AH2 undergoes

photosensitised oxidation in the presence of NA indicating that the photostability of

ascorbic acid is affected by NA

isc

130

68 INTERACTION OF ΑLPHA-TOCOPHEROL WITH ASCORBIC ACID

TP is an unstable compound and its oxidation by air results in the formation of an

epoxide which than produces a quinone that is inactive (Connors et al 1986) TP is

destroyed by sun light and artificial light containing the wavelengths in the UV region

(Ball 2006) It interacts with other antioxidants such as ascorbic acid (AH2) according to

the following reactions

TPndashO + AH2 rarr TP + AHmiddot (611)

2 AHmiddot rarr A + AH2 (612)

TP + AHmiddot rarr TPndashO + AH2 (613)

The tocopheroxyl radical (TPndashO) reacts with AH2 to regenerate TP and form the

ascorbyl radical (AHmiddot) (611) This radical undergoes further reactions as described in

equations (64) and (65) (Traber 2007) It may also disproportionate back to A and AH2

(612) TP reacts with AHmiddot to produce again the TPndashO radical and AH2 Thus in the

presence of light TP leads to the oxidation of AH2 which is ultimately regenerated in the

reaction (Davies et al 1991) Ascorbic acid has the ability to act as a co-antioxidant with

the TPndashO to regenerate TP (Packer et al 1979 2002) In this manner AH2 and TP act

synergistically to function in a redox cycle and AH2 is stabilized in the cream

formulations and microemulsions (Rozman and Gasperlin 2007 Rozman et al 2009)

69 EFFECT OF CARBON CHAIN LENGTH OF EMULSIFYING AGENT

The graphs of kobs for the photodegradation of AH2 in the presence of RF NA and

TP versus the carbon chain length of emulsifying agents are shown in Fig 26 It appears

that the photodegradation of AH2 in the presence of all the three vitamins in the creams

lies in the order

131

Fig 26 Plots of k for photodegradation of ascorbic acid in creams (10ndash18) against

carbon chain length of emulsifier () Stearic acid () palmitic acid

() myristic acid

00

05

10

15

20

25

k

(Mndash

1 m

inndash

1)

00

05

10

15

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash

1 m

inndash

1)

132

myristic acid gt stearic acid gt palmitic acid

The same order of emulsifying agents has been observed in the absence of the

added vitamins (Section 57) The polar character of these acids (Yao et al 2009) on the

basis of their carbon chain length may play a part in the photostability of AH2 The

greater stability of AH2 in creams in the presence of palmitic acid (Fig 26) may be due to

the interaction of AH2 with palmitic acid as discussed in Section 57 Ascorbic acid-6-

palmitate is known to be an antioxidant in cosmetic preparations (Lee et al 2009) and

food products (Doores 2002)

610 EFFECT OF VISCOSITY OF CREAMS

The plots of kobs for the degradation of AH2 in the presence of the highest

concentration of vitamins versus reciprocal of the viscosity of creams (Table 21) are

linear (Fig 27) and indicate that the increase in cream viscosity leads to a decrease in the

rate of degradation of AH2 The slopes of the plots indicate the effect of viscosity on the

interaction of AH2 with other vitamins in the order

riboflavin gt nicotinamide gt alpha-tocopherol

The relatively slow rate of degradation of AH2 in creams containing palmitic acid may be

due to the interaction of AH2 with the vitamins as well as palmitic acid (Lee et al 2009)

Thus viscosity is an important factor in the stability of AH2 in cream formulations and

may affect its rate of interaction with other vitamins It has been suggested that an

increase in the viscosity of the medium makes access to air at the surface more difficult to

prevent the oxidation of a drug (Wallwork and Grant 1977) This is in agreement with

the photolysis of AH2 in aqueous and organic solvents cream formulations (Chapter 5)

and aerobic oxidation of Ah2 in syrups (Blaug and Hajratwala 1972)

133

Table 21 Average viscosity of cream formulations containing different emulsifying

agents and glycerin as humectant (25 plusmn 1 ordmC) and the photodegradation rate

constants of AH2

Cream No Emulsifying

agent

Viscosityab

(mPa s)

kobs times 103c

10 (RF)

13 (NA)

16 (TP)

Stearic acid 9000 089

101

038

11 (RF)

14 (NA)

17 (TP)

Palmitic acid 8600 086

094

034

12 (RF)

15 (NA)

18 (TP)

Myristic acid 7200 127

115

051

a plusmn10

b Average viscosity of creams containing the individual vitamins (RF NA TP)

c The values have been obtained in the presence of highest concentration of the

vitamins

134

00

05

10

15

20

25

30

100 110 120 130 140

Viscosity (mPa s)ndash1

times 103

kob

s (m

inndash1)

Fig 27 Plots of kobs in the presence of highest concentration of vitamins versus

reciprocal of the viscosity of creams () riboflavin

( ) nicotinamide (- - -- - -) alpha-tocopherol

135

611 DEGRADATION OF ASCORBIC ACID IN THE PRESENCE OF OTHER

VITAMINS IN THE DARK

In order to observe the effect of riboflavin nicotinamide and alpha-tocopherol on

the degradation of AH2 in the creams stored in the dark the AH2 contents of the creams

were assayed at appropriate intervals (Table 22) The apparent first-order rate constants

determined from the kinetic plots (Fig 28) for the degradation of AH2 in the presence of

the highest concentrations of the individual vitamins in cream formulations (10ndash18) are

reported in Table 23 These rate constants indicate that the overall degradation of AH2 in

the presence of the highest concentration of the individual vitamins (RF NA and TP) is

about 70 times slower than that obtained on the exposure of creams to UV irradiation

This decrease in the rate of degradation of AH2 in the creams is the same as observed in

the case of AH2 alone In the absence of light the degradation of AH2 occurs due to

chemical oxidation (Section 132) and does not appear to be affected by the presence of

riboflavin and nicotinamide as indicated by the comparisons of the values of kobs in the

presence and absence of these vitamins (Table 15 and 23) In the presence of alpha-

tocopherol the degradation is slower than that in the presence of riboflavin and

nicotinamide This may be due to some interaction of AH2 and alpha-tocopherol causing

stabilisation of AH2 in the creams

As observed in the case of AH2 degradation alone in creams in the dark the AH2

degradation in the presence of the highest concentrations of other vitamins also occurs in

the same order of emulsifying agents (Fig 29)

myristic acid gt stearic acid gt palmitic acid

136

Table 22 Degradation of ascorbic acid in cream formulations in the dark in presence of

highest concentration of other vitamins

Concentration of ascorbic acid (mg 2g) Cream

No Time (days) 0 10 20 40 60 80

10e (RF) 375 285 233 171 110 69

11e (RF) 374 341 281 221 148 113

12e (RF) 372 259 203 130 89 59

13e (NA) 365 330 255 187 126 81

14e (NA) 370 321 289 219 159 109

15e (NA) 366 289 249 159 110 63

16e (TP) 377 359 321 261 211 159

17e (TP) 377 366 333 275 228 191

18e (TP) 373 361 304 252 200 167

137

02

07

12

17lo

g c

on

cen

tra

tio

n (

mg

)

10-12Riboflavin

02

07

12

17

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

13-15Nicotinamide

10

12

14

16

18

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

16-18Alpha-Tocopherol

Fig 28 First-order plots for the degradation of ascorbic acid in the dark in presence of

other vitamins using the emulsifying agents (minusminusminusminus) Stearic acid

(minus minusminus minus) palmitic acid (----) myristic acid

138

Table 23 First-order rate constants (kobs) for the degradation of ascorbic acid in presence

of other vitamins in cream formulations in the dark

Cream

formulation

Other

vitaminc

kobs times 102

(dayndash1

)ab

10e RF 204

(0995)

11e RF 156

(0992)

12e RF 222

(0992)

13e NA 189

(0995)

14e NA 151

(0993)

15e NA 214

(0995)

16e TP 100

(0994)

17e TP 088

(0995)

18e TP 105

(0993)

a The values in parenthesis are correlation coefficients and range from 0991ndash0996 due to

some variations in AH2 distribution in the creams

b The values of rate constants are relative and depend on specific experimental

conditions

c Vitamin concentrations (andashe) are as given in Table 16

The estimated error is plusmn5

139

Riboflavin

Nicotinamide

Alpha-Tocopherol

00

10

20

30

12 14 16 18Carbon chain length

ko

bs times

10

2 (

da

yndash1)

Fig 29 Plots of kobs for degradation of ascorbic acid in the dark in creams (10ndash18)

against carbon chain length of the emulsifier () Stearic acid () palmitic acid

() myristic acid

140

This indicates that the rate of degradation of AH2 is slowest in the creams containing

palmitic acid as the emulsifying agent The reason for AH2 degradation in the dark in this

order has already been explained in section 512

CHAPTER VII

STABILIZATION OF

ASCORBIC ACID WITH

CITRIC ACID TARTARIC

ACID AND BORIC ACID IN

CREAM FORMULATIONS

142

71 INTRODUCTION

Ascorbic acid is an ingredient of cosmetic preparations (Section 51) and is

sensitive to light (Rowe et al 2009 Sweetman 2009 British Pharmacopoeia 2009)

degrading to dehydroascorbic acid on UV irradiation by photooxidation (Kitagawa

1968) The photosensitivity of ascorbic acid makes it unstable in pharmaceutical and

cosmetic preparations (DeRitter 1982) The present work is an attempt to study the

photodegradation of ascorbic acid in cream formulations in the presence of certain

compounds (eg citric acid tartaric acid and boric acid) to investigate their role in the

stabilization of the vitamin on exposure to light and in the dark Citric acid and tartaric

acid are used as an antioxidant synergist (Rowe et al 2009) and boric acid is a

complexing agent for hydroxy compounds (Ahmad et al 2009cd)

72 CREAM FORMULATIONS

The details of the various cream formulations used in this study are given in Table

24 and the results obtained on the photodegradation of ascorbic acid in the presence of

stabilizing agents in these formulations are discussed in the following sections

143

Table 24 Composition of cream formulations containing ascorbic acid (2) and

stabilizers

Ingredients Cream

No SA PA MA CA GL PG EG AH2 CTa TA

b BA

c PH DW

19 a + minus minus + + minus minus + a minus minus + +

b + minus minus + + minus minus + b minus minus + +

c + minus minus + + minus minus + c minus minus + +

20 a minus + minus + + minus minus + a minus minus + +

b minus + minus + + minus minus + b minus minus + +

c minus + minus + + minus minus + c minus minus + +

21 a minus minus + + + minus minus + a minus minus + +

b minus minus + + + minus minus + b minus minus + +

c minus minus + + + minus minus + c minus minus + +

22 a + minus minus + + minus minus + minus a minus + +

b + minus minus + + minus minus + minus b minus + +

c + minus minus + + minus minus + minus c minus + +

23 a minus + minus + + minus minus + minus a minus + +

b minus + minus + + minus minus + minus b minus + +

c minus + minus + + minus minus + minus c minus + +

24 a minus minus + + + minus minus + minus a minus + +

b minus minus + + + minus minus + minus b minus + +

c minus minus + + + minus minus + minus c minus + +

25 a + minus minus + + minus minus + minus minus a + +

b + minus minus + + minus minus + minus minus b + +

c + minus minus + + minus minus + minus minus c + +

26 a minus + minus + + minus minus + minus minus a + +

b minus + minus + + minus minus + minus minus b + +

c minus + minus + + minus minus + minus minus c + +

27 a minus minus + + + minus minus + minus minus a + +

b minus minus + + + minus minus + minus minus b + +

c minus minus + + + minus minus + minus minus c + +

144

Table 24 continued

28 a + minus minus + minus + minus + minus minus a + +

b + minus minus + minus + minus + minus minus b + +

c + minus minus + minus + minus + minus minus c + +

29 a minus + minus + minus + minus + minus minus a + +

b minus + minus + minus + minus + minus minus b + +

c minus + minus + minus + minus + minus minus c + +

30 a minus minus + + minus + minus + minus minus a + +

b minus minus + + minus + minus + minus minus b + +

c minus minus + + minus + minus + minus minus c + +

31 a + minus minus + minus minus + + minus minus a + +

b + minus minus + minus minus + + minus minus b + +

c + minus minus + minus minus + + minus minus c + +

32 a minus + minus + minus minus + + minus minus a + +

b minus + minus + minus minus + + minus minus b + +

c minus + minus + minus minus + + minus minus c + +

33 a minus minus + + minus minus + + minus minus a + +

b minus minus + + minus minus + + minus minus b + +

c minus minus + + minus minus + + minus minus c + +

SA = stearic acid PA = palmitic acid MA = myristic acid CA = cetyl alcohol

AH2 = ascorbic acid GL = glycerin PG = propylene glycol EG = ethylene glycol

PH = potassium hydroxide DW = distilled water CT = citric acid TA = tartaric acid

BA = boric acid

a CT (g ) a = 01 b = 02 c = 04

b TA (g ) a = 01 b = 02 c = 04

c BA (g ) a = 01 b = 02 c = 04

145

73 PRODUCTS OF ASCORBIC ACID PHOTODEGRADATION

The photodegradation of AH2 in cream formulations leads to the formation of

DHA as detected by TLC and reported earlier in the photolysis of AH2 in aqueous

solutions (Vaid et al 2006) and cream formulations (Sections 52 and 63) AH2 and

DHA in the methanolic extracts of the degraded creams were identified by comparison of

their Rf and color of the spots with those of the reference standards DHA is also

biologically active (Gardner 1972 Doores 2002) but its further degradation to 23-

diketo-gulonic acid (DGA) results in the loss of vitamin activity (Section 132)

However this product has not been detected in the present cream formulations

Therefore the creams may still possess their biological efficacy

74 SPECTRAL CHANGES IN PHOTODEGRADED CREAMS

In order to observe the spectral changes in photodegraded creams in the presence

of stabilizing agents the absorption spectra of the methanolic extracts of a degraded

cream were determined The spectra show a gradual loss of absorbance around 245 nm

due to the oxidation of AH2 to DHA on UV irradiation and similar to that shown for the

photodegradation of AH2 alone in Fig 5 DHA has negligible absorbance around 245 nm

(Davies et al 1991) and therefore it does not interfere with the absorbance of AH2 in

methanolic solutions The spectral changes and loss of absorbance around 245 nm in

methanolic solution depend on the extent of photooxidation of AH2 in a particular cream

75 ASSAY OF ASCORBIC ACID IN CREAMS

The UV spectrophotometric method (Zeng et al 2005) has previously been

applied to the determination of AH2 in cream formulations (Section 54) The absorbance

of the methanolic extracts of creams containing AH2 during photodegradation was used

146

to determine the concentration of AH2 The method was validated in the presence of citric

acid (CT) tartaric acid (TA) and boric acid (BA) before its application to the evaluation

of the kinetics of AH2 degradation in cream formulations The recovery of AH2 in creams

has been found to be in the range of 90ndash96 and is similar to that reported in Table 7

The reproducibility of the method lies within plusmn5 The assay data on the degradation of

AH2 in various creams in the presence of the stabilizing agents are reported in Table 25

76 KINETICS OF PHOTODEGRADATION

The effect of CT TA and BA as stabilizing agents on the photodegradation of

AH2 was studied by adding 01ndash04 of each compound to the cream formulations (19ndash

33) at pH 60 This concentration range is normally used for the stabilization of drugs in

pharmaceutical preparations (Im-Emsap et al 2002) The apparent first-order rate

constants (kobs) determined from the plots of log concentration versus time (Fig 30ndash34)

are reported in Table 26 The second-order rate constants (k) determined from the plots

of kobs versus concentration of the individual compounds (Fig 35ndash36) are given in Table

27 The values of k indicate the rate of inhibition of photodegradation of AH2 by each

compound

77 EFFECT OF STABILIZING AGENTS

In order to compare the effectiveness of CT TA and BA as stabilizing agents for

AH2 plots of k versus carbon chain length of the emulsifying agents were constructed

(Fig 37) The k values for the interaction of these compounds with AH2 are in the order

citric acid gt tartaric acid gt boric acid

The curves indicate that the highest interaction of these compounds with AH2 is in the

order

147

Table 25 Photodegradation of ascorbic acid in cream formulations in the presence of

stabilizers

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(min) a b c

0 374 378 379

60 362 362 372

120 349 355 367

210 333 335 349

19 (CT)

300 319 322 336

400 296 309 324

0 381 378 380

60 368 370 369

120 355 363 364

210 344 345 355

20 (CT)

300 328 335 341

400 312 319 331

21 (CT) 0 368 370 374

60 355 356 360

120 340 344 343

210 321 322 333

300 296 299 315

400 272 285 299

148

Table 25 continued

0 375 374 378

60 363 363 368

120 352 354 362

210 329 335 345

22 (TA)

300 307 314 333

400 292 299 313

0 370 377 374

60 364 365 368

120 352 357 357

210 332 344 349

23 (TA)

300 317 330 335

400 301 310 322

24 (TA) 0 376 379 377

60 367 369 368

120 351 348 352

210 325 330 344

300 306 317 326

400 284 294 310

149

Table 25 continued

0 370 375 380

60 356 362 359

120 331 339 344

210 311 318 330

25 (BA)

300 279 288 305

400 260 269 283

0 377 375 370

60 364 363 361

120 351 353 351

210 331 332 337

26 (BA)

300 323 324 325

400 301 307 313

27 (BA) 0 380 377 375

60 369 368 366

120 333 338 341

210 305 313 318

300 292 294 304

400 262 266 281

150

Table 25 continued

0 373 376 378

60 348 349 360

120 329 336 339

210 315 312 323

28 (BA)

300 282 283 299

400 249 264 280

0 370 373 380

60 358 355 367

120 343 346 356

210 325 329 347

29 (BA)

300 307 312 325

400 287 295 315

30 (BA) 0 369 375 372

60 353 358 362

120 321 330 335

210 283 294 303

300 265 281 293

400 242 254 270

151

Table 25 continued

0 374 376 379

60 348 366 352

120 324 340 337

210 303 319 322

31 (BA)

300 275 289 293

400 243 260 275

0 370 374 375

60 355 354 366

120 339 344 345

210 313 319 330

32 (BA)

300 288 297 308

400 261 271 290

33 (BA) 0 377 380 377

60 357 361 367

120 324 335 339

210 288 294 307

300 270 280 293

400 233 248 265

Creams 19ndash27 contain glycerin 28ndash30 contain propylene glycol and 31ndash33 contain

ethylene glycol as humectants

152

a

b

c

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

19

ab

c

14

15

16

log

co

nce

ntr

ati

on

(m

g)

20

a

b

c

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

21

Fig 30 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and citric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

153

a

b

c

14

15

16lo

g c

on

cen

tra

tio

n (

mg

)

22

a

b

c

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

23

a

b

c

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

24

Fig 31 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and tartaric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

154

ab

c

13

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

25

abc

13

14

15

16

0 60 120 180 240 300 360 420

log

co

nce

ntr

ati

on

(m

g)

26

ab

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

27

Fig 32 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing glycerin and boric acid (a) 01 (b) 02 (c) 04

Palmitic acid

Stearic acid

Myristic acid

155

a

b

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

28

ab

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

29

a

b

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

30

Fig 33 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing propylene glycol and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

156

a

b

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

31

ab

c

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

32

a

b

c

13

14

15

16

0 60 120 180 240 300 360 420Time (minutes)

log

co

nce

ntr

ati

on

(m

g)

33

Fig 34 First-order plots for the photodegradation of ascorbic acid in cream formulations

containing ethylene glycol and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

157

Table 26 Apparent first-order rate constants (kobs) for the photodegradation of ascorbic

acid in presence of different stabilizers in cream formulations

kobs times 103 (min

ndash1)ab

Cream

formulation Stabilizer

c

a b c

19 CT 057

(0995)

050

(0992)

041

(0991)

20 CT 049

(0996)

043

(0995)

034

(0993)

21 CT 076

(0995)

067

(0995)

055

(0992)

22 TA 065

(0995)

058

(0995)

046

(0991)

23 TA 054

(0994)

047

(0993)

038

(0994)

24 TA 072

(0996)

063

(0992)

049

(0991)

25 BA 091

(0994)

086

(0995)

071

(0993)

26 BA 055

(0994)

050

(0993)

042

(0993)

27 BA 095

(0995)

089

(0992)

074

(0996)

28 BA 097

(0995)

088

(0992)

075

(0993)

29 BA 064

(0994)

057

(0991)

047

(0993)

30 BA 110

(0994)

100

(0996)

084

(0992)

31 BA 105

(0995)

094

(0994)

078

(0992)

32 BA 088

(0994)

079

(0993)

066

(0993)

33 BA 120

(0995)

108

(0993)

091

(0993) a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental conditions

including the light intensity

c Stabilizer concentrations (andashc) are as given in Table 24

The estimated error is plusmn5

158

00

04

08

12

00 04 08 12 16 20

Citric acid concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

19-21

00

04

08

12

00 05 10 15 20 25

Tartaric acid concentration (M times 102)

ko

bs times

10

3 (

min

ndash1)

22-24

Fig 35 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against citric acid (19ndash21) and tartaric acid concentrations (22ndash24) in cream

formulations

159

00

04

08

12k

ob

s times

10

3 (

min

ndash1)

25-27

00

04

08

12

00 20 40 60

ko

bs times

10

3 (

min

ndash1)

28-30

00

04

08

12

00 20 40 60

Boric acid concentration (M times 102)

kob

s times

10

3 (

min

ndash1)

30-33

Fig 36 Plots of first-order rate constants (kobs) for the photodegradation of ascorbic acid

against boric acid concentrations in cream formulations (25ndash33)

Propylene glycol

Glycerin

Ethylene glycol

160

Table 27 First-order rate constants (k0)a for the photodegradation of ascorbic acid in the

absence of stabilizers and second-order rate constants (k) for the interaction of

ascorbic acid with CT TA and BA

Cream

formulation Stabilizers

k0 times 103

(minndash1

)

k times 102

(Mndash1

minndash1

)

Correlation

coefficient

19 CT 062 111 0991

20 CT 053 103 0994

21 CT 082 145 0995

22 TA 071 092 0995

23 TA 059 080 0993

24 TA 080 118 0996

25 BA 098 041 0994

26 BA 059 026 0994

27 BA 102 044 0995

28 BA 104 046 0992

29 BA 069 033 0995

30 BA 118 054 0994

31 BA 113 053 0995

32 BA 095 045 0995

33 BA 129 060 0993

a The variations in the values of k0 are due to the presence of different emulsifying agents

in cream formulations

161

00

04

08

12

16

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash1

min

ndash1)

18-33

a

b

e

cd

Fig 37 Plots of k for photodegradation of ascorbic acid in creams (19ndash33) against

carbon chain length of emulsifier () Stearic acid () palmitic acid ()

myristic acid (a) CT (b) TA (c) BA with glycerin (d) BA with propylene

glycol (e) BA with ethylene glycol

162

myristic acid gt stearic acid gt palmitic acid

In the case of myristic acid and stearic acid it may be explained on the basis of the

decreasing polarity (Yao et al 2009) It is interesting to observe the lowest rates of

interaction of these compounds in the creams containing palmitic acid This could be due

to the interaction of AH2 with palmitic acid to form a palmitate derivative in addition to

its interaction with the individual stabilizing agents CT and TA are known to act as

antioxidant synergists (Rowe et al 2009 Sweetman 2009) and in this capacity may

inhibit the photooxidation of AH2 as indicated by the values of the degradation rate

constants in the presence of these compounds The addition of CT to nutritional

supplements is known to inhibit the oxidation of AH2 (Doores 2002) Boric acid forms a

complex with AH2 (Rivlin 2007) and there by may inhibit its degradation Boric acid

may also interact with glycerin added to the creams as a humectant and form a complex

(Rowe et al 2009) This may influence its interaction and stabilizing effect on AH2 in

creams as indicated by the lower k values compared to those in the presence of CT and

TA It has further been observed that the k values for BA are greater in propylene glycol

and ethylene glycol compared to those in glycerin (Table 27) Again this may be due to

greater interaction of BA with glycerin compared to other humectants in the creams

78 DEGRADATION OF ASCORBIC ACID IN PRESENCE OF STABILIZING

AGENTS IN THE DARK

An important factor in the formulation of cosmetic preparations is to ensure the

chemical and photostability of the active ingredient by the use of appropriate stabilizing

agents The choice of these agents would largely depend on the nature and

physicochemical characteristics of the active ingredient AH2 possesses a redox system

163

and can be easily oxidized by air or light In order to observe the effect of CT TA and

BA on the stability of AH2 the cream formulations containing the individual compounds

were stored in the dark for a period of about three months and the rate of degradation of

AH2 was determined The assay data are reported in Table 28 and the kinetic plots are

shown in Fig 38ndash42 The values of apparent first-order rate constants for the degradation

of AH2 in the presence of the stabilizing agents are reported in Table 29 The second

order-rate constants for the interaction of CT TA and BA with AH2 are reported in Table

30 (Fig 43ndash44) The plots of k against the carbon chain length of the emulsifiers are

shown in Fig 45 The kinetic data indicate the same pattern of rates of degradation and

interaction of AH2 with these compounds as observed in the presence of light except that

the rates are much slower in the dark Thus the stabilizing agents are equally effective in

inhibiting the rate of degradation of AH2 in the dark The effect of emulsifying agents and

the humectants on the rate of degradation of AH2 in the presence of the stabilizers has

been discussed in the above Section 77

79 EFFECT OF ADDITIVES ON TRANSMISSION OF ASCORBIC ACID

In order to observe the effect of additives (citric tartaric and boric acids) on the

transmission characteristics of ascorbic acid (0002 mg100 ml) in methanol containing

the highest concentration of the additives (004) used in this study the transmission

spectra were measured It has been found that these additives produce a hypsochromic

shift in the absorption maximum of ascorbic acid This may result in the reduction of the

fraction of light absorbed by ascorbic acid to the extent of about 10 and thus influence

the rate of photodegradation reactions However since all the additives produce similar

effects the rate constants can be considered on a comparative basis

164

Table 28 Degradation of ascorbic acid in cream formulations in the presence of

stabilizers in the dark

Concentration of ascorbic acid (mg 2g) Cream

No

Time

(days) a b c

0 374 378 379

10 355 346 362

20 326 328 342

40 293 297 322

19 (CT)

60 264 269 295

80 241 245 262

0 381 378 380

10 361 364 372

20 339 350 348

40 309 312 330

20 (CT)

60 279 286 301

80 260 266 282

21 (CT) 0 368 370 374

10 342 346 364

20 310 321 348

40 278 282 313

60 249 251 278

80 217 228 249

165

Table 28 continued

0 375 374 378

10 339 344 351

20 317 326 336

40 282 288 306

22 (TA)

60 251 258 280

80 222 235 252

0 370 377 374

10 340 354 355

20 332 336 343

40 297 303 310

23 (TA)

60 266 282 294

80 238 248 267

24 (TA) 0 376 379 377

10 341 339 350

20 306 319 323

40 263 284 279

60 223 241 249

80 196 202 223

166

Table 28 continued

0 370 375 380

10 331 341 334

20 287 289 301

40 225 247 245

25 (BA)

60 189 185 214

80 141 154 170

0 377 375 370

10 355 357 349

20 326 314 324

40 264 267 286

26 (BA)

60 232 238 254

80 189 199 211

27 (BA) 0 380 377 375

10 346 339 337

20 309 288 301

40 233 241 260

60 192 196 211

80 140 147 163

167

Table 28 continued

0 373 376 378

10 314 322 333

20 267 281 305

40 217 233 253

28 (BA)

60 167 177 204

80 122 135 151

0 370 373 380

10 336 329 343

20 283 277 306

40 233 243 267

29 (BA)

60 189 190 217

80 144 154 173

30 (BA) 0 369 375 372

10 308 319 329

20 255 275 310

40 210 226 244

60 158 163 191

80 113 131 147

168

Table 28 continued

0 374 376 379

10 303 311 329

20 266 260 289

40 211 219 239

31 (BA)

60 155 158 178

80 112 121 149

0 370 374 375

10 314 323 339

20 276 280 305

40 222 233 258

32 (BA)

60 172 187 193

80 126 136 162

33 (BA) 0 377 380 377

10 308 306 320

20 254 265 280

40 205 214 237

60 144 155 175

80 107 118 138

169

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

19

abc

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

20

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

21

Fig 38 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing citric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

170

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

22

ab

c

12

13

14

15

16

log

co

nce

ntr

ati

on

(m

g)

23

ab

c

12

13

14

15

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

24

Fig 39 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing tartaric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

171

a

b

c

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

25

abc

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

26

ab

c

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

27

Fig 40 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing glycerin and boric acid (a) 01 (b) 02 (c) 04

Stearic acid

Palmitic acid

Myristic acid

172

a

b

c

10

12

14

16

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

28

ab

c

10

12

14

16

0 20 40 60 80

log

co

nce

ntr

ati

on

(m

g)

29

a

b

c

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

30

Fig 41 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing propylene glycol and boric acid (a) 01 (b) 02 (c)

04

Palmitic acid

Stearic acid

Myristic acid

173

ab

c

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

31

ab

c

08

10

12

14

16

log

co

nce

ntr

ati

on

(m

g)

32

a

b

c

08

10

12

14

16

0 20 40 60 80Time (days)

log

co

nce

ntr

ati

on

(m

g)

33

Fig 42 First-order plots for the degradation of ascorbic acid in the dark in cream

formulations containing ethylene glycol and boric acid (a) 01 (b) 02 (c)

04

Myristic acid

Palmitic acid

Stearic acid

174

Table 29 Apparent first-order rate constants (kobs) for the degradation of ascorbic acid in

presence of different stabilizers in cream formulations in the dark

kobs times 102 (day

ndash1)ab

Cream

formulation Stabilizer

c

a b c

19 CT 055

(0994)

052

(0992)

044

(0991)

20 CT 048

(0995)

046

(0995)

038

(0992)

21 CT 064

(0994)

061

(0995)

052

(0994)

22 TA 063

(0994)

058

(0995)

049

(0996)

23 TA 054

(0995)

050

(0995)

041

(0994)

24 TA 081

(0995)

075

(0993)

066

(0995)

25 BA 118

(0996)

113

(0994)

097

(0994)

26 BA 087

(0995)

079

(0993)

068

(0994)

27 BA 124

(0995)

114

(0994)

101

(0993)

28 BA 134

(0995)

124

(0996)

110

(0992)

29 BA 116

(0996)

108

(0992)

096

(0995)

30 BA 142

(0993)

131

(0995)

115

(0995)

31 BA 145

(0995)

137

(0992)

117

(0995)

32 BA 130

(0996)

120

(0993)

107

(0994)

33 BA 153

(0995)

141

(0994)

122

(0994) a The values in parenthesis are correlation coefficients

b The values of rate constants are relative and depend on specific experimental

conditions

c Stabilizer concentrations (andashc) are as given in Table 24

The estimated error is plusmn5

175

176

Table 30 First-order rate constants (k0)a for the degradation of ascorbic acid in the

absence of stabilizers and second-order rate constants (k) for the chemical

interaction of ascorbic acid with CT TA and BA in the dark

Cream

formulation Stabilizers

k0 times 102

(dayndash1

)

k times 102

(Mndash1

dayndash1

)

Correlation

coefficient

19 CT 060 797 0996

20 CT 052 723 0995

21 CT 069 850 0994

22 TA 068 710 0996

23 TA 058 636 0994

24 TA 086 758 0994

25 BA 126 444 0993

26 BA 092 375 0992

27 BA 131 480 0991

28 BA 141 488 0993

29 BA 122 418 0994

30 BA 149 531 0991

31 BA 155 578 0996

32 BA 137 472 0994

33 BA 163 627 0996

a The variations in the values of k0 are due to the presence of different emulsifying agents

in cream formulations

177

00

04

08

12

00 04 08 12 16 20

Citric acid concentration (M times 102)

kob

s times

10

2 (

da

yndash1)

19-21

00

04

08

12

00 05 10 15 20 25

Tartaric acid concentration (M times 102)

kob

s times

10

2 (

da

yndash1)

22-24

Fig 35 Plots of first-order rate constants (kobs) for the degradation of ascorbic acid in the

dark against citric acid (19ndash21) and tartaric acid (22ndash24) concentrations in

cream formulations

178

00

10

20

00 20 40 60

kob

s times

10

3 (

min

ndash1)

25-27

00

10

20

00 20 40 60

kob

s times

10

3 (

min

ndash1)

28-30

00

10

20

00 20 40 60

Boric acid concentration (M times 102)

kob

s times

10

3 (

min

ndash1)

30-33

Fig 44 Plots of first-order rate constants (kobs) for the degradation of ascorbic acid in the

dark against boric acid concentrations in cream formulations (25ndash33)

Glycerin

Propylene glycol

Ethylene glycol

179

00

04

08

12

12 14 16 18

Carbon chain length

k

times 1

02 (

Mndash

1 d

ayndash

1)

18-33

b

a

e

dc

Fig 45 Plots of k for degradation of ascorbic acid in the dark in creams (19ndash33) against

carbon chain length of emulsifier () Stearic acid () palmitic acid ()

myristic acid (a) CT (b) TA (c) BA with glycerin (d) BA with propylene

glycol (e) BA with ethylene glycol

CONCLUSIONS

AND

SUGGESTIONS

180

CONCLUSIONS

The main conclusions of the present study on the photodegradation of the ascorbic

acid in organic solvents and cream formulations are as follows

1 Identification of Photodegradation Products

The photodegradation of ascorbic acid in aqueous organic solvents and

laboratory prepared oil-in-water cream preparations on UV irradiation leads to the

formation of dehydroascorbic acid No further degradation products of dehydroascorbic

acid have been detected under the present experimental conditions The product was

identified by comparison of its Rf value and color of the spot with those of the authentic

compound by thin-layer chromatography and spectral changes

2 Assay of Ascorbic Acid

Ascorbic acid in aqueous organic solvents and cream preparations was assayed

in acidified methanolic solutions (pH 20) at 245 nm using a UV spectrophotometric

method Ascorbic acid in combination with other vitamins (riboflavin nicotinamide and

alpha-tocopherol) was assayed by the official iodimetric method due to interference by

these vitamins at the analytical wavelength Both analytical methods were validated

under the experimental conditions employed before their application to the assay of

ascorbic acid The recoveries of ascorbic acid in cream preparations are in the range of

90ndash96 and the reproducibility of both methods are within plusmn5 The F test and the t test

show that there is no significant difference between the precision of the two methods and

therefore these methods can be applied to the assay of ascorbic acid in cream

preparations with comparable results

181

3 Kinetics of Photodegradation

a) Photodegradation of ascorbic acid in organic solvents

Ascorbic acid degradation follows apparent first-order kinetics in aqueous

organic solvents A plot of the first-order rate constants (log kobs) versus solvent dielectric

constant is linear with positive slope indicating an increase in the rate with dielectric

constant On the contrary a plot of kobs verses reciprocal of solvent viscosity is linear with

a positive slope showing a decrease in the rate with solvent viscosity Thus the rate of

photodegradation of ascorbic acid (an oxidizable drug) depends on the solvent

characteristics

b) Photodegradation of ascorbic acid in cream preparations

Ascorbic acid has been found to follow apparent first-order kinetics in cream

preparations and the rate of degradation is affected by the following factors

i Effect of concentration

An apparent linear relationship has been observed between log kobs and

concentration (05ndash25) of ascorbic acid in a cream preparation Thus the rate of

degradation of ascorbic acid appears to be faster at a lower concentration

compared to that of a higher concentration on exposure to the same intensity of

light

ii Effect of carbon chain length of the emulsifying agent

The plots of kobs verses carbon chain length of the emulsifying agent show that the

photodegradation of ascorbic acid is affected in the order myristic acid gt stearic

acid gt palmitic acid This is predominantly due to the interaction of ascorbic acid

with palmitic acid and the carbon chain length (measure of relative polar

182

character) of the emulsifying acid probably does not play a part in the

photodegradation kinetics of ascorbic acid in creams This is evident from the

non-linear relationship between the rate constants for ascorbic acid degradation

and the carbon chain length of the emulsifying acids

iii Effect of viscosity

The values of kobs for the photodegradation of ascorbic acid in cream preparations

are in the order of humectant ethylene glycol gt propylene glycol gt glycerin

showing that the rates of degradation are influenced by the viscosity of the

humectant and decrease with an increase in the viscosity as observed in the case

of organic solvents

iv Effect of pH

The log kndashpH profiles for the photodegradation of ascorbic acid in creams

represent sigmoid type curves indicating an increase in the rate of oxidation of the

molecule with ionization (pH 42ndash70 557ndash999) The AHndash species appears to

be more susceptible to oxidation than the non-ionized molecule in the pH range

studied

v Effect of redox potential

The values of kobs show that the rate of photooxidation of ascorbic acid is

influenced by its redox potential which varies with pH The greater photostability

of ascorbic acid at pH 5ndash6 compared to that at pH 7 and above is due to its lower

rate of oxidation-reduction in the lower range The increase in the rate of

photooxidation with pH is due to a corresponding increase in the redox potential

of ascorbic acid

183

c) Photodegradation of ascorbic acid in the presence of other vitamins (riboflavin

nicotinamide alpha-tocopherol) in cream preparations

The photodegradation of ascorbic acid is affected by the presence of other

vitamins in creams The kinetic data on the photochemical interactions indicate that

riboflavin and nicotinamide act as photosensitizers in the degradation of ascorbic acid

and have an adverse effect on the photostability of the vitamin in creams Whereas

alpha-tocopherol exerts an inhibitory effect on the degradation of ascorbic acid by acting

as a redox partner in the creams Thus a combination of ascorbic acid and alpha-

tocopherol has a synergistic effect on the stabilization of ascorbic acid in creams These

vitamins do not appear to influence the rate of degradation of ascorbic acid in the dark

d) Photodegradation of ascorbic acid in the presence of citric acid tartaric acid and

boric acid in cream preparations

The rate of photodegradation of ascorbic acid in creams has been found to be

inhibited by the addition of compounds such as citric acid tartaric acid and boric acid in

creams These compounds show a stabilizing effect on the photodegradation of ascorbic

acid in the order citric acid gt tartaric acid gt boric acid The lower effect of boric acid

may be due to its interaction with the emulsifying agents and humectants Boric acid

exerts this effect by complex formation with ascorbic acid Citric acid and tartaric acid

are antioxidant synergists and in combination with ascorbic acid may exert a stabilizing

effect on its degradation

184

Salient Features of the Work

In the present work an attempt has been made to study the effects of solvent

characteristics formulation factors particularly the emulsifying agents in terms of the

carbon chain length and humectants in terms of viscosity medium pH drug

concentration redox potential and interactions with other vitamins and stabilizers on the

kinetics of photodegradation of ascorbic acid in cream preparations The study may

provide useful information to improve the photostability and efficacy of ascorbic acid in

cream preparations

SUGGESTIONS

The present work may provide guidelines for a systematic study of the stability of

drug substances in cream ointment preparations and the evaluation of the influence of

formulation variables such as emulsifying agents and humectants concentration pH

polarity viscosity redox potential on the rate of degradation and stabilization of drug

substances This may enable the formulator in the judicious design of formulations that

have improved stability and efficacy for therapeutic use The kinetic parameters may

throw light on the comparative stability of the preparations and help in the choice of

appropriate formulation ingredients

REFERENCES

186

Abe Y Horii H Taniguchi S (1986) A theoretical study of the oxidation mechanism

of triose reductone in reference to L-ascorbic acid Can J Chem 64 360-365

Ahmad I (1968) A Study of the Degradation of Riboflavin and Related Compounds

PhD Thesis University of London London

Ahmad I Rapson HDC (1990) Multicomponent spectrophotometric assay of

riboflavin and photoproducts J Pharm Biomed Anal 8 217-223

Ahmad I Vaid FHM (2006) Photochemistry of flavins in aqueous and organic

solvents In Silva E Edwards AM Eds Flavins Photochemistry and

Photobiology Royal Society of Chemistry Cambridge Chap 2

Ahmad I Tollin G (1981) Solvent effect on flavin electron transfer reactions

Biochemistry 20 5925-5928

Ahmad I Jamil N Zoha SMS (1973) Statistical evaluation of antioxidants used in

the manufacture of ascorbic acid tablets J Sci Univ Kar 2 269-287

Ahmad I Fasihullah Q Vaid FHM (2004b) A study of simultaneous photolysis and

photoaddition reactions of riboflavin in aqueous solution J Photochem Photobiol B

Biol 75 13-20

Ahmad I Fasihullah Q Vaid FHM (2005) Effect of phosphate buffer on

photodegradation reactions of riboflavin in aqueous solution J Photochem

Photobiol B Biol 78 229-234

Ahmad I Fasihullah Q Vaid FHM (2006a) Effect of light intensity and wavelength

on photodegradation reactions of riboflavin in aqueous solution J Photochem

Photobiol B Biol 82 21-27

187

Ahmad I Fasihullah Q Vaid F H M (2006b) Photolysis of formylmethylflavin in

aqueous and organic solvents Photochem Photobiol Sci 5 680-685

Ahmad I Ahmed S Sheraz MA Vaid FHM (2008) Effect of borate buffer on the

photolysis of riboflavin in aqueous solution J Photochem Photobiol B Biol 93 82-

87

Ahmad I Ahmed S Sheraz MA Vaid FHM (2009c) Borate toxicity effect on

drug stability and analytical applications In Chung MP Ed Handbook on

Borates Chemistry Production and Applications Nova Science Publishers Inc

New York in press

Ahmad I Sheraz MA Ahmed S Vaid FHM (2009d) Analytical applications of

borates Mater Sci Res J in press

Ahmad I Fasihullah Q Noor A Ansari IA Ali QNM (2004a) Photolysis of

riboflavin in aqueous solution a kinetic study Int J Pharm 280 199-208

Ahmad I Ahmed S Sheraz MA Aminuddin M Vaid FHM (2009a) Effect of

caffeine complexation on the photolysis of riboflavin in aqueous solution a kinetic

study Chem Pharm Bull in press

Ahmad I Ahmed S Sheraz MA Vaid FHM Ansari IA (2009b) Effect of

divalent ions on photodegradation kinetics and pathways of riboflavin in aqueous

solution Photochem Photobiol Sci accepted

Ahmed S (2009) Effect of Complexing Agents on the Photodegradation of Riboflavin

in Aqueous Solution PhD Thesis Baqai Medical University Karachi

Ahmad T (1978) Studies on the Stability of Sulphacetamide and Degradation Products

PhD Thesis University of Karachi Karachi

188

Ahmad T Ahmad I (1983) The effect of antioxidants on the photolysis of aqueous

sulfacetamide and sulfanilamide solutions Pol J Pharmacol Pharm 35 69-75

Albini A Fasani E Eds (1998) Drugs Photochemistry and Photostability The Royal

Society of Chemistry Cambridge

Allwood MC (1984a) Factors influencing the stability of ascorbic acid in total

parenteral nutrition infusions J Clin Hosp Pharm 9 75-85

Allwood MC (1984b) Compatibility and stability of TPN mixtures in big bags J Clin

Pharm Ther 9 181-198

Allwood MC Kearney MCJ (1998) Compatibility and stability of additives in

parenteral nutrition admixtures Nutrition 14 697-706

Al-Meshal IA Hassan MMA (1982) Ascorbic Acid In Florey K Ed Analytical

Profiles of Drug Substances Vol 11 Academic Press New York pp 45-78

Anderson NH Johnston D McLelland MA Munden P (1991) Photostability

testing of drug substances and drug products in UK pharmaceutical laboratories J

Pharm Biomed Anal 9 443-449

Ansari IA (2002) Photolysis and Interactions of Cyanocobalamin with some B-

Vitamins and Ascorbic Acid in Parenteral Solutions PhD Thesis University of

Karachi Karachi

Ansari IA Vaid Vaid HM Ahmad I (2004) Spectral Study of Photolysis of

Aqueous Cyanocobalamin Solutions in Presence of Vitamins B and C Pak J Pharm

Sci 17 83-93

Arcus CL Zilva SS (1940) The photochemical decomposition of L-ascorbic acid II

Biochem J 34 61-66

189

Arger CB Rupp D Lo P (1996) Preservation of dispersed systems In Liberman

HA Rieger MM Banker GS Eds Pharmaceutical Dosage Forms Disperse

Systems 2nd ed vol1 Marcel Dekker New York Chap9

Asard H May J Smirnoff N Eds (2004) Vitamin C Function and Biochemistry in

Animals and Plants BIOS Scientific Publishers New York

Asker AF Canady D Cobb C (1985) Influence of DL-methionine on the

photostability of ascorbic acid solutions Drug Dev Ind Pharm 11 2109-2125

Ault RG Baird DK Carrington HC Haworth WN Herbert R Hirst EL

Percival EGV Smith F Stacey M (1933) Synthesis of d- and of l-ascorbic acid

and of analogous substances J Chem Soc 1419-1423

Austria R Semenzato A Bettero A (1997) Stability of vitamin C derivatives in

solution and topical formulations J Pharm Biomed Anal 15 795-801

Baertschi SW Alsante KM (2005) Stress testing The chemistry of drug degradation

In Baertschi SW Ed Pharmaceutical Stress testing Predicting Drug Degradation

Taylor amp Francis London Chap 3

Bailey CW Bright JR Jasper JJ (1945) A study of the binary system nicotinamide-

ascorbic acid J Am Chem Soc 67 1184-1186

Ball GFM (2006) Vitamins in Foods Analysis Bioavailability and Stability CRC

Press Boca Raton FL Chap 15

Banhegyi G Marcolongo P Puskas F Fulceri R Mandl J Benedetti A (1998)

Dehydroascorbate and ascorbate transport in rat liver microsomal vesicles J Biol

Chem 273 2758-2762

190

Bardolph EM Taylor RH (1997) Sailors scurvy science and authority J R Soc

Med 90 238

Barel AO Paye M Maibach HI Eds (2001) Handbook of Cosmetic Science and

Technology Marcel Dekker New York

Barry BW (1983) Dermatological Formulations Percutaneous Absorption Marcel

Dekker New York

Barry BW (2002) Transdermal drug delivery In Aulton ME Ed Pharmaceutics

The Science of Dosage Form Design Churchill Livingstone London Chap 33

Basu TK Temple NJ Garg ML (1999) Antioxidants in Human Health and

Disease CABI Publishing Oxford

Becher P (1965) Emulsions Theory and Practice Reinhold New York p 1

Beissenhirtz M Scheller F Lisdat F (2003) Immobilized cytochrome c sensor in

organicaqueous media for the characterization of hydrophilic and hydrophobic

antioxidants Electroanalysis 15 1425-1435

Berlett BS Stadtman ER (1997) Protein oxidation in aging disease and oxidative

stress J Biol Chem 272 20313-20316

Betageri G Prabhu S (2002) Semisolid preparations In Swarbrick J Boylan JC

Eds Encyclopedia of Pharmaceutical Technology 2nd ed Marcel Dekker New

York pp 2436-2457

Bielski BHJ (1982) Chemistry of ascorbic acid radicals In Seib PA Tolbert BM

Eds Ascorbic Acid Chemistry Metabolism and Uses Advances in Chemistry

Series American Chemical Society Washington DC pp 81-100

191

Bielski BHJ Allen AO Schwartz HA (1981) Mechanism of disproportionation of

ascorbate radicals J Am Chem Soc 103 3516-3518

Billany M (2002) Suspensions and emulsions In Aulton ME Ed Pharmaceutics

The Science of Dosage Form Design 2nd ed Churchill Livingstone London Chap

23

Bisby RH Morgan CG Hamblett I Gorman AA (1999) Quenching of singlet

oxygen by trolox C ascorbate and amino acids effect of pH and temperature J Phys

Chem 103 7454-7459

Bissett DL (2006) Anti-aging skin care formulations In Draelos ZD Thaman LA

Eds Cosmetic Formulation of Skin Care Products Taylor amp Francis Group New

York Chap 11

Blaugh SM Hajratwala B (1972) Kinetics of aerobic oxidation of ascorbic acid J

Pharm Sci 61 556-562

Block LH (1996) Pharmaceutical emulsions and microemulsions In Lieberman HA

Rieger MM Banker GS Eds Pharmaceutical Dosage Forms Disperse Systems

Vol 2 2nd ed Chap 2

Bolliger HR Konig A (1969) Water-soluble vitamins In Stahl E Ed Thin-layer

Chromatography Springer-Verlag Berlin pp 304-306

Bonwstra JA De Graaff A Gooris GS Nijsse J Wiechers JW Van Aelst AC

(2003) Water distribution and related morphology in human stratum corneum at

different hydration levels J Invest Dermatol 120 750-758

Bors SR (2005) Scurvy Thomas Dunne Books St Martinrsquos Griffen New York

192

Bors W Buettner GR (1997) The vitamin C radical and its reactions In Packer L

Fuchs J Eds Vitamin C in Health and Disease Marcel Dekker New York Chap

4

Braun L Puskas F Csala M Meszaros G Mandl J Banhegyi G (1997)

Ascorbate as a substrate for glycolysis or gluconeogenesis evidence for an interorgan

ascorbate cycle Free Radic Biol Med 23 804-808

British Pharmacopoeia (2009) Her Majestyrsquos Stationary Office London Electronic

version

Buettner G (1993) The pecking order of free radicals and antioxidants lipid

peroxidation α-tocopherol and ascorbate Arch Biochem Biphys 300 535-543

Buettner GR Schafer FQ (2004) Ascorbate as an antioxidant In Asard H May

JM Smirnoff N Eds Vitamin C Function and Biochemistry in Animals and

Plants BIOS Scientific Publishers Oxford Chap 10

Burke KE (2006) Topical nutritional antioxidants In Draelos ZD Thaman LA

Eds Cosmetic Formulation of Skin Care Products Taylor amp Francis Group New

York Chap 21

Burns JJ Ed (1961) Vitamin C Ann NY Acad Sci 92 1

Byrom P Turnbull JH (1967) Excited states of flavin coenzymes II Anaerobic

oxidation of amino acids by excited riboflavin derivatives Photochem Photobiol 6

125-131

Cadenas E Packer L (2002) Handbook of Antioxidants 2nd ed Marcel Dekker New

York Chap 7-9

193

Capellmann M Bolt HM (1992) Simultaneous determination of ascorbic acid and

dehydroascorbic acid by HPLC with postcolumn derivatisation and fluorometric

detection Fresenius J Anal Chem 342 462-466

Cairns WL Metzler DE (1971) Photochemical degradation of flavins VI A new

photoproduct and its use in studying photolytic mechanism J Am Chem Soc 93

2772-2777

Carlotti ME Ugazio E Sapino S Peira E Gallarate M (2006) Photodegradation

of retinol and anti-aging effectiveness of two commercial emulsions J Cosmet Sci

57 261-277

Carpenter K (1986) The History of Scurvy and Vitamin C Cambridge University Press

Cambridge

Carr AC Zhu BZ Frei B (2000) Potential antiatherogenic mechanisms of ascorbate

(vitamin C) and alpha-tocopherol (vitamin E) Circ Res 87 349-354

Carstensen JT (1972) Theory of Pharmaceutical Systems Academic Press New York

pp 211-214

Chaudhri SK Jain NK (2009) History of cosmetics Asian J Pharm 3 164-167

Chessex P Lovoic JC Roulean T Brochu P St Louis P Levy E Al Varez F

(2002) Photooxidation of parenteral multivitamins induced hepatic steatosis in a

neonatal guinea pig model of intravenous infusion Pediatr Res 52 958-963

Connors KA Amidon GL Stella VJ (1986) Chemical Stability of Pharmaceuticals

A Handbook for Pharmacists 2nd ed Wiley New York pp 105-106 142 208-220

194

Dabrowski K Huiterleitner S (1989) Application of a simultaneous assay of ascorbic

acid dehydroascorbic acid and ascorbic sulfate in biological matter Analyst

(London) 114 83-87

Dale JK Booth RE (1976) In Hoover JE Ed Dispensing of Medication 8th ed

Mack Easton Pennsylvania pp 479-481

Darr D Dunston S Faust H Pinnell S (1996) Effectiveness of antioxidants (vitamin

C and E) with and without sunscreens as topical photoprotectants Acta Derm

Venereol 76 264-268

Davey MW Montagu MV Inze D Sanmartin M Kanellis A Smirnoff N

Benzie IJJ Strain JJ Favell D Fletcher J (2000) Plant L-ascorbic acid

chemistry function metabolism bioavailability and effect of processing J Sci Food

Agr 80 825-860

Davies MB Austin J Partridge DA (1991) Vitamin C Its Chemistry and

Biochemistry The Royal Society of Chemistry Cambridge

De La Rochette A Birlouez-Aragon I Silva E Morliere P (2003) Advanced

glycation endproducts as UVA photosensitizers of tryptophan and ascorbic acid

consequences for the lens Biochem Biophys Acta 1621 235-241

De La Rochette A Silva E Birlouez-Aragon I Mancini M Edwards AM

Morliere P (2000) Riboflavin photodegradation and photosensitization effects are

highly dependent on oxygen and ascorbate concentration Photochem Photobiol 72

815-820

195

De Vries H Beijersbergen Van Henegouwen GMJ Huff FA (1984) Photochemical

decomposition of chloramphenicol in a 025 eye drop and in a therapeutic

intraocular concentration Int J Pharm 20 265-271

Dean RT Fu S Stocker R Davies MJ (1997) Biochemistry and pathology of

radicalmediated protein oxidation Biochem J 324 1-18

DeRitter E (1982) Vitamins in pharmaceutical formulations J Pharm Sci 71 1073-

1096

Deutsch JC (1998a) Ascorbic acid oxidation by hydrogen peroxide Anal Biochem

255 1-7

Deutsch JC (1998b)Spontaneous hydrolysis and dehydration of dehydroascorbic acid

in aqueous solution Anal Biochem 260 223-229

Deutsch JC (1998c)oxygen-accepting antioxidants which arise during ascorbate

oxidation Anal Biochem 265 238-245

Dodsun KY Young ER Soliman AGM (1992) Determination of total vitamin C

in various food matrices by liquid chromatography and fluorescence detection J

AOAC Int 75 887-891

Dollery C Ed (1999) Therapeutic Drugs 2nd ed Vol 1 Churchill Livingstone

London pp A213-A216

Doores S (2002) pH control agents and acidulants In Branen LA Davidson PM

Salminen S Thorngate JH Eds Food Additives Marcel Dekker New York p

637

Eberlein-Koumlnig B Ring J (2005) Relevance of vitamins C and E in cutaneous

photoprotection J Cosmet Dermatol 4 4-9

196

Ehrlich M Rao J Pabby A Goldman MP (2006) Improvement in the appearance

of wrinkles with topical transforming growth factor beta(1) and l-ascorbic acid

Dermatol Surg 32 618-625

EIRI Board (2007) Profitable Small Scale Manufacture of Cosmetics Engineers India

Research Institute Nai Sarak Delhi

Eitenmiller RR Ye L Landen WOJr (2008) Vitamin Analysis for the Health and

Food Sciences 2nd ed CRC Press Boca Raton FL Chap 5

Elia S (2009) Nutrition In Kumar P Clark M Eds Kumar and Clarkrsquos Clinical

Medicine 7th ed Elsevier Limited Chap 5

Elmore AR (2005) Final report of the safety assessment of L-ascorbic acid calcium

ascorbate magnesium ascorbate magnesium ascorbyl phosphate sodium ascorbate

and sodium ascorbyl phosphate as used in cosmetics Int J Toxicol 24 51-111

Erb C Nau-Staudt K Flammer J Nau W (2004) Ascorbic acid as a free radical

scavenger in porcine and bovine aqueous humour Ophthal Res 36 38-42

Espinal-Perez LE Moncada B Castanedo-Cazares JP (2004) A double-blind

randomized trial of 5 ascorbic acid vs 4 hydroquinone in melasma Int J

Dermatol 43 604-607

Fall HH Petering HG (1956) Metabolic inhibitors 1 67-Dimethyl-9-

formylmethylisoalloxazine67-dimethyl-9-(2-hydroxyethyl)-isoalloxazine and

derivatives J Am Chem Soc 78 377-381

Farahmand S Tajerzadeh H Farboud ES (2006) Formulation and evaluation of a

vitamin C multiple emulsion Pharm Dev Technol 11 255-261

197

Fasani E Albini A (2005) Photostability Stress Testing Predicting Drug Degradation

Taylor amp Francis London Chap 10

Fasihullah Q (1988) Photolysis of Flavins in Aqueous and Organic Solvents PhD

Thesis University of Karachi Karachi

Fasman GD Ed (1976) CRC Handbook of Biochemistry and Molecular Biology 3rd

ed Physical Chemical Data Vol 1 CRC Press Cleveland Ohio pp 122-130

Ferreira SLC Bandeira MLSF Lemos VA Dos Santos HC Spinola Costa

AC De Jesus DS (1997) Sensitive spectrophotometric determination of ascorbic

acid in fruit juices and pharmaceutical formulations using 2-(5-bromo-2-pyridylazo)-

5-diethylaminophenol (Br-PADAP) Anal Bioanal Chem 357 1174-1178

Fessenden RW Verma NC (1978) A time-resolved electron spin resonance study of

the oxidation of ascorbic acid by hydroxyl radical Biophys J 24 413-430

Finholt P Paulssen RB Higuchi T (1963) Rate of anaerobic degradation of ascorbic

acid in aqueous solution J Pharm Sci 52 948-954

Florence AT Attwood D (2006) Drug Stability Physicochemical Principles of

Pharmacy 4th ed Pharmaceutical Press London p 411

Flynn GL (2002) Cutaneous and transdermal delivery-processes and systems of

delivery In Banker GS and Rhodes CT Eds Modern Pharmaceutics Marcel

Dekker New York Chap 8

Foote CS (1991) Definition of Type I and Type II Photosensitized oxidation

Photochem Photobiol 54 659

Fornaro A Coicher N (1998) L-ascorbic acid complexation and redox reactions with

some transition metal ions Quni Nova 21 642-650

198

Fraga CG Motchink PA Wyrobek AJ Rempel DM Ames BN (1996)

Smoking and low antioxidant levels increase oxidative damage to sperm DNA

Mutat Res 351 199-203

Fraga CG Motchnik PA Shigenaga MK Helbock HJ Jacob RA Ames BN

(1991) Ascorbic acid protects against endogenous oxidative DNA damage in human

sperm Proc Natl Acad Sci USA 88 11003-11006

Frenich AG Torres ME Vega AB Vidal JL Bolanos PP (2005) Determination

of ascorbic acid and carotenoids in food commodities by liquid chromatography with

mass spectrometry detection J Agric Food Chem 53 7371-7376

Fung HL (1990) Drug stability In Banker GS Rhodes CT Eds Modern

Pharmaceutics 2nd ed Marcel Dekker New York Chap 6

Gallarate M Carlotti ME Trotta M Bovo S (1999) On the stability of ascorbic

acid in emulsified systems for topical and cosmetic use Int J Pharm 188 233-241

Ganshirt H Malzacher A (1960) Separation of several vitamins of the B group and C

by chromatography Naturwiss 47 279-280

Gardner WH (1972) Acidulants in food processing In Furia TE Ed Handbook of

Food Additives 2nd ed Vol 1 CRC Press Cleveland OH pp 225ndash270

Garrett ER (1967) Kinetics and mechanisms in stability of drugs In Bean HS

Beckett AH Carless JE Eds Advances in Pharmaceutical Sciences Vol 2

Academic Press London Chap 1

Garti N Aserin A (1996) Pharmaceutical emulsions double emulsions and

microemulsions In Benita S Ed Microencapsulation Methods and Industrial

Applications Marcel Dekker New York pp 411-534

199

Gensler M Rossmann A Schmidt HL (1995) Detection of added L-ascorbic acid in

fruit juice by isotope ratio mass spectrometry J Agric Food Chem 43 2662-2666

Gibbons E Allwood MC Neal T Hardy G (2001) Degradation of dehydroascorbic

acid in parenteral nutrition mixtures J Pharm Biomed Anal 25 605-611

Guitton C Gros P Comtat M (2007) Direct electrochemical measurements in dermo-

cosmetic creams In Alegret Merkoci Eds Comprehensive Analytical Chemistry

Elsevier Amsterdam 49 e59-e68

Guttman DE Brooke D (1963) Solution phase interaction of nicotinamide with

ascorbic acid J Pharm Sci 52 941-945

Gyorgy P Pearson WN Eds (1967) Vitamin Methods Vol VII Academic Press

New York pp 27-49

Hagg M Ylikoski S Kumpulainen J (1994) Vitamin C and α- and β-carotene in

vegetables consumed in Finland during 1988-1989 and 1992-1993 J Food Compos

Anal 7 252-259

Hagg M Ylikoski S Kumpulainen A (1995) Vitamin C content in fruits and berries

consumed in Finland J Food Compos Anal 8 12-20

Hall NK Chapman TM Kim HJ Min DB (2009) Antioxidant mechanisms of

Trolox and ascorbic acid on the oxidation of riboflavin in milk under light Food

Chem in press doi 101016jfoodchem200905017

Halliwell B (1996) Vitamin C antioxidant or pro-oxidant in vivo Free Radic Res 25

439-454

200

Halliwell B Whiteman M (1997) Antioxidant and prooxidant properties of vitamin C

In Packer L Fuchs J Eds Vitamin C in Health and Disease Marcel Dekker New

York pp 25-94

Halver JE Felton SP (2001) Analytical enigmas in assaying for vitamers C In

Dabrowski K Ed Ascorbic Acid in Aquatic Organisms CRC Press Boca Raton

Chap 3

Hanck A Ed (1982) Vitamin C New Clinical Applications in Immunology Lipid

Metabolism and Cancer Hans Huber Bern

Hashmi MH (1973) Assay of Vitamins in Pharmaceutical Preparations John Wiley

New York Chap 13

Hatchard CG Parker CA (1956) A new sensitive chemical actinometer II Potassium

ferrioxalate as a standard chemical actinometer Proc R Soc London Ser A A235

518-536

Heber GK Markovic B Hayes A (2006) An immunohistological study of anhydrous

topical ascorbic acid compositions on ex vivo human skin J Cosmet Dermatol 5

150-156

Heelis PF Parsons BJ Phillips GO McKellar JF (1981) The flavin sensitized

photooxidation of ascorbic acid a continuous and flash photolysis study Photochem

Photobiol 37 7-13

Henry JA Ed (1997) The New Guide to Medicines and Drugs The British Medical

Association 4th ed Dorling Kindersley London p 434

Hickey S Roberts H (2004) Ascorbate-The Science of Vitamin C Electronic Version

(ISBN 1-4116-0724-4)

201

Higdon JV Frei B (2002) The Antioxidant Vitamin C and E AOAC Press Arlington

VA pp 1-16

Hirst EL Zilva SS (1933) Ascorbic acid as the anti-scorbutic factor Biochem J 27

1271-1278

Ho AHL Puri A Sugden JK (1994) Effect of sweetening agents on the light

stability of aqueous solutions of L-ascorbic acid Int J Pharm 107 199-203

Holmstrom B Oster G (1961) Riboflavin as an electron donor in photochemical

reactions J Am Chem Soc 83 1867-1871

Homann P Gaffron H (1964) Photochemistry and metal catalyzes studies in a flavin

sensitized oxidation of ascorbic acid Photochem Photobiol 3 499-515

Hughes DE (1985) Irreversible reaction kinetics of the aerobic oxidation of ascorbic

acid Anal Chem 57 555-558

Humbert PG Haftek M Creidi P Lapiegravere C Nusgens B Richard A Schmitt D

Rougier A Zahouani H (2003) Topical ascorbic acid on photoaged skin Clinical

topographical and ultrastructural evaluation double-blind study vs placebo Exp

Dermatol 12 237-244

ICH Harmonized Tripartite Guideline (1997) QIB Photostability Testing of New Drug

Substances and Products Federal Register 62 27115-27122

Ichikawa M Ide N Shiraishi S Ono K (2005) Effect of various halide salts on the

incompatibility of cyanocobalamin and ascorbic acid in aqueous solution Chem

Pharm Bull 53 688-690

202

Ihara H Shino Y Aoki Y Hashizume N Minegishi N (2000) A simple and rapid

method for the routine assay of total ascorbic acid in serum and plasma using

ascorbate oxidase and o-phenylenediamine J Nutr Sci Vitaminol 46 321-324

Im-Emsap W Paeratakul O Siepmann J (2002) Disperse Systems In Banker GS

Rhodes CT Ed Modern Pharmaceutics Maecel Dekker New York Chap 9

Irache JM Ezpeleta I Vega FA (1993) HPLC determination of antioxidant

synergists and ascorbic acid in some fatty pharmaceuticals cosmetics and food

Chromatographia 35 232-23

Iwase H Ono I (1994) Determination of ascorbic acid in human plasma by high-

performance liquid chromatography with electrochemical detection using a

hydroxyapatite cartridge for precolumn deproteinization J Chromatogr B Biomed

Appl 655 195-200

Jain S Tiwari AK Jain NK (2006) Topical products In Jain NK Ed

Pharmaceutical Product Development CBS Publishers New Delhi Chap 7

Jamil N Ahmad I (1984) Moisture content of ascorbic acid tablets stored at various

temperatures and humidities Pak J Sci Ind Res 27 252

Jamil N Ahmad I Beg AE (1980a) The effect of UV and tungsten light on physical

characteristics of ascorbic acid tablets Laboratory prepared samples IV Kar Univ

J Sci 8 77-87

Jamil N Ahmad I Beg AE (1980b) The effect of UV and tungsten light on physical

characteristics of ascorbic acid tablets Commercial samples III Kar Univ J Sci 8

117-126

203

Jentzsch A Aikens P (2005) RetiSTARTM

for cosmetic formulations-stabilized retinol

In Rosen MR Ed Delivery System Handbook for Personal Care and Cosmetic

Products Technology Applications and Formulations William Andrew Publishing

Norwich NY pp 862-866

Johnston CS Steinberg FM Rucker RB (2007) Ascorbic Acid In Zempleni J

Rucker RB McCormick DB Suttie JW (Eds) Handbook of Vitamins 4th ed

CRC Press Boca Raton FL Chap 15

Jung MY Kim SM Kim SY (1995) Riboflavin-sensitized photooxidation of

ascorbic acid kinetics and amino acid effects Food Chem 53 397-403

Kall MA Anderson CJ (1999) Improved method for simultaneous determination of

ascorbic acid and dehydroascorbic acid isoascorbic acid and dehydroisoascorbic acid

in food and biological samples J Chromatogr B Biomed Sci Appl 730 101-111

Kampfenkel K Van Montagu M Inze D (1995) Extraction and determination of

ascorbate and dehydroascorbate from plant tissue Anal Biochem 225 165-167

Kaplan DL Moloney SJ Troy WR Dickens MS Pinnell SR (1989) A new

stabilized ascorbic acid solution percutaneous absorption and effect on relative

collagen synthesis J Cutan Aging Cosmet Derm I 115-121

Karatepe M (2004) Simultaneous determination of ascorbic acid and free

malondialdehyde in human serum by HPLC-UV LC GC (North America) 22 362-

365

Karlsen J (1996) Light-activated drugs and drug formulations in drug targeting In

Tonneson HH Ed The Photostability of Drugs and Formulations Taylor amp

Francis London Chap 8

204

Kassem MA Kassem AA Ammar HO (1969a) Studies on the stability of

injectable L-ascorbic acid solutions I Effect of pH solvent light and container

Pharm Acta Helv 44 611-623

Kassem MA Kassem AA Ammar HO (1969b) Studies on the stability of

injectable L-ascorbic acid solutions II Effect of metal ions and oxygen content of

solvent water Pharm Acta Helv 44 667-675

Kassem MA Kassem AA Ammar HO (1972) Studies on the stability of injectable

L-ascorbic acid solutions 3 Effect of metal-complexing agents Pharm Acta Helv

47 89-97

Kaushansky K Kipps TJ (2006) Hematopoietic agents growth factors minerals and

vitamins In Brunton L Ed Goodman amp Gilmans The Pharmacologic Basis of

Therapeutics 11th ed digital ed McGraw-Hill Companies USA Chap 53

Kearney MCJ Allwood MC Martin H Neal T Hardy G (1998) The influence

of amino acid source on the stability of ascorbic acid in TPN mixtures Nutrition 14

173-178

Kim H Kirshenbaum LJ Rosenithol I Riesz P (1993) Photosensitized formation

of ascorbate radicals by riboflavin an ESR study Photochem Photobiol 57 777-

784

Kimoto K Terada S Yamaguchi T (1997) Analysis of ascorbic acid

dehydroascorbic acid and transformation products by ion-pairing high-performance

liquid chromatography with multi-wavelength ultraviolet and electrochemical

detection Methods Enzymol 279 3-12

205

King CG Burns JJ Ed (1975) Second Conference on Vitamin C Ann N Y Acad

Sci 258

Kitagawa Y (1968) The effects of ultraviolet rays on vitamin C Eiyo To Shokuryo 20

416-421 through CA (1968) 68 111560f

Klick S Muijselaar PG Waterval J EichingerT Korn C Gerding TK Debets

AJ de Griend CS van den Beld C Somsen GW De Jong GJ (2005)Toward

a generic approach for stress testing of drug substances and drug products Pharm

Tech 29 48-66

Konings EJ (2006) Water-soluble vitamins J AOAC Int 89 285-288

Kwon BM Foote CS (1988) Chemistry of singlet oxygen 50 Hydroperoxide

intermediates in the photooxygenation of ascorbic acid J Am Chem Soc 110

6582-6583

Kwon BM Foote CS Khan SI (1989) Photooxygenation of ascorbic acid

derivatives and model compounds J Am Chem Soc 111 1854-1860

Laborie S Lavoie JC Chessex P (1998) Paradoxical role of ascorbic acid and

riboflavin in solutions of total parenteral nutrition implication in photoinduced

peroxide generation Pediatr Res 43 601-601

Laborie S Lavoie JC Pineault M Chessex P (1999) Protecting solutions of

parenteral nutrition from peroxidation JPEN 23 104-108

Laborie S Lavoie JC Pineault M Chessex P (2000) Contribution of multivitamin

air and light in the generation of peroxides in adult and neonatal parenteral nutrition

solutions Ann Pharmacother 34440-445

206

Laborie S Lavoie JC Rouleau T Chessex P (2002) Multivitamin solutions for

enteral supplementation a source of peroxides Nutrition 18 470-473s

Lachman L DeLuca P Akers MJ (1986) Kinetic principles and stability testing In

Lachman L Liberman HA Kanig JL Eds The Theory and Practice of

Industrial Pharmacy 3rd ed Lea amp Febiger Philadelphia Chap 26

Laidler KJ (1987) Chemical Kinetics 3rd ed Harper and Row New York pp 183-

185

Lambert WE de Leenheer A (1992) In Nollet LML Ed Food Analysis by HPLC

Marcel Dekker New York Chap 10

Lavoie JC Chessex P Rouleau T Migneault D Comte B (2004) Light-induced

byproducts of vitamin C in multivitamin solutions Clin Chem 50 135-140

Lee JS Kim JW Han SH Chang IS Kang HH Lee OS Oh SG Suh KD

(2004) The stabilization of L-ascorbic acid in aqueous solution and water-in-oil-in-

water double emulsion by controlling pH and electrolyte concentration J Cosmet

Sci 55 1-12

Lee KM Kwon JY Lee KW Lee HJ (2009) Ascorbic acid 6-palmitate

suppresses gap-junctional intercellular communication through phosphorylation of

connexin 43 via activation of the MEK-ERK pathway Mutat Res 660 51-56

Leacutevecircque N Mac-Mary S Muret P Makki S Aubin F Kantelip JP Heusegravele C

SS Humbert P (2005) Evaluation of a sunscreen photoprotective effect by ascorbic

acid assessment in human dermis using microdialysis and gas chromatography mass

spectrometry Exp Dermatol 14 176-181

207

Levine M (1986) New concepts in the biology and biochemistry of ascorbic acid N

Engl J Med 314 892-902

Lewin S (1976) Vitamin C Its Molecular Biology and Medical Potential Academic

Press New York

Liau LS Lee BL New AL Ong CN (1993) Determination of plasma ascorbic

acid by high-performance liquid chromatography with ultraviolet and electrochemical

detection J Chromatogr 612 63-70

Lin JY Selim MA Shea CR Grichnik JM Omar MM Monteiro-Riviere NA

Pinnell SR (2003) UV photoprotection by combination topical antioxidants vitamin

C and vitamin E J Am Acad Dermatol 48 866-874

Lin FH Lin JY Gupta RD Tournas JA Burch JA Selim MA Monteiro-

Riviere NA Grichnik JM Zielinski J Pinnell SR (2005) Ferulic acid

stabilizes a solution of vitamins C and E and doubles its photoprotection of skin J

Invest Dermatol 125 826-832

Lindahl T (1993) Instability and decay of the primary structure of DNA Nature 362

709-715

Litner CJ (1973) Drug Product Stability In Cooper MS Ed Quality Control in

Pharmaceutical Industry Vol 2 Academic Press New York p 185

Liu TZ Chin N Kiser MD Bigler WN (1982) Specific spectrophotometry of

ascorbic acid in serum or plasma by use of ascorbate oxidase Clin Chem 28 2225-

2228

Lu GW Flynn GL (2009) Cutaneous and transdermal delivery-processes and

systems of delivery In Florence AT Siepmann J Eds Modern Pharmaceutics-

208

Applications and Advances Vol 2 5th ed Informa Healthcare Inc New York

Chap 3

Lykkesfeldt J (2000) Determination of ascorbic acid and dehydroascorbic acid in

biological samples by high-performance liquid chromatography using subtraction

methods reliable reduction with tris [2-carboxyethyl] phosphine hydrochloride Anal

Biochem 282 89-93

Macek TJ (1960) Stability problems with some vitamins in pharmaceuticals Am J

Pharm 132 433-455

Mader WJ (1961) Vitamins In Higuchi T Brochmann-Hanssen E Eds

Pharmaceutical Analysis Interscience New York pp 689-693

Maggi N Pasqualucci CR Ballotta R Sensi P (1966) Rifampicin a new orally

active rifamycin Chemotherapy 11 285-292

Maia AM Baby AR Pinto CA Yasaka WJ Suenaga E Kaneko TM Velasco

MV (2006) Influence of sodium metabisulfite and glutathione on the stability of

vitamin C in OW emulsion and extemporaneous aqueous gel Int J Pharm 322

130-135

Mancini M Edwards AM Becker MI De Ioannes A Silva E (2000) Reactivity

of monoclonal antibodies against a tryptophan-riboflavin adduct toward irradiated

and non-irradiated bovine-eye-lens protein fractions an indicator of in vivo visible

light mediated phototransformations J Photochem Photobiol B Biol 55 9-15

Manoharan M Schwille PO (1994) Measurement of ascorbic acid in human plasma

and urine by high performance liquid chromatography Results in healthy subjects

209

and patients with idiopathic calcium urolithiasis J Chromatogr B Biomed Appl

654 134-139

Margolis SA Schmidt HL (1996) Measurement of ascorbic acid in human plasma

and serum stability intralaboratory repeatability and interlaboratory reproducibility

Clin Chem 42 1257-1262

Mason P (2007) Dietary Supplements 3rd ed Pharmaceutical Press pp 330-339

Massad WA Bertolotti S Garcia N (2004) Kinetics and mechanism of the vitamin

B2-sensitized photooxidation of isoproterenol Photochem Photobiol 79 428-433

Matsubayashi T Sakaeda T Kita T Kurimoto Y Nakamura T Nishiguchi K

Fujita T Kamiyama F Yamamoto A Okumura K (2003) Intradermal

concentration of hydroquinone after application of hydroquinone ointments is higher

than its cytotoxic concentration Biol Pharm Bull 26 1365-1367

McBride MM Moore WM (1967) The photochemistry of riboflavin II

Polarographic studies on the hydroxyethyl and formylmethyl analogues of riboflavin

Photochem Photobiol 6 103-113

McDowell LR (2000) Vitamins in Animal and Human Nutrition 2nd ed Iowa State

University Press Chap 15

Meister A (1994) Glutathione-ascorbic acid antioxidant system in animals J Biol

Chem 269 9397-9400

Meister A (1995) Mitochondrial changes associated with glutathione deficiency

Biochim Biophys Acta 1271 35-42

Min DB Boff JM (2002) Chemistry and reaction of singlet oxygen in foods

CRFSFS 1 58-72

210

Moeslinger T Brunner M Volf I Spieckermann PG (1995) Spectrophotometric

determination of ascorbic acid and dehydroascorbic acid Clin Chem 41 1177-1181

Moffat AC Osselton MD Widdop B (2004) Clarkersquos Analysis of Drugs and

Poisons 3rd ed Pharmaceutical Press London pp 649-650

Moore DE (2004) Standardization of kinetic studies of photodegradation reactions In

Tonnesen HH Ed Photostability of Drugs and Drug Formulations 2nd ed CRC

Press Boca Raton FL Chap 3

Moura T Gaudy D Jacob M Cassanas G (1994) pH influence on the stability of

ascorbic acid spray-drying solutions Pharm Acta Helv 69 77-80

Murray JC Burch JA Streilein RD Iannacchione MA Hall RP Pinnell SR

(2008) A topical antioxidant solution containing vitamins C and E stabilized by

ferulic acid provides protection for human skin against damage caused by ultraviolet

irradiation J Am Acad Dermatol 59 418-425

Mushran SP Agarwal MC (1977) Mechanistic studies on the oxidation of ascorbic

acid 5 J Sci Res (India) 36 274-283

Nerlo H Palak W Tomaszek J (1968) Stabilization of vitamin preparations II

Syrup of vitamin D3 and its stabilization Acta Polon Pharm 25 183-188

Niki E Noguchi N (1997) Protection of human low-density lipoprotein from oxidative

modification by vitamin C In Packer L Fuchs J Eds Vitamin C in Health and

Disease Marcel Dekker New York pp 183-192

Nishikimi M Fukuyama R Minoshima S Shimizu N Yagi K (1994) Cloning and

chromosomal mapping of the human nonfunctional gene for L-gulono-gamma-

211

lactone oxidase the enzyme for L-ascorbic acid biosynthesis missing in man J Biol

Chem 269 13685-13688

Njus D Wigle M Kelly PM Kipp BH Schlegel HB (2001) Mechanism of

ascorbic acid oxidation by Cytochrome B561 Biochemistry 40 11905-11911

Nusgens BV Humbert P Rougier A Colige AC Haftek M Lambert CA

Richard A Creidi P Lapiegravere CM (2001) Topically applied vitamin C enhances

the mRNA level of collagens I and III their processing enzymes and tissue inhibitor

of matrix metalloproteinase 1 in the human dermis J Invest Dermatol 116 853-859

Ogata Y Kosugi Y (1969) Solvent effect on the autooxidation of L-ascorbic acid

Tetrahedron 25 1055-1062

OrsquoKeefe T (2001) Ascorbic acid and stable ascorbate esters as sources of vitamin C in

aquaculture feeds ASA Technical Bulletin AQ48 wwwsoyaquaorg

OrsquoNeil MJ Ed (2001) The Merck Index 13th ed Merck amp Co Inc Rahway NJ

USA Electronic version

Ortega GR Delgado JN (1998) Vitamins and related compounds In Wilson and

Gisvoldrsquos Textbook of Organic Medicinal and Pharmaceutical Chemistry 10th ed

Lippincott-Raven Philadelphia Chap 27

Ortwerth BJ Monnier VM (1997) Protein glycation by the oxidation products of

ascorbic acid In Packer L Fuchs J Eds Vitamin C in Health and Disease Marcel

Dekker New York pp 123ndash142

Ozer O Muguet V Roy E Grossiord JL Seiller M (2000) Stability study of

WOW viscosified multiple emulsions Drug Dev Ind Pharm 26 1185-1189

Packer I Fuchs J (1999) Vitamin C in Health and Disease Marcel Dekker New York

212

Packer JE Slater TF Wilson RL (1979) Direct observation of a free radical

interaction between vitamin E and vitamin C Nature 278 737-738

Packer L Traber MG Kraemer K Frei B (2002) The Antioxidant Vitamins C and

E AOCS Press Illinois

Padayatty SJ Katz A Wang Y Eck P Kwon O Lee JH Chen S Corpe C

Dutta A Dutta SK Levine M (2003) Vitamin C as an antioxidant Evaluation of

its role in disease prevention J Am Coll Nutr 22 18-35

Parker CA (1953) A new sensitive chemical actinometer I Some trials with potassium

ferrioxalate Proc Roy Soc (Lond) A220 104-116

Parker JN Parker PM (2003) Antioxidants ICON Group International

Pastore P Rizzetto T Curcuruto O Cin MD Zaramella A Martin D (2001)

Characterization of dehydroascorbic acid solutions by liquid chromatography mass

spectrometry Rapid Commun Mass Spectrom 15 2051-2057

Pelletier O (1985) In Augustin J Klein BP Becker D Venugopal PB Eds

Methods of Vitamins Assay Wiley New York pp 303-347

Peyrat-Maillard MN Bonnely S Rondini L Berset C (2001) Effect of vitamin E

and vitamin C on the antioxidant activity of malt rootlets extracts Lebensmittel-

Wissenschaft Und-Technological 34 176-182

Pinnell SR (2003) Cutaneous photodamage oxidative stress and topical antioxidant

protection J Am Acad Dermatol 48 1-19

Pinnell SR Madey DL (1998) Topical vitamin C in skin care Aesthet Surg J 18

468-470

213

Pinnell SR Yang H Omar M Monteiro-Riviere N DeBuys HV Walker LC

Wang Y Levine M (2001) Topical L-ascorbic acid percutaneous absorption

studies Dermatol Surg 27 137-142

Placzek M Gaube S Kerkmann U Gilbertz KP Herzinger T Haen E Przybilla

B (2005) Ultraviolet B-induced DNA damage in human epidermis is modified by

the antioxidants ascorbic acid and D-alpha-tocopherol J Invest Dermatol 124 304-

307

Puskas F Gergely PJr Banki K Perl A (2000) Stimulation of the pentose

phosphate pathway and glutathione levels by dehydroascorbate the oxidized form of

vitamin C FASEB J 14 1352-1361

Puskas F Gergely P Niland B Banki K Perl A (2002) Differential regulation of

hydrogen peroxide and Fas-dependent apoptosis pathways by dehydroascorbate the

oxidized form of vitamin C Antioxid Redox Signal 4 357-369

Racz I (1989) Drug Formulation Wiley New York pp 121-122

Ramu A Mehta MM (2000) The riboflavin-mediated photooxidation of doxorubicin

Cancer Chemoth Pharm 46 449-458

Raschke T Koop U Duumlsing HJ Filbry A Sauermann K Jaspers S Wenck H

Wittern KP (2004) Topical activity of ascorbic acid from in vitro optimization to in

vivo efficacy Skin Pharmacol Physiol 17 200-206

Reed RA Harmon P Manas D Wasylaschuk W Galli C Biddell R Bergquist

PA Hunke W Templeton AC Ip D (2003) The role of excipients and package

components in the photostability of liquid formulations PDA J Pharm Sci Tech 57

351-368

214

Reichstein T Griissner A Oppcnuuer R (1933) Synthese der d- und l-Ascorbinsaumlure

(C-Vitamin) Helv Chim Acta 16 1019-1033

Riehl J Maupin C Layloff T (1995) On the choice of a photolysis source for the

photostability testing of pharmaceuticals Pharmacop Forum 21 1654-1663

Rivlin RS (2007) Riboflavin (vitamin B2) In Zempleni J Rucker RB McCormick

DB Suttie JW Eds Handbook of Vitamins 4th ed Taylor amp Francis CRC

Press Boca Raton FL p 241

Rogers AR Yacomeni JA (1971) The effect of pH on the aerobic degradation of

ascorbic acid solutions J Pharm Pharmacol 23 218S

Rosen MR Ed (2005) Delivery System Handbook for Personal Care and Cosmetic

Products-Technology Applications and Formulations William Andrew Publishing

Norwich NY

Rosenberg HR (1945) Chemistry and Physiology of the Vitamins Interscience New

York pp 289-338

Roth HJ Eger K Troschutz R (1991) Pharmaceutical Chemistry Vol 2 Drug

Analysis Ellis Horwood London pp 282-288

Rowe RC Sheskey PJ Quinn ME (2009) Handbook of Pharmaceutical Excipients

6th ed Pharmaceutical Press London pp 43-47 181-183 731-732

Roacutezanowska M Bober A Burke JM Sarna T (1997) The role of retinal pigment

epithelium melanin in photoinduced oxidation of ascorbate Photochem Photobiol

65 472-479

Rozman B Gasperlin M (2007) Stability of vitamins C and E in topical

microemulsions for combined antioxidant therapy Drug Deliv 14 235-245

215

Rozman B Gasperlin M Tinois-Tessoneaud E Pirot F Falson F (2009)

Simultaneous absorption of vitamins C and E from topical microemulsions using

reconstructed human epidermis as a skin model Eur J Pharm Biopharm 72 69-75

Ruumlmelin A Fauth U Halmagyi M (1999) Determination of ascorbic acid in plasma

and urine by high performance liquid chromatography with ultraviolet detection Clin

Chem Lab Med 37 533-536

Rumsey SC Levine M (2000) Vitamin C In Song WO Beecher GR

Eitenmiller RR Eds Modern Analytical Methodologies in Fat- and Water-Soluble

Vitamins Wiley New York Chap 13

Saari JC Baker EM Sauberlich HE (1967) Thin-layer chromatographic separation

of the oxidative degradation products of ascorbic acid Anal Biochem 18 173-177

Salkić M Keran H Jašić M (2007) Direct spectrophotometric determination of l-

ascorbic acid in the presence of potassium cyanide Agric Conspec Sci 72 371-375

Salvador A Chisvert A (2007) Analysis of Cosmetic Products Elsevier Amsterdam

Sattar A DeMan JM Alexander JC (1977) Light-induced degradation of vitamins

II Kinetic studies on ascorbic acid decomposition in solution Can Inst Food Sci

Technol J 10 65-68

Satoh K Sakagami H Terasaka H Ida Y Fujisawa S (2000) Interaction between

hydroquinone and ascorbic acid derivatives quenching effects of organic solvents

Antican Res 20 1577-1581

Sauberlich HE (1994a) Pharmacology of vitamin C Annu Rev Nutr 14 371-391

Sauberlich HE (1994b) Vitamin C and Cancer In Kritchevsky D Carroll K Eds

Nutrition and Disease Update Cancer AOCS Press Champaign IL pp 111-172

216

Sauermann K Jaspers S Koop U Wenck H (2004) Topically applied vitamin C

increases the density of dermal papillae in aged human skin BMC Dermatol 4 13

Sawicka J (1991) The influence of excipients and technological process on

cholecalciferol stability and its liberation from tablets Pharmazie 46 519-521

Scheuplein RJ Blank IH (1973) Mechanism of percutaneous absorption IV

Penetration of non-electrolytes (alcohols) from aqueous solutions and from pure

liquids J Invest Dermatol 60 286-296

Schick B (1943) Scurvy and pine needles Science 98 241-242

Schuman Jorns M Schollnhammer G Hemmerich P (1975) Intramolecular addition

of the riboflavin side chain Anion-catalysed neutral photochemistry Eur J

Biochem 57 35-48

Seib PA Tolbert BM (1982) Ascorbic Acid Chemistry Metabolism and Uses

Advances in Chemistry Series 200 American Chemical Society Washington DC

Sequeira F Vozone C (2000) Photostability studies of drug substances and products

Practical implications of the ICH guideline Pharm Tech 24 30-35

Shacter E (2000) Quantification and significance of protein oxidation in biological

samples Drug Metab Rev 32 307-326

Shah VP (1997) FDA Guidance for Industry Document Nonsterile Semisolid Dosage

Forms Centre for Drug Evaluation and Research Rockville MD pp 1-40

Sheraz MA Ahmed S Ahmad S (2007) Developments in the clinical and food

analysis of vitamin C J Baqai Med Univ 10 19-24

217

Shindo Y Witt E Han D Packer L (1994) Dose-response effect of acute ultraviolet

irradiation on antioxidants and molecular markers of oxidation in murine epidermis

and dermis J Invest Dermatol 102 470-475

Sibi M Batcu A Gheorghiu I (1953) Vitamin oxidation-reduction effect II Effect of

light on the oxidation-reduction reactions of lactoflavin Studii cercetari stiinf

(Romania) 4 291-305 through CA (1956) 50 14816g

Sidhu DS Sugden JK (1992) Effect of food dyes in the photostability of aqueous

solutions of L-ascorbic acid Int J Pharm 83 263-266

Silva E Quina FH (2006) Photoinduced processes in the eye lens do flavins really

play a role In Silva E Edwards AM Eds Flavins Photochemisry and

Photobiology The Royal Society of Chemistry Cambridge Chap 7

Silva E Ugarte P Andrade A Edwards AM (1994) Riboflavin-sensitized photo-

processes of tryptophan J Photochem Photobiol B Biol 23 43-48

Sim AK (1972) Ascorbic acid-A scurvy past and present Chem Indust 8 160-165

Singh S (2006) Stability testing during product development In Jain NK ED

Pharmaceutical Product Development CBS Publishers New Delhi Chap 9

Singh S Bakshi M (2000) Guidance on conduct of stress testing to determine inherent

stability of drugs Pharm Tech 24 1-14

Sinko PJ (2006) Chemical Kinetics and Stability Martinrsquos Physical Pharmacy and

Pharmaceutical Sciences 5th ed Lippincott Williams amp Wilkins Baltimore Chap

8 15 18

218

Smith JL Canham JE Kirkland WD Wells PA (1988) Effect of intralipid amino

acids container temperature and duration of storage on vitamin stability in total

parenteral nutrition admixtures J Parenter Enteral Nutr 12 478-483

Speek AJ Schrijver J Schreurs WHP (1984) Fluorimetric determination of total

vitamin C and total isovitamin C in food stuffs and beverages by high-performance

liquid chromatography with precolumn derivatization J Agric Food Chem 32 352-

355Spratley JE Hellstroumlm SO Mattsson CK Pais-Clemente M (2001)

Topical ascorbic acid reduces myringosclerosis in perforated tympanic membranes A

study in the rat Ann Otol Rhinol Laryngol 110 585-591

Spiclin P Homer M Zupancic A Gasperlin M (2003) Sodium ascorbyl phosphate

in topical microemulsions Int J Pharm 256 65-73

Stadtman ER (1991) Ascorbic acid and oxidative inactivation of proteins Am J Clin

Nutr 54 1125S-1128S

Stone I (1969) The Healing Factor Vitamin C Against Disease Grosset and Dunlap

New York

Sugden JK (1985) Photostability of cosmetic materials Int J Cosmet Sci 7 165-173

Swarbrick J Rubino JT Rubino OP (2006) Coarse Dispersions In Hendrickson

R Ed Remington The Science and Practice of Pharmacy 21st ed Lippincott

Williams amp Wilkins Baltimore Chap22

Sweetman SC Ed (2009) Martindale The Complete Drug Reference 36th ed

Pharmaceutical Press London Electronic version

219

Szent-Gyoumlrgi A (1928) Observation on function of peroxidase systems and chemistry

of adrenal cortex description of new carbohydrate derivative Biochem J 22 1387-

1389

Szymula M (2005) The influence of ascorbic acid on the rheological properties of the

microemulsion region of the SDSpentanolwater system J Cosmet Sci 56 267-

277

Taqui Khan MM Martell AE (1967) Metal ion and metal chelate catalyzed oxidation

of ascorbic acid by molecular oxygen 1 Cupric acid and ferric ion catalyzed

oxidation J Am Chem Soc 89 4176-4185

Tangney CC (1988) Analysis of vitamin C in biological samples with an emphasis on

recent chromatographic techniques Prog Clin Biol Res 259 331-362

Tatsumi K Ichikawa H Wada S (1992) Flavin-sensitized photooxidation of

substituted phenols in natural water J Contam Hydrol 9 207-219

Tessier F Birlouez-Aragon I Tjani C Guilland JC (1996) Validation of a

micromethod for determining oxidized and reduced vitamin C in plasma by HPLC-

fluorescence Int J Vitam Nutr Res 66 166-170

Thatcher SR Mansfield RK Miller RB Davis CW Baertschi SW (2001a)

Pharmaceutical photostability a technical and practical interpretation of the ICH

guideline and its application to pharmaceutical stability Part 1 Pharm Tech 25 98-

110

Thatcher SR Mansfield RK Miller RB Davis CW Baertschi SW (2001b)

Pharmaceutical photostability a technical and practical interpretation of the ICH

220

guideline and its application to pharmaceutical stability Part 2 Pharm Tech 25 50-

62

Thiele JJ Dreher F Packer L (2000) Antioxidant defense systems in skin In

Elsner P Maibach HI Eds Cosmeceuticals- Drugs vs Cosmetics Marcel

Dekker New York Chap 11

Thomas DP (1997) Sailors scurvy and science J R Soc Med 90 50-54

Thomson CO Trenerry VC (1995) A rapid method for the determination of total L-

ascorbic acid in fruits and vegetables by micellar electrokinetic capillary

chromatography Food Chem 53 43-50

Tonnesen HH (1991) Photochemical degradation of components in drug formulations

1 An approach to the standardization of degradation studies Pharmazie 46 263-265

Tonnesen HH (2002) Photodecomposition of Drugs In Swarbrick J Boylan JC

Eds Encyclopedia of Pharmaceutical Technology Vol 3 Marcel Dekker New

York pp 2197-2203

Tonnesen HH Ed (2004) The Photostability of Drugs and Drug Formulations

Culinary and Hospitality Industry Publications Services Weimex TX

Tonnesen HH Karlsen J (1997) A comment on photostability testing according to

ICH guideline calibration of light sources PharmEuropa 9 735-736

Tonnesen HH Moore DE (1993) Photochemical degradation of components in drug

formulations Pharm Technol Int 5 27-33

Tournas JA Lin FH Burch JA Selim MA Monteiro-Riviere NA Zielinski

JE Pinnell SR (2006) Ubiquinone idebenone and kinetin provide ineffective

221

photoprotection to skin when compared to a topical antioxidant combination of

vitamins C and E with ferulic acid J Invest Dermatol 126 1185-1187

Traber MG (2007) Vitamin E In Zempleni J Rucker RB McCormick DB

Suttie JW Eds Handbook of Vitamins 4th ed Taylor amp Francis CRC Press

Boca Raton FL Chap 4

Traikovich SS (1999) Use of topical ascorbic acid and its effect on photodamaged skin

topography Arch Otolaryngol Head Neck Surg 125 1091-1098

Treadwell GE Cairns WL Metzler DE (1968) Photochemical degradation of

flavins V Chromatographic studies of the products of photolysis of riboflavin J

Chromatogr 35 376-388

Truswell AS (2003) ABC of Nutrition 4th ed BMJ Publishing Group Chap 10

Tsao CS Young M (1985) Analysis of ascorbic acid derivatives by high-performance

liquid chromatography with electrochemical detection J Chromatogr 13 855-856

Underberg WJM (1978) Oxidative degradation of pharmaceutically important

phenothiazines III Kinetics and mechanism of promethazine oxidation J Pharm Sci

67 1133-1138

Urso ML Clarkson PM (2003) Oxidative stress exercise and antioxidant

supplementation Toxicology 189 41-54

Vaid FHM (1998) Photolysis and Interaction of Thiamine Hydrochloride with

Riboflavin in Aqueous Solution PhD Thesis University of Karachi Karachi

Vaid FHM Shaikh RH Ansari IA Ahmad I (2005) Spectral study of the

photolysis of aqueous thiamine hydrochloride and ascorbic acid solution in the

presence and absence of riboflavin J Chem Soc Pak 27 227-232

222

Vaid F H M Shaikh RH Ansari I A Ahmad I (2006) Chromatographic study of

the photolysis of aqueous thiamine hydrochloride and ascorbic acid solutions in the

presence and absence of riboflavin J Chem Soc Pak 28 481-464

Valvani SC (2000) Industrial stability testing in the United States and computerization

of stability data In Carstensen JT Rhodes CT Eds Drug Stability Principles

and Practices 3rd

ed Marcel Dekker New York pp 534-536

Vanderslice JT Higgs DJ (1988) Chromatographic separation of ascorbic acid

isoascorbic acid dehydroascorbic acid and dehydroisoascorbic acid and their

quantitation in food products J Micronutr Anal 4 109-118

Vanderslice JT Higgs DJ Hayes JM Block G (1990) Ascorbic acid and

dehydroascorbic acid content of foods-as-eaten J Food Comp Anal 3 105-118

Vanderslice JT Higgs DJ (1993) Quantitative determination of ascorbic acid

dehydroascorbic acid by HPLC in foods and other matrices J Nutr Biochem 4 184-

190

Varvaresou A Tsirivas E Iakovou K Gikas E Papathomas Z Vonaparti A

Panderi I (2006) Development and validation of a reversed-phase ion-pair liquid

chromatography method for the determination of magnesium ascorbyl phosphate and

melatonin in cosmetic creams Anal Chim Acta 573-574 284-290

Verma KK Jain A Verma A Chaurasia A (1991) Spectrophotometric

determination of ascorbic acid in pharmaceuticals by background correction and flow

injection Analyst (London) 116 641-645

Vimaladevi M (2005) Textbook of Cosmetics CBS Publishers New Dehli Chap 2

and 17

223

Wallwork SC Grant DJW (1977) Physical Chemistry for Students of Pharmacy and

Biology 3rd ed Longmann New York pp 502-505

Waugh WA King CG (1932) Isolation and identification of vitamin C J Biol

Chem 97 325-331

Wechtersbach L Cigic B (2007) Reduction of dehydroascorbic acid at low pH J

Biochem Biophys Methods 70 767-772

Weiner N (1996) Introduction In Liberman HA Rieger MM Banker GS Eds

Pharmaceutical Dosage Forms Disperse Systems 2nd ed vol1 Marcel Dekker

New York Chap1

Werner W (1949) The reaction of l-ascorbic and d-isoascorbic acid with nicotinic acid

and its amide J Org Chem 14 22-26

Wiechers JW (2003) Formulating for Efficacy Proceedings of the 2003 IFSCC

Conference Seoul Korea

Wiechers JW (2005) Optimizing skin delivery of active ingredients from emulsions

from theory to practice In Rosen MR Ed Delivery System Handbook for

Personal Care and Cosmetic Products-Technology Applications and Formulations

William Andrew Inc Norwich NY Chap 20

Wille JJ (2005) Thixogel-Novel topical delivery systems for hydrophobic plant actives

In Rosen MR Ed Delivery System Handbook for Personal Care and Cosmetic

Products-Technology Applications and Formulations William Andrew Inc

Norwich NY Chap 36

224

Wright JL (1986) Vitamin A monograph In Connors KA Amidon GL Stella

VJ Eds Chemical Stability of Pharmaceuticals A Handbook for Pharmacists 2nd

ed Wiley New York pp 791-803

Yao AA Wathelet B Thonart P (2009) Effect of protective compounds on the

survival electrolyte leakage and lipid degradation of freeze-dried Weissella

paramesenteroides LC11 during storage J Microbiol Biotechnol 19 810-817

Yeh SY Lach JL (1961) Stability of morphine in aqueous solutions III Kinetics of

morphine degradation in aqueous solution J Pharm Sci 50 35-42

Yoshimura A Ohno T (1988) Lumiflavin-sensitized photooxygenation of indole

Photochem Photobiol 48 561-565

Yoshioka S Stella VJ (2000) Stability of Drugs and Dosage Forms Kluwer

Academic Plenum Publishers New York Chap 2

Zaeslein C (1982) Vitamins in the Field of Medicine Hoffman La Roche Basle pp

89-96

Zeng W Martinuzzi F MacGregor A (2005) Development and application of a novel

UV method for the analysis of ascorbic acid J Pharm Biomed Anal 36 1107-1111

Zhang GF Chen HY (2000) Chemiluminescence studies of the oxidation of ascorbic

acid with copper (II) catalyzed by halide anions and its applications to the

determination of halide anions and ascorbic acid Anal Sci 16 1317-1321

Zhang L Lerner S Rustrum WV Hofmann GA (1999) Electroporation-mediated

topical delivery of vitamin C for cosmetic applications Bioelectrochem Bioenerg

48 453-461

225

Zilva SS (1932) The non-specificity of the phenolindophenol reducing capacity of

lemon juice and its fractions as a measure of their antiscorbutic activity Biochem J

26 1624-1627

226

AUTHORrsquoS PUBLICATIONS

The author obtained his B Pharm degree in 2003 and joined the post graduate

program securing an M Phil degree in Pharmaceutics in 2006 from Baqai Medical

University He is a co-author of following publications

CHAPTER IN BOOK

1 Chapter on ldquoBorate Toxicity Effect on Drug Stability and Analytical

Applicationsrdquo by Iqbal Ahmad Sofia Ahmed MA Sheraz and Faiyaz H M

Vaid In Handbook on Borates Chemistry Production and Applications (MP

Chung Ed) Nova Science Publishers Inc NY USA (in press)

PAPERS PUBLISHED

INTERNATIONAL

2 Iqbal Ahmad Sofia Ahmed MA Sheraz and Faiyaz HM Vaid ldquoEffect of Borate

Buffer on the Photolysis of Riboflavin in Aqueous Solutionrdquo Journal of

Photochemistry and Photobiology B Biology 93 82-87 (2008)

3 Iqbal Ahmad Sofia Ahmed MA Sheraz M Aminuddin and Faiyaz HM Vaid

ldquoEffect of Caffeine Complexation on the Photolysis of Riboflavin in Aqueous

Solution A Kinetic Studyrdquo Chemical and Pharmaceutical Bulletin 57 (2009)

published online September 14 2009

4 Iqbal Ahmad MA Sheraz Sofia Ahmed and Faiyaz HM Vaid ldquoAnalytical

Applications of Boratesrdquo Materials Science Research Journal (in press)

5 Iqbal Ahmad Sofia Ahmed MA Sheraz Kefi Iqbal and Faiyaz HM Vaid

ldquoPharmacological Aspects of Boratesrdquo International Journal of Medical and

Biological Frontiers (in press)

6 Iqbal Ahmad Sofia Ahmed MA Sheraz Faiyaz HM Vaid and Izhar A Ansari

ldquoEffect of Divalent Ions on Photodegradation Kinetics and Pathways of

Riboflavin in Aqueous Solutionrdquo Photochemical and Photobiological Sciences

accepted

227

NATIONAL

7 Sofia Ahmed MA Sheraz and Iqbal Ahmad ldquoAdvances in Antioxidant Activity of

Vitamin Erdquo Journal of Baqai Medical University 10 13-18 (2007)

8 MA Sheraz Sofia Ahmed and Iqbal Ahmad ldquoDevelopments in the Clinical and

Food Analysis of Vitamin Crdquo Journal of Baqai Medical University 10 19-24

(2007)

9 A Azmi SNH Naqvi M Usman MA Sheraz and Sofia Ahmed ldquoPancreatic

Glucagon in Certain Ungulates Comparative Study of Extraction and

Bioassayrdquo Pakistan Journal of Entomology 20 23-28 (2005)

10 Iqbal Ahmad Sofia Ahmed MA Sheraz Faiyaz HM Vaid and S Hasan

ldquoAdvances in Biochemical Functions and the Photochemistry of Flavins and

Flavoproteinsrdquo Pakistan Journal of Pharmaceutical Sciences in press

11 MA Sheraz Sofia Ahmed and Iqbal Ahmad ldquoEffect of Borates on the Stability of

Chemical and Pharmaceutical Compoundsrdquo Journal of Baqai Medical University

accepted

PAPERS SUBMITTED

12 Iqbal Ahmad MA Sheraz Sofia Ahmed Riaz H Shaikh and Faiyaz HM Vaid

ldquoPhotostability of Ascorbic Acid in Organic Solvents and Cream Formulationsrdquo

Chemical and Pharmaceutical Bulletin

13 Iqbal Ahmad MA Sheraz Sofia Ahmed Riaz H Shaikh and Faiyaz HM Vaid

ldquoPhotochemical Interaction of Ascorbic Acid with Riboflavin Nicotinamide and

Alpha-Tocopherol in Cream Formulationsrdquo Journal of Cosmetic Science

14 Iqbal Ahmad Kefi Iqbal Sofia Ahmed MA Sheraz ldquoApplications of Laser Flash

Photolysis Spectroscopy and Electron Microscopy in Photopolymerization and

Development of Glass Ionomer Dental Cementsrdquo Materials Science Research

Journal

15 Sofia Ahmed MA Sheraz M Aminuddin I Ahmad and Faiyaz HM Vaid ldquoA

Rapid Titrimetric Assay for Quantitation of Vitamin B1 in Neat and

Pharmaceutical Preparationsrdquo Pakistan Journal of Pharmaceutical Sciences

• 01 SZ-786
• 02 SZ-title
• 03 SZ-Certificate
• 04 SZ-Abstract
• 05 SZ-Acknowledgement
• 06 SZ-Dedication
• 07 SZ-Contents
• 08 SZ-Chapter 1
• 09 SZ-Chapter 2
• 10 SZ-Chapter 3
• 11 SZ-Object of Present Investigation
• 12 SZ-Chapter 4
• 13 SZ-Chapter 5
• 14 SZ-Chapter 6
• 15 SZ-Chapter 7
• 16 SZ-Conclusion
• 17 SZ-References
• 18 SZ-Authors Publications