novel rp-hplc method for the simultaneous estimation of thiamine
TRANSCRIPT
Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242
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226
NOVEL RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF THIAMINE
MONONITRATE, CALCIUM PANTOTHENATE, L-CYSTINE AND PARA AMINO BENZOIC ACID
IN MULTI VITAMIN DOSAGE FORMS
Thamma Narendra kumar a*, R. Sreenivasulu b, Dr. NSV Raju a, Useni Reddy Mallub and D. Sandeepa
a Genovo Development Services Ltd. (R&D), Bommasandra Industrial Estate, Bengaluru-560099, Karnataka, India,b
Department of Chemistry, Sri Krishnadevaraya University, Anantapur, Andhra Pradesh-515003,
ISSN:2249-5347 IJSID
International Journal of Science Innovations and Discoveries An International peer
Review Journal for Science
Research Article Available online through www.ijsidonline.info
Received: 10.08.2011
Modified: 16.10.2011
Published: 27.10.2011
Keywords: Simultaneous estimation; Vitamins; High-performance liquid chromatography; Pharmaceutical preparation.
*Corresponding Author
Address:
Name: Tamma Narendra Kumar
Place: Bangalore, India
E-mail: [email protected]
ABSTRACT
Objectives: Thiamine Mononitrate, Calcium Pantothenate, L-Cystine and Para
Amino Benzoic acid are essential dietary components for animals and humans.
The main objective of this research is to develop a simple, accurate RP-HPLC
method for the quantification of Thiamine Mononitrate, Calcium Pantothenate, L-
Cystine and Para Amino Benzoic acid in drug substances as well as drug product.
Methods: Chromatographic separation was achieved by using Phenomenex
Synergi Max-RP 80 A° 150 × 4.6 mm; 4.0 µm column, a gradient programme of
mobile phase A & B are used, Mobile phase-A is 100 % buffer (dissolved 3 g of 1-
Hexane sulphonic acid sodium salt and 2 ml of Orthophosphoric acid in 1000 ml of
Milli-Q water). Then filtered the solution through 0.45 µm nylon filter and
degassed. Whereas Mobile phase-B 100 % Methanol. The flow rate of the mobile
phase is 1.0 ml/min. Column temperature maintained at 25°C. Injection volume is
10 µl and run time is 35 minutes. Analytes absorbance was measured at 210 nm.
Results/Conclusion: The developed method was validated as per ICH guidelines
with respect to specificity, precision, linearity, accuracy, robustness and system
suitability. Satisfactory results found from method validation and the method is
applicable for determination of assay of Thiamine Mononitrate, Calcium
Pantothenate, L-Cystine and Para Amino Benzoic acid in drug substances and
different pharmaceutical dosage forms, Nutritional supplements, Multi vitamin
preparations.
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INTRODUCTION
Vitamins represent a group of various compounds, both chemically and analytically, because they
comprise a wide range of bio molecules (Fig. 1). They may be present in several chemically diverse but
biologically inter convertible forms. Their common properties reside solely in fact that they are essential
dietary components for animals and/or humans [1–4]. They are needed in relatively small amounts to
sustain life and good health. Currently many vitamin supplements such as multivitamin tablets are
available for the prevention and control of Avitaminosis but also for treating some other diseases.
L-cystine (1-6) is a naturally occurring amino acid that is classified as a protein amino acid. One of
the main functions of L-cystine is the promotion of stomach lining health and also the correction of
situations where the absorption of essential nutrients from food sources takes place. Many people are
able to obtain as much of this protein source as they need without taking any type of supplement. L-
cystine can be found in a number of foods ranging from meats to dairy and vegetable, Chicken, turkey and
pork are all good sources of L-cysteine. L-cystine as cysteine can be obtained from eggs and milk. One of
the largest applications is the production of flavors. For example, the reaction of cysteine with sugars in a
Maillard reaction yields meat flavors. it is used for permanent wave applications predominantly in Asia.
Again the cysteine is used for breaking up the disulfide bonds in the hair's keratin.
Thiamine (7-26) or thiamin or vitamin B1 is a water-soluble vitamin of the B complex. First named
aneurin for the detrimental neurological effects if not present in the diet. Its phosphate derivatives are
involved in many cellular processes. The best-characterized form is thiamine pyrophosphate (TPP), a
coenzyme in the catabolism of sugars and amino acids. In yeast, TPP is also required in the first step of
alcoholic fermentation. All living organisms use thiamine in their biochemistry, but it is only synthesized
in bacteria, fungi, and plants. There is still much research devoted to elucidating the exact mechanisms by
which thiamine deficiency leads to the specific symptoms observed. New thiamine phosphate derivatives
have recently been discovered, emphasizing the complexity of thiamine metabolism. Thiamine
derivatives with improved pharmacokinetics have been discovered and are to be considered more
effective in alleviating the symptoms of thiamine deficiency and other thiamine-related conditions such
as impaired glucose metabolism in diabetes. These compounds include allithiamine, prosultiamine,
fursultiamine, benfotiamine and sulbutiamine, among others.
Pantothenic acid (27-38), also called pantothenate or vitamin B5, it is a water-soluble vitamin. For
many animals, pantothenic acid is an essential nutrient. Animals require pantothenic acid to synthesize
coenzyme-A (CoA), as well as to synthesize and metabolize proteins, carbohydrates and fats. It is
commonly found as its alcohol analog, the provitamin panthenol, and as calcium pantothenate.
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Pantothenic acid is an ingredient in some hair and skin care products. It plays a huge role in the
functioning of the enzymes in the human body. Some of its major functions are converting food into
energy, stimulating growth, reproduction, and many other normal bodily processes. It is essential in
human growth, reproduction and many other normal bodily processes. One of its major role in the body
is to help in the metabolism and break down of carbohydrates, fats and proteins for the production of
energy in the body. Pantothenic acid or vitamin B5 also produces enzymes and helps maintain accurate
communication between the central nervous system and the brain. It is also required for the production
of steroid hormones and hormones of the adrenal gland.
Para-amino benzoic acid (39-43) (PABA) is a naturally occurring substance that is often used in
sunscreen products. PABA is sometimes called vitamin Bx, but it is not a true vitamin. PABA overdose
occurs when someone accidentally or intentionally takes more than the normal or recommended amount
of this substance. PABA is used to improve the protein used in the body, it relates to red blood cell
formation as well as assisting the manufacture of folic acid in the intestines. Para-aminobenzoic acid is
used in sunscreen preparations since it can help protect the skin against ultra-violet radiation. It has been
linked to hair growth as well as reversing the graying of hair, but these results are disappointing. Oral
supplements of PABA can make the skin less sensitive to sun damage.
Thiamine Mononitrate Calcium Pantothenate
L-cystine Para amino benzoic acid
Figure–1: Structure of analytes
UV-Vis spectrophotometry, Fluorimetry, Chemiluminiscence, Capillary electrophoresis ,
Microbiology and High-performance liquid chromatography have been proposed for the determination of
vitamins. Nevertheless, there is no single analytical approach to determine vitamins within a complicated
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matrix in a single run. In this work, we developed and optimised a high performance liquid
chromatographic method using diode array detection for determination water and fat-soluble vitamins at
a single run. The method was successfully applied to the determination of vitamins in Nutritional
supplements, Multivitamin preparations, Pharmaceutical preparations, etc.
MATERIALS AND METHODS
Chemicals and reagents
Capsules and Standards components were supplied by Medreich India limited. All solvents were
HPLC grade and High purity water was prepared by using Millipore Milli-Q plus water purification
system (Millipore, Milford, MA, USA).
Equipment
The Shimadzu UFLC system used consists of a pump, auto sampler and a PDA detector. The output
signal was monitored and processed by using LC solutions software.
Chromatographic conditions
The method was developed using Phenomenex Synergi Max-RP 80 A°, 150 × 4.6 mm; 4.0 µm
column. A gradient programme of Mobile phase A & B are used , Mobile phase A is 100 % buffer, which is
prepared by dissolving 3 g of 1-Hexane sulphonic acid sodium salt in 1000 ml of Milli-Q water , then
added 2 ml of Ortho phosphoric acid and mixed well. Then filtered the solution through 0.45 µm nylon
filter and degassed. Whereas Mobile phase B was 100 % Methanol. The flow rate of the mobile phase was
1.0 ml/min. The column temperature was maintained at 25oC and the wavelength was monitored at 210
nm. The injection volume was 10 µl. The run time is fixed to 35 minutes.
Preparation of stock solutions
A stock solution was prepared by transferring accurately weighed about 60 mg of Calcium
Pantothenate, 20 mg of L-Cystine, 30 mg of Para Amino Benzoic acid and 60 mg of Thiamine Mononitrate
working standard into a 100 ml volumetric flask, added 10 ml of 1N HCl, sonicated for 5 min, then added
5 ml of Methanol, sonicated for 5 min and then added 55 ml of purified water, sonicated to dissolve, then
made up to the volume with purified water and mixed well.
Preparation of sample solutions
Twenty capsules were weighed, then transferred the powder sample into a petri dish and weighed
empty capsule, then calculated the average fill weight of the capsule. Weighed about 410 mg of capsule
powder (equivalent to 60 mg of Thiamine Mononitrate, 60 mg of Calcium Pantothenate, 30 mg of Para
Amino Benzoic acid and 20 mg of L-Cystine) then transferred in to a 100 ml volumetric flask, 10 ml of 1N
HCl was added, sonicated for 5 min, then added 5 ml of Methanol, sonicated for 5 minutes and then added
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55 ml of purified water, sonicated for 30 minutes to dissolve, then made up to the volume with purified
water and mixed well. This solution was centrifuged at 3500 rpm for 10 min and collected the
supernatant clear solution.
Calculation of % analyte:
a. Calculation for % Calcium Pantothenate:
TRCP WCP 100 PCP -----------x-----------x-----------x-----------x A SRCP 100 TW LCP
b. Calculation for % L - Cystine:
TRLC WLC 100 PLC -----------x-----------x-----------x-----------x A SRLC 100 TW LLC
c. Calculation for % Para Amino Benzoic Acid:
TRPB WPB 100 PPB -----------x-----------x-----------x-----------x A SRPB 100 TW LPB
d. Calculation for % Thiamine Mononitrate:
TRTM WTM 100 PTM -----------x-----------x-----------x-----------x A SRTM 100 TW LTM
Where, TRCP = Calcium Pantothenate response from test preparation; TRLC = L - Cystine response
from test preparation; TRPB = Para amino benzoic acid response from test preparation; TRTM = Thiamine
Mononitrate response from test preparation; SRCP = Calcium Pantothenate average response from
standard preparation; SRLC = L - Cystine average response from standard preparation; SRPB = Para amino
benzoic acid average response from standard preparation; SRTM = Thiamine Mononitrate average
response from standard preparation; WCP = Calcium Pantothenate working standard weight in mg, for
standard preparation; WLC = L - Cystine average working standard weight in mg, for standard
preparation; WPB = Para amino benzoic acid working standard weight in mg, for standard preparation;
WTM = Thiamine Mononitrate working standard weight in mg, for standard preparation; PCP = Calcium
Pantothenate working standard purity in %, on as such basis; PLC = L - Cystine average working standard
purity in %, on as such basis; PPB = Para amino benzoic acid working standard purity in %, on as such
basis; PTM = Thiamine Mononitrate working standard purity in %, on as such basis; LCP = Label claim of
Calcium Pantothenate; LLC = Label claim L - Cystine average; LPB = Label claim Para amino benzoic acid;
LTM = Label claim Thiamine Mononitrate; A = Average weight of test sample and TW = Test weight taken
in mg
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RESULTS AND DISCUSSION
Method development and optimization
Different brand of HPLC columns were tried for getting the symmetrical peak for all the actives. In
these trails found that Phenomenex Synergi Max-RP 80 A°, 150 × 4.6 mm; 4.0 µm column is the best
suitable for the peak shapes as well as for responses of the actives. By this column obtained superior peak
shape and separation because of the unique property of fully porous silica support with bonded phase of
C12 with TMS end capping. This column is the best suite for the separation of basic compounds. Diluent
was optimized based on the physical properties of the actives to get the better stability in the solution
form with superior extraction of the actives from excipients in to the solution form.
Table – 1: System Suitability Results
Standard inj N°
Active Ingredient
Calcium
Pantothenate L – Cystine
Para Amino
Benzoic Acid
Thiamine
Mononitrate
1 3591923 1592561 15353893 16694727 2 3564911 1594309 15365712 16171727 3 3574612 1601222 15352225 16353795 4 3571210 1599206 15398240 16340249 5 3568934 1601830 15415350 16281353
Mean 3574318 1597826 15377084 16368370
%RSD 0.3 0.3 0.2 1.2 Theoretical Plate count 4316 5453 4779 170077
Tailing Factor 1.4 1.1 1.5 1.2 Precision Results (% RSD of % assay)
Sample N°
Active Ingredient
Calcium
Pantothenate L – Cystine
Para Amino
Benzoic Acid
Thiamine
Mononitrate
1 99.5 99.5 100.3 99.6 2 99.5 99.9 100.3 99.7 3 99.4 99.6 99.6 99.5 4 99.5 99.5 100.1 99.5 5 99.5 99.9 100.1 99.6 6 99.1 99.7 100.2 99.5
Mean 99.4 99.7 100.1 99.6
%RSD 0.2 0.2 0.3 0.1 Comparison b/n Precision Intermediate Precision Results (Response % RSD)
%RSD Calcium
Pantothenate L – Cystine
Para Amino
Benzoic Acid
Thiamine
Mononitrate
Precision 0.2 0.2 0.3 0.1 Intermediate Precision 0.5 0.2 0.2 0.3
Results of forced degradation studies
A study was conducted to demonstrate the effective separation of degradants from the active
ingredients. Mixture of drug substance with placebo was subjected to the following stress conditions to
induce degradation.
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Table – 2: Stress study conditions and results
Stress Study Stress Condition
% Degradation from Drug Product
Calcium
Pantothenate L-Cystine
Para Amino
Benzoic acid
Thiamine
Mononitrate
Acid Hydrolysis 0.1N HCl solution for about 2 hrs
at 60°C 12.2 1.3 3.0 3.5
Base Hydrolysis 0.1N NaOH solution for about 2 hrs 30 min at 60°C
12.5 3.0 3.1 2.6
Oxidation 1% H2O2 for 2 hrs at 25°C 4.1 3.6 6.0 4.8 Aqueous
Hydrolysis Purified water for about 6 hrs at 60°C
4.0 3.6 2.7 5.7
Fluorescent Light Exposure
Sun-light of 1.2 Million Lux Hours
2.7 2.1 1.5 3.6
UV Light Exposure
UV-light of 200 Watts h/m2 2.4 1.7 1.5 2.7
Thermal Dry heat at 105°C for 24hrs 4.6 3.3 2.8 5.7 Humidity 90% RH at 25°C for 7 days 3.2 2.5 1.7 3.4
For all active peaks peak purity was evaluated by using the Photodiode array detector and purity of peaks
was passed.
VALIDATION OF THE METHOD
Specificity: Specificity is the ability of the method to measure the analyte response in the presence of its
degradants. The specificity of the developed method was carried in the presence of its degradants. Stress
studies were performed on dosage form to provide an indication of the stability-indicating property and
specificity of the proposed method. Intentional degradation was attempted with stress condition of UV
light (200 watts h/m2), Sun light (1.2 Million Lux Hours), Humidity (90% RH at 25°C), Dry heat (105oC),
Acid (0.1N HCl), Base (0.1N NaOH) and Oxidation (1% H2O2) to evaluate the ability of the proposed
method to separate Calcium Pantothenate, L-Cystine, Para Amino Benzoic acid and Thiamine
Mononitrate from its degradation products. For heat, study period was 24 hrs whereas for hydrolytic,
acid, base and oxidation study period was about 2 hrs. Peak purity test was carried out by using PDA
detector in stress samples.
In the stressed samples % degradant products were calculated and reported.
In the standard and sample chromatograms before 2 minutes one peak will appear and this peak
no need of quantification and it originates from the salt form of the materials have been used.
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Figure – 2: Blank chromatogram
Figure – 3: Standard chromatogram
Figure – 4: Placebo chromatogram
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Figure – 5: Sample chromatogram with peak purity evaluation
Figure – 6: Purity plot of Calcium Pantothenate
Figure – 7: Purity plot of L – Cystine
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Figure – 8: Purity plot of Para amino benzoic acid
Figure – 9: Purity plot of Thiamine Mononitrate
Precision: The precision of the method verified by Repeatability and Intermediate precision.
Repeatability was checked by injecting six individual weights of placebo mixed with API (Calcium
Pantothenate, L-Cystine, Para Amino Benzoic acid and Thiamine Mononitrate). % RSD of assay results for
each ingredient was calculated. The intermediate precision of the assay method was evaluated by
different analysts and performing the analysis on different days and with different HPLC instruments and
columns.
The % RSD of assay of Calcium Pantothenate, L-Cystine, Para Amino Benzoic acid and Thiamine
Mononitrate during the assay method precision study was 0.2 %, 0.2 %, 0.3 %, and 0.1 % respectively.
The % RSD of the assay results obtained in the intermediate precision study was within the limit (0.5, 0.2,
0.2, and 0.3) conforming good precision of the method. The % RSD values were represented in table-1.
Accuracy: Accuracy of the assay method was evaluated with dosage form equivalent to about 50% to
150% of the target assay of the Calcium Pantothenate, L-Cystine, Para Amino Benzoic acid and Thiamine
Mononitrate. The percentages of recovery at each level were calculated. The percentage recovery of
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Calcium Pantothenate, L-Cystine, Para Amino Benzoic acid and Thiamine Mononitrate was ranged from
98.2 to 101.5%. The % recovery values for Calcium Pantothenate, L-Cystine, Para Amino Benzoic acid and
Thiamine Mononitrate are represented in table-3.
Table – 3: Recovery of active ingredients - summary table
Level
Calcium
Pantothenate L – Cystine
Para Amino Benzoic
Acid
Thiamine
Mononitrate
%
Recovery
%
RSD
%
Recovery
%
RSD % Recovery
%
RSD % Recovery
%
RSD
50% 99.5 0.3 99.9 1.1 99.0 0.7 98.2 0.4 25% 98.8 0.4 100.4 0.6 99.3 0.2 98.2 0.4 50% 99.0 0.2 100.6 0.9 101.5 0.5 100.8 0.3
100% 98.7 0.2 100.9 0.1 98.3 0.0 100.8 0.2 150% 98.8 0.3 101.0 0.2 99.9 0.1 100.6 0.2
Linearity: Linearity of test solutions for the assay method was verified in the range of Calcium
Pantothenate 300, 600, 900 µg/ml, L-Cystine 100, 200, 300 µg/ml, Para Amino Benzoic acid 150, 250,
450 µg/ml, Thiamine Mononitrate 300, 600, 900 µg/ml respectively. The peak area versus concentration
data was treated by least-squares linear regression analysis.
Linearity calibration plot for the assay method was obtained over the calibration ranges tested, i.e.
Calcium Pantothenate 300-900 µg/ml, L-Cystine 100-300 µg/ml, Para Amino Benzoic acid 150-450
µg/ml, Thiamine Mononitrate 300-900 µg/ml respectively and correlation coefficient obtained was
greater than 0.999. The result shows that an excellent correlation existed between the peak area and
concentration of the analyte.
Linearity chromatograms, graphs and results were represented in figure-10, figure-11 and table-4
respectively.
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Figure-10: Linearity chromatograms of four ingredients
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Table – 4: Linearity Regression Summary Table
Active ingredient name Calcium
Pantothenate L – Cystine
Para Amino
Benzoic Acid
Thiamine
Mononitrate
Lin
ea
rity
So
luti
on
s
Re
spo
ns
e
Level – 1 (50%) 1659535 784171 7737695 7672519 Level – 2 (75%) 2702541 1252070 12412698 12536931
Level – 3 (100%) 3364230 1572471 15987709 16798448 Level – 4 (120%) 4038990 1931182 18609074 21433362 Level – 5 (140%) 4746170 2218895 22052848 25615005 Level – 6 (150%) 5028976 2395662 23441163 27734450
Re
gre
ss
ion
Re
sult
s Slope 5543.037981 7987.176329 51640.87933 33639.47941
Intercept 61611.26695 7678.721728 286708.7734 2751597.782 Correlation Coefficient 0.999 0.999 0.999 0.999
Standard Error 68892.55769 26164.82623 291318.456 377888.9883
Figure-11: Linearity graphs Robustness: To determine the robustness of the developed method, experimental conditions were
deliberately altered. To study the effect of flow rate on resolution; flow was changed by + 0.1 units from
0.9 to1.l ml/min instead of 1.0ml/min. The effect of the column temperature on resolution was studied at
+ 5°C units from 20°C and 30oC instead of 25oC. The effect of filters was conducted using two different
filters namely, 0.45µm PVDF filter and 0.45µm Nylon 66 filter.
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In all the deliberate varied chromatographic conditions (flow rate, column temperature and
change of filters), the resolution between all pairs of compounds was greater than 2.0, tailing factor for
the components was less than 2.0 and the theoretical plate count was more than 5000. 0.45 µm PVDF
filter and 0.45 µm Nylon 66 filters are found to be suitable for filtration.
Table –5: Robustness Parameter Summary table – Flow rate variation
Peak Name
Flow Rate Variation (± 0.1 mL/min)
Low (0.9 mL/min) Actual (1.0 mL/min) High (1.1 mL/min)
TF %RSD TF %RSD TF %RSD
Calcium Pantothenate 1.2 0.5 1.2 0.9 1.2 0.2 L – Cystine 0.9 0.1 0.9 0.3 0.9 0.1
Para Amino Benzoic Acid 1.3 0.1 1.3 0.2 1.3 0.2 Thiamine Mononitrate 1.4 0.1 1.4 0.4 1.5 0.2
Table –6: Robustness Parameter Summary table – Column oven temperature variation
Peak Name
Column oven temperature variation (± 5°C)
Low (20°C) Actual (25°C) High (30°C)
TF %RSD TF %RSD TF %RSD Calcium Pantothenate 1.2 0.4 1.2 0.9 1.2 0.2
L – Cystine 1.0 0.1 0.9 0.3 0.8 0.1 Para Amino Benzoic Acid 1.3 0.1 1.3 0.2 1.3 0.1
Thiamine Mononitrate 1.4 0.1 1.4 0.4 1.5 0.1
TF signifies the tailing factor of respective peak and %RSD signifies the % relative standard deviation of
respective peak response from five replicate injections.
Table –7: Filter validation parameter Summary table
Active Ingredient Name
Filter validation
Similarity factor
0.45 µm PVDF Filter 0.45 µm Nylon 66 Filter Calcium Pantothenate 0.98 0.98
L – Cystine 0.99 0.99 Para Amino Benzoic Acid 1.00 0.99
Thiamine Mononitrate 1.00 0.99
Solution stability: Solution stability in the assay method was carried out by leaving both the test
solution of sample and reference standard in tightly capped volumetric flasks at room temperature for 1
day and after 2 days.
The % RSD of the assay of actives during solution stability experiments were within 1%. No
significant changes were observed in the content during solution stability experiments. The solution
stability experiment data confirms that the sample solutions used during assay were stable for 2 days.
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CONCLUSION
The simple reverse phase LC method developed for quantitative analysis of actives in
pharmaceutical dosage forms is precise, accurate, linear, robust and specific. Satisfactory results were
obtained from validation of the method. The method is stability indicating and can be used for routine
analysis of production samples and stability samples of actives in pharmaceutical dosage forms.
REFERENCES
1. Martens, Jürgen; Offermanns, Heribert; Scherberich, Paul (1981), "Facile Synthesis of Racemic Cysteine", Angew.
Chem. Int. Ed. Engl. 20 (8): 668,
2. Karlheinz Drauz, Ian Grayson, Axel Kleemann, Hans-Peter Krimmer, Wolfgang Leuchtenberger, Christoph Weckbecker
"Amino Acids"in Ullmann's Encyclopedia of Industrial Chemistry 2007, Wiley-VCH, Weinheim.
3. Lill, Roland; Mühlenhoff, Ulrich (2006), "Iron-Sulfur Protein Biogenesis in Eukaryotes: Components and Mechanisms",
Ann. Rev. Cell Dev. Biol. 22: 457–86,.
4. Lippard, Stephen J.; Berg, Jeremy M. (1994), Principles of Bioinorganic Chemistry, Mill Valley, CA: University Science
Books,
5. Baker, David H.; Czarnecki-Maulden, Gail L. (1987), "Pharmacologic role of cysteine in ameliorating or exacerbating
mineral toxicities", J. Nutr. 117 (6): 1003–10,
6. Sprince, Herbert; Parker, Clarence M.; Smith, George G.; Gonzales, Leon J. (1974), "Protection against Acetaldehyde
Toxicity in the rat by L-cystine, thiamine and L-2-Methylthiazolidine-4-carboxylic acid", Inflam. Res. 4 (2): 125–30,
7. R.E. Austic and M.L. Scott, Nutritional deficiency diseases, in Diseases of poultry, ed. by M.S. Hofstad, Iowa State
University Press, Ames, Iowa, USA ISBN 0-8138-0430-2, p. 50.
8. National Research Council. 1996. Nutrient Requirements of Beef Cattle, Seventh Revised Ed. Washington, D.C.:
National Academy Press.
9. Balk, L; Hägerroth, PA; Akerman, G; Hanson, M; Tjärnlund, U; Hansson, T; Hallgrimsson, GT; Zebühr, Y et al. (2009).
"Wild birds of declining European species are dying from a thiamine deficiency syndrome". Proc Natl Acad Sci U S A
106 (29): 12001–12006.
10. Bettendorff L, Peeters M., Jouan C., Wins P., Schoffeniels E. (1991). "Determination of thiamin and its phosphate esters
in cultured neurons and astrocytes using an ion-pair reversed-phase high-performance liquid chromatographic
method". Anal. Biochem 198 (1): 52–59.
11. Losa R, Sierra MI, Fernández A, Blanco D, Buesa JM. (2005). "Determination of thiamine and its phosphorylated forms
in human plasma, erythrocytes and urine by HPLC and fluorescence detection: a preliminary study on cancer
patients". J Pharm Biomed Anal 37 (5): 1025–1029.
12. Lu J, Frank EL. (May 2008). "Rapid HPLC measurement of thiamine and its phosphate esters in whole blood". Clin
Chem. 54 (5): 901–906.
13. Shabangi M, Sutton JA. (2005). "Separation of thiamin and its phosphate esters by capillary zone electrophoresis and
its application to the analysis of water-soluble vitamins". Journal of Pharmaceutical and Biomedical Analysis 38 (1):
66–71. doi:10.1016/j.jpba.2004.11.061.
Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242
International Journal of Science Innovations and Discoveries Vol 1, Issue 2, September-October 2011
241
14. Slater, PV (1978). "Thiamine Responsive Megaloblastic Anemia with severe diabetes mellitus and sensorineural
deafness (TRMA)". The Australian nurses' journal 7 (11): 40–3. PMID 249270.
15. Kopriva, V; Bilkovic, R; Licko, T (Dec 1977). "Tumours of the small intestine (author's transl)". Ceskoslovenska
gastroenterologie a vyziva 31 (8): 549–53. ISSN 0009-0565.
16. Beissel, J (Dec 1977). "The role of right catheterization in valvular prosthesis surveillance (author's transl)". Annales
de cardiologie et d'angeiologie 26 (6): 587–9. ISSN 0003-3928.
17. Butterworth RF. Pyruvate dehydrogenase deficiency disorders. In: McCandless DW, ed. Cerebral Energy Metabolism
and Metabolic Encephalopathy. Plenum Publishing Corp.; 1985.
18. Blass JP. Inborn errors of pyruvate metabolism. In: Stanbury JB, Wyngaarden JB, Frederckson DS et al., eds. Metabolic
Basis of Inherited Disease. 5th ed. New York: McGraw-Hill, 1983.
19. Djoenaidi W, Notermans SL, Gabreëls-Festen AA, Lilisantoso AH, Sudanawidjaja A (1995). "Experimentally induced
beriberi polyneuropathy in chickens". Electromyogr Clin Neurophysiol 35 (1): 53–60.
20. Bruce WR, Furrer R, Shangari N, O’Brien PJ, Medline A, Wang Y (2003). "Marginal dietary thiamin deficiency induces
the formation of colonic aberrant crypt foci (ACF) in rats". Cancer Lett 202 (2): 125–129.
21. Langlais PJ (1995). "Pathogenesis of diencephalic lesions in an experimental model of Wernicke's encephalopathy".
Metab Brain Dis 10 (1): 31–44.
22. Frederikse PH, Zigler SJ Jr, Farnsworth PN, Carper DA (2000). "Prion protein expression in mammalian lenses". Curr
Eye Res 20 (2): 137–43.
23. Kale, S; Ulas, G; Song, J; Brudvig, GW; Furey, W; Jordan, F (2008). "Efficient coupling of catalysis and dynamics in the
E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex". Proc Natl Acad Sci U S A 105 (4):
1158–1163.
24. Kluger, R; Tittmann, K (2008). "Thiamin diphosphate catalysis: enzymic and nonenzymic covalent intermediates".
Chem Rev 108 (6): 1797–1833.
25. Makarchikov, AF; Lakaye, B; Gulyai, IE; Czerniecki, J; Coumans, B; Wins, P; Grisar, T; Bettendorff, L (2003). "Thiamine
triphosphate and thiamine triphosphatase activities: from bacteria to mammals". Cell Mol Life Sci 60 (7): 1477–1488.
26. Bettendorff L. and Wins P. (2009). "Thiamin diphosphate in biological chemistry : new aspects of thiamin metabolism,
especially triphosphate derivatives acting other than as cofactors". FEBS J. 276 (11): 2917–2925.
27. Combs, G. F. Jr. The Vitamins: Fundamental Aspects in Nutrition and Health. 2nd Edition. Ithaca, NY: Elsevier Academic
Press; 1998; pg.374
28. Etensel, B., Özkıscık, S., Özkara, E., Serbest, Y. A., Yazıcı, M., Gürsoy, H. (2007) The protective effect of dexpanthenol on
testicular atrophy at 60th day following experimental testicular torsion. Pediatric Surgery International. 23: 271-275.
29. Etensel, B., Özkıscık, S., Özkara, E., Karul, A., Öztan, O., Yazıcı, M., Gürsoy, H. (2007). Dexpanthenol attenuates lipid
peroxidation and testicular damage at experimental ischemia and reperfusion injury. Pediatric Surgery International.
23: 177-181.
30. Abdelatif, M., Yakoot, M., Etmaan, M. (2008). Safety and efficacy of a new honey ointment on diabetic foot ulcers: a
prospective pilot study. Journal of Wound Care. 17.3:108-110.
31. Naruta, E., Buko, V. (2001). Hypolipidemic effect of pantothenic acid derivatives in mice with hypothalamic obesity
induced by aurothioglucose. Experimental and Toxologic Pathology. 53: 393-398.
Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242
International Journal of Science Innovations and Discoveries Vol 1, Issue 2, September-October 2011
242
32. Weimann, B. J., Hermann, D. (1999). Studies on wound healing: Effects of calcium D-pantothenate on the migration,
proliferation and protein synthesis of human dermal fibroblasts in culture. International Journal for Vitamin and
Nutrition Research. 69.2: 113-119.
33. G. David Novelli (1953). "Metabolic Functions of Pantothenic Acid". Physiol Rev 33 (4): 525–43.
34. Shun Ishibashi, Margrit Schwarz, Philip K. Frykman, Joachim Herz and David W. Russell (1996). "Disruption of
Cholesterol 7-Hydroxylase Gene in Mice, I. Postnatal lethality reversed by bile acid and vitamin supplementation". J.
Biol. Chem. 271 (30): 18017–18023.
35. C. Smith, W. Song (1996). "Comparative nutrition of pantothenic acid". The Journal of Nutritional Biochemistry 7 (6):
312–321.
36. Paul F. Fenton2, George R. Cowgill, Marie A. Stone and Doris H. Justice (1950). "The Nutrition of the Mouse, VIII.
Studies on Pantothenic Acid, Biotin, Inositol and P-Aminobenzoic Acid". Journal of Nutrition 42 (2): 257–269.
37. Leung L (1995). "Pantothenic acid deficiency as the pathogenesis of acne vulgaris". Med Hypotheses 44 (6): 490–2.
38. Leung L (1997). "A stone that kills two birds: how pantothenic acid unveils the mysteries of acne vulgaris and
obesity". J Orthomol Med 12 (2): 99–114.
39. "Para-aminobenzoic acid poisoning". National Institute of Health: National Library of Medicine. 2007. Retrieved 2007-
06-19.
40. Para-aminobenzoic acid: MedlinePlus Medical Encyclopedia
41. "Compound Summary on PubChem". PubChem. National Institute of Health: National Library of Medicine. 2006.
Retrieved 2006-04-05.
42. Melanoma Madness The scientific flap over sunscreens and skin cancer -- Chemical studies, Science News Online,
6/6/98 (accessed 10/1/2009, 2009)
43. Osgood, Pauline J.; Moss, Stephen H.; Davies, David J. G. (1982). "The Sensitization of Near-Ultraviolet Radiation Killing
of Mammalian Cells by the Sunscreen Agent Para-aminobenzoic Acid". Journal of Investigative Dermatology 79 (6):
354.