http://www.rasayanjournal.com

Vol.3, No.1 (2010), 94-99

ISSN: 0974-1496 CODEN: RJCABP

LAMIVUDINE, ZIDOVUDINE AND NEVIRAPINE D. Anantha Kumar et al.

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

*1Pharmaceutical Analysis and Quality assurance Division, A.U.College of Pharmaceutical Sciences, Andhra University, Visakhapatnam-530 003, India.

2Organic 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 23±10 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 µg/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 non-nucleoside 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 (3′azido-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.

Vol.3, No.1 (2010), 94-99

LAMIVUDINE, ZIDOVUDINE AND NEVIRAPINE D. Anantha Kumar et al.

95

EXPERIMENTAL Chromatographic conditions A Jasco HPLC instrument equipped with a HiQ Sil C18 V (250mmX4.6mm; 5µ) analytical column, a PU 2080 pump, a Rheodyne 7725 sample injector with a 20 µl loop and a UV-2075 detector was employed for this analysis. Jasco’s 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 0.45µ 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 µg/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 µg/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 µg/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 µg/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 µg/ml. The contents of the flasks were mixed well and made up to the volume with the mobile phase. Each of these solutions (20 µL) 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.

Vol.3, No.1 (2010), 94-99

LAMIVUDINE, ZIDOVUDINE AND NEVIRAPINE D. Anantha Kumar et al.

96

RESULTS AND DISCUSSION The present study was aimed at developing a rapid, sensitive, precise and accurate HPLC method for the simultaneous determination of lamivudine, zidovudine and nevirapine in pharmaceutical dosage forms. In order to effect analysis of the component peaks under isocratic conditions, mixtures of acetonitrile and potassium dihydrogen ortho-phosphate buffer in different combinations were tested as mobile phase on a Hi Q Sil C18 stationary phase. A binary mixture of acetonitrile and 0.015 M potassium dihydrogen ortho-phosphate (pH 5.0) in a ratio of 55:45 v/v was proved to be the most suitable of all the combinations since the chromatographic peaks obtained were better defined and resolved and almost free from tailing. A flow rate of 1.0 ml/min of the mobile phase was found to be suitable in the study range of 0.8 -1.2 ml/min. The retention times for lamivudine, zidovudine, nevirapine and the internal standard carbamazepine were found to be 2.45, 2.93 3.72 and 4.76 respectively. A Typical chromatogram showing the separation is given in Fig.1. The method was validated by determining the specificity, linearity, precision and accuracy, LOD, LOQ, robustness, ruggedness and system suitability as per ICH guidelines33. The chromatograms recorded for the drug matrix (mixture of drug and excipients) showed almost no excipient peaks within the retention time range of 10 min. All the three drugs were suitably resolved from internal standard as shown in Fig-1.This indicates that, the method is specific and selective for their simultaneous determination. Six point calibration curves constructed with the working standard dilutions (n=3) were found to be linear (r2>0.9991) for each of the analytes in the concentration range of 0.5-10 µg/ml. The results are presented in Table-1. Both precision and accuracy were determined with standard quality control samples prepared in triplicates at different concentration levels covering the linearity range. The repeatability and intermediate precision are reported as % RSD in Table-2 and the minimum variation in the % RSD indicates that the present method is precise. The accuracy of the proposed method was assessed by adding known amount of the drug to a drug solution of known concentration and subjecting the samples to the proposed HPLC method. All solutions were prepared and analysed in triplicate. The above procedure is adopted for all the three drugs and a high recovery values obtained (Table-3) indicate that the proposed method is highly accurate. All the three drugs were suitably recovered from the tablet dosage forms by the proposed method and the results are shown in Table-4. The method specificity was assessed by studying the chromatograms obtained for a mixture of the drugs and the common excipients. As none of the excipients interfered with the analytes of interest, the method was found to be suitable for analysing the commercial formulation of these antiretroviraldrugs. System suitability parameters were studied with six replicates of working standard solution (containing 10 µg of each drug) and the values are presented in Table-5.

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, Physician’s Desk Reference, Edn.56, Medical Economics Company Inc,

Montvale (2003).

Vol.3, No.1 (2010), 94-99

LAMIVUDINE, ZIDOVUDINE AND NEVIRAPINE D. Anantha Kumar et al.

97

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. Savaşer, S. Goraler, A. Taşoz, 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 Lamivudine Zidovudine Nevirapine

Range (µg/ml) 0.5-10 0.5-10 0.5-10

Vol.3, No.1 (2010), 94-99

LAMIVUDINE, ZIDOVUDINE AND NEVIRAPINE D. Anantha Kumar et al.

98

Regression equation y= 0.2469X-0.0397 y= 0.2552X-0.0349 y= 0.1745X-0.0016

Correlation coefficient(r2) 0.9994 0.9995 0.9991

Table-2: Precision of the proposed method

Measured Conc.±S.D.(n=3) µg/ml % C.V.

Drug Concentration taken µg/ml

Intra day Inter day Intra day Inter day

1 0.94±0.76 0.91±1.21 0.81 1.25

4 4.02±0.62 3.95±0.68 0.68 0.71 Lamivudine

8 7.92±0.86 7.95±0.94 0.85 0.99

1 0.98±0.74 0.97±0.88 0.82 0.86

4 4.11±1.86 4.15±1.41 1.58 1.41 Zidovudine

8 8.02±0.99 7.97±1.36 0.99 1.51

1 1.09±0.61 1.01±1.91 0.55 1.88

4 3.99±0.94 3.99±1.13 0.93 1.14 Nevirapine

8 7.99±1.06 7.96±1.10 1.04 1.08

Table-3: Recoveries of drugs by the proposed HPLC method

Drug Concentration µg/ml Measured conc.±S.D.(n=3) µg/ml % C.V.

0.75 0.73 ± 1.01 1.03

2.25 2.25 ± 0.25 0.25 Lamivudine

4.5 4.43 ± 0.86 0.87

1.5 1.46 ± 0.66 0.75

4.5 4.36 ± 1.47 1.62 Zidovudine

9 8.9 ± 0.80 0.92

1 0.91 ± 1.73 1.89

3 2.95 ± 1.51 1.53 Nevirapine

6 5.88 ± 0.29 0.32

Vol.3, No.1 (2010), 94-99

LAMIVUDINE, ZIDOVUDINE AND NEVIRAPINE D. Anantha Kumar et al.

99

Table-4: Recoveries of lamivudine, zidovudine and Nevirapine in commercial formulation by proposed HPLC method

Table-5: System suitability parameters of lamivudine, zidovudine and Nevirapine

Fig.-1: Typical Chromatogram for the separation of Lamivudine, Zidovudine and Nevirapine with internal standard

Carbamazepine (Received: 7 November 2009 Accepted: 27 November 2009 RJC-482)

Parameter

Lamivudine

Zidovudine

Nevirapine

Relative retention time 0 1.17 1.07 Capacity factor 2.28 2.79 3.6

Theoretical plates 4384 4020 4411 HETP 57 X 10-6 62 X 10-6 56 X 10-6

Tailing Factor 0.94 1.06 1.40

Formulation Duovir-N Labeled amount (mg) Mean±S.D(amount recovered)

(n=3) Mean ± S.D

(% recovered)

Lamivudine Zidovudine Nevirapine

150 300 200

148.6±0.11 298.6±0.03 199.3±0.01

99.06±0.34 99.50±0.64 99.70±0.33