Escolar Documentos
Profissional Documentos
Cultura Documentos
com
SIMULTANEOUS DETERMINATION OF LAMIVUDINE, ZIDOVUDINE AND NEVIRAPINE IN TABLET DOSAGE FORMS BY RP-HPLC METHOD
D. Anantha Kumar1, M.V. Naveen Babu1, J.V.L.N. Seshagiri Rao*1 and V. Jayathirtha Rao2
*1
Pharmaceutical Analysis and Quality assurance Division, A.U.College of Pharmaceutical Sciences, Andhra University, Visakhapatnam-530 003, India. 2 Organic Division-II, Indian Institute of Chemical Technology, Hyderabad-500 007, India. Email: jvlns@yahoo.com
ABSTRACT A rapid, accurate and reproducible RP-HPLC method has been developed for the simultaneous determination of lamivudine, zidovudine and nevirapine in tablet dosage forms. The separation was effected on a HiQ Sil C18V column using a mobile phase consisting of 0.015 M potassium dihydrogen orthophosphate (pH 5.0) and acetonitrile (45:55 v/v) at a flow rate of 1 ml/min. The detection was carried out at 270 nm and a column temperature of 2310 C was maintained throughout the study. Carbamazipine was used as the internal standard for this study. The retention times for lamivudine, zidovudine and nevirapine were found to be 2.45, 2.93 and 3.72 min. respectively. The calibration curves were linear in the concentration range of 0.5-10 g/ml for all the three drugs. The proposed method was validated as per USP guidelines and the method was found to be suitable for the routine quality control analysis of the drugs in tablet dosage forms. Key words: Lamivudine, Zidovudine, Nevirapine, RP- HPLC, Tablets
INTRODUCTION
The fixed dose combinations of nucleoside reverse transcriptase inhibitors, protease inhibitors and nonnucleoside reverse transcriptase inhibitors are effective in the first line therapy of HIV infection by acting at various stages of viral replication. Nucleoside reverse transcriptase inhibitors like lamivudine ((-) 2,3- dideoxy, 3'- thiacytidine) and zidovudine (3azido-3-deoxythymidine) act at an early stage in the HIV life cycle by blocking the action of reverse transcriptase, which is essential for the conversion of viral RNA to proviral DNA and subsequent viral replication. Both lamivudine and zidovudine are phosphorylated intracellularly to their active triphosphate metabolites. These metabolites cause inhibition of reverse transcriptase via DNA chain termination after incorporation of nucleoside analogue. Nevirapine (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido [3,2-b:2',3'-e][1,4] diazepin-6-one ), a nucleoside reverse transcriptase inhibitor of HIV-1 blocks both the RNA dependent and DNA dependent DNA polymerase activity. All the three drugs are official in I.P1 and are indexed in other sources 2,3 also.
A literature survey reveals the report of analytical methods4-31 for the determination of these drugs individually and in combination with one another in biological samples and in their dosage forms based on HPLC, HPTLC and LC-MS/MS. Only one HPLC method has been reported for the simultaneous determination lamivudine, zidovudine and nevirapine in plasma samples32. Hence, the authors have attempted to develop a rapid, precise and accurate method for the estimation of these drugs in dosage forms. The proposed method employs a mobile phase consisting of 0.015 M potassium dihydrogen ortho-phosphate (PH 5.0) and acetonitrile which shows good peak shapes with appreciable resolution.
EXPERIMENTAL
Chromatographic conditions A Jasco HPLC instrument equipped with a HiQ Sil C18 V (250mmX4.6mm; 5) analytical column, a PU 2080 pump, a Rheodyne 7725 sample injector with a 20 l loop and a UV-2075 detector was employed for this analysis. Jascos Borwin software was used for the quantification of peaks. A Bandelin sonerex sonicator was used for enhancing dissolution of the compounds. A Digisun DI 707 digital pH meter was used for pH adjustments. Drug Samples and Solvents Reference standard samples of lamivudine, zidovudine and nevirapine were gifted by Well Quest Laboratories, Hyderabad and the internal standard carbamazepine by MSN Laboratories, Hyderabad. A commercial sample of Duovir-N tablets (Cipla, India) containing lamivudine (150mg), zidovudine (300mg) and nevirapine (200mg) were purchased from local market. HPLC grade acetonitrile and methanol and AR grade potassium dihydrogen ortho-phosophate were purchased from Merck Ltd., India. Water used in this study was prepared by Millipore milli Q (Bedford, USA) water purification system. A mobile phase consisting of a mixture of 0.015 M potassium dihydrogen ortho-phosphate (pH 5.0) and acetonitrile in a ratio of 45:55 v/v was prepared and filtered through a 0.45 membrane filter and degassed by sonication before use. Drug and Internal Standard solutions Stock solutions of lamivudine, zidovudine and nevirapine were separately prepared by dissolving 25 mg of each drug with a small quantity of methanol in separate 25 ml volumetric flasks. The contents were sonicated for about 15 min and then made up to the volume with methanol. Working standard solutions were prepared from the individual stock solutions in the concentration range of 0.5-10 g/ml with the mobile phase as diluent. Stock solution of carbamazepine (internal standard) was prepared by dissolving 25 mg of the drug in a 25 ml volumetric flask in methanol with sonication for about 15 min. From this, a working standard solution of 50 g/ml was prepared by diluting with the mobile phase. Calibration curve Separate calibration curves were constructed for the three drugs by the following method. Six different dilutions each consisting of a definite concentration of each of the three drugs in the concentration range of 0.5-10 g/ml were prepared. The dilutions were made using the mobile phase. Suitable aliquot of the internal standard solution was added uniformly to all the dilutions to get a final concentration of 5 g/ml in each mixture sample. The calibration lines were constructed by plotting the peak area ratios of the analyte to internal standard against the concentration of the drug. The regression parameters of each drug are presented in Table 1. The results are highly reproducible with a correlation coefficient greater than 0.9991. Estimation of the drugs from tablet dosage forms Ten tablets of Duovir-N were weighed and finely powdered. A quantity of the powder equivalent to one tablet content was accurately weighed, transferred into a 100 ml volumetric flask containing methanol. The above mixture was sonicated for about 15 min. for complete mixing. This solution was filtered through Whatman No.1 filter paper. From the filtrate different aliquots were taken in separate 10 ml volumetric flasks. These solutions were spiked with a suitable volume of the internal standard solution uniformly, such that the concentration of the internal standard in each dilution was 5 g/ml. The contents of the flasks were mixed well and made up to the volume with the mobile phase. Each of these solutions (20 L) was then injected three times into the column. The mean peak area ratios of the drug to the internal standard of three such determinations for each drug were calculated and the drug contents in the tablets were quantified using the respective regression equations obtained for the reference samples.
95
CONCLUSION
The proposed RP-HPLC method is rapid, precise and accurate and can be used for the routine quality control analysis for simultaneous determination of lamivudine, zidovudine, and nevirapine in their tablet dosage forms.
ACKNOWLEDGEMENTS
The authors are thankful to M/s. Well Quest Laboratories, Hyderabad and MSN Laboratories, Hyderabad for providing gift samples of the drugs for the study. The authors are also thankful to IICT, Hyderabad for providing necessary laboratory facilities.
REFERENCES
1. Indian Pharmacopoeia 1996 (Addendum 2002). Govt. of India, Ministry of Health and Family Welfare, Controller of Publications, Delhi (2002). 2. S.C. Sweetman, Martindale, The Complete Drug Reference, Edn. 33, Pharmaceutical Press, London (2002). 3. N.J. Montvale, Physicians Desk Reference, Edn.56, Medical Economics Company Inc, Montvale (2003).
LAMIVUDINE, ZIDOVUDINE AND NEVIRAPINE
96
4. W.V. Caufield, J.T. Stewart, Chromatographia., 54, 561 (2001). 5. I. Schrive and J.C. Plasse, J. Chromatogr. B., 657,233 (1994). 6. R.P.G. Heeswijk, R.M.W. Hoetelmans, P.L. Meenhorst, J.W.Mulder and J.H. Beijsen, J. Chromatogr. B., 713, 395 (1998). 7. G. Bahrami, S. Mirzaeei, A. Kiani, B. Mohammadi, J. Chromatogr. B. Analyt. Technol. Biomed. Life. Sci., 823,213 (2005). 8. E.K. Kano, CH.D.R. Serra, E.E. Koono, S.S. Andrade, V. Porta, Int. J. Pharm., 297,73 (2005). 9. Y. Hassan, A. Mohammed and M. Hefnaun., Anal. Lett., 36, 2527 (2003). 10. A. Sibel, B. Ozkan, Uslu, J.Liq.Chrom.Rel.Tech., 25, 1447 (2002). 11. D.G. Sharnkar and D.V.S.P. Kumar, Asian. J. Chem., 17, 2575 (2005). 12. R.M. Lopez, L. Pou, M.R. Gomez, I. Ruiz and J. Monterde, J. Chromatogr. B Biomed. Sci. Appl, 751, 371 (2001). 13. QC. T. Tougas, K. Cohen, R. Lee, P. Meagan, M. Corson and T. Much nickz,J. Chromatogr. Sci., 38, 246 (2000). 14. J.W. Pav, L.S. Rowland, D.J. Korpalski, J. Pharm. Biomed. Anal., 20, 91 (1998). 15. P.G. Rolf, G.V. Heeswijka, M. Richard, W. Hoetelmansa, P.L. Meenhorstb, J.W. Mulderb and J.H. Beijnena, J. Chromatog. B., 713,395 (1998). 16. E. Marchei, L. Valvo, R. Pacifici, M. Pelligrini, G. Tossini and P. Zuccaro, J. Pharm. Biomed. Anal. 29, 1081 (2002). 17. R. Geetha, A.K. Hemanthkumar, V.K. Swami and S. Sowmya, J. Chromatogr. B., 843, 339 (2006). 18. K.B. Kenney, S.A. Wring, R.M. Carr, G.N. Wells and J.A. Dunn, J. Pharm. Biomed. Anal., 22, 967 (2000). 19. N. Erk, Pharmazie , 59,106 (2004). 20. R. Sekar, S. Azhaguvel, J. Pharm. Biomed. Anal., 39,653 (2005). 21. M.S. Palled, P.M.N. Rajesh, M. Chatter and A.R. Bhat, Indian J. Pharm. Sci., 67,110 (2005). 22. N.Kapoor, S. Khandavilli, R. Panchagnula, J. Pharm. Biomed. Anal., 41,761 (2006). 23. S.B. Wankhede, K.R. Gupta,S.G. Wadodkar, Indian J. Pharm. Sci., 67, 96 (2005). 24. N.Kapoor, S. Khandavilli, R. Panchagnula, Analytica. Chemica. Acta., 570, 41 (2006). 25. M. Sarkar, S. Khandavilli, R. Panchagnula, J. Chromatogr. B. Analyt. Technol. Biomed. Lif.e Sci., 830,349 (2006). 26. S.Anbazhagan, N. Indumathy, P. Shanmugapandiyan and S.K. Sridhar, J. Pharm. Biomed. Anal., 39,801 (2005). 27. A. Savaer, S. Goraler, A. Taoz, B. Uslu, H. Lingeman and S. A. Ozkan., Chromatographia., 65,259 (2007). 28. P. Djurdjevia,A. Larban, S. Markovic and M.J. Stankor, Analytical Letters., 37,2649 (2004). 29. N.L. Rezk, R. Rechard, Tidwell and A.D.M Kashuba, J. Chromatogr. B., 774, 79 (2002). 30. G. Aymard, M. Legrand, N. Trichereau and B. Diquet, J. Chromatogr. B, 744, 227 (2000). 31. C.P. Verweij-van Wissen, R.E. Aarnoutse and D.M. Burger, J. Chromatogr. B Analyt. Technol. Biomed. Life. Sci., 816,121 (2005). 32. B. Fan, J.T. Stewart, J. Pharm. Biomed. Anal., 28,903 (2002). 33. Validation of analytical procedures: Text and Methodology, I.C.H Hormonised Tripartite Guideline Q2 (R1), Commission of the European Communities (2005)
Table-1: Regression characteristics of the proposed HPLC method Parameter Range (g/ml)
LAMIVUDINE, ZIDOVUDINE AND NEVIRAPINE
Lamivudine 0.5-10 97
Zidovudine 0.5-10
Nevirapine 0.5-10
D. Anantha Kumar et al.
y= 0.2469X-0.0397 0.9994
y= 0.2552X-0.0349 0.9995
y= 0.1745X-0.0016 0.9991
Table-2: Precision of the proposed method Measured Conc.S.D.(n=3) g/ml Intra day 1 Lamivudine 4 8 1 Zidovudine 4 8 1 Nevirapine 4 8 0.940.76 4.020.62 7.920.86 0.980.74 4.111.86 8.020.99 1.090.61 3.990.94 7.991.06 Inter day 0.911.21 3.950.68 7.950.94 0.970.88 4.151.41 7.971.36 1.011.91 3.991.13 7.961.10 % C.V. Intra day 0.81 0.68 0.85 0.82 1.58 0.99 0.55 0.93 1.04 Inter day 1.25 0.71 0.99 0.86 1.41 1.51 1.88 1.14 1.08
Drug
Table-3: Recoveries of drugs by the proposed HPLC method Drug Concentration g/ml 0.75 Lamivudine 2.25 4.5 1.5 Zidovudine 4.5 9 1 Nevirapine 3 6 Measured conc.S.D.(n=3) g/ml 0.73 1.01 2.25 0.25 4.43 0.86 1.46 0.66 4.36 1.47 8.9 0.80 0.91 1.73 2.95 1.51 5.88 0.29 % C.V. 1.03 0.25 0.87 0.75 1.62 0.92 1.89 1.53 0.32
98
Table-4: Recoveries of lamivudine, zidovudine and Nevirapine in commercial formulation by proposed HPLC method Parameter Relative retention time Capacity factor Theoretical plates HETP Tailing Factor Lamivudine 0 2.28 4384 57 X 10-6 0.94 Zidovudine 1.17 2.79 4020 62 X 10-6 1.06 Nevirapine 1.07 3.6 4411 56 X 10-6 1.40
Table-5: System suitability parameters of lamivudine, zidovudine and Nevirapine Formulation Duovir-N Lamivudine Zidovudine Nevirapine Labeled amount (mg) 150 300 200 MeanS.D(amount recovered) (n=3) 148.60.11 298.60.03 199.30.01 Mean S.D (% recovered) 99.060.34 99.500.64 99.700.33
Fig.-1: Typical Chromatogram for the separation of Lamivudine, Zidovudine and Nevirapine with internal standard Carbamazepine
RJC-482)
99