Journal of Chromatography B, 830 (2006) 349–354
Development and validation of RP-HPLC and ultraviolet
spectrophotometric methods of analysis for the quantitative
estimation of antiretroviral drugs in pharmaceutical dosage forms
Mahua Sarkar, Sateesh Khandavilli, Ramesh Panchagnula∗
Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER),
Sector 67, S.A.S Nagar, Punjab 160 062, India
Received 18 March 2005; accepted 11 November 2005
Available online 5 December 2005
Abstract
A high-performance liquid chromatographic and an UV spectrophotometric method were developed and validated for the quantitative deter-
mination of three antiretroviral drugs viz. Lamivudine, Stavudine and Nevirapine that constitute one of the first line regimens in antiretroviral
therapy. The different analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD) and limit of
quantification (LOQ) were determined according to International Conference on Harmonization ICH Q2B guidelines. Chromatography was carried
out by isocratic technique on a reversed-phase C-18 SYMMETRY column with mobile phase based and optimized depending on the polarity of
the molecules. The UV spectrophotometric determinations were performed at 270, 265 and 313nm for Lamivudine, Stavudine and Nevirapine,
respectively. The linearity of the calibration curves for each analyte in the desired concentration range is good (r2> 0.999) by both the HPLC
and UV methods. Both the methods were accurate and precise with recoveries in the range of 97 and 103% for all the three drugs and relative
standard deviation (R.S.D.) <5%. Moreover, the accuracy and precision obtained with HPLC correlated well with the UV method which implied
that UV spectroscopy can be a cheap, reliable and less time consuming alternative for chromatographic analysis. The proposed methods are highly
sensitive, precise and accurate and hence were successfully applied for the reliable quantification of API content in the commercial formulations
of Lamivudine, Stavudine and Nevirapine.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Lamivudine; Stavudine; Nevirapine; UV spectrophotometry; RP-HPLC
1. Introduction
One of the deadliest and unmanageable chronic health
catastrophes is HIV/AIDS. It requires lifelong treatment with
potent life saving essential drugs that include nucleoside reverse
transcriptase inhibitors, non nucleoside reverse transcriptase
inhibitors and protease inhibitors. Amongst these Lamivu-
dine (3TC: 2,3-dideoxy-3-thiacytidine), Stavudine (d4T:
2,3-didehydro-3-doexythymidine) and Nevirapine (NVP)
constitute first line therapy. Combination of these three drugs
into fixed dose combinations (FDCs) has been an essential
∗Corresponding author at: School of Biomedical Sciences, University of
Ulster, Cromore Road, Coleraine BT52 1SA, UK. Tel.: +44 28 7032 4128;
fax: +44 28 20324965.
E-mail address: r.panchagnula@ulster.ac.uk (R. Panchagnula).
constituent of the Highly Active Anti-retroviral (HAART)
therapy. Lamivudine (Fig. 1a) is a nucleoside analog having
potent in vitro and in vivo inhibitory activity against HIV
reverse transcriptase. Lamivudine specifically refers to the
(−) enantiomer of the cis racemate and is marketed as tablets
in different strengths. Stavudine (Fig. 1b) is chemically a
thymidine nucleoside analogue. It has a complete and less
variable oral absorption as compared to other nucleoside
analogs.
Nevirapine (NVP) chemically 11-cyclopropyl-5,11-dihydro-
4-methyl-6H-dipyrido [3,2-b: 2,3-e] [1,4] diazepin-6-one is a
non-nucleoside inhibitor of DNA and RNA dependent DNA
polymerase (Fig. 1c). It is a known agent for the treatment of
infection by HIV-1 (human immunodeficiency virus, type 1),
which acts through non-competitive inhibition of HIV-1 reverse
transcriptase [1]. The three antiretroviral drugs are official only
in Indian Pharmacopoeia (IP, Addendum 2002).
1570-0232/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jchromb.2005.11.014
