IJPRD, 2011; Vol 4(06): August-2012 ( ) International Standard Serial Number
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1 IJPRD, 2011; Vol 4(06): August-2012 ( ) International Standard Serial Number DEVELOPMENT AND VALIDATION OF ANALYTICAL METHOD FOR SIMULTANEOUS ESTIMATION OF METFORMIN HYDROCHLORIDE AND ALPHA LIPOIC ACID IN BULK DOSAGE FORM USING UV-VISIBLE SPECTROPHOTOMETRY Alagar raja M 1*, Swathi M 1 David Banji 1, Rao K.N.V 1, Swathi A 1, Deepthi K 1 1 Department of pharmaceutical Analysis and Quality Assurance, Nalanda college of Pharmacy, Charlapally, Nalgonda. Andhra Pradesh, India ABSTRACT A simple, precise, rapid and selective spectroscopic method has been developed for simultaneous estimation of metformin hydrochloride and Alpha lipoic acid in bulk dosage form. The method employed simultaneous equation method for Analysis using methanol as a solvent the two wavelengths 236nm and 215nm were selected for estimation of metformin hydrochloride and α- lipoic acid respectively. Linearity was observed in the concentration range of 1-9µg/ml for both drugs. The recovery studies ascertained the accuracy of the proposed method and the results were validated as per ICH guidelines. The method can be employed for estimation of pharmaceutical formulations with low interference from any other excipients and diluents. KEYWORDS : Metformin hydrochloride, Alpha lipoic acid, Simultaneous equation, etc. Correspondence to Author Alagar raja M Department of pharmaceutical Analysis and Quality Assurance, Nalanda college of Pharmacy, Charlapally, Nalgonda. Andhra Pradesh, India rajampharman_1982@rediffmail.com INTRODUCTION Metformin hydrochloride is biguanide group of diabetic drug chemically it is N, N-dimetylimido dicarbonimic diamide hydrochloride; its main use is to reduce blood sugar levels. They do not cause insulin release presence of some insulin is essential for their action. It suppresses hepatic gluconeogenesis and glucose output from liver, other hexoges, amino acids and vitamin B 12. The main use of metformin is in the use of diabetes mellitus. [1-2] The second drug alpha lipoic acid is a potent antioxidant chemically it is (1, 2-dithiolan-3-yl) pentanoic acid. It has been shown to lower blood glucose in diabetic animals. Α-lipoic acid enhances glucose uptake and GLUT1 and GLUT4 translocation in 3T3-L1 adipocytes and L6 myotubes mimicking insulin action. [1-2] 102
2 Alpha lipoic acid is a cofactor of mitochondrial dehydrogenase complexes in clinical trials, and it also improves glucose metabolism in patients with type-2 diabetes. Furthermore, this compound has proven beneficial in treatment of diabetic neuropathy. The chemical structure of metformin hydrochloride and alpha lipoic acid are shown in figure no: 1 Literature survey reveals that various spectrophotometric and HPLC methods[3-12] have been developed for the drugs individually for metformin and alpha lipoic acid but there is no records available for combination that is the simultaneous determination of metformin Hcl and alpha lipoic acid in bulk dosage form and also till it s not in any pharmaceutical dosage form in market. In this communication we propose aim of present study is that a fast, very simple and accurate derivative spectrophotometric method for estimation of metformin hydrochloride and alpha lipoic acid in bulk dosage forms. Figure no: 1 Metformin Hydrochloride Alpha lipoic acid MATERIALS AND METHODS Chemicals and reagents: A double beam UV-Visible spectrophotometer Elico SL-196 model and shimadzu-1601 model with 1cm matched quartz cell. Metformin hydrochloride and Alpha lipoic acid pure drugs used for the development of analytical method, were gifted by Life science Pharma Pvt ltd, Pondicherry respectively methanol and water used as solvents of AR guide (Merck), India, for the analytical purpose. Preparation of standard and stock solutions: Standard and stock solutions of metformin hydrochloride and alpha lipoic acid was prepared by dissolving 10mg of drug in 100ml of methanol in a volumetric flask to get a concentration of 100µg/ml. An appropriate aliquot portions of 1,2,3,4 and 5ml of metformin hydrochloride standard stock solutions were transferred to separate 10ml volumetric flasks and volume was made up to the mark to obtain concentrations 10, 20, 30, 40, and 50 µg/ml of metformin. The same procedure followed to obtain concentrations 10,20,30,40, and 50µg/ml of alpha lipoic acid. Drug solutions were scanned separately between 200nm to 400nm. The spectrum of both drugs was recorded fig 2-3 and two wavelengths 236 nm (λ max of MET) and 215nm (λ max of ALA) were selected for further study. Method: Different aliquots were taken from the stock solutions and diluted with the same solvent to prepare a series of concentrations.the absorbance of these solutions were measured at 236 nm and 215 nm for MET and ALA respectively and calibration curves were plotted at selected wavelengths. The optical characteristics and linearity data is shown in table no: 1, the overlain spectra of MET and ALA are shown in figure no: 4 The simultaneous equation in two variables C1 and C2 were framed by using E (1 1cm) A1 = (83) C1+ (47) C2 I A2 = (72) C1+ (92) C2 II Where C1 and C2 are the concentrations of MET and ALA measured in g/100ml in the solutions at selected wavelengths that is 236 nm and 215nm respectively. 103
3 Figure 2: UV Spectrum for Metformin Hydrochloride Figure 3: UV Spectrum for Alpha lipoic acid 104
4 Figure 4: Overlain UV spectra for Metformin & α Lipoic acid Validation of the method (ICH guidelines, 2005) [13] The method was validated with a reference to accuracy, precision, linearity and ruggedness. Accuracy: The accuracy of the prepared method was assessed by recovery studies at three different levels that are 80% 100% 120%. The recovery studies were carried out by bulk drugs the resulting solutions were re-analysed by proposed methods and the results are shown in table no: 3 Precision: Precision of the method was studied as intra-day, interday and repeatability. Intra -day study was performed by analysing the three different concentrations of drug for three times in the same day. Interday precision was performed by analysing three different concentration of the drug for three days in a week. Repeatability was performed by analysing same concentration of drugs for six times. The results are shown in table no: 4 Ruggedness: Ruggedness of the proposed method is determined by analysis of aliquots from homogeneous slot by different analysts using similar operational and environmental conditions. The results are shown in table no: 4 105
5 Table 1: Optical characteristics and linearity data Parameters MET ALA Absorption maximum(nm) Beers law limit(µg/ml) Correlation coefficient Intercept (c) Slope (m) *Met = Metformin Hcl Ala = Alpha Lipoic Acid Table: 2 E (1%, 1cm) for MET and ALA E(1% 1cm) at 553nm +_ SD E(1% 1cm) at 215nm +_ SD MET ALA MET ALA Ax 1 = 83 ± 0.43 Ay 1 = 47 ± 0.38 Ax 1 = 71.2 ± 0.75 Ay 1 = 92.8 ± 0.56 Table 3: Results from accuracy studies Drug name Concentration Amount recovered % Recovery 80% (n=3) MET 100%(n=3) %(n=3) %(n=3) ALA 100%(n=3) %(n=3) Table 3: Results from precision and ruggedness Parameters MET ALA Precision (%RSD) Intra-day (n=3) Inter-day (n=3) Repeatability (n=6) Ruggedness (%RSD) Analyst 1 (n=3) Analyst 2 (n=3) RESULTS AND DISCUSSION The proposed two wavelengths 236 nm (λ max for MET) and 215nm (λ max for ALA) were selected for analysis of the bulk drugs in methanol. The Beers-lamberts law concentration range is 1-9µg/ml for both drugs with coefficient correlation 0.99 and --- respectively. % estimation of MET and ALA 99.8% and 98% respectively by using simultaneous equation method with standard deviation of <2. The results did not show any statistical difference between analysts suggesting that methods developed were rugged. The results of precision and ruggedness are shown in table no: 3 and statistical data proves validity of the method compares as per ICH guidelines. CONCLUSION Thus the proposed method for the two wavelength estimation of metformin hydrochloride and alpha lipoic acid in bulk dosage forms to be rapid, sensitive, simple, accurate And economical, high percentage of recovery shows that the method is free from the interference of excipients therefore 106
6 the method can be useful in routine quality control of these drugs in future analysis. ACKNOWLEDGEMENT The authors express their gratitude to Life science Pharma Pvt ltd, Pondicherry. (India) for the gift sample of Pure Metformin Hydrochloride and Alpha lipoic acid. Thanks also extended to Principal & management, Nalanda College of pharmacy, Nalgonda, for providing necessary facilities and constant support REFERENCES 1. Indian pharmacopeia ministry of health and family welfare, government of India, Newdelhi vol 1&3, 2007 p- 154, K.D.Tripathi, Essentials of medical pharmacology, 2008, 6th edition, p safaa M Raid, Mamdoun R REZK, G hada Y Mahmoud and Abdel-Aziz, EI. Bayoumi Abdel Aleem, International Journal of comprehensive Pharmacy, 2012, 5, (01). 4. P.Umapathi, J.Ayyappan, S.Darlin Quine, Trop J pharm Res, 11 (1); 107, D Nagavalli, S. Vijayashanthi, S. Jagan, R. Lakshmisundram, International research Journal of pharmacy, 2011, 2 (12), P.C Bhamare, Dr S.B Bari, Dr S.Natarajan, A.A. Patil, S.H Patil, P.T. Shirode, A journal of biochemical and pharmaceutical research issue 2 (vol1) Kaushelendra Mishra, Himesh soni, Govind nayak, Sita sharan Patel and A.K Singhai, E- Journal of chemistry, 8(3), , Madhukar. A, A. Prince, Vijay Kumar, Sangeeva.Y, Jagadeeshwar.K, D. Raghuprathap, International Journal of Pharmacy and Pharmaceutical science, vol 3, issue 3, Ayyappan.J, Umapathi.P, Darlin Quine.S, International journal of pharmacy and pharmaceutical sciences, vol 3, suppl 5, Mubeen. G, Khalikha noor and Vimala MN, International Journal of chemtech research, vol 2, issue , P.N. Dhabala, C.R Seervi, International journal of chem. Tech research, vol 2, issue , S. Poongothai, R. Ilavarasan, C.M. Karunakaran, International Journal of Pharmacy and Pharmaceutical sciences, Issn , vol 2, ICH, Q2 (R1), Harmonized tripartite guideline, Validation of analytical procedures: text and methodology, International Conference on Harmonization ICH, Geneva, Nov 2005 ***** 107
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