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Chapter-I General Introduction
1
GENERAL INTRODUCTION
1.01 Introduction to Drugs
A drug, broadly speaking, is any substance that, when absorbed into the body
of a living organism, alters normal bodily function. Pharmacology defines a drug as "a
chemical substance used in the treatment, cure, prevention, or diagnosis of disease or
used to enhance physical or mental well-being. The methods of quality control and
conditions of their storage are the important contents placed in many text books of
pharmaceutical chemistry [1-8]. Drugs are synthesized in bulk and used for their
therapeutic effects in pharmaceutical formulations. These biologically active chemical
substances are generally formulated into convenient dosage forms such as tablets,
capsules, dry syrups, liquid orals, creams or ointments, parenterals (injections in dry
or liquid forms) lotions, dusting powders, aerosols, metered dose inhalers and dry
powder inhalers etc. These formulations deliver the drug substances in a stable,
nontoxic and acceptable form, ensuing its bioavailability and therapeutic activity.
According to the chemical structure and therapeutic action, the drugs may be
classified as follows.
(i)Chemotherapeutic agents
Chemotherapeutic agents are used to kill the invading organisms without
harmful effects on the tissues of the patient. They may be sub-divided into various
classes such as antibacterial, trypanocides, antiprotozoals, antifungals, anthelmintics,
antiseptics, antitubercular, antilepral drugs, antineoplastic agents, disinfectants and
antiviral drugs. Levofloxacin antibiotic has been chosen in the present investigation.
Chapter-I General Introduction
2
(ii) Pharmacodynamic agents
Pharmacodynamic agents have certain effects on animal organs but are not
specific remedies for particular diseases. They may be further subdivided into
different classes like central nervous system modifiers, adrenergic stimulants,
blocking agents, cholinergic, anticholinergic agents, cardio-vascular agents, diuretics,
anti-inflammatory agents, immuno suppressive agents, antispasmodics,
antihypertensive, antidepressants, antihistamines, anticoagulants and antipsycotic
agents. Hormones (steroidal and nonsteroidal) production to desirable extent is also
essential.
(a) Antihypertensive agents: Antihypertensive drugs are used to control blood
pressure. These are classified into various types such as peripheral antiadrenergic,
centrally acting agents, direct vasodilators, ganglionicblocking agents, β-adrenergic
blockers, calcium channel blockers, angiotensin converting enzyme
inhibitors,antagonists and miscellaneous. Trandolapril, an antihypertensive agent is
chosen for the analysis.
(b) Antidepressants: An antidepressant is a psychiatric medication used for
alleviating major depression and dysthymia (milder depression). These are classified
as selective serotonin reuptake inhibitors, neither serotonin nor epinephrine reuptake
inhibitors, noradrenergic and specific serotonergic antidepressants [9], norepinephrine
reuptake inhibitors, nor epinephrine-dopamine reuptake inhibitors, tricyclic
antidepressants, monoamine oxidase inhibitors. Novel antidepressants specifically
affect serotonin and other neurotransmitter.
Chapter-I General Introduction
3
(c) Corticosteroids: The cortex or outer portion of the adrenal gland is one of the
endocrine structures most essential for normal metabolic function. The vital role of
the adrenal cortex is due to its ability to produce a group of steroidal hormones [10].
In addition to the naturally occurring corticosteroids many synthetic steroids with
similar properties have been introduced. The pharmaceutical properties of
corticosteroids also make them useful in the treatment of rheumatoid arthritis,
bronchial asthma and bronchial hypersensitivity [11]. The anti-inflammatory
corticoids represented by hydrocortisone and related synthetic analogs have gained an
unchallenged position in modern therapeutic practice. The therapeutic effect of
steroids depends on their stability [12, 13].
(d)Antiemetic: These drugs are suitably used to treat motion sickness and the side
effects of opioid analgesics, general anaesthetics and chemotherapy directed against
cancer. Antiemetic drugs block messages to the part of the brain that controls nausea
and vomiting. Ondansetron HCl, an antiemetic drug is chosen for the present
investigation.
(e) Antimigraine: These drugs are used for the treatment of the acute migraine
attacks. They work by narrowing blood vessels in the brain, stopping pain signals
from being sent to the brain, and stopping the release of certain natural substances that
cause pain, nausea, and other symptoms of migraine. Naratriptan hydrochloride, an
antimigraine drug is selected for the investigation.
1.02 Quantitative analysis
Chemical analysis may be stated as the application of a process or a series of
processes in order to identify, quantify a substance, the components of a solution of
Chapter-I General Introduction
4
mixture, or the determination of the structures of chemical compounds. Such methods
are to be validated demonstrating the accuracy, precision, and specificity, limit of
detection, quantification, linearity range and interferences. The validation of
analytical procedures is an important part of the registration application for a new
drug [14, 15]. The International Chemical Harmonization (ICH) has harmonized the
requirements in two guidelines [16, 17]. These guidelines serve as a basis worldwide
both for regulatory authorities and industry in proper validation. Quantification can be
achieved by the introduction of more refined and sensitive methods of physiochemical
analysis [18-19] such as colorimetry, spectrophotometry covering UV, visible and IR
regions, fluorimetry or turbidimetry, NMR and Mass, and chromatography [20-28]
that enables one to assay of drugs more accurately and with the smallest consumption
of the analyte, reagent and time. The good manufacturing practices provide minimum
quality standards for production of pharmaceuticals as well as their ingredients [29].
Every country has legislation [31] on bulk drugs and their pharmaceutical
formulations that sets standard and obligatory quality indices for them. These
regulations are presented in separate article general and specific relating to individual
drugs, and are published in the form of book called “Pharmacopoeia” (e.g. Indian
Pharmacopoeia, IP [32], United States Pharmacopoeia USP [33], European
Pharmacopoeia EP [34], United Kingdom, BP [35], Martindale Extra Pharmacopoeia
[36], Merck Index [37], etc.). Complete quantitative analysis of a sample actually
involves the following steps.
(a) Preparation of sample solution for analytical investigation: Sample solutions for
the analytical investigation can be prepared by dissolving finely powder of the tablets
or granules of the capsules in suitable solvent for the samples in solid state.
Chapter-I General Introduction
5
(b) Conversion of the analyte into a measurable form: This step is a vital one, in
developing any analytical method. Particularly, while dealing with the interferences in
pharmaceutical products by spectrophotometry, one should have sound knowledge
about the chemical and structural factors of the analyte and interfering molecules, and
also in the selection of appropriate chromogenic reagent, which should form the
colored product with the analyte molecule, and not with the foreign molecule. Any
possible interference from foreign matter is if expected during the course of the color
development, an appropriate clean up procedure should be adopted prior to the
analysis.
(c) Measurements: The measurement step in an analysis can be carried out by
chemical, physical or biological means. An important feature of modern
pharmaceutical chemistry is the introduction of more refined and sensitive methods of
physico-chemical analysis such as spectroscopy and chromatography that enable one
to assay the drugs more accurately and with the smallest consumption of the analyte,
reagents and time. The modern methods of choice (HPLC, GLC, NMR and Mass
spectroscopy) for assay involve sophisticated equipment, which are very costly and
pose problems of maintenance. Hence they are not in the reach of most laboratories
and small-scale industries. The visible spectrophotometric (or colorimetric) or
fluorimetric methods are very simple, cheap and easy to carry out.
(d) Calculation and Interpretation of Measurements: In spectrophotometric methods
absorbance is directly proportional to the concentration of the analyte in the solution.
Since errors can be made in any measurement, the analytical chemist must consider
this possibility in interpreting his results. The methods of statistics are commonly
used and are especially useful in expressing the significance of analytical data.
Chapter – I Part- A High Performance Liquid Chromatography
6
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
1.03 High performance liquid chromatography (HPLC)
High Performance Liquid Chromatography (HPLC) [38-39] is a simple, fast,
specific, precise and highly accurate analytical technique that is used for the
separation and determination of organic and inorganic solutes in any samples
especially biological, pharmaceutical, food, environmental, industrial etc. In HPLC,
separations are achieved by partition, adsorption or ion exchange, according to the
nature of the interactions between the solute and the stationary phase, which may arise
from hydrogen bonding, Vander walls forces, electrostatic forces or hydrophobic
forces or basing on the size of the particles [40]. Reversed phase HPLC (RP-HPLC or
RPC) has a non-polar stationary phase and an aqueous, moderately polar mobile
phase. One common stationary phase is silica which has been treated with RMe2SiCl,
where R is a straight chain alkyl group such as C18H37 or C8H17. With these stationary
phases, retention time is longer for molecules which are more non-polar, while polar
molecules elute more readily. An investigator can increase retention time by adding
more water to the mobile phase; thereby making the affinity of the hydrophobic
analyte for the hydrophobic stationary phase stronger relative to the now more
hydrophilic mobile phase. Similarly, an investigator can decrease retention time by
adding more organic solvent to the eluent. Structural properties of the analyte
molecule play an important role in its retention characteristics. In general, an analyte
with a larger hydrophobic surface area (C-H, C-C, and generally non-polar atomic
bonds, such as S-S and others) results in a longer retention time because it increases
the molecule's non-polar surface area, which is non-interacting with the water
Chapter – I Part- A High Performance Liquid Chromatography
7
structure. On the other hand, polar groups, such as -OH, -NH2, COO- or -NH3
+ reduce
retention as they are well integrated into water.
1.04 HPLC Method Validation
Validation of an analytical method is the process by which it is established by
laboratory studies, that the performance characteristics of the method meet the
requirements for the intended analytical application. The validation of the assay
procedure is carried out using the following parameters. Analytical methods should be
used within good manufacturing practice and good laboratory practice environments,
and must be developed using the protocols set out in the International Conference on
Harmonization (ICH) guidelines [41-42]. The US Food and Drug Administration
(FDA) [43] and US Pharmacopoeia (USP) [44] both refer to ICH guidelines. The
most widely applied validation characteristics are accuracy, precision (repeatability
and intermediate precision), specificity, detection limit, quantitation limit, linearity,
range, robustness and stability of analytical solutions. Method validation must have a
written and approved protocol prior to use [45].
(a)Precision: According to the ICH guide lines, precision should be performed at two
different levels - repeatability and intermediate precision. Repeatability is an
indication of how easy it is for an operator in a laboratory to obtain the same result for
the same batch of material using the same method at different times using the same
equipment and reagents. It should be determined from a minimum of six
determinations covering the specified range of the procedure or from a minimum of
six determinations at 100% of the test or target concentration. Intermediate precision
results from variations such as different days, analysts and equipment. In determining
Chapter – I Part- A High Performance Liquid Chromatography
8
intermediate precision, experimental design should be employed so that the effects (if
any) of the individual variables can be monitored. Precision criteria for an assay
method are that the instrument precision and the intra-assay precision (RSD) will be
≤2%.
% Relative Standard Deviation= (Standard deviation / mean) x 100
(b)Accuracy: The accuracy of an analytical procedure expresses the closeness of
agreement between the value which is accepted either as a conventional true value or
an accepted reference value and the value found. Accuracy is usually determined by
measuring a known amount of standard material under a variety of conditions but
preferably in the formulation, bulk material or intermediate product to ensure that
other components do not interfere with the analytical method. For assay methods,
spiked samples are prepared in triplicate at three levels across a range of 50-150% of
the target concentration. The per cent recovery should then be calculated. The
accuracy criterion for an assay method is that the mean recovery will be 100±2% at
each concentration across the range of 80-120% of the target concentration. To
document accuracy, ICH guidelines regarding methodology recommend collecting
data from a minimum of nine determinations across a minimum of three concentration
levels covering the specified range.
% Error = [(Measured value -True value) / True value] x100
(c) Limit of Detection (LOD) & Limit of Quantitation (LOQ): The limit of detection
(LOD) is defined as the lowest concentration of an analyte in a sample that can be
detected, not quantified. It is expressed as a concentration at a specified signal: noise
ratio usually 3:1. The limit of quantitation (LOQ) is defined as the lowest
Chapter – I Part- A High Performance Liquid Chromatography
9
concentration of an analyte in a sample that can be determined with acceptable
precision and accuracy under the stated operational conditions of the method. The
ICH has recommended a signal: noise ratio 10:1. LOD and LOQ may also be
calculated based on the standard deviation of the response (SD) and the slope of the
calibration curve(s) at levels approximating the LOD according to the formulae
LOD = 3Sa / b LOQ = 10Sa / b
(d) Linearity and Range: The range of an analytical procedure is the interval between
the upper and lower concentration (amounts) of analyte in the sample (including these
concentrations) for which it has been demonstrated that the analytical procedure has a
suitable level of precision, accuracy and linearity. The range of the method is validated
by verifying that the analytical method provides acceptable precision, accuracy and
linearity when applied to samples containing analyte at the extremes of the range as well
as within the range.
(e) Ruggedness: The ruggedness of an analytical method is the degree of
reproducibility of test results obtained by the analysis of the same samples under a
variety of conditions, such as different laboratories, different analysts, different
instruments, different lots of reagents, different elapsed assay times, different assay
temperatures, different days, etc.
(f) Robustness: Robustness measures the capacity of an analytical method to remain
unaffected by small but deliberate variations in method parameters. Parameters that
should be investigated are percent organic content in the mobile phase, pH of the
mobile phase, buffer concentration, and temperature and injection volume.
Chapter – I Part- A High Performance Liquid Chromatography
10
1.05 Chromatographic Parameters
(a) Resolution: Chromatographers measure the quality of separation by resolution Rf
of adjacent bands
21
12 )(2
WW
ttRf
t1 and t2 are retention times of the first and second adjacent bands ;W1 and W2 are
base line band width.
(b) Capacity factor (k): It is the measure of how well the sample molecules are
retained by the column during an isocratic separation. It is affected by the solvent
composition, separation, aging and temperature of separation.
O
OR
t
tt'K
Where tR = Band retention time and tO = Column dead volume
(c) Column Efficiency (N): It is called as the number of theoretical plates. It
measures the band spreading of a peak. When band spread in smaller, the number of
theoretical plates is higher. It indicates a good column and system performance.
Column performance can be defined in terms of values of N
Column efficiency (N)=16 (tR/W)2
Plate height H=N/L (length)
(d) Peak Asymmetry/Peak Tailing: Peak with poor symmetry can result in (i)
Inaccurate plate number and resolution measurement (ii) imprecise quantization (iii)
Degraded resolution and undetected minor bands in the peak tail (iv) Poor retention
Chapter – I Part- A High Performance Liquid Chromatography
11
reproducibility. Increased peak asymmetry value, k>1.5 the sign that the column
should be changed
(e) Selectivity: It measures relative retention of two components. Selectivity is the
function of chromatographic surface (column), melting point and temperature.
01
02
'
1
'
2
VV
VV
k
k
(f) Quantization: A critical requirement for quantitative methods is ability to
measure wide range of sample concentration with a linear response for each analyte.
To achieve the best result with an HPLC method, it is necessary to understand and
have a control of the factors that affect quantization. Calculating the following values
are used to access overall system performance.
1. Theoretical plates : n = 16 (t / W)2
2. Plates per meter : N = n / L
3. Height equivalent to theoretical
Plate (HEPT) : L/n
4. Resolution : R = 2 (t2 – t1) / (W2 + W1)
5. Peak asymmetry : T = W 0.05 / 2f
Where α = Relative retention
t2 = Retention time of the second peak measured from point of injection
t1 = Retention time of the first peak measured from point of injection
Chapter – I Part – B Visible Spectrophotometry
12
VISIBLE SPECTROPHOTOMETRY
1.06Visible Spectrophotometry
Among the several instrumental techniques available for the assay of drugs,
usually visible spectroscopic techniques [46-49] are simple and less expensive. The
fundamental principle of visible spectrophotometry lies in that light of a definite
interval of wavelength passes through a cell with a colored solution or solvent and
falls on the photoelectric cell that converts the radiant energy into electrical energy
measured by a galvanometer. Photometric methods of analysis are based on
measuring light absorption of molecules in a solution, utilizing the principle that the
amount of light absorbed by a substance in solution is proportional to the intensity of
incident light and to the concentration or number of the absorbing species in the path
of the beam. Beer‟s-Lambert law or simply Beer‟s law is expressed as the equation.
log10 (I0/I) = -1og10 T = acl = A
Where Io is the intensity of incident light, I is the intensity of transmitted light,
'T' is the transmittance, 'a' is a constant factor characteristic of a solute, 'l' is the path
length through an absorbing solution, „c‟ is the concentration of absorbing substance
and 'A‟ is absorbance (extinction or optical density). The constant a, called
absorptivity, the absorption coefficient or the extinction coefficient, specifies a
characteristic property of the absorbing substance and is a function of its wavelength.
Its units depend on the concentration and path length units employed. When
concentration is expressed in moles per liter and path length is expressed in
centimeters, the constant is known as molar absorptivity formerly called the molecular
Chapter – I Part – B Visible Spectrophotometry
13
or molar extinction coefficient and is used as a physical constant for absorbing species
under standard conditions. It has a unit of liters per mole per centimeter (lt. mole-1
.
cm-1
) and designated as Є. Absorbance = Є.C.l, this expression show that there is a
linear relationship between the absorbance and the concentration of a given solution,
if the path length and the wavelength of radiation are kept constant.
The objective of the present investigation is to develop a simple, accurate,
precise, rapid, and sensitive spectrophotometric method to determine the amount of
drug in bulk and pharmaceutical formulations. Spectrophotometric methods of
analysis depend on measuring the amount of radiant energy of a particular wavelength
absorbed/emitted by the sample. The important characteristics of the developed
method should be accurate, precise, low cost and less time.
1.07 Chemistry of chromogenic reagents
Chemical methods used to prepare suitable colored solutions are usually
called chromogenic reactions and the color forming reagents are known as
chromogenic reagents. The preparation of the colored solution is as important as the
measurement; hence a careful attention is extremely important while preparing a
colored solution. In spectrophotometric analysis the use of a soluble colorless
chromogenic reagent is desirable. If the reagent possesses some self color, the
preferential solvent extraction of the colored constituent by an immiscible solvent
eliminates the additive effect of the reagent to the resultant color. The preliminary
importance in quantitative analysis of drugs involve the knowledge of the functional
groups either acidic or basic nature present in the drug molecules(Table: 1.01, P: 46-
48), chemical reactions such as redox, substitution, addition, elimination,
Chapter – I Part – B Visible Spectrophotometry
14
rearrangement and complexaction ect between an analyte (drug) or its converted form
with preliminary treatment (cationic, anionic, oxidized, reduced ect.) and a reagent or
its converted form (cationic, anionic, electrophlic, nucleophlic, oxidized and reduced
ect.) with preliminary treatment to produce color. An electrophlic reagent i.e cation,
dipolar molecule, or molecule that has atoms with incomplete octet, is a species
having electron deficient atom or center. The nucleophlic reagent is electron rich.
Various reactions may involve the formation of three main intermediates namely free
radicals, carbonium ions (C+ )and carbanion (C
-)which then react with the reagents to
form products. The analytical application of each reagent has been discussed in detail
separately. Under proposed experimental conditions, the methods M1 to M19 refer to
the serial number. The alphabets „a‟ and „b‟ refer to the different dyes/oxidants used
in the present investigation. The selectivity and sensitivity of the visible spectroscopic
methods depends only on the nature of chemical reactions based on functional groups
present in the drug with suitable chromogenic agent involved in color development.
I. Dyes in ion association complex formation: Methods M1(a), M1(b), M1(c) , M1(d)
M2(a) , M2(b)) and M17.
Ion-Ion association complex or adduct is a special form of molecular complex
resulting from two oppositely charged components extractable into organic solvents
from aqueous phase at suitable pH. One component is a chromogen (dye or metal
complex) possessing charge (cationic or anionic nature) and so insoluble in organic
solvents and the other is colorless, possessing opposite charge (anionic or cationic) to
that of chromogen. The ion-ion association complex extraction has been applied to the
estimation of numerous compounds; possessing basic moieties (secondary or tertiary
aliphatic amino groups) by using an acid dye as a reagent and a chlorinated solvent as
Chapter – I Part – B Visible Spectrophotometry
15
an extractant. The structure of the species formed may depend upon the experimental
conditions (concentration of the components, pH of the aqueous phase). The color can
be altered or intensified upon acidification or re-extracted into a buffer. The presence
of hydrophilic substituents such as -OH or -COOH often prevents extraction of the
complex into organic phase. The selectivity of the reaction may increase by using
appropriate organic solvent (extractant) which depends upon the polarities of the
amine and of the dye. Chemical features of some acidic and basic dyes used in ion
association complex formation are listed in Table: 1.02, P: 49-51.
Simple extractive spectrophotometric methods have been developed for the
estimation of several drugs having basic /acidic moiety either in pure and
pharmaceutical dosage forms [50-60]. These methods are based on the formation of
ion-pair complex of the drug with acidic / basic dye. Alfuzosin [50] is estimated in
both pure and pharmaceutical dosage forms with acidic dye bromocresol green (BCG)
in acidic condition, followed by its extraction in organic solvent (chloroform). R.
Kalaichelvi et al [51] have developed an extractive spectrophometric method for the
assay of pantoprazole sodium either in pure form or in pharmaceutical solid dosage
form using bromothymol blue in aqueous acidic medium. The extracted complexes
showed absorbance maxima at 428 nm. Ashour [52] et al reported a method to
determine alfuzosin hydrochloride either in pure form or in pharmaceutical
formulations. S V Muralimohan Rao et al [53] have reported an extractive
spectrophometric method for the assay of bromhexine HCl both in pure form and in
pharmaceutical solid dosage form using acidic dyes such as TPooo, Napthalene Blue
and Azocarmine. A simple and sensitive extractive spectrophotometric method is
described [54] for determination of buspirone. Two spectrophotometric methods have
Chapter – I Part – B Visible Spectrophotometry
16
been developed [55] for the determination of quetiapine fumarate (QTF) in pure form
and in its dosage forms. These methods are based on the formation of ion-pair
complex between the drug and two sulphonthalein acidic dyes, namely, bromophenol
blue and thymol blue, followed by the measurement of absorbance at 410 and 380nm,
respectively. Extractive spectrophotometric determination of omeprazole is developed
[56] using acidic dyes- bromophenol blue and orange G - as ion-pairing agents in
aqueous medium (pH 7.0 and 6.0, respectively). A spectrophotometric method has
been developed by H. Abdine et al [57] for the determination of cinnarizine in
pharmaceutical preparations. Abdel-Aziz M [58] developed a spectrophotometric
method for the determination of guanethidine sulphate, guanfacine hydrochloride,
guanoclor sulphate, guanoxan sulphate and debrisoquine sulphate which involves ion-
pair formation of the selected compounds with bromocresol purple at pH 3.8. Two
acid dye reagents have been utilized for spectrophotometric determination of
triamterene [59] in pure form and in pharmaceutical preparations. The dyes used are
bromophenol blue (BPB), and bromothymol blue (BTS). They form a chloroform-
soluble, coloured ion association complex with triamterene, at pH 3.4 and 3.2 using
bromo phenyl blue and bromo thymole blue respectively. Krishna and Sankar [60]
have reported four simple and sensitive ion-pairing spectrophotometric methods have
been described for the assay of gemifloxacin mesylate (GFX) either in pure form or in
pharmaceutical formulations. In the present investigations, ARS, BTB, MO and
TPooo (Method M1(a), M1(b), M1(c) M1(d)) have been used as acidic dyes for the
formation of ion –ion association complex with the selected drugs NTT, LEF, OND
and TDP. TDP and LEF form ion association complexes with basic dyes MB and
SAF-O, which are extractable into chloroform from the aqueous phase and the max
Chapter – I Part – B Visible Spectrophotometry
17
and max values obtained with the two basic dyes with the responded drug LEF and
TDP are compiled in Chapter - III and Chapter - V.
II. Fe (III)/o-Phenanthroline: Method M3
Ferric salt, Fe (III) converts into a ferrous salt, Fe (II) by reduction process and
the reduced for of iron can be easily detected by the usual reagent o-phenanthroline
[61], bipyridyl or triazine [62].
3Fe+2
+ 2 [Fe (CN)6]-3
Fe3 [Fe(CN)6]2
The reaction between o-Phen and Fe (II) forms a red complex. Each of the
nitrogen atom in 1, 10-phenanthroline has an unshared air of electrons that can be
shared with iron (II). Three such molecules of the organic compound attach
themselves to the metallic ion to form a blood-red complex ion. The iron (II) can be
oxidized to iron (III), and the later ion also forms a complex with three molecules o-
phen. Based on its complexing tendency and oxidizing properties, ferric salt is
suggested in the estimation of several drugs. P.Nagaraju et.al [63] have developed
three spectrophotometric methods for the determination of Atorvastatin calcium in
pure and its pharmaceutical formulations. These methods are based on the oxidation
of Atorvastatin calcium by ferric chloride in presence of o-phenanthroline or 2,
2'bipyridyl or potassium ferricyanide. Kanakapura [64] has described two
spectrophotometric methods for the determination of etamsylate in bulk and in
capsule formulations. These methods are based on the oxidation of the drug with
ferric chloride in neutral medium and subsequent chelation of the resulting iron(II)
with 1,10-phenanthroline and with 2,2‟-bipyridyl. A simple, sensitive and accurate
Chapter – I Part – B Visible Spectrophotometry
18
spectrophotometric method [65] for the determination of Levodopa , Carbidopa and
-Methyldopa at the ppb level has been developed. This method is based on the
oxidation of the drugs by Fe(III), using a Fe(III)-o-Phenanthroline mixture and is
followed by the formation of a highly stable orange-red coloured tris-complex [Fe(II)-
(o-Phenanthroline)3]2+
in a moderate acidic medium (pH=5.0±0.2) which exhibits an
absorption maximum at =510 nm. Alaa S. Amin [66] et.al have reported two
methods for assaying domperidone (I) and metoclopramide (II) in a bulk sample and
in dosage forms are investigated. These methods are based on the oxidation of I
and/or II by Fe3+
in the presence of o-phenanthroline or bipyridyl. The formation of
tris-complex upon reactions with Fe3+
-o-phen and/or Fe3+
-bipy mixture in an acetate
buffer solution of the optimum pH-values is demonstrated. Ayman A Gouda and
Wafaa S Hassan [67] have described three simple, sensitive and reproducible
spectrophotometric assay methods for the determination of etodolac in pure form and
in pharmaceutical formulations. Two of the methods are based on the oxidation of
etodolac by Fe3+
in the presence of o-phenanthroline (o-phen) or bipyridyl (bipy). The
formation of the tris-complex on reaction with Fe3+
-o-phen and/or Fe3+
-bipy mixtures
in acetate buffer solution at optimum pH is demonstrated at 510 and 520 nm with o-
phen and bipy. Third method is based on the oxidation of etodolac by Fe3+
in acidic
medium, and the subsequent interaction of iron(II) with ferricyanide to form Prussian
blue, with the product exhibiting an absorption maximum at 726 nm. Ragaa El-Shiekh
[68] have reported three spectrophotometric methods for the determination of
pipazethate hydrochloride (PiCl) in pure form and in pharmaceutical formulations are
described. The first and second methods are based on the oxidation of the drug by
Fe3+
in the presence of o-phenanthroline (o-phen) or bipyridyl (bipy). The formation
of tris-complex upon reactions with Fe3+
-o-phen and/or Fe3+
-bipy mixture in an
Chapter – I Part – B Visible Spectrophotometry
19
acetate buffer solution of the optimum pH values is demonstrated at 510 and 522 nm,
respectively, with o-phen and bipy. The third method is based on the reduction of
Fe(III) in acid medium and subsequent interaction of Fe(II) with ferricyanide to form
Prussian blue, which exhibits an absorption maximum at 750 nm. In the present
investigations, (Method M3), the selected drug LEF is treated with access of Fe (III)
salt under specified experimental conditions. Acting as oxidant Fe (III) undergoes
reduction to Fe (II) in oxidizing (LEF) which corresponds to the drug concentration.
Fe (II) is estimated by the usual reagent for divalent iron, o-Phenanthroline. The
details of the investigations, scheme of reactions are compiled in chapter III.
III. Brucine – Periodate: Method M4
Brucine (2, 3 – dimethoxystrychnine) under acidic conditions has been sued as
an effective reagent for spectrophotometric determination of nitrates and nitrites,
cerium, manganese, cadmium and platinum. Several modifications have been
introduced for the spectrophotometric determination of nitrites and nitrates using this
reagent. Brucine can also been used for the spectrophotometric determination of
halides and cysteine and as an indicator in redox titration. Brucine forms a 1:1 colored
complex with p–dimethylamino cinnamaldehyde under acidic conditions
Sodium metaperiodate is an effective oxidant for converting methyl
substituted p-dihydroxy phenols to o-quinones and is also color stabilizer. brucine-
periodate reagent for spectrophotometric determination of tryptophan and some
sulphur compounds and for tetracyclines, chlorophenicol and streptomycin
.According to them, periodate converts most electron rich portion of the coupler
(tryptophan and other mentioned compounds) to yield 1-mono substituted
Chapter – I Part – B Visible Spectrophotometry
20
bruciquinone derivatives with an absorption maximum at 500-510 nm as the colored
species. Brucine – periodate reagent gave colored species with the compounds
containing either primary or secondary aliphatic amino and aromatic primary amine
groups. A simple, accurate and reproducible UV-Visible spectrophotometric method
[69] established for the assay of ceftiofur (CEFT) based on oxidative coupling of
CEFT with brucine/IO4. Determination of CEFT in bulk form and in pharmaceutical
formulations has also been incorporated. A spectrophotometric method has been
developed for the determination of nicorandil [70] in drug formulations and biological
fluids. This method is based on the reaction of the drug with brucine–sulphanilic acid
reagent in sulphuric acid medium producing a yellow-coloured product, which
absorbs maximally at 410 nm. On the basis of this observation, the author has
developed a specific method for the assay of NTT (Method M4) in bulk samples and
dosage forms. The details of the spectrophotometric investigations of the
corresponding drug are incorporated in chapter II.
IV. MBTH – Oxidant: Methods M5(a), M5(b), M5(c) and M5(d)
3-Methyl-2-benzothiazolinone hydrazone Hydrochloride (MBTH) is
synthesized by Besthorn [71]. The first procedure described by for the determination
of aldehydes, with which MBTH condenses to give a blue cation. This technique is
later improved, allowing more sensitive determinations. The reaction is applied to the
analysis of aliphatic aldehydes and the detection of the aldehyde groups in tissue and
collagen. Under reaction conditions, MBTH loses two electrons and one proton on
oxidation, forming the electrophilic intermediate, which has been postulated to be the
active coupling species. The intermediate reacts with amine or phenol by electrophilic
attack on the most nucleophilic site on the aromatic ring of amine or phenol (i.e., para
Chapter – I Part – B Visible Spectrophotometry
21
or ortho position) and the intermediate is spontaneously oxidized in the presence of
oxidant to form the colored species. MBTH also forms a strongly electrophilic
diazonium salt when acted upon by an oxidizing agent. These properties led the way
to colorimetric determinations based on the formation of formazans. Glyoxal reacts
with MBTH in the presence of acetic acid giving yellow diazine, which allows its
determination in the presence of unsubstituted monoaldehydes, when oxidant is
present. Phenol is so determined using the oxidant cerium (IV) ammonium sulphate.
This reaction is extended to miscellaneous other phenols, using various oxidants [72]
and an automated method MBTH can be used for the determination of polyhydroxy
compounds aromatic amines [73], aliphatic and alicyclic amines. Azodyes, stilbenes
and Schiff bases as well as pyrrole derivatives also react with MBTH under oxidative
conditions. This reaction is extended to the determination of bilirubin and its
oxidation products such as urobilin and biliverdin. Ferric chloride has been mostly
used as an oxidant for the determination of aromatic and heterocyclic amines by (in
neutral conditions) and (in acidic conditions). Other oxidants such as periodate (acidic
conditions), ammonium persulphate (alkaline conditions) and potassium dichromate
(acidic conditions) are employed for the determination of ethylenic compounds and
primary alcohols (after oxidation with Ruthenium tetraoxide. Sastry et al [74]
reviewed various aspects of MBTH chemistry in pharmaceutical analysis. Malipatil
et. al [75] have developed a spectrophotometric method in which oseltamivir
phosphate formed a green coloured chromogen when treated with MBTH in the
presence of oxidant ferric chloride. Vijaya Raja [76] reported a method which is based
on the oxidation of 3-methylbenzothiazolin-2-one hydrazone (MBTH) by ferric
chloride followed by its coupling with the drug producing colored complex measured
at 670nm in acidic medium. Adefovir dipivoxil [77] is subjected to acid hydrolysis
Chapter – I Part – B Visible Spectrophotometry
22
and the hydrolysed product used for the estimation. A simple and sensitive kinetic
method is described for the determination of ketoprofen [78] in pure form,
pharmaceuticals and biological fluids. Rekha Rajeevkumar et.al [79] have reported a
visible spectrophotometeric method for the quantitative estimation of Moprolol in
bulk drug and pharmaceutical preparations which is based on the reaction of Moprolol
with 1%w/v 3-methyl-2- benzthiazolinone hydrazone hydrochloride (MBTH) reagent
in presence of 2%w/v of ferric chloride salt to give a green colored chromogen with
absorption maximum at 629nm. Prakash et.al [80] have developed a method which is
based on oxidation followed by coupling of 3 methyl-2-benzothiazolinone hydrazone
(MBTH) with Ganciclovir in presence of ferric chloride to form bluish green color
chromogen and exhibiting absorption maximum at 611.8 nm and obeying beers law
in the concentration range of 50 – 250 μg/ml respectively. Vijaya Raja et.al [81] have
described a spectrophotometric method for the determination of bromhexine
hydrochloride in bulk and formulations which is based on oxidation of 3-
methylbenzothiazolinone-2- hydrazone by ferric chloride followed by its coupling
with the drug in acidic medium forming an intense green colored chromogen with
absorbance maxima at 630nm. Malipatil et al [82] have developed a
spectrophotometric method for the determination of Citicoline with MBTH. Lakshmi
et al [83] reported a spectrophotometric method utilizing the reaction of manidipine
with 3-methyl-2-benzothiazoniumhydrazone hydrochloride (MBTH) in the presence
of ferric chloride. A novel-coupling reagent is used for the simple and sensitive
spectrophotometric determination of caffeine (CF) and theophylline (TP) [84] in pure
or pharmaceutical formulations. In the present investigation the selected drugs NTT,
LEF, OND and TDP which possesses amino group, produced an oxidative coupling
product with MBTH in the presence of oxidants such as Ce (IV), NaIO4 and IBDA.
Chapter – I Part – B Visible Spectrophotometry
23
The probable sequence of reactions based on analogy is presented in the respective
chapters of the corresponding drugs.
V. Redox reactions- Folin Ciocalteu [FC] reagent : Method M6
Heteropolyacid complexes are formed by the combination of orthophosphoric
acid and periodic, molybdic, vanadic, tungstic and molybdovanadic acids. Treatment
of complexes with reducing agents result in the formation of the corresponding
reduction products, which are blue in color (eg: molybdenum blue from
phosphomolybdate, tungsten blue from phosphotungstate). This reaction is the basis
of several methods suggested for the determination of phosphate. Various reducing
agents have been used for the reduction of heteropolyacids. Stannous chloride is most
widely used one among several reducing agents (1,2, 4-aminonaphthol sulphonic acid
, ascorbic acid , hydrazine, ferrous sulphate ], p-amino phenol hydrochloride
,thiosulphate and sulphite ,thiourea, pyrogallol and metol. Among the various
heteropolyacids, phosphomolybdo tungstic acid, the well-known Folin-Ciocalteu
reagent (F.C reagent) is preferred by a number of workers for the determination of
drugs [85-87]. The wavelength of maximum absorption and stability of the blue
colored reduction product and the sensitivity and reproducibility of the reaction are
dependent upon pH, composition of the heteropolyacid complex, nature and
concentration of the reducing agent, temperature and time. Allopurinol [88], caffeine ,
pentazocine [89], oxymetazoline, isoxsuprine, orciprenaline, pholedrin, vitamin–K
and rutin [90] are some typical examples of drugs estimates in this manner. A
spectrophotometric method for the estimation of hydralazine hydrochloride [91] in
bulk drug and in their tablet formulations is described. The described method is based
on the formation of blue colored chromogen due to the reaction of hydralazine
Chapter – I Part – B Visible Spectrophotometry
24
hydrochloride with Folin Ciocalteu reagent in presence of alkali, which exhibits
maximum wavelength at 640 nm. A reproducible colorimetric method has been
developed for the estimation of Memantine by Jagathi [92] in bulk and in
pharmaceutical formulations. This method is based on the reduction of
Folin‐Ciocalteau reagent by the drug and the reduced species posses a characteristic
intense blue color (λmax 760 nm). Singh et.al [93] have reported a method for the
determination of ajmaline and brucine based on the development of blue coloured
product due to reduction of tungstate and/or molybdate in Folin Ciocalteu‟s reagent
by ajmaline and brucine in alkaline medium. Mohamed Abd El-Ghaffar [94] has
reported a spectrophotometric method for the determination of ritodrine hydrochloride
(RTH) either in pure form or dosage forms using Folin-Ciocalteu reagent producing
blue chromogen which is measured at 760 nm. Prakash S Sarsambi [95] et.al have
described a spectrophotometric method for the determination of which is based on
reduction of Ganciclovir, an acyclic guanosine analog used in the treatment of AIDS
using Folin-Ciocalteu reagent in presence of alkali to form intense blue color
chromogen exhibiting absorption maximum at 764.7nm. A spectrophotometric
method [96] has been developed for the estimation of diacerein in Pharmaceutical
dosage forms. A spectrophotometric method for the determination of penicillins [97]
(ampicillin, amoxycillin and carbenicillin) using Folin-Ciocalteu reagent (FC reagent)
is described. Basavaiah K et.al [98] have described a spectrophotometric method for
the determination of acyclovir in bulk drug and in formulations. A spectrophotometric
method is described for the determination of amoxicillin and ampicillin by Dhruv et.al
[99] using Folin-Ciocalteu reagent. The above method has been used in the
determination of NTT in the present investigations. The details of the investigation
have been incorporated in Chapters II.
Chapter – I Part – B Visible Spectrophotometry
25
VI. N-Bromosuccinimide (NBS) as an oxidant: Method M7
N-Bromosuccinimide contains weakly bound bromine and is used for
brominations and dehydrogenation in organic chemistry. The reagent behaves as a
mild oxidising agent and converts primary and secondary alcohols to the
corresponding aldehydes and ketones. It has been used as quantitative oxidizing agent
for hydrazines for thiourea and some of its derivatives for isoniazid, and for the ene-
diol group in ascorbic acid. The reagent is highly selective oxidant and after all the
compound being titrated has been oxidized a slight excess gives a blue color with
potassium iodide - starch or decolorises methyl red either of which can be used as the
end point detector. By the titrimetric procedure analyte can be estimated only at
milligram level. After completion of the reaction with analyte the unreacted NBS can
be determined using visible spectrophotometric method. The reacted NBS (NBS
originally added - NBS unreacted) corresponds to the analyte present in the
microgram level. Two reagents (CB) [100], PMAP-SA[101] have been used in the
literature for the colorimetric determination of NBS. A spectrophotometric method is
described for the determination of the commonly used antimycobacterial drugs such
as Isoniazid and Rifampicin[102] in their pure forms, pharmaceutical preparations and
biological fluids based on the reaction of drugs with N-bromosuccinimide (NBS),
then the excess of NBS is reacted with KI, the liberating iodine is determined at wave
length 572 nm. Ibrahim et.al [103] have reported a method which is based on the
reaction with N-bromosuccinimide (NBS) and subsequent reaction of the remaining
NBS with fluorescein (FLC) to give a pink colored product that is measured at 518
nm. A sensitive spectrophotometric method is presented [104] for the assay of
pantoprazole sodium sesqui hydrate (PNT) in bulk drug and in formulations using N-
Chapter – I Part – B Visible Spectrophotometry
26
bromosuccinimide (NBS) and two dyes, methyl orange and indigo carmine, as
reagents. This method involves the addition of a known excess of NBS to PNT in acid
medium, followed by determination of unreacted oxidant by reacting with a fixed
amount of either methyl orange and measuring the absorbance at 520 nm (method A)
or indigo carmine and measuring the absorbance at 610 nm. In the present
investigations the author proposed a simple selective and sensitive indirect
spectrophotometric method using NBS/CB for the assay of OND (Method M7). This
method involves two steps. First step involves the oxidation of OND with NBS. The
second step in the procedure is the quantitative decolorisation of CB by the unreacted
NBS. The probable sequence of reactions in two steps (drug – NBS, NBS - CB) based
on analogy are present in the schemes of the corresponding chapters. The details of
these investigations are incorporated in chapter IV of the individual drugs.
VIII. Condensation reaction - (a)Isatin - Sulphuric acid : Method M8
Isatin (1H – indole – 2, 3 – dione) is first obtained by Erdman and Laurnet as a
product from the oxidation of indigo by nitric and chromic acids. It usually exists in
two tautomeric forms (lactam and lactim). Isatin under alkaline conditions hydrolyze
giving o-amino benzene keto acid, which condenses with acetone giving 2 – methyl
cinchonic acid. The blue color of indophenin dye formation (pfitzinger reaction)
involving isatin and thiophene in presence of sulphuric acid is due to the formation of
compounds related to indigo [105-106]. Under acidic conditions isatin reacts with
proline or pyrrole to give colored condensation product. Isatin also produce a
fluorogenic derivative when reacted with tryptophan, which has been used for its
detection by TLC [107-108]. In the present investigation, a visible spectrophotometric
method has been developed for NTT, which possesses carbazone moiety by using
Chapter – I Part – B Visible Spectrophotometry
27
isatin and sulphuric acid in acetic acid medium. The details of these investigations
are presented in chapter II.
b) Vanillin: Method M9
It is well known that aromatic aldehydes form colored condensation product
(Schiff base) with aromatic primary amines in particular. It has been observed by
suitable alteration of experimental conditions, others such as hydrazine and its mono
substituted derivatives, primary alkyl amines, amino acids [109] converted to pyrrole
derivatives [110,111]. Primary heterocyclic amines and m-diphenol[112] also develop
color with aromatic aldehydes. Enoche Florence Oga [113] has described a method
for the determination of isoniazid in pure form and in pharmaceutical formulations.
Two simple and sensitive spectrophotometric methods have been developed for the
estimation of Famciclovir [114] in bulk and tablet dosage form which are based on the
condensation reaction of Famciclovir with carbonyl reagents such as p-
dimethylaminobenzaldehyde (PDAB) and vanillin in acidic condition to form orange
yellow colored chromogen with absorption maxima at 480 nm and 470 nm
respectively. Manikya Sastry et. al [115] developed a spectrophotomeric method for
the determination of Lerccnidipine HCl using vanillin as a chromogenic reagent
having maximum absorbance at 600nm. A new spectrophotometric method [116] has
been examined for the determination of the tranexamic acid by derivatization with
vanillin. A colorimetric method is developed for the quantitative determination of
hydralazine hydrochloride in dosage forms by Bala et.al [117]. Three
spectrophotometric methods have been developed for the quantitative estimation of
nitazoxanide [118] in bulk drug and pharmaceutical formulations. These methods are
based on the reaction of reduced nitazoxanide with p-dimethylaminobenzaldehyde, p-
Chapter – I Part – B Visible Spectrophotometry
28
dimethyl amino cinnamaldehyde and vanillin in acidic conditions to form pink,
orange red, and orange yellow coloured chromogens with absorption maxima at 559
nm, 534.5 nm, and 475 nm respectively. These observations have led to application of
aromatic aldehyde, Vanillin (p-hydroxy-m-methoxy benzaldehyde) as analytical
reagent for the analysis of pharmaceutical dosage forms. In the present investigation
the selected drugs NTT, it is observed that Vanillin under certain established
experimental conditions produce color of maximum intensity in methanol with the
selected drug NTT and the results of these investigations are presented in chapters II.
IX. Ortho nitro benzaldehyde: Method M10
It is well known that aromatic aldehydes form colored product due to
condensation with aromatic primary amines in particular. It has been observed by
suitable alternation of experimental conditions, others such as hydrazine and its mono
substituted derivative, primary alkyl amines, amino acids converted to pyrrole
derivatives, indole derivatives primary hetero cyclic amines and m-diphenol develop
color with aromatic aldehydes. These observations have led to numerous applications
of aromatic aldehydes such as p-dimethyl amino benzaldehyde (PDAB), p-dimethyl
amino cinnamaldehyde (PDAC), ortho notro benzaldehyde (ONB) as analytical
reagents. In the present investigation, NTT responded to condensation reaction with
ONB in the presence of Conc. H2SO4. The details of the investigation of the
corresponding drug (NTT) are incorporated in chapter II.
X. Poly acid complexes - AMV /H2SO4 : Method M11
The orthovanadate ion [VO4]-3
occurs only at very high pH. It is such a strong
base that the first step in its protonation, forming [HVO4]-2
, is already complete at pH
Chapter – I Part – B Visible Spectrophotometry
29
12. When the pH is gradually lowered to one successive protonation takes place that
ultimately leads to the formation of the pale yellow cationic species, usually
formulated as VO-1
2 Due to the great tendency of vanadate to oligomerize, the
protonated monomers [HVO4]-2
, [H2VO4]-1
, and VO+
2 (except at very low pH) are
predominant only in highly diluted solution. The chemistry of vanadium is
complicated. It forms compounds corresponding to oxidation numbers +2 to +5. The
most stable and commonly encountered compounds of Molybdenum are derived from
its oxide [VO4]-3
.The vanadium compounds corresponding to the oxidation states
ranging from +2 to +5 are mostly complexes species. The isopolyanionic or hetero
polyanionic species of vanadium undergo reduction to coloured vanadium species
with certain bioactive compounds. The max values of reduction products vary from
600nm – 850 nm depending upon the reaction conditions (nature and strength of acid
or base medium, temperature, time) nature of poly acid (very efficient if the
composition of hetero acids are more) and nature of reducing agent (analyte).
“Vanadium greenish Blue” is the result of mild reduction of an acidified solution,
which contains V(VI), either as an iso-or a hetero polymolybdate anion (or even
alkaline conditions). Wieslawa Misiuk and Ewa Kleszezewska [119] have reported a
spectrophotometric method for the determination of Prothipendyl HCl on reacting
with ammonium metavanadate forming a colored oxidation product exhibiting
maximum absorbance at maximum wavelength 374nm. In the present investigation,
TDP responded to condensation reaction with AMV in the presence of Conc. H2SO4.
The details of the investigation of the corresponding drug (TDP) are incorporated in
Chapter V.
Chapter – I Part – B Visible Spectrophotometry
30
XI. NaIO4 / Phenyl hydrazine hydrochloride (PHH)/[Fe(CN)6]-3
:Method M12
Periodic acid oxidation [120] is applicable to compounds having two hydroxyl
groups or a hydroxyl and an amino group attached to adjacent carbon atoms and are
characterized by the cleavage of the carbon-carbon bond. If the hydroxyl groups or a
hydroxyl and an amino group are not vicinal, no oxidation takes place. This selectivity,
which is the outstanding characteristic of periodic acid oxidation, adopts this reaction for
the presence of vicinal hydroxyl groups and hydroxyl and amino groups. Carbonyl
compounds in which the carbonyl group is adjacent to a second carbonyl (α-diketone) or
hydroxyl (α-ketol) group are also oxidized.
Sodium metaperiodate (IO4-) is considerably soluble in water (12.62g/100mL,
25oC). The solubility of sodium metaperiodate is greatly reduced in alkaline solution
because of the formation of disodium metaperodate (Na2H3IO6). This effect occurs at
pH>5.0.Aqueous solution of sodium metaperiodate at pH 4.0 or below is the most suitable
one as the oxidant. The rapid and generally quantitative nature of the reaction recommends
for a very wide variety of analytical applications. Certain analytical procedures have been
developed for the determination of aldehydes utilizing periodate oxidation. Different
reagents are used in developing the spectrophotometric methods for their determination.
Amoung several reagents used for the determination of aldehydes in particular
formaldehyde (existing or formed through some preliminary treatment such as periodate
oxidation of compounds possessing vicinal aminol, diol or ketol), the reagent like
schryver‟s appear to yield highly sensitive and stable chromogen with formaldehyde
especially. This method avoid the distillation or diffusion step and permit the determination
of the liberated formaldehyde directly in the reaction medium colorimetrically by oxidative
coupling reaction with schryver reaction with PHH and hexacyanoferrate (III) and this
Chapter – I Part – B Visible Spectrophotometry
31
method has been applied for the determination of doxorubicin [121]. In the present
investigation, LEF responded to oxidative coupling reaction with PHH in the presence of
hexacyanoferrate (III) giving formazan dye. The details of the investigation of the
corresponding drug (LEF) are incorporated in chapter III.
XII. Ce (IV) / 2, 4 DNP: Method M13
Condensation reaction is one in which two molecules join together with the
loss of a water molecule in the process. In this case, that small molecule is water. In
terms of mechanisms, this is a nucleophilic addition-elimination reaction. 2, 4-
dinitrophenylhydrazine is often abbreviated to 2,4-DNP or 2,4-DNPH. A solution of
2,4-dinitrophenylhydrazine in a mixture of methanol and sulphuric acid is known as
Brady's reagent. The 2,4-dinitrophenylhydrazine first adds across the carbon-oxygen
double bond to give an intermediate compound which then loses a molecule of water.
The ketone functional group can also take part in auto condensation reactions, which
eliminate water. A spectrophotometric method has been proposed by Nagaraja and
Aswinee Kumar [122] for the determination of four phenolic drugs; salbutamol,
ritodrine, amoxicillin and isoxsuprine. The method is based on the oxidation of 2, 4-
dinitrophenylhydrazine and coupling of the oxidized product with drugs to give
intensely colored chromogen. A simple spectrophotometric method is developed and
validated for the determination of Tolperisone [123] in bulk and its dosage forms. The
proposed method is based on the interaction of the drug with 2,4-
dinitrophenylhydrazine in the presence of an acid catalyst, followed by treatment with
a methanolic solution of potassium hydroxide; an intensely colored chromogen is
formed that is measured in dimethyl formamide as the diluting solvent at 520 nm.
Osama [124] developed a spectrophotometric method for the determination of
Chapter – I Part – B Visible Spectrophotometry
32
azithromycin (AZ), clarithromycin (CLA), and roxithromycin (ROX) in bulk powders
and their dosage forms.
XIII. Coordination complex formation with Cobalt thiocyanate: Method M14
Cobalt thiocyanate (CTC) (formed by combination of ammonium thiocyanate
and cobalt nitrate) has been proved to be a valuable chromogenic reagent for the
detection and determination of amino compounds. The colored species formed is the
coordination complex of the drug (electron donor) and the central metal atom of
cobalt thiocyanate which is extractable into nitrobenzene from aqueous solution. The
basis in the present investigation is the formation of the blue colored complexes by
LEF (the presence of cyclic tertiary amine group) when treated with CTC. The
probable sequences of reactions based on analogy are presented in the individual
chapters III.
XIV. p-CA + CHCl3 -Charge-transfer Complex formation reactions: Method M15
It is well known that electron donors and electron acceptors can interact in
solution to form intensively colored charge-transfer complexes. These complexes are
usually characterized by absorption bands, not present in either reagent, assigned to an
intermolecular charge-transfer transition [125-126]. Some substituted quinones such
as p-benzoquinone chlorimide [127], 2,5-dichloro benzoquinone[128] and p-N-
methylbenzoquinone monoamine[129], 2,3-dichloro-5,6-dicyano-p-benzoquinone
DDQ[130-131], chloranilic acid[132] and chloranil have been used in the studies of
amines. Two spectrophotometric methods have been proposed [133] for the
determination of milrinone in pharmaceutical formulations based on the charge
transfer complexation reaction of milrinone with 2, 3-Dichloro-5, 6-
Chapter – I Part – B Visible Spectrophotometry
33
Dicyanobenzoquinone, DDQ and p-Chloranilic Acid (pCA). Two Spectrophotometric
procedures are presented by M. Walash et. al. [134] for the determination of two
commonly used H2-receptor antagonists nizatidine (I) and ranitidine hydrochloride
(II). The methods are based mainly on charge transfer complexation reaction of these
drugs with p-chloranilic acid or 2, 3 dichloro-5, 6-dicyanoquinone (DDQ).
Kanakapura Basavaiah et.al. [135] reported two visible spectrophotometric methods
for the determination of bupropion hydrochloride in pharmaceuticals and spiked
human urine. These methods are based on charge transfer complexation reaction of
bupropion base (BUP) as n-electron donor with either p-chloranilic acid or 2, 3-
dichloro-5, 6-dicyano-1, 4-benzoquinone (DDQ) as π-acceptors to give highly colored
radical anion species.
Rahman and Haque [136] have developed two spectrophotometric methods
are based on the charge transfer complexation reaction of the drug with p-chloranilic
acid (pCA) in 1, 4-dioxan-methanol medium, 2, 3-dichloro 5, 6-dicyano 1, 4-
benzoquinone (DDQ) in acetonitrile-1,4 dioxane medium. Two spectrophotometric
methods have been proposed [137] for the determination of lisinopril in pure form and
pharmaceutical formulations. The methods are based on the charge transfer
complexation reaction of the drug with 7, 7, 8, 8, tetracyanoquinodimethane (TCNQ)
and p-chloranilic acid (pCA) in polar media.
Hisham E. Abdellate [138] have developed two spectrophotometric methods
for the determination of perindopril which are based on the reaction of this drug as n-
electron donor with 2,3-dichloro-5,6-dicyano-p-benzoquinone(DDQ)-7,7,8,8
tetracyanoquinodimethane (TCNQ), tetracyanoethylene (TCNE), chloranil (CL) and
p-chloranilic acid (p-CA) as π-acceptors to give highly coloured complex species.
Chapter – I Part – B Visible Spectrophotometry
34
Chloranil, a strong electron acceptor can form charge-transfer complex with a variety
of electron donors, including the above drugs, containing amino-group. Studies
showed that tetrachloro-p-benzoquinone (p-chloranil), acting as an electron acceptor,
could be used for determination of aliphatic and aromatic amines as well as some
amino acids. Taking these considerations into account, NTT, LEF, OND and TDP
drug molecules, which have amino groups, can act as an electron donor and react with
p-chloranil. The proposed mechanism for the reaction between these drugs and p-
chloranil to produce the charge transfer complex are showed in respective chapters
XV. H2O2 / per acids +MO: Method M16
Tertiary amines can be convertedto amine by oxidation. Hydrogen peroxide is
often used, but peracids are also important reagents for this purpose.In the attack by
hydrogen peroxide there is first formed a trialkyl ammonium peroxide,the
decomposition of this complex probably involves an attack by the OH +
moiety of
H2O2.
R3N + H2O2 R3N.H2O2 R3N+
- OH
The positive ion of the oxidisedform of the drug is associated with negative ion of the
methyl orange molecule to form ion-ion associated complex which is extracted in to
chloroform solvent.
XVII. Condensation reactions with 4-AP, INH: Method M18 and Method M19
The ketone functional group can also take part in auto condensation reactions
with elimination of water molecule. These reactions will produce an aromatic
molecule from a starting point of an aliphatic, straight chain molecule. Polymerization
Chapter – I Part – B Visible Spectrophotometry
35
across the carbonyl group is a very uncommon reaction, particularly compared to the
similar group in aldehydes. Carbonyl compounds, ketosteroids can be determined
through the formation of hydrazones or schiff bases with Isoniazid (INH) and 4-
aminoantipyrine (4-AP) [139]. The reaction with hydrazines is rather general,
condensation with hydrazides and amines may lead to more selective colorimetric or
fluorimetric determinations. In the present investigation 4-AP and INH have been
used for the determination of LEF and OND which possess keto group and the details
concerned are presented individually Chapter-III and Chapter – IV. Statement
showing functional groups/moieties in the selected drugs, methods proposed,
reagents, types of reactions involved are presented in Table: 1.03, P: 52-55.
1.08 Method Development and Optimization
In developing a quantitative method for determining an unknown
concentration of a given substance by absorption spectrophotometry, the first step will
be the selection of analytical wavelength at which absorption measurements are made.
The analytical wavelength can be chosen either from literature or experimentally by
means of scanning with a spectrophotometer. In order to enhance the sensitivity of the
method and signal to noise ratio, the wavelength of maximum absorbance is chosen as
analytical wavelength. Absorption spectrum is a graphical representation of the
amount of light absorbed by a substance at definite wavelengths. To plot a curve, the
values of wavelength in the visible region are laid off along the x- axis and the
absorbance values on y-axis. A characteristic of an absorption spectrum is a position
of the peaks of light absorption by the substance and also the intensity of absorption,
which is determined by the absorptivity at definite wavelength. After selection of the
analytical wavelength, the chromogenic reagent and the absorbing product must be
Chapter – I Part – B Visible Spectrophotometry
36
stable for a considerable period of time. Now a series of standard solutions are
prepared under the optimum conditions, and the absorbance values are measured at
λmax, these values are plotted against the concentration in μg/ml.
In each type of basic reaction, the colored species is formed or the final color
of the reaction mixture whose absorbance is measured and thus the sensitivity of the
method, rate of color formation and stability is affected by the concentration of the
reagent in the solution, nature of solvent, temperature, pH of the medium, order of
addition of reactants and intervals between additions. For simple systems having no
interaction between variables, the one variable at a time strategy appears to be simple,
efficient and effective to establish the optimum conditions [140]. The one variable at a
time approach requires all variables but one to be held constant while a univariate
search is carried out on the variable of interest. The details of fixing optimum
conditions used in different procedures of present investigations are furnished in
subsequent chapters.
1.09 Statistical Treatment of Analytical Data
1.09 (i) Calibration: Calibration is one of the most important steps in bioactive
compound analysis. A good precision and accuracy can only be obtained when a good
calibration procedure is used. In the spectrophotometric methods, the concentration of
a sample cannot be measured directly, but is determined using another physical
measuring quantity 'y (absorbance of a solution). An unambiguous empirical or
theoretical relationship can be shown between this quantity and the concentration of
analyte.
Chapter – I Part – B Visible Spectrophotometry
37
The calibration between y = g (x) is directly useful and yields by inversion of
the analytical calculation function. The calibration function can be obtained by fitting
an adequate mathematical model through the experimental data. The most convenient
calibration function is linear, goes through the origin and is applicable over a wide
dynamic range. In practice, however, many deviations from this ideal calibration line
may occur. For the majority of analytical techniques the analyst uses the calibration
equation.
Y = a + bx
In calibration univariate regression is applied, which means that all observations are
dependent upon a single variable x.
1.09(ii) Linear Regression- Method of least squares
Least-squares regression analysis [141,142] can be used to describe the
relationship between response (y) and concentration (x). The relationship can be
represented by the general function Y = f (x, a, b1, ……. bm ) Where a, b1......, bm
are the parameters of the function.
We adopt the convention that the x values relate to the controlled on
independent variable (e.g. the concentration of a standard) and the y values to the
dependent variable (the response measurements). This means that the x values have
no error. On the condition that the errors made in preparing the standards are
significantly smaller than the measuring error (which is usually the case in analytical
problems). The values of the unknown parameters a, b1, ... bm must be estimated in
such a way that the model fits the experimental data points (xi, yi) as well as possible.
Chapter – I Part – B Visible Spectrophotometry
38
The true relationship between x and y is considered to be given by a straight line. The
relation between each observation pair (Xi, Yi) can be represented as
Yi = + Xi + ei
The signal yi is composed of deterministic component predicted by linear
model and a random component ei. One must now find the estimates of a and b of the
two values and . This can be done by calculating the values a and b for which ei2 is
minimal. The component ei represent the differences between the observed yi values
and the predicted yi values by the model. The ei are called the residuals, a and b are
the intercept and slope respectively.
2
1 1
2
1 11
n
i
n
i
ii
n
i
n
i
ii
n
i
ii
xxn
yxyxn
b 2
1 1
2
1 11
2
1
n
i
n
i
ii
n
i
n
i
ii
n
i
ii
n
i
i
xxn
yxxxy
a
1.09(iii) Standard error on estimation ( Se)
The standard error on estimation is a measure of the difference between
experimental and computed values of the dependent variable. It can be represented by
the following equation,
)2/()(1
2
nyySn
i
iie
Yi, and yi, are the observed and predicted values, respectively. Standard deviations on
slopes (Sb) and intercepts (Sa) are quoted less frequently, even though they are used to
Chapter – I Part – B Visible Spectrophotometry
39
evaluate proportional differences between or among methods as well as to compute
the independent variables such as concentration etc. It is important to understand how
uncertainties in the slope are influenced by the controllable properties of the data set
such as the number and range of data points and also how properties of data sets can
be designed to optimize the confidence in such data.
1.09(iv) Standard deviation on slope ( Sb)
The standard deviation on slope is proportional to standard error of estimate
and inversely proportional to the range and square root of the number of data points.
)2(
)(1
2
n
yy
S
n
i
ii
b
Where Xi is the arithmetic mean of xi values
1.09(v) Standard deviation on intercept, Sa
Intercept values of least squares fits of data are often to evaluate additive
errors between or among different methods
)2(
)(1
2
n
yy
S
n
i
ii
a
1.09(vi) Correlation coefficient (r)
To establish whether there is a linear relationship between two variables xi and
yi, use Pearson‟s correlation coefficient r. The value of r must lie between +1 and -1;
n
i
ii xx1
2)(
1
n
i
ii xx1
2)(
1
n
i
ii xx1
2)(
1
n
xn
i
i1
2
Chapter – I Part – B Visible Spectrophotometry
40
the nearer it to ±1, the greater the probability that a definite linear relationship exists
between the variables x and y, values close to +1 indicate positive correlation and
values close to -1 indicate negative correlation.Valuer of r that towards zero indicate
that x and y are not lineary related.
222 )1/()()(
)1/())((
nyyxx
nyyxx
rn
ii
n
ii
1.09(vii) Selectivity of the method
Matrix and interference effects may disturb the determination of an analyte.
Some of the excipients, incipients and additives present in pharmaceutical
formulations may sometimes interfere in the assay of drug and in such instances
appropriate separation procedure is to be adopted initially. The selectivity of the
method is ascertained by studying the effect of a wide range of excipients and other
additives usually present in the pharmaceutical formulations to be determined under
optimum conditions. Initially, interference studies are carried out by the determination
of fixed concentration of the drug several times by the optimum procedure in the
presence of a suitable (1-100 fold) molar excess of the foreign compound under
investigation and its effect on the absorbance of the solution is noticed. The foreign
compound is considered to be interfering at these concentrations if it constantly
produces an error of less than 3.0% in the absorbance produced in pure solution.
Chapter – I Part – B Visible Spectrophotometry
41
1.09(viii) Linearity and Sensitivity of the method
Knowledge of the sensitivity of the color is important and the following terms
are commonly employed for expressing sensitivity. According to Beer's law
A= c t
The absorbance (A) is proportional to the concentration (c) of the absorbing
species, if absorptivity () and thickness of the medium (t) are constant. When c is in
moles per litre, the constant is called Molar absorptivity. Beer's law limits and max
values are expressed as g.ml-1
and l mole-1
.cm-1
, respectively. Sandell's sensitivity
[143] refers to the number of g of the drug to be determined, converted to the colored
product, which in a column solution of cross section 1 cm2 shows an absorbance of
0.001 (expressed as g.cm-2
).
1.09(ix) Limit of Detection and Limit of Quantification
The limit of detection (LOD) [144] of an analytical method may be defined as
the concentration, which gives rise to an instrument signal that is significantly
different from the blank. For spectroscopic techniques or other methods that rely upon
a calibration curve for quantitative measurements, the IUPAC approach employs the
standard deviation of the intercept (Sa), which may be related to LOD and the slope of
calibration curve, b, by
LOD = 3Sa / b LOQ=10Sa / b
Chapter – I Part – B Visible Spectrophotometry
42
1.09(x) Ringbom's Plots
The relative concentration error depends inversely upon the product
absorbance and transmittance. The relative error increases at the extremes of the
transmittance scale. The slope of plot 'C‟ versus T, i.e. Ringbom plot [145,146] gives
relative error coefficient (i.e. plot of log C T) The main limitations of Ringbom plot
is that it provides no concerning the concentration range of good precision unless it is
combined with T versus T relation. The above expression is valid whether Beer's
law is followed or not.
1.09(xi) Precision and accuracy
The purpose of carrying out a determination is to obtain a valid estimate of a
'true' value. When one considers the criteria according to which an analytical
procedure is selected, precision and accuracy [147] are usually the first time to come
to mind. Precision and accuracy together determine the error of an individual
determination. They are among the most important critical for judging analytical
procedures by their results.
Precision
Precision refers to the reproducibility of measurement within a set, i.e., to the
scatter of dispersion of a set about its central value. One of the most common
statistical terms employed is the standard deviation of a population of observation.
Standard deviation is the square root of, the sum of squares of deviations of individual
results for the mean, divided by one less than the number of results in the set. The
standard deviation S, is given by
Chapter – I Part – B Visible Spectrophotometry
43
n
i
i xxn
S1
2)(1
1
Standard deviation has the same units as the property being measured. The
square of standard deviation is called Variance (S2). Relative standard deviation is the
standard deviation expressed as a fraction of the, mean, i.e. S/x. It is sometimes
multiplied by 100 and expressed as a percent relative standard deviation. It becomes a
more reliable expression of precision.
% Relative standard deviation = S X 100/x
Accuracy
Accuracy normally refers to the difference between the mean x, of the set of
results and the true or correct value for the quantity measured. According to IUPAC
accuracy relates to the difference between results (or mean) and the true value. For
analytical methods, there are two possible ways of determining the accuracy, absolute
method and comparative method.
a) Absolute method
The test for accuracy of the method is carried out by taking varying amounts
of the constituents and proceeding according to specified instructions. The difference
between the means of an adequate number of results and amount of constituent
actually present, usually expressed as parts hundred (%) is termed as % error.
The constituent in question will be determined in the presence of other
substances, and it will therefore be necessary to know the effect of these upon the
Chapter – I Part – B Visible Spectrophotometry
44
determination. This will require testing the influence of a large number of probable
compounds in the chosen samples, each varying amounts. In a few instances, the
accuracy of the method controlled by separations (usually solvent extraction or
chromatography technique) involved.
b) Comparative method
In the analysis of pharmaceutical formulations (or solid laboratory prepared
samples of desired composition), the content of the constituent sought (expressed as
percent recovery) has been determined by two or more (proposed and official or
reference) supposedly "accurate" methods of essentially different character can
usually be accepted as indicating the absence of an appreciable determinate error.
1.10 Comparison of Results
To evaluate the accuracy of the method, one often compares [148] the method
being investigated of 'test method' with an existing method called the 'reference
method.
a) Student t-test
Student t-test is used to compare the means of two related (paired) samples
analysed by reference and test methods. It gives answer to the correctness of the null
hypothesis with a certain confidence such as 95% or 99%. If the number of pairs (n)
are small than 30, the condition of normality of x is required or at least the normality
of the difference (di). If this is the case the quantity
ns
dt
d
i
/
Chapter – I Part – B Visible Spectrophotometry
45
If this is the case the quantity has a student t-distribution with (n-1) degrees of
freedom, where di =XR (Reference method) – XT. (Test method) and Sd is the
standard deviation (S).
b) F- test
By the F-test we can test the significance of the difference in variances of
reference and test methods. Let us suppose that one carried out n1 replicate
measurements by test methods and n2 replicate measurements by using reference
method. If the null hypothesis is true, then the estimates ST2 (variance of the test
method) and SR2 (variance of reference method) do not differ very much and their
ratio should not differ much from unity. Infact, one uses the ratio of variances.
F = ST2
/ SR2
It is conventional to calculate the F - ratio by dividing the larger variance by the
smallest variance in order to obtain a value equal or larger than unity. If the
calculated F - value is smaller than F - value from the table, one can conclude that the
procedures are not significantly different in precision at given confidence level.
46
Table 1.01
Functional groups imparting acidic nature
1.Functional groups imparting acidic nature to a substance
i Carboxylic acid - COOH v
Enediol
- C (OH) = C (OH)
I
ii
Imide
- CO
- CO
N - H
vi
Phenolic Hydroxyl
OH
iii
Thiol
- SH
vii
Sulphonic acid
- SO3H
iv Enol
C = C (OH)
Cond…..
47
Table 1.01
Functional groups imparting basic nature
2. Functional groups imparting basic nature to a substance
i Primary amino group (R1 = R
2 = H)
ii Secondary amino group (R1 = H, R
2= alkyl)
iii Tertiary amino group (R1 = R
2 = alkyl)
The tertiary nitrogen is the necessary element in the molecules of alkaloids, heterocyclic compounds and mono & di substituted
hydrazine derivatives R2 – HN – NHR
1
ON
R1
R2
N
R1
R2
N
R1
R2 - (CH2)n - N
R1
R2
R,
,
and
Cond…..
48
Table 1.01
Functional groups imparting neither acidic nor basic nature
3.Functional groups which impart neither acidic nor basic nature to a substance
i Aldehyde
R-CHO vii
Ester
- COOR
ii Keto R-CO-R
1
viii
Lactam
NH
O
iii
Hydroxy methyl
R-CH2-OH
ix
Olefinic
C = C
iv
Nitroso
- N = O x Acetylenic - C C -
v
Methoxy
-O-CH3
vi
Ether
R-O-R1
Cond…..
49
Table 1.02
Chemical features of some acidic dyes used in ion association complex formation
S.No Dye name / CI No. Chemical category Structure Chemical name
1
Alizarine Red S
(ARS) / 58005
Anthraquinone dye
NaO3S
HO
O
O
2-Anthrcene sulphonic acid
- 9,10-dihydro-3,4-
dihydroxy
-9,10-dioxo, mono sodium
salt
2 BTB Triphenyl methane BrH3C
CHMe2
H3C
Br
Me2HC
HO OH
HC
O
SO2
Dibromo thymol sulphone
phthalein
Cond…
50
Table 1.02
Chemical features of some acidic dyes used in ion association complex formation
S.No Dye name / CI No. Chemical category Structure Chemical name
3 MO Azodye
N NNaO3S N(CH3)2
4
Tropaeoline ooo
(Tpooo) / 14600
Azodye
NNNaO3S OH
Benzenesulphonic acid,4[
(4-hydroxy-1-naphthalenyl)
azo]-, mono sodium salt
Cond…
51
Table 1.02
Chemical features of some basic dyes used in ion association complex formation
Sl.No Dye name / CI No. Chemical category Structure Chemical name
3
Safranine-O
(SFNO)/ 50240
Azines
N
NH3C
H2N
CH3
NH2Cl-
Dimethyl pheno Safranin
4
Methylene Blue
(MB)/ 52015
Thaizines
N
SCH3
N N+
CH3
CH3
Cl-
CH3
Tetra methyl thionin
52
Table 1.03
STATEMENT SHOWING FUNCTIONAL GROUPS /MOIETIES EXPLOITED IN THE SELECTED DRUGS, METHODS PROPOSED,
REAGENTS, TYPES OF REACTIONS INVOLVED –DRUG WISE
Drug Estimated/ Chapter
No.
Proposed
Method
Reagents used for the
exploitation of functional
group/ moiety
Type of reaction
involved
Functional group/moiety
in the drug exploited
Naratriptan HCl/Chapter –
II
M1(a) ARS+0.1M HCl+CHCl3 Ion-Ion Association Tertiary Nitrogen
M1(b) BTB+0.1M HCl+CHCl3 -----------do---------- -----------do----------
M1(c) MO+0.1M HCl+CHCl3 -----------do---------- -----------do----------
M1(d) TPooo+0.1M HCl+CHCl3 -----------do---------- -----------do----------
M3 Brucine+NaIO4 Oxidative Coupling Secondary nitrogen
M5(a) MBTH+Ce(IV) Oxidative Coupling -----------do----------
M5(b) MBTH+ NaIO4 -----------do---------- -----------do----------
M5(c) MBTH+ NaIO4 +AcOH -----------do---------- -----------do----------
M5(d) MBTH+IBDA -----------do---------- -----------do----------
M6 FC-Reg.+Na2CO3 Redox Reaction -----------do----------
M8 Isatin+H2SO4 Condensation -----------do----------
M9 Vanillin+H2SO4 -----------do---------- -----------do----------
M10 ONB + H2SO4 -----------do---------- -----------do----------
M15 p-CA+ CHCl3 Charge Transfer Tertiary Nitrogen
M16 MO+Ce(IV)+ H2SO4 Redox/Association -----------do----------
M17 PA+ CHCl3 Ion-Ion Association -----------do----------
Cond…
53
Table 1.03
STATEMENT SHOWING FUNCTIONAL GROUPS /MOIETIES EXPLOITED IN THE SELECTED DRUGS, METHODS PROPOSED,
REAGENTS, TYPES OF REACTIONS INVOLVED –DRUG WISE
Drug Estimated/ Chapter
No.
Proposed
Method
Reagents used for the
exploitation of functional
group/ moiety
Type of reaction
involved
Functional group/moiety
in the drug exploited
Levofloxacin
/Chapter – III
M1(a) ARS+0.1M HCl+CHCl3 Ion-Ion Association Tertiary Nitrogen
M1(d) TPooo+0.1M HCl+ CHCl3 -----------do---------- -----------do----------
M2(a) MB+ Buffer + CHCl3 Ion-Ion Association -----------do----------
M3 Fe(III)+O-PHEN Redox Reaction Aromaticring
M5(c) MBTH+NaIO4 + AcOH -----------do---------- -----------do----------
M12 PHH+NaIO4 +[Fe(CN)6]-3
Condensation Keto
M14 CTC+Nitrobebzene Complex formation Secondary nitrogen
M17 PA+ CHCl3 Ion-Ion Association Secondary nitrogen
M18 4-AP+ H2SO4 Condensation Tertiary Nitrogen
Cond…
54
Table 1.03
STATEMENT SHOWING FUNCTIONAL GROUPS /MOIETIES EXPLOITED IN THE SELECTED DRUGS, METHODS PROPOSED,
REAGENTS, TYPE OF REACTIONS INVOLVED –DRUG WISE
Drug Estimated/ Chapter
No.
Proposed
Method
Reagents used for the
exploitation of functional
group/ moiety
Type of reaction
involved
Functional group/moiety
in the drug exploited
Ondansetron
HCl/Chapter – IV
M1a ARS+0.1M HCl+CHCl3 Ion-Ion Association Tertiary Nitrogen
M1b BTB+0.1M HCl+ CHCl3 -----------do---------- -----------do----------
M5a MBTH+ Ce(IV) Oxidative Coupling Nitrogen in Imidazol
M5c MBTH+ NaIO4+AcOH Oxidative Coupling -----------do----------
M7 NBS+CA Redox Reaction
M15 p-CA+ CHCl3 Charge Transfer Tertiary Nitrogen
M16 H2O2+MO Redox/Association -----------do----------
M17 PA+Buffer+ CHCl3 Ion-Ion Association -----------do----------
M18 4-AP+MeOH Condensation Keto Group
M19 INH+MeOH -----------do---------- Keto Group
Cond…
55
Table 1.03
STATEMENT SHOWING FUNCTIONAL GROUPS /MOIETIES EXPLOITED IN THE SELECTED DRUGS, METHODS PROPOSED,
REAGENTS, TYPE OF REACTIONS INVOLVED –DRUG WISE
Drug Estimated/ Chapter
No.
Proposed
Method
Reagents used for the
exploitation of functional
group/ moiety
Type of reaction
involved
Functional group/moiety
in the drug exploited
Trandolapril
/Chapter – IV
M1a ARS+0.1M HCl+CHCl3 Ion-Ion Association Tertiary Nitrogen
M1b BTB+0.1M HCl+ CHCl3 -----------do---------- -----------do----------
M1c MO+0.1M HCl+ CHCl3 -----------do---------- -----------do----------
M1d TPooo+0.1M HCl+ CHCl3 -----------do---------- -----------do----------
M2a MB+ Buffer + CHCl3 Ion-Ion Association Carboxylic Acid
M2b SFN-O+ Buffer + CHCl3 -----------do---------- -----------do----------
M5a MBTH+Ce(IV) Oxidative Coupling Basic Nitrogen
M11 AMV+H2SO4 Redox Reaction -----------do----------
M15 p-CA+CHCl3 Charge Transfer Tertiary Nitrogen
M17 PA+ CHCl3 Ion-Ion Association Tertiary Nitrogen
Chapter – I Part- C Derivative Spectrophotometriy
56
DERIVATIVE SPECTROPHOTOMETRY
1.11 Introduction to Derivative Spectrophotometry
The quantitative investigations of broad spectra are frequently difficult, especially
where the measurement of small absorbance is concerned. But this difficulty is overcome
by derivative spectrophotometry (DS), due to the increased resolution of spectral bands,
allowing the detection and location of the wavelengths of poorly resolved components of
complex spectra and reducing the effect of spectral background interferences [149].
Derivative spectrophotometry is widely applied in inorganic and organic analysis,
toxicology and clinical analysis, analysis of pharmaceutical products, amino acids and
proteins, in analysis of food and in environmental chemistry. General analytical
applications of UV-visible derivative spectrophotometry have been reviewed [150-151].
The main characteristic of derivative spectrophotometry, the enhancement of the
resolution of overlapping spectral bands, is the consequence of differentiation which
discriminates against broad bands in favour of a sharp peak to an extent which increases
parallel to the derivative order [152].
1.12Pharmaceutical analysis by Derivative spectrophotometry
Due to its increased selectivity and sensitivity compared to classical
spectrophotometry, DS (Derivative spectrophotometry) is especially widely applied in
analytical chemistry for the determination of pharmaceutical compounds and trace
elements of similar chemical properties present in mixtures at different concentration
levels. For the purpose, the first and the second order derivative are usually used,
although in some cases higher-order derivatives provide more reliable results [153-
Chapter – I Part- C Derivative Spectrophotometriy
57
154]. Methods for the determination of organic substances by the DS technique have
been developed mainly for application in the analysis of pharmaceuticals and/or
clinically and biochemically interesting systems. The interference of the formulation
excipients or other UV-absorbing components, such as co-formulated drugs and
degradation products, usual in conventional UV- spectrophotometry can be
successfully eliminated by the DS technique.
A variety of procedures that render the DS determination of drugs more
specific and sensitive, regardless of whether they are determined as single compounds
or in mixtures, have been published. First-and second-order DS methods have been
proposed for the assay of the anti inflammatory drugs such as fentiazac, flufenamic
acid, tiaprofenic acid and proquaone [155], matronidazole in tablets [156],
Carboplaton [157], anthralin in ointments [158] and paracetamol in blood sera [159].
Aspirin, phenacetin and caffeine in analgesic tablets have been determined by zero
crossing derivative spectrophotometry [160]. Chlorpheniraminemaleate, codeine
phosphate and ephedrine hydrochloride [161] have been estimated without separation
using second-order DS. For quality control of pharmaceutical preparations containing
clozapine derivative spectrophotometric method is suitable for different levels of the
drug [162]. Simultaneous determination of atropine sulfate and morphine
hydrochloride in their binary mixture [163] using spectrophotomtric methods is
proposed by Dinc et al. A method for the determination of cetrizine
dihydrochloride[164] in pharmaceuticals by first, second, third and fourth-order
derivation spectrophotometry is described using “Peak-Peak” (P-P), and “Peak-Zero”
(P-O) measurements. First and second derivative spectrophotometry is applied for the
simultaneous determination of amoxycillin and clavulanic acid[165] in
Chapter – I Part- C Derivative Spectrophotometriy
58
pharmaceutical preparations. A simple and rapid derivative spectrophotometric assay
procedure is described for the analysis of casteine, acetaminophen and
propyphenazone [166] in tablet formulations. Caffeine [167] content is determined in
cola, coffee and tea by second and third order derivative spectrophotometry without
using any separation or background correction technique and reagent.
Second derivative spectrophotometric determination of trimethoprime (TMP)
and sulfamethoxazole (SM) in the presence of hydroxyl propyl-β-cyclodextrin (HP-β-
CD) has been reported [168]. A fast and accurate method for the determination of
dropenidol in the presence of methylparaben and propylparaben is developed using
derivative spectrophotometry [169]. Derivative spectrophotometry in the
determination of phenyl-β-naphthlaine (PBN) used as an antioxidant in rubber
mixtures have been described [170]. Simple, fast and reliable derivative
spectrophotometric methods are developed for determination of indapamide [171] in
bulk and pharmaceutical dosage forms. A derivative spectrophotometric method is
developed for the three binary mixtures of pseudophedrine with fexofenadine (mix.I),
cetrizine (mix.II) and loratidine (mix.III) [172]. Derivative procedures reported for
vitamin mixtures are concerned with pyridoxine hydrochloride and thiamine
hydrochloride in tablets (first and third order) [173], vitamins B6 B1 and B12 uridine 5-
triphosphate in injections (second order) [174], and sodium salicylate, thiamine
hydrochloride and ascorbic acid in visalicyl tablets (first and second order). First-
derivative measurements have been used to determine benzimidazole and cinnamate,
as well as benzophenone derivatives in order to characterize sun-screens in cosmetic
formulations [175]. First- and second-order DS have been described for evaluating
bilirubin, albumin and oxyhemoglobin in amnionic fluid [176]. First-order DS is used
Chapter – I Part- C Derivative Spectrophotometriy
59
for the determination of intact ceftazidime cefuroxime sodium and cefotaxime sodium
in the presence of their degradation products [177]. For a simultaneous determination
of acetaminophen and Phenobarbital after their extraction from the corresponding
suppositories with borate buffer, pH 10, a first-order DS method is developed [178].
Simultaneous analysis of a ternary mixture containing etamizole, paracetamol and
caffeine [179] is carried out by derivative spectrophotometry. First DS methods for
determination of triamterene and hydrochlorothiazide respectively in combined tablets
have also been described [180]. A first order DS method has been developed for the
simultaneous determination of rifamycin SV sodium and lidocaine hydrochloride in
injection solutions [181]. The simultaneous determination of ethinyl estradiol and
norgestrel in tablets utilizing first-order DS has been reported [182]. A method for the
simultaneous determination of melatoninpyridoxine combination in tablets by the
zero-crossing technique of the first and second-order DS has been reported [183].
This method is successfully applied for the determination of both drugs present in
laboratory prepared mixtures and in tablets [184]. For the evaluation of diclofenac and
benzyl alcohol as an excipient in injectable formulations, the first- and second-order
DS method using the zero-crossing technique has been described [185]. Three new
methods (first-order DS, ratio spectra DS and Vierordt’s method) for the quantitative
analysis of tablet formulations containing pseudoephedrine hydrochloride and
triprolidine hydrochloride are developed and compared [186]. A rapid, simple and
direct assay procedure based on first-order DS using zero-crossing and peak-to-base
measurements for the determination of dextromethorphan HBr and bromhexine HCl
has also been developed[187]. A simple and economical DS procedure is developed
for the simultaneous determination of indomethacin and paracepamol in combined
dosage forms [188]. Applying the zero-crossing technique of the second-order DS a
Chapter – I Part- C Derivative Spectrophotometriy
60
method for the determination of 1,4-benzodiazepin, midazolam[189] and
lorazepam[190] in tablets is developed. A second-order derivative UV
spectrophotometric method for determination of vitamin C [191] content in a variety
of natural samples is described. A second-order derivative spectrophotometric
method for the determination of bifonazole in the presence of methyl-and propyl p-
hydroxybenzoate as preservatives has been developed [192].
UV derivative spectrophotometric method to quantify Losartan potassium
[193] in pharmaceutical formulations is developed. A procedure has been developed
for the fourth derivative spectrophotometric determination of
tetramethyldithiocarbamate [194-195]. Paracetamol and phenoprobamate are
determined by first-order derivative spectrum [196], mixtures of cocaine, procaine
and lidocaine in pulver samples by second-order derivative spectrum [197],
paracetamol and caffeine in tablets by first- and second-order derivative spectrum
[198], acetylsalicylic acid and free salicylic acid in sustained release tablets [199],
cetrimide and chlorhexidine glyconate in antiseptic solutions by first-order derivative
spectrum [200] have been described. Fourth-order derivative spectrum procedures
have been used for the determination of clopramide and pindolol in tablets [201],
lidocaine hydrochloride and 5-nitrox in liquid formulations [202]. Derivative
spectrophotometric methods have been described for the assay of phenobarbitone in
mixtures with oxyphenonium bromide and meprobramate, paracetamol, or acetyl
salicylic acid [203], procaine hydrochloride with benzoic acid, pyridoxine
hydrochloride, 4-aminobenzoic acid [204], sulfanilamide and sulfadiazine [205] and
sulfamethoxazole and trimethoprim[206].