International Journal of Chemical and Analytical Science ISSN: Research Article

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1 International Journal of Chemical and Analytical Science ISSN: Research Article Method Development and Validation of Vitamin B Complex Injection in Pharmaceutical Dosage form by Using High Performance Liquid Chromatography Technique Deepti kiranmayi Malladi* 1, Venkateswararao. P 2, Sudhakar Babu A. M. S 3, Divya. B 1, Ramya Krishna. N 1 1 Department of Pharmaceutical Analysis, A.M. Reddy Memorial College of Pharmacy, Narasaraopet, Guntur, Andhra Pradesh, India. 2 Department of Pharmaceutical Chemistry, A.M. Reddy Memorial College of Pharmacy, Narasaraopet, Guntur, Andhra Pradesh, India. 3 Department of Pharmaceutics, A.M. Reddy Memorial College of Pharmacy, Narasaraopet, Guntur, Andhra Pradesh, India. A reversed-phase High performance liquid chromatographic method (HPLC) has been developed and validated for the routine analysis of Thiamine hydrochloride, Riboflavin-5-phosphate, D-Panthenol, Nicotinamide and Pyridoxine hydrochloride in injection dosage form. HPLC separation of the vitamins was performed on Inertsil ODS 3V ( mm, 5µ) column using gradient elution with ph 3 Sodium heptane sulphonate buffer as mobile phase A and methanol as mobile phase B at a flow-rate of 1.0mL/min. Detection was performed using U.V detector at 210 nm. The method was linear in the ranges of , 20-60, , , µg/ml for thiamine hydrochloride, riboflavin -5 phosphate,d-panthenol, pyridoxine hydrochloride and nicotinamide, respectively. The average recoveries of thiamine hydrochloride, riboflavin-5-phosphate, D-Panthenol, Pyridoxine hydrochloride and Nicotinamide were 100.0, 100.3, 99.8, 99.4 and 101.1% respectively. The method has been successfully applied to the simultaneous determination of Thiamine hydrochloride, Riboflavin-5-phosphate, D-Panthenol, Nicotinamide and Pyridoxine hydrochloride in injection dosage form. Keywords: RP-HPLC, Thiamine hydrochloride, Riboflavin-5-phosphate,D-Panthenol, Nicotinamide and Pyridoxine hydrochloride. INTRODUCTION Vitamins are micronutrients that are essential to life, and many of them play an important role in regulation of many functions. Extensive literature survey reveals that several analytical methods have been reported for the estimation of Vitamin B Complex which includes use of capillary zone electrophoresis in the determination B vitamins in pharmaceutical products 1, Thin-layer chromatography and fibre-optic fluorimetric quantization of thiamine, riboflavin and niacin 2, Determination of vitamins in food based on supercritical fluid extraction prior to micellar electrokinetic capillary chromatographic analyses of individual vitamins 3, Simultaneous determination of water-soluble vitamins excreted in human urine after eating an overdose of vitamin pills by a HPLC method coupled with a solid phase extraction 4,Simultaneous determination of nicotinamide, pyridoxine hydrochloride, thiamine mononitrate and riboflavin in multivitamin with minerals tablets by reversedphase ion-pair high performance liquid chromatography 5, validated HPLC method for the determination of B-complex vitamins in pharmaceuticals and biological fluids after solid phase extraction 6, High-performance liquid chromatography/electrospray ionization-mass spectrometry for simultaneous determination of taurine and 10 watersoluble vitamins in multivitamin tablets 7,Spectrophotometric determination of ternary mixtures of thiamin, riboflavin and pyridoxal in pharmaceutical and human plasma by leastsquares support vector machines 8,Simultaneous Determination of Four Water-Soluble Vitamins in Fortified Infant Foods by Ultra-Performance Liquid Chromatography Coupled with Triple Quadrupole Mass Spectrometry 9, Simultaneous determination of some water-soluble vitamins and preservatives in multivitamin syrup by validated stability-indicating high-performance liquid chromatography method 10,The present paper describes a new quantitative reversed-phase high-performance liquid chromatographic method, coupled with prominence UV-VIS detector, as an alternative technique for quality control of products. The purpose of this investigation was to develop and validate a method using a simple, rapid, sensitive, precise, accurate and specific reversed phase HPLC assay. Hence, this method can be used for the analysis of large number of samples. MATERIALS AND METHODS Chemicals and solvents The reference sample of Vitamin B Complex was supplied by Pharma med Pvt Ltd., Hyderabad. HPLC grade water and Methanol were purchased from E. Merck (India) Ltd., Ahmedabad. Sodium Dihydrogen phosphate and orthophosphoric acid of AR Grade was obtained from S.D. Fine Chemicals Ltd., Mumbai. Instrument The HPLC system is of Waters, Alliance 2695 with 2489 UV- VIS detector, HPLC Pump LC-20AT pump and Injector Loop rheodyne, model No Data acquisition was performed by the Empower software. Chromatographic Conditions Chromatographic analysis was performed on a inertsil ODS 3Vcolumn with 250 x 4.6mm internal diameter and 5µm particle size. Heptane sulphonate buffer (ph 3.0) is used as mobile phase A and methanol is used as mobile phase B at a flow rate of 0.9 ml/min. The mobile phase was degassed and filtered through 0.45 µ filter under vacuum before pumping Corresponding Author: Deepti Kiranmayi Malladi, Department of Pharmaceutical Analysis, A.M. Reddy Memorial College of Pharmacy, Narasaraopet, Guntur, Andhra Pradesh, India; < Deeptikiranmayi010@gmail.com > Received ; Accepted

2 into HPLC system. The effluent was monitored by UV detection at 210 nm. Preparation of ph 3 sulphonate buffer Dissolve 1 g of sodium-1-heptane sulphonate in small amount of distilled water transferred in to 1000 ml beaker and make up to final volume with water. Adjusted the ph to 3.0 with ortho phosphoric acid. Preparation of mobile phase ph 3 sulphonate buffer was used as mobile phase A and methanol was used as mobile phase B. The solution was degassed in an ultrasonic water bath for 5 minutes and filtered through 0.45 µ filter under vacuum. Preparation of Vitamin B Complex standard and sample Solutions Preparation of standard stock solution-1(riboflavin) Accurately weigh and transfer 33 mg of Riboflavin working standard into a 100ml clean dry volumetric flask add about 60 ml of Diluent and sonicate to dissolve it completely and make volume up to the mark with the same diluents. Preparation of standard stock solution-2 Accurately weigh and transfer 25 mg of nicotinamide, pyridoxine, D-panthenol and 50mg of Thiamine working standards into 50 ml volumetric flask add about 20ml of diluent and sonicate to dissolve it completely and make volume up to mark with diluents. Preparation of standard Solution Take 4ml of standard stock solution-1 and 5ml of standard stock solution-2into 25ml volumetric flask and dilute to volume and mix well. Sample Solution Preparation Take 2ml of Vitamin B complex injection into 100 ml clean dry volumetric flask add about 60 ml of diluent shake well and make volume up to the mark with the same diluent. Sample Solution Preparation (Nicotinamide) Take 5ml of Vitamin B complex injection into 100 ml clean dry volumetric flask add about 60 ml of diluent shake well and make volume up to the mark with the same diluent. Table 1: Assay studies Drug Standard area Sample area Labelled claim Purity% Nicotinamide mg 99.5 Pyridoxine mg 99.9 Riboflavin mg 70.5 D-Panthenol mg 99.0 Thiamine mg 96.2 Table 2: System suitability parameters Results Drug Thiamine Riboflavine D-Panthenol Pyridoxine Nicotinamide Peak Area Retention time Theoretical plates Tailing Factor Table 3: Linearity studies of Riboflavin L1-50% Slope L1-60% Y-intercept L1-80% % of Y-intercept L1-100% Correlation coefficient L1-120% r L1-140% L1-150% Table 4: Linearity studies of D-panthenol L1-50% Slope L1-60% Y-intercept L1-80% % of Y-intercept 0.97 L1-100% Correlation coefficient L1-120% r L1-140% L1-150% RESULTS Method development and optimization Heptane sulphonate buffer (ph 3.0) as mobile phase A and methanol as mobile phase B was found to be the most suitable for ideal separation of Vitamin B Complex. The solvent mixture was filtered through a 0.45 µ membrane filter and sonicated before use. It was pumped through the column at a flow rate of 1 ml/min. The column was maintained at ambient temperature. The column was equilibrated by pumping the mobile phase through the column for at least 30 min prior to the injection of the drug solution. The detection of the drug was monitored at 210 nm. The run time was set at 30 min. Under these optimized chromatographic conditions the 1676

3 retention time obtained for the drug was the retention time is min for Riboflavin-5-Phosphate, min for D- Panthenol, min for Nicotinamide, min for Pyridoxine Hcl, min for Thiamine Hcl. Validation Procedures The method validation was carried out according to the recommendations for analytical method validation System suitability System-suitability tests are an integral part of method development and are used to ensure adequate performance of the chromatographic system. Retention time (tr), number of theoretical plates (N) and tailing factor (T) were evaluated the drug. The results which are given in Table.2 were within acceptable limits. Table 5: Linearity studies of Nicotinamide L1-50% Slope L1-60% Y-intercept L1-80% % of Y-intercept 1.07 L1-100% Correlation coefficient L1-120% r L1-140% L1-150% Table 6: Linearity studies of Pyrdoxinde L1-50% Slope L1-60% Y-intercept L1-80% % of Y-intercept L1-100% Correlation coefficient L1-120% r L1-140% L1-150% Table 7: Linearity studies of Thiamine L1-50% Slope L1-60% Y-intercept L1-80% % of Y-intercept 0.70 L1-100% Correlation coefficient L1-120% r L1-140% L1-150% Table 8: Results of Accuracy Drugs name Thiamine Riboflavin Nicotinamide D-Panthenol Pyridoxine Amount of drug added(mg) Amount of drug recovered(mg) %Recovered Mean recovery %

4 Table 9: Results of precision Drug Thiamine Riboflavin D-Panthenol Pyridoxine Nicotinamide Precision Precision Precision Precision Precision Precision Table 10: Calculated %RSD Sample % Assay Riboflavin D-Panthenol Nicotinamide Pyridoxine Thiamine Mean Statistical SD analysis %RSD Linearity Preparation of stock solution Accurately weigh and transfer 57mg of riboflavin, 100mg of pyridoxine, and 100mg of D-panthenol and 208 mg of thiamine working standards into a 100ml clean dry volumetric flask sonic ate to dissolve it completely and make up the volume up to the mark with water. Fig.1: Calliberation curve of Riboflavin Preparation of Level-3 4ml of stock solution has taken in 50ml of volumetric flask dilute up to the mark with water. Preparation of Level-4 5ml of stock solution has taken in 50ml of volumetric flask dilute up to the mark with diluent. Preparation of Level-5 3ml of stock solution has taken in 25ml of volumetric flask dilute up to the mark with diluent water. Preparation of Level ml of stock solution has taken in 25ml of volumetric flask dilute up to the mark with diluent water. Preparation of Level-7 3ml of stock solution has taken in 20ml of volumetric flask dilute up to the mark with diluent water. Evaluation was performed with UV detector at 210 nm. The peak area was recorded for all the peaks and calibration graph was obtained by plotting peak area versus concentration of Vitamin B Complex. The plot of peak area against concentration of Vitamin B Complex was found to be linear. The calibration data of Vitamin B Complex is given in Table.3-7 and the calibration curves of Vitamin B Complex were shown in Fig.1-5. Fig.2: Calliberation curve of D-Panthenol Preparation of Level-1 5ml of stock solution taken in 100ml of volumetric flask dilute up to the mark with water. Preparation of Level-2 6ml of stock solution has taken in 100ml of volumetric flask dilute up to the mark with water. Fig.3: Calliberation curve of Nicotinamide Accuracy Accuracy was performed in triplicate after spiking pure drug equivalent to 50, 100, and 150% of the standard concentration of Vitamin B Complex. The results obtained (Table.4) indicate 1678

5 that recovery was excellent, not less than 100% ± 2. The results indicate the method is highly accurate for determination of the Vitamin B Complex. feasible method, including linearity, accuracy, precision and specificity the chromatographic system allows the analysis of a large number of samples in a short period of time. Therefore, the method is suitable for analysis of large samples during routine analysis of formulation. Fig. 4. Calliberation curve of Pyridoxine Precision Sample preparation (For Riboflavin, Pyridoxine, Thiamine and D-panthenol) 2ml of vitamin B Complex injection is taken into 100ml volumetric flask diluted upto volume with water. Sample preparation (For Nicotinamide) 5ml of vitamin B Complex injection is taken into 100ml volumetric flask diluted upto volume with water. Fig. 7. Chromatogram of sample CONCLUSION Platinum on gamma alumina was synthesized by a simple chemical route to use it as a catalyst and was characterized by XRD, SEM, TEM, AFM, BET and FTIR analysis. XRD and TEM analysis shows the presence of nanostructured particles in the sample. This platinum on gamma alumina shows good catalytic activity for the isomerization of light naphtha components to dimethyl butanes, methyl pentanes, cyclo pentanes and cyclohexanes,, which were absent in the original feed stock. ACKNOWLEDGEMENT Fig. 5. Calliberation curve of Thiamine Fig. 6. Chromatogram of standard The above preparation was performed six determinations and introduced into chromatographic system. %RSD was calculated presented in Table.10. DISCUSSION A rapid and simple RP-HPLC method for determination of Vitamin B Complex has been developed and validated. The RP-HPLC method involved a ph 3.0 heptane sulphonate buffer as mobile phase A and Methanol as mobile phase B accomplished at 210nm at a flow-rate of 1 ml/min and the injection volume was 5µl. This chromatographic assay fulfilled all the requirements to be identified as a reliable and The authors whole heartedly wish to thank Pharma med Ltd, Hyderabad for providing the necessary facilities to carry out my work. REFERENCES 1. Huopalahti and Sunell J. Use of capillary zone electrophoresis in the determination B vitamins in pharmaceutical products, Journal of Chromatography, 636(1), 1993, Navas Diaz A, Guirado Paniagua A and Garcia Sánchez F. Thin-layer chromatography and fibre-optic fluorimetric quantization of thiamine, riboflavin and niacin Journal of Chromatography A, Volume 655, Issue 1, 26 November, Buskov S, Møller P, Sørensen H, Sørensen J.C, and Sørensen S. Determination of vitamins in food based on supercritical fluid extraction prior to micellar electrokinetic capillary chromatographic analyses of individual vitamins, Journal of Chromatography A, vol. 802, no. 1, 1998, Cho CM et al. Simultaneous determination of water-soluble vitamins excreted in human urine after eating an overdose of vitamin pills by a HPLC method coupled with a solid phase extraction, Talanta, Apr 3, 51(4), 2000, Ke li. Simultaneous determination of Nicotinamide, Pyridoxine hydrochloride, Thiamine mononitrate and Riboflavin in multivitamin with minerals tablets by reversed-phase ion-pair high performance liquid chromatography, Biomedical Chromatography, Volume 16, Issue 8, December 2002, pages Chatzimichalakis P F, Samanidou V F, Verpoorte R, and Papadoyannis I N. Development of a validated HPLC method for the determination of B-complex vitamins in pharmaceuticals and biological fluids after solid phase extraction, Journal of Separation Science, Volume 27, No. 14, 2004, pp Chen Z, Chen B, and Yao S. High-performance liquid chromatography/electrospray ionization-mass spectrometry for simultaneous determination of Taurine and 10 water-soluble vitamins in multivitamin tablets, Analytica Chemica Acta, Vol.569, No.1-2, 2006, pp Niazi J, Zolgharnein and Afiuni-Zadeh S. Spectrophotometric determination of ternary mixtures of thiamin, riboflavin and pyridoxal 1679

6 in pharmaceutical and human plasma by least-squares support vector machines, Analytical Sciences, 23(11), 2007, Baiyi Lu ann Yiping Ren. Simultaneous Determination of Four Water- Soluble Vitamins in Fortified Infant Foods by Ultra-Performance Liquid Chromatography Coupled with Triple Quadrupole Mass Spectrometry, Journal of Chromatographic Science, 46, Vidović S, Stojanović B, Veljković J, Pražić-Arsić L, Roglić G, and Manojlović D, Simultaneous determination of some water-soluble vitamins and preservatives in multivitamin syrup by validated stabilityindicating high-performance liquid chromatography method, Journal of Chromatography A, 1202, 2, 2008, ICH: Q2B, Analytical validation-methodology, ICH: Q2A, Text on validation of analytical procedure, ICH: Q2 (R1), Validation of analytical procedures: text and methodology, Source of support: Nil, Conflict of interest: None Declared 1680

IJRPC 2013, 3(2) Nagamallika et al. ISSN: INTERNATIONAL JOURNAL OF RESEARCH IN PHARMACY AND CHEMISTRY

IJRPC 2013, 3(2) Nagamallika et al. ISSN: INTERNATIONAL JOURNAL OF RESEARCH IN PHARMACY AND CHEMISTRY INTERNATIONAL JOURNAL OF RESEARCH IN PHARMACY AND CHEMISTRY Available online at www.ijrpc.com Research Article A VALIDATED STABILITY INDICATING RP-UPLC METHOD FOR SIMULTANEOUS DETERMINATION OF WATER SOLUBLE