novel rp-hplc method for the simultaneous estimation of thiamine

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

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.



228

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



229

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



230

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


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


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.



233

Figure – 2: Blank chromatogram

Figure – 3: Standard chromatogram

Figure – 4: Placebo chromatogram



234

Figure – 5: Sample chromatogram with peak purity evaluation

Figure – 6: Purity plot of Calcium Pantothenate

Figure – 7: Purity plot of L – Cystine



235

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



236

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


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.



237

Figure-10: Linearity chromatograms of four ingredients



238

Table – 4: Linearity Regression Summary Table

Active ingredient name Calcium


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.



239

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.



240

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.

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