DETERMINATION OF LAMIVUDINE AND TENOFOVIR IN PHARMACEUTICAL DOSAGE FORM BY SIMULTANEOUS AND Q- ANALYSIS UV-SPECTROPHOTOMETRIC METHOD

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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Imran et al. SJIF Impact Factor 2.786 Volume 4, Issue 03, 688-696.

1 WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Imran et al. SJIF Impact Factor Volume 4, Issue 03, Research Article ISSN DETERMINATION OF LAMIVUDINE AND TENOFOVIR IN PHARMACEUTICAL DOSAGE FORM BY SIMULTANEOUS AND Q- ANALYSIS UV-SPECTROPHOTOMETRIC METHOD Mohammed Imran Anees* and Mirza Shahed Baig Dept. of Pharmaceutical Chemistry, Y.B. Chavan College of Pharmacy. Dr. Rafiq Zakaria Campus, Rauza Bagh, Aurangabad , Maharashtra, India. Article Received on 12 Dec 2014, Revised on 05 Jan 2015 Accepted on 30 Jan 2015 *Correspondence for Author Mohammed Imran Anees Dept. of Pharmaceutical Chemistry, Y.B. Chavan College of Pharmacy. Dr. Rafiq Zakaria Campus, Rauza Bagh, Aurangabad , Maharashtra, India ABSTRACT Two simple, accurate, precise, methods were developed for the estimation of Tenofovir and Lamivudine in Tablet dosage form. Both the drugs are used against the HIV infection as reverse transcriptase inhibitors. Method A is Simultaneous equation method, wavelength selected for Quantitation are nm and nm for Tenofovir (TEN) and Lamivudine (LAM) respectively which are the λ max of both the drugs. Method B is Q Analysis method, wavelength selected were nm (λ max of TEN) and nm (Isobastic point) for the analysis. In both the methods linearity for detector response was observed in the concentration range of microgram/ml for TEN and LAM respectively. The results of bulk drug analysis for Simultaneous equation method was found to be 99.37% S.D for LAM and 99.94% S.D for TEN and results obtained for Q Analysis method was ± SD for LAM and ± SD for TEN. The proposed methods were successfully applied for the Simultaneous determination of both the drugs in commercial tablet preparation. The results of the analysis have been validated statistically and by recovery studies according to ICH guidelines. KEYWORDS: Tenofovir, Lamivudine, Simultaneous Equation method and Q-Analysis method, UV Spectrophotometric method. Vol 4, Issue 03,

2 INTRODUCTION Lamivudine Chemically it is (2R, 5S)-4-Amino1 [2-(Hydroxy methyl)-1, 3-oxathiolan-5yl]- 2(1H)-Pyrimidinedione. It is used in HIV infection. The principal mode of action of 3TC-TP inhibition of RT via DNA chain termination after incorporation of the nucleotide analogue. LAMIVUDINE Tenofovir is 1-(6-aminopurin-9-yl) propan-2-yl-oxymethyl phosphonic acid. It is nucleotide analogue Reverse Transcriptase inhibitor (NRTI). TENOFOVIR DISPROXIL FUMARATE Both Tenofovir and Lamivudine are official in IP and are marketed as combined tablet dosage formulation in the Ratio is 300:300 mg LAM: TEN and were named as TENVIR-L as brand name which was produced by Cipla Ltd. Literature survey revealed that a number of methods have been reported for estimation of both the drugs individually and in combination of other drugs, but not a single method is reported for the estimation of both the drugs simultaneously. Present work describes two precise, accurate, and reproducible methods for simultaneous estimation of TEN and LAM in tablet formulation. MATERIAL AND METHODS Instruments: UV-VISIBLE SPECTROPHOTOMETER (DOUBLE BEAM) Make: Jasco Model: UV V-630 Spectrophotometer Spectral Bandwidth: 2nm Vol 4, Issue 03,

3 Materials: Standard gift sample of Tenofovir and Lamivudine were provided by Cipla Ltd and Combined dose Tablet (Tenvir-L: 300 mg Lamivudine and 300 mg Tenofovir; manufactured by Cipla Pvt. Ltd.), were purchased from local market for analysis. Solvent used: Double distilled water used as solvent. Stock solution: Stock solution of both the drugs 300mcg/ml is prepared by dissolving 30mg each drug in 100ml volumetric flask and the volume is make up by Double distilled water. Method A - Simultaneous equation method In this method, the stock solution of both the drugs 100mcg/ml is prepared by dissolving 30mg each drug in 100ml volumetric flask and the volume is makeup by Double distilled water, by appropriate dilution of standard stock solutions of both the drugs to 30mcg/ml dilution respectively is scanned in the spectrum mode from 400 nm to 200 nm. The absorption spectra thus obtained is selected for analysis, from the overlain spectra of both the drugs (fig.1), wavelength selected for Quantitation are nm and nm for LAM and TEN respectively which are the λ max of both the drugs. The calibration curves for TEN and LAM was plotted in the concentration range of mcg/ml exhibiting the Beer s and Lamberts range (table 1, 2). The concentration of individual drug present in the mixture was determined by using the simultaneous equation calculations. C LAM = A 271 x α (TEN) 260 A 260 x α (TEN) 271 α( TEN) 260 x α(lam) 271 α(lam) 271 x α(ten) 260 A 260 x α(ten) 271 A 271 x α(ten) 260 C TEN = α(lam) 271 x α(ten) α( TEN) 260 x α(lam) 271 Fig 1: Overlain Spectra of LAM and TEN Vol 4, Issue 03,

4 Absorbance Imran et al. Fig 2: Calibration Curve of LAM Table 1: Calibration Curve Table of LAM Conc. In mcg/ml Absorbance Calibration curve 1.6 y = x R 2 = Concentration Fig 3: Calibration Curve of TEN Table 2: Calibration Curve Table of TEN Conc. in mcg/ml Absorbance Vol 4, Issue 03,

5 Table no 3: Statistical Parameters of LAM and TEN in Tablet Table no 4: Recovery Studies of LAM and TEN in Tablet Level of % Recovery Amount present (mg/tab) Amount of standard added (mg) Total amount recovered (mg) %Recovery LAM TEN LAM TEN LAM TEN LAM TEN Method B - Q Analysis method For the selection of Analytical wavelength, solution of TEN and LAM (30 mcg/ml, each) were prepared separately by appropriate dilution of standard stock solution and scanned in the spectrum mode from 400 nm to 200 nm. From overlain spectra of both the drugs (fig.4), wavelengths selected were nm and nm (Isobastic point) for the analysis. The Q values of both the drugs were determined at the selected wavelength. The Q value is the ratio of Absorbance of std.1 at nm to the Absorbance of std.2 at nm. Molar Absorptivities for both the drugs were calculated by Absorbance of std. at nm with the concentration in gm/lit. A set of two simultaneous equations obtained by using Q values are given below. Component Mean Standard Co-efficient of Standard Deviation Variation Error LAM % TEN % C lam = Q 0 Q ten / Q lam Q ten lam (1) X A / a C Ten = Q 0 Q lam / Q ten Q lam ten (2) X A / a C lam, C ten were concentration of LAM and TEN, respectively. The concentration of LAM and TEN in sample was determined by using the equation (1) and (2). Fig no 4: Overlain Spectra of LAM & TEN showing Isobastic Point (Q method) Vol 4, Issue 03,

6 The Q value and Molar absorptivities for both drugs were calculated as follows: Absorbance of Std.1 at 260 nm Q LAM = Absorbance of Std.1 at 269 nm Absorbance of Std.2 at 260 nm Q TEN = Absorbance of Std.2 at 269 nm Absorbance of Std.1 at 269 nm A LAM = Concentration of Std.1 in gms/lit Absorbance of Std.2 at 269 nm A TEN = Concentration of Std.2 in gms/lit Table No 7: Analysis of LAM and TEN in Tablet. n=5 Component Label Claim (mg/tablet) Drug Content (%) ± SD ± SEM LAM ± TEN ± Table No 8: Accuracy Data of LAM and TEN in Tablet Level of % Recovery Amount present (mg/tab) Amount of standard added (mg) Total amount recovered (mg) %Recovery LAM TEN LAM TEN LAM TEN LAM TEN Table no 9: Validation Parameters of LAM and TEN Method characteristics Lamivudine Tenofovir Linearity 5-60 µg/ml 5-60 µg/ml Regressions equation y = y = Correlation coefficient LOD(µg/mL) LOQ(µg/mL) Vol 4, Issue 03,

7 Precision (RSD, %) Intraday (n=3) Interday (n=3) Specificity Specific Specific RESULTS AND DISCUSSION The methods discussed in the present work provide a convenient and accurate way for simultaneous analysis of LAM and TEN. In simultaneous equation method wavelength selected for Quantitation were nm for LAM and nm for TEN. In Q-Analysis method the wavelength selected were nm and nm (Isobastic point). In both the methods linearity for detector response was observed in the concentration range of mcg/ml for LAM and TEN, both. In method A, concentration of individual drug present in the mixture was determined against calibration curve in Quantitation mode. In method B, Q values were calculated for both the drugs at selected wavelengths and substituted in equations for determining the concentration of LAM and TEN in tablet sample solution. Percent label claim for LAM in tablet analysis by both the methods was found in the range of ± for LAM and ± for TEN. Standard deviation and coefficient of variance for five determination of tablet sample, by both the methods was found to be less than indicating precision of both the methods. Accuracy of both the methods was ascertained by recovery studies and the results are expressed as % recovery. Percent recovery was found in the range of for LAM and for TEN, values of standard deviation and coefficient of variation was satisfactorily low indicating the accuracy of both the methods. LOD of LAM was and for LAM and TEN and LOQ was and 1.56 for LAM and TEN respectively. The result of analysis shows that the developed methods are accurate, precise, reproducible and economical and can be employed for routine quality control analysis of Lamivudine and Tenofovir in combined dose tablet formulation. ACKNOWLEDGEMENTS The authors are very much thankful to the Chairman, Mrs. Fatma Rafiq Zakaria, Maulana Azad Educational Trust, Dr Rafiq Zakaria Campus for providing necessary facilities for the project work. The authors are also thankful to Cipla Pvt. Ltd, for providing gift samples of Lamivudine and Tenofovir. Vol 4, Issue 03,

8 REFERENCES 1. Instruction Manual User s System Guide UV-630 Jasco Spectrophotometer (P/N: D). JASCO Corporation, Analytical instrument division, Tokyo, Japan. 2. Indian Pharmacopoeia. Govt. of India, health ministry & family welfare, published by IP commission Ghaziabad, 2007; 2: pp 673, Beckett AH, Stenlake JB. Practical Pharmaceutical chemistry. CBS Publishers, New Delhi. 4 th edition: pp Skoog DA, Holler J, Crouch R.Instrumental analysis.cengage learning India (P) Ltd, New Delhi, 2007; Skoog D.R., Hollar F.J.,Nieman 2005 T.A. Principles of Instrumental Analysis. 5th edition. Thomson Brooks/Cole; 1-3,300, Jeffery, G.H., Bassat, J. Mendham, J. Denny, R.C. 1989: Vogel s Textbook of Qualitative Chemical Analysis. 5thedition. Atkin. ELB Swithlongman Publication. Harlow, B. K. Sharma. Instrumental methods of chemical analysis. 2000, Introduction to analytical chemistry. 19th edition. Goel publishing house; , U.S. Pharmacopoeia. United States Pharmacopoeial Convention, Inc, (1994); International Conference on Harmonization. Draft Guideline on Validation of 10. Analytical Procedures, 1995: Definitions and Terminology. Federal Register. 60: Reviewer Guidance, Validation of Chromatographic Methods 1994, Center for Drug Evaluation and Research, Food and Drug Administration Guideline for Submitting Samples and Analytical Data for Methods Validation, Food and Drug Administration. 13. Bekett A.H., Stenlake J.B. Practicle Pharmaceutical chemistry.2004, 4th edition. CBS Publishers New Delhi; Part-II: Kemp W. Organic Spectroscopy. 2nd edition. ELBS publication (1989); Chatwal and Anand. Instrumental methods of chemical analysis. 2007, 5th edition. Himalaya Publishing House, 2; 149-2: Sharma B.K., in: Spectroscopy. 6thedition.Goel Publication Co. Meerut, (1983); Tony Owen. Fundamentals of modern UV-visible spectroscopy Agilent technologies Kasture A.V, Wadodkar S.G., Mahadik K.R., More H.N. 1998, Pharmaceutical Analysis (Instrumental Methods). Nirali Prakashan. Pune, 2: Vol 4, Issue 03,

9 19. Howell J.A. Handbook of Instrumental techniques for Analytical Chemistry. 1st edition (2004); British pharmacopeia, 6th edition, 2010; Vol II, Vol III, London: Her Majesty s Stationary Office The Merck Index An encyclopedia of chemicals, drugs and biologicals Merck and Co. Inc., whitehouse station, NJ, USA,14th edition, 2006, British pharmacopeia, 6 th edition, 2010; Vol I, Vol III, London: Her Majesty s Stationary Office Vol 4, Issue 03,

Determination of Lamivudine and Tenofovir in Pharmaceutical dosage form by Area under Curve and Multicomponant UV Spectrophotometric method

ISSN: 2319-7706 Volume 4 Number 2 (2015) pp. 756-763 http://www.ijcmas.com Original Research Article Determination of Lamivudine and Tenofovir in Pharmaceutical dosage form by Area under Curve and Multicomponant