0102 2011 019

Tamma Narendra Kumar et al.
, IJSID 2011, 1 (2), 226-242
ISSN:2249-5347
IJSID
International Journal of Science Innovations and Discoveries
Research Article
An International peer Review Journal for Science
Available online through www.ijsidonline.info
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,
Received: 10.08.2011 Modified: 16.10.2011 Published: 27.10.2011 Keywords: Simultaneous
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, LCystine 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 m column, a gradient programme of
estimation; Vitamins; High-performance liquid chromatography; Pharmaceutical preparation.

*Corresponding Author
mobile phase A & B are used, Mobile phase-A is 100 % buffer (dissolved 3 g of 1Hexane sulphonic acid sodium salt and 2 ml of Orthophosphoric acid in 1000 ml of Milli-Q water). Then filtered the solution through 0.45 m 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 25C. Injection volume is 10 l and run time is 35 minutes. Analytes absorbance was measured at 210 nm. Results/Conclusion: The developed method was validated as per ICH guidelines
Address: Name: Tamma Narendra Kumar Place: Bangalore, India E-mail:
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.
International Journal of Science Innovations and Discoveries Vol 1, Issue 2, September-October 2011
narendrakumar.thamma@gmail.com
226
Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242
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
[14].
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. Lcystine 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.
227
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 Figure1: 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
228
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 m 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 m 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 l. 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
229
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: WLC 100 PLC TRLC -----------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: WTM 100 PTM TRTM -----------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
230
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 m 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
Active Ingredient Calcium Para Amino Thiamine L Cystine Pantothenate Benzoic Acid 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) Active Ingredient Sample N Calcium Para Amino Thiamine L Cystine Pantothenate Benzoic Acid 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) Calcium Para Amino Thiamine %RSD L Cystine Pantothenate Benzoic Acid Mononitrate Precision 0.2 0.2 0.3 0.1 Intermediate Precision 0.5 0.2 0.2 0.3 Standard inj N
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.
231
Table 2: Stress study conditions and results

% Degradation from Drug Product Stress Study Stress Condition Calcium Pantothenate 12.2 12.5 4.1 4.0 2.7 2.4 4.6 3.2 L-Cystine 1.3 3.0 3.6 3.6 2.1 1.7 3.3 2.5 Para Amino Benzoic acid 3.0 3.1 6.0 2.7 1.5 1.5 2.8 1.7 Thiamine Mononitrate 3.5 2.6 4.8 5.7 3.6 2.7 5.7 3.4
Acid Hydrolysis Base Hydrolysis Oxidation Aqueous Hydrolysis Fluorescent Light Exposure UV Light Exposure Thermal Humidity
0.1N HCl solution for about 2 hrs at 60C 0.1N NaOH solution for about 2 hrs 30 min at 60C 1% H2O2 for 2 hrs at 25C Purified water for about 6 hrs at 60C Sun-light of 1.2 Million Lux Hours UV-light of 200 Watts h/m2 Dry heat at 105C for 24hrs 90% RH at 25C for 7 days
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 25C), 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.
232
Figure 2: Blank chromatogram
Figure 3: Standard chromatogram
Figure 4: Placebo chromatogram
233
Figure 5: Sample chromatogram with peak purity evaluation
Figure 6: Purity plot of Calcium Pantothenate
Figure 7: Purity plot of L Cystine
234
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
235
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 50% 25% 50% 100% 150% Calcium Pantothenate % % Recovery RSD 99.5 0.3 98.8 0.4 99.0 0.2 98.7 0.2 98.8 0.3 L Cystine % Recovery 99.9 100.4 100.6 100.9 101.0 % RSD 1.1 0.6 0.9 0.1 0.2 Para Amino Benzoic Acid % % Recovery RSD 99.0 0.7 99.3 0.2 101.5 0.5 98.3 0.0 99.9 0.1 Thiamine Mononitrate % Recovery 98.2 98.2 100.8 100.8 100.6 % RSD 0.4 0.4 0.3 0.2 0.2
Linearity: Linearity of test solutions for the assay method was verified in the range of Calcium Pantothenate 300, 600, 900 g/ml, L-Cystine 100, 200, 300 g/ml, Para Amino Benzoic acid 150, 250, 450 g/ml, Thiamine Mononitrate 300, 600, 900 g/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 g/ml, L-Cystine 100-300 g/ml, Para Amino Benzoic acid 150-450 g/ml, Thiamine Mononitrate 300-900 g/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.
236
Figure-10: Linearity chromatograms of four ingredients
237
Table 4: Linearity Regression Summary Table Active ingredient name

Level 1 (50%) Level 2 (75%) Level 3 (100%) Level 4 (120%) Level 5 (140%) Level 6 (150%) Slope Intercept Correlation Coefficient Standard Error Linearity Solutions Response
Calcium Pantothenate
1659535 2702541 3364230 4038990 4746170 5028976 5543.037981 61611.26695 0.999 68892.55769
L Cystine
784171 1252070 1572471 1931182 2218895 2395662 7987.176329 7678.721728 0.999 26164.82623
Para Amino Benzoic Acid

7737695 12412698 15987709 18609074 22052848 23441163 51640.87933 286708.7734 0.999 291318.456
Thiamine Mononitrate
7672519 12536931 16798448 21433362 25615005 27734450 33639.47941 2751597.782 0.999 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 + 5C units from 20C and 30oC instead of 25oC. The effect of filters was conducted using two different filters namely, 0.45m PVDF filter and 0.45m Nylon 66 filter.
Regress ion Results
238
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 m PVDF filter and 0.45 m Nylon 66 filters are found to be suitable for filtration. Table 5: Robustness Parameter Summary table Flow rate variation
Peak Name
Calcium Pantothenate L Cystine Para Amino Benzoic Acid Thiamine Mononitrate
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
1.2 0.9 1.3 1.4 0.5 0.1 0.1 0.1 1.2 0.9 1.3 1.4 0.9 0.3 0.2 0.4 1.2 0.9 1.3 1.5 0.2 0.1 0.2 0.2
Table 6: Robustness Parameter Summary table Column oven temperature variation

Peak Name
Column oven temperature variation ( 5C) Low (20C) Actual (25C) High (30C) TF %RSD TF %RSD TF %RSD
1.2 1.0 1.3 1.4 0.4 0.1 0.1 0.1 1.2 0.9 1.3 1.4 0.9 0.3 0.2 0.4 1.2 0.8 1.3 1.5 0.2 0.1 0.1 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 m PVDF Filter 0.45 m Nylon 66 Filter
0.98 0.99 1.00 1.00 0.98 0.99 0.99 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.
239
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, Jrgen; 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; Mhlenhoff, Ulrich (2006), "Iron-Sulfur Protein Biogenesis in Eukaryotes: Components and Mechanisms", Ann. Rev. Cell Dev. Biol. 22: 45786,. 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): 100310, 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): 12530, 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; Hgerroth, PA; Akerman, G; Hanson, M; Tjrnlund, U; Hansson, T; Hallgrimsson, GT; Zebhr, 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): 1200112006. 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): 5259. 11. Losa R, Sierra MI, Fernndez 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): 10251029. 12. Lu J, Frank EL. (May 2008). "Rapid HPLC measurement of thiamine and its phosphate esters in whole blood". Clin Chem. 54 (5): 901906. 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): 6671. doi:10.1016/j.jpba.2004.11.061. International Journal of Science Innovations and Discoveries Vol 1, Issue 2, September-October 2011
240
Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242 14. Slater, PV (1978). "Thiamine Responsive Megaloblastic Anemia with severe diabetes mellitus and sensorineural deafness (TRMA)". The Australian nurses' journal 7 (11): 403. 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): 54953. 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): 5879. 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, Gabrels-Festen AA, Lilisantoso AH, Sudanawidjaja A (1995). "Experimentally induced beriberi polyneuropathy in chickens". Electromyogr Clin Neurophysiol 35 (1): 5360. 20. Bruce WR, Furrer R, Shangari N, OBrien PJ, Medline A, Wang Y (2003). "Marginal dietary thiamin deciency induces the formation of colonic aberrant crypt foci (ACF) in rats". Cancer Lett 202 (2): 125129. 21. Langlais PJ (1995). "Pathogenesis of diencephalic lesions in an experimental model of Wernicke's encephalopathy". Metab Brain Dis 10 (1): 3144. 22. Frederikse PH, Zigler SJ Jr, Farnsworth PN, Carper DA (2000). "Prion protein expression in mammalian lenses". Curr Eye Res 20 (2): 13743. 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): 11581163. 24. Kluger, R; Tittmann, K (2008). "Thiamin diphosphate catalysis: enzymic and nonenzymic covalent intermediates". Chem Rev 108 (6): 17971833.
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): 14771488.
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): 29172925. 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., zksck, S., zkara, E., Serbest, Y. A., Yazc, M., Grsoy, 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., zksck, S., zkara, E., Karul, A., ztan, O., Yazc, M., Grsoy, 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. International Journal of Science Innovations and Discoveries Vol 1, Issue 2, September-October 2011
241
Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242 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): 52543. 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): 1801718023. 35. C. Smith, W. Song (1996). "Comparative nutrition of pantothenic acid". The Journal of Nutritional Biochemistry 7 (6): 312321. 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): 257269. 37. Leung L (1995). "Pantothenic acid deficiency as the pathogenesis of acne vulgaris". Med Hypotheses 44 (6): 4902. 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): 99114. 39. "Para-aminobenzoic acid poisoning". National Institute of Health: National Library of Medicine. 2007. Retrieved 200706-19. 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.
242

0102 2011 019

Enviado por

Dados do documento

Título original

Direitos autorais

Formatos disponíveis

Compartilhar este documento

Compartilhar ou incorporar documento

Opções de compartilhamento

Você considera este documento útil?

Este conteúdo é inapropriado?

Direitos autorais:

Formatos disponíveis

0102 2011 019

Enviado por

Direitos autorais:

Formatos disponíveis

Tamma Narendra Kumar et al.

, IJSID 2011, 1 (2), 226-242

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.

Address: Name: Tamma Narendra Kumar Place: Bangalore, India E-mail:

Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242

They are needed in relatively small amounts to

also called pantothenate or vitamin B5, it is a water-soluble vitamin. For

Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242

(PABA) is a naturally occurring substance that is often used in

Para amino benzoic acid Figure1: Structure of analytes

Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242

Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242

Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242

Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242

Table 2: Stress study conditions and results

Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242

Figure 2: Blank chromatogram

Figure 3: Standard chromatogram

Figure 4: Placebo chromatogram

Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242

Figure 5: Sample chromatogram with peak purity evaluation

Figure 6: Purity plot of Calcium Pantothenate

Figure 7: Purity plot of L Cystine

Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242

Figure 8: Purity plot of Para amino benzoic acid

Figure 9: Purity plot of Thiamine Mononitrate

Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242

Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242

Figure-10: Linearity chromatograms of four ingredients

Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242

Table 4: Linearity Regression Summary Table Active ingredient name

Para Amino Benzoic Acid

Regress ion Results

Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242

Table 6: Robustness Parameter Summary table Column oven temperature variation

Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242

Você também pode gostar