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1 Available online at International Journal of Analytical and Bioanalytical Chemistry Universal Research Publications. All rights reserved ISSN Original Article VALIDATED RP- HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF METFORMIN HYDROCHLORIDE AND BENFOTIAMINE IN BULK DRUG AND IN PHARMACEUTICAL DOSAGE FORM Mihirkumar G. Patel, Pravin O. Patil*, Sanjay B. Bari Department of Quality Assurance, H.R. Patel Institute of Pharmaceutical Education and Research, Shirpur, Dist: Dhule (M.S.) India Addresses for correspondence* H. R. Patel Institute of Pharmaceutical Education and Research, Shirpur Dist: Dhule (M.S.) India Tel/fax: , Received 20 July 2012; accepted 28 July 2012 Abstract A new simple, precise, accurate, selective and economical RP-HPLC method has been developed and validated for simultaneous estimation of Metformin Hydrochloride (MET) and Benfotiamine (BEN) in tablet dosage form. The method was carried out on a Waters column C-18 (250 mm x 4.6 mm, 5 µm) with a mobile phase consisting of water (ph 3.2 adjusted with ortho-phosphoric acid) and acetonitrile (75:25 v/v); at a flow rate of 0.8 ml min -1 with run time of 10 min. Detection was carried out at 254 nm. The retention time for MET and BEN was found to be and min, respectively. The MET and BEN followed linearity in the concentration range of µg ml -1 and µg ml -1 with r 2 = 0.999, respectively. The amounts of both drugs estimated by proposed method were found to be in good agreement with label claim. The developed method was validated for precision, accuracy, sensitivity, robustness and ruggedness. The LOD and LOQ were found to be 0.49 and 0.19 µg for MET and 1.6 and 0.65 µg for BEN respectively. The developed method can be used for routine analysis of titled drugs in tablet formulation Universal Research Publications. All rights reserved Keywords: Metformin Hydrochloride; Benfotiamine; RP-HPLC, Validation. 23] method for estimation of benfotiamine in pharmaceutical formulation. Combination product of metformin hydrochloride (MET) and benfotiamine (BEN) is Benforce M tablet which used as a neutraceuticals [24]. The chemical structures of both drugs are shown in (Figure 1, 2). Introduction Metformin hydrochloride (MET) is an oral anti-diabetic drug and is chemically N,N dimethylimidodicarbon -imidic diamide hydrochloride (1,1-dimehylbiguanide hydrochloride) [1] which acts by suppressing excessive hepatic glucose production and improving glucose clearance, its predominant effect is to decrease fasting plasma glucose [2]. It is official in Indian Pharmacopoeia [3], British Pharmacopoeia [4], European Pharmacopoeia [5] and United States Pharmacopeia [6]. A literature survey revealed spectrophotometry [7-9], HPLC [10-14], LC- MS/MS [15] and Ion-pairing HPLC [16] methods for estimation of MET in pharmaceutical formulation. Benfotiamine (BEN) is a synthetic derivative of thiamine (vitamin B-1) and is chemically N-((4-amino-2-methyl- 5- pyrimidinyl) methyl)-n-(4-hydroxy-2- mercapto-1-methyl- 1 butenyl)formamide- S-benzoate dihydrogen phosphate [1]which show beneficial effects on general nerve health, neuropathy, retinopathy, peripheral neuropathy, general ageing [17-21] A literature survey revealed RP-HPLC [ To the best of our knowledge, few literature methods were reported for estimation of MET and BEN individually. but no literatures have been found for simultaneous determination of MET and BEN in pharmaceutical preparations. The present paper describes a precise, accurate, specific, sensitive and cost effective RP-HPLC method for the simultaneous determination of MET and BEN in the tablet dosage form and validation of the same as per the ICH guidelines [25-27]. Materials and Methods Chemicals Metformin hydrochloride and Benfotiamine were obtained from Rajat Pharmachem. Pvt. ltd, Ankleshwar and Akhil

2 Figure 1: Chemical Structure of Metformin Hydrochloride Figure 2: Chemical Structure of Benfotiamine. Health care, Baroda, India as a gift sample respectively. Ortho-phosphoric acid (AR Grade), acetonitrile (HPLC Grade), was purchased from Merck (India) Ltd., Worli, Mumbai, India. Tablet formulation (Benforce- M) was purchased from Indian market, containing MET (500 mg), BEN (75 mg). HPLC Grade water (Milli-Q) was used throughout the experiment. Instrumentation Analysis was performed on chromatographic system of HPLC LC-2010C HT Shimadzu with LC Solution equipped with Shimadzu SPD-M20A Prominence Diode array detector. HPLC instrument was controlled by LC Solution software. Chromatographic Conditions A Waters column C 18, 5 μ (250 X 4.6 mm) was used for chromatographic separation. The mobile phase composed of water (ph 3.2 adjusted with ortho-phosphoric acid) and acetonitrile (75:25 v/v); at a flow rate of 0.8 ml min -1 with run time of 10 min. Mobile phase and sample solutions were filtered through a 0.45 µm membrane filter and degassed. The detection of both drugs was carried out at 254 nm. Preparation of Diluent 1000 ml of HPLC Grade water was adjusted to ph 3.2 with o-phosphoric acid. Preparation of Standard Stock Solutions Standard stock solutions of 2000 µg ml -1 of MET and 300 µg ml -1 of BEN were prepared separately using diluent. The stock solution of MET was diluted with diluent to give working standard solutions containing µg ml -1 concentrations, similarly the BEN stock solution was diluted with diluent to give working standard solutions in the range µg ml -1. These standard solutions were injected into HPLC column and calibration curves were plotted by taking drug peak areas vs concentrations. Analysis of marketed tablet formulation To determine the content of MET and BEN in tablets (Brand name: Benforce M, label claim: MET 500 mg, BEN 75 mg per tablet), twenty tablets were weighed, their mean weight determined and finally powdered. An accurately weighed tablet powder equivalent to 500 mg of MET and 75 mg BEN was transfer into 100 ml volumetric flask containing 50 ml of diluent, sonicate for 10 minute and volume was made up to the mark with diluent, the resulting solution was filtered using 0.45 µm filter (Mill filter, Milford, MA). From filtrate, 8 ml of solution was transferred into 100 ml volumetric flask and volume was made up to mark with diluent to obtain the concentration of 400 µg ml -1 MET and 60 µg ml -1 of BEN was subjected to propose method and the amount of MET and BEN were determined. Validation of Method The HPLC method was validated in accordance with ICH guidelines. Precision The precision of the analytical method was studied by analysis of multiple sampling of homogeneous sample. Precision was estimated by repeatability and the repeatability was assessed by analyzing six injection of a homogeneous sample of 400 µg ml -1 of MET and 60 µg ml -1 of BEN. The value of precision of repeatability along with intra-day and inter day using three different concentrations 300 µg ml -1, 400 µg ml -1 and 500 µg ml -1 of MET and 45 µg ml -1, 60 µg ml -1 and 75 µg ml -1 of BEN respectively, in triplicate for three consecutive days. Accuracy The different between theoretical added amount and practically achieved amount is called accuracy of analytical method. Accuracy was determined at three different level 80 %, 100 % and 120 % of the target concentration 400 µg ml -1 of MET and 60 µg ml -1 of BEN in triplicate. Specificity The specificity of an analytical method may defined as the ability to detect the analyte peak in the presence of the analyte by product, or other inactive components, such as dosage form excipient or impurities. Limit of detection (LOD) and Limit of quantification (LOQ) Limit of detection and limit of quantification were estimated from signal to noise ratio. LOD is the lowest concentration resulting in a peak area of three times the baseline noise and the equation is LOD = 3.3 x ASD/S. LOQ is the lowest concentration that provide signal to noise ratio more than 10 and the equation is LOQ = 10 x ASD/S, where ASD is the average standard deviation and S is the slope of the line. Robustness Robustness was performed by deliberately changing the chromatographic conditions. The important parameter to be studied was the resolution factor between two peaks. Robustness of the method was carried out by deliberately made small variation in the flow rate, ph of mobile phase, organic phase ratio and column oven temperature by using 400 µg ml -1 of MET and 60 µg ml -1 solution of BEN, respectively. 197

3 Ruggedness Ruggedness was determined between two different labs, different analyst, different instrument and columns. Ruggedness of the method was performed by injecting 400 µg ml -1 of MET and 60 µg ml -1 solution of BEN, respectively. Results and Discussion Selection of Chromatographic Conditions and Optimization of Mobile Phase Mobile phase was optimized to separate MET and BEN using Waters column C 18, 5 μ (250 X 4.6 mm). Initially, phosphate buffer ph 3.5 and acetonitrile in the various proportions were tried as mobile phase but the broad peaks for both the drugs were observed. Therefore, we select Water (ph 3.2 adjusted ortho-phosphoric acid) and acetonitrile as a mobile phase composition in 75:25 % v/v ratio. Good resolution and symmetric peaks were obtained for both drugs when the ph of the mobile phase was adjusted to 3.2. The flow rate of the mobile phase was 0.8 ml min -1. Under optimum chromatographic conditions, the retention time for MET and BEN was found to be and respectively when the detection was carried out at 254 nm. A typical chromatogram of two drugs is shown in (Figure 3). Table 1: Linearity Studies PARAMETER MET BEN Linearity [µg ml -1 ] Linearity Equation Y = 5481X Y = 26543X Slope Intercept Correlation Coefficient Figure 4: Calibration curve of MET Correlation Coefficient = 0.999, Slope = 5481, Intercept = Figure 3: Typical Chromatogram of MET and BEN Linearity The linearity was determined at five levels over the range of 50 % to 150 % of standard concentration in a diluent and calibration curve constructed by plotting peak area against the respective concentrations. The linearity of MET and BEN followed in the concentration range of µg ml -1 and µg ml -1, respectively. Each sample solution was chromatographed in triplicate and the mean peak area for MET and BEN calculated. The results are shown in Table 1, (Figure 4,5). Figure 5: Calibration curve of BEN Correlation Coefficient = 0.999, Slope = 26543, Intercept = Precision The precision of this method was evaluated by calculating the % RSD of the peak area of six replicate injections. The RSD for repeatability of MET and BEN was found to be 0.55 and 1.27 %, respectively. The RSD values for intraday precision for MET and BEN was found to be in the range and %, respectively and the inter-day precision for MET and BEN was found to be in the range and %, respectively. The assay method precision acceptance criteria set in the validation was RSD 2.0%. Results of precision study are shown in Table 2. Table 2: Results of Precision studies Conc. [ g/ml] MET Amount Found in µg ml -1 [n= 3] SD % RSD Conc. [ g/ml] 198 BEN Amount Found in µg ml -1 [n= 3] SD %RSD Intra day Precision Inter day Precision

4 Table 3: Results of Recovery Studies Drugs Label claim (mg) Amount of standard drug added (%) % DrugRecovered [ n = 3] % R.S.D MET BEN Table-4: Assay Result of Marketed Formulation Tablet Amount Content taken [µg/ml] Label claim (mg) % Label claim [n = 6] %RSD MET BEN Specificity and Selectivity The specificity of the method was checked for the interference of retention time of tablet formulation and standard drug under optimized condition. Specificity of the method was determined by comparing the chromatogram obtained from tablet formulation and standard drug. As there was no interference of excipient and the retention time of standard drugs and tablet formulation were same, so method was specific and selective. Accuracy Accuracy was evaluated as percentage relative error between the found mean concentration and added concentration for MET and BEN. The result obtained for three different concentration levels i.e. 80 %, 100 % and 120 % showed acceptable % recoveries in the range of 99.65% % for MET and 99.94% % for BEN which suggests that accuracy is excellent for both drugs. The results are shown in Table 3. Sensitivity The limit of detection (LOD) and limit of quantification (LOQ) for MET and BEN was found to be 0.01 and 0.42 µg and 0.03 and 1.28 µg, respectively. The low values of LOD and LOQ indicates high sensitivity of the method. Robustness and Ruggedness study The Robustness of the method evaluated by changing the chromatographic condition and results were examined. Ruggedness test was determined between two different analyst and instrument under same environmental condition. The percentage RSD was below 2.0%, Showed robustness and ruggedness of method. Analysis of marketed tablet formulation Experimental results of the amount of MET and BEN in tablet, expressed as a percentage of label claim were in good agreement and the amounts were found to be 99.1 % and %respectively. A good agreement of result indicates that there was no interference from any of the excipients which was normally present in tablet. The results are shown in Table 4. System Suitability Test according to USP 2009, system suitability tests were an integral part of liquid chromatographic methods in the course of optimizing the conditions of the proposed method. System suitability tests were used to verify that the resolution and reproducibility were adequate for the performed. The parameter of these tests is column efficiency (number of theoretical plate), tailing factor, resolution, peak asymmetry and capacity factor were calculated for standard solutions. The results obtained from validation of the methods and system suitability studies are summarized in Table 5. Table 5: Summary of system suitability study Parameter MET BEN Retention time [t R ] Theoretical plates [N] Resolution [Rs] Asymmetry [T] Conclusion The proposed new RP-HPLC method provide simple, fast, accurate, precise, reproducible and economical approach for the simultaneous identification and quantification that can be used for the quality control of the metformin hydrochloride and benfotiamine in tablet formulation in routine quality control laboratories and the method was validated as per ICH guidelines. Acknowledgement The authors wish to express their gratitude to the Principal and management, H.R. Patel Institute of Pharmaceutical Education and Research, Shirpur (M.S.), India for providing the necessary facilities to carry out this research work. References 1. The Merck Index An Encyclopedia of Chemicals, Drugs and Biological, 14 th edn, Merck Research Laboratories, Whitehouse Station, New Jersey, USA, (2006) p.1038, A. Oanal, Eur. J. Med. Chem. 44 (2009) Indian Pharmacopoeia, Govt. of India, Ministry of Health & Family Welfare, published by Indian Pharmacopoeial convention, Ghaziabad,Vol. II, (2010) p British Pharmacopoeia, The Stationary Office Medicinal and Pharmaceutical Substances, Vol. III, London, (2009) p European Pharmacopoeia, Council of Europe, France, 6 rd edn, Vol. 2, (2008) p United States Pharmacopoeia-32, National Formulary- 27, United States Pharmacopoeial Convention, Rockville, Vol. II. (2009), p S. S. Patil, C. G Bonde, Int. J. Chem. Tech. Res. 1 (2009) S. D. Jadhav, A. V. Chandewar, R. L. Bakal, Int. J. Pharm. Rec. Res. 2 (2011)

5 9. K. P. D Adhania, P. A. Nadpara, Y. K. Agrawal, Int. J. Pharm. Sci. Res. 2 (2011) A. K. Madhukar, A. M. Prince, Y. K. Sanjava, Int. J. Pharm. Pharm. Sci. 3 (2011) P. K. Shaoo, R. K. Sharma, S. C. Chaturvedi, Ind. J. Pham. Sci. 70 (2008) P. C. Bhamare, S. B. Bari, A. A. Patil, Int. J. Chem. Tech. Res. 3 (2011) K. S. Lakshmi, T. S. Rajesh, S. H. Sharma, Ind. J. Pham. Pharm. Sci. 1 (2009) U. R. Mallu, V. R. P. Reddy, S. P. Penumajji, J. Pharm. Res. 4 (2011) P. K. Sengupta, U. B. Bhaumik, A. S. Ghosh, Chromatographia. 69 (2009) B. U. Rao, A. P. Nikalje, Afr. J. Pharm. Pharmacol. 5 (2011) U. R. Charrondiere, D. M. Gott, G. E. N. Kass, European Food Safety authority Journal 864(2008) X. Pan, N. Gong, J. Zhao, A Journal of neurology, 133 (2010) D. Laew, Int. J.Clin. Pharmacol. Ther. 34 (1996) M. L. Volvert, S. Seyen, M. Piette, BMC Pharmacology. 8 (2008) Benfotiamine Monograph, Alternative Medicine Review, 11(2006) S. Poongothai, R. Ilavarasan, C. Karrunakaran, Int. J. Pham. Pharm. Sci. 2 (2010) Source of support: H.R. Patel Institute of Pharmaceutical Education and Research, Shirpur (M.S.), India; Conflict of interest: None declared 200

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