Development and Validation of a Gradient RP-HPLC Method for Simultaneous Estimation of Lamivudine, Tenofovir Disoproxil Fumarate, and Dolutegravir in Combined Pharmaceutical Formulation

Research Article

Abstract

A novel gradient reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for simultaneous estimation of three antiretroviral drugs - Lamivudine, Tenofovir Disoproxil Fumarate, and Dolutegravir in combined tablet formulation. The chromatographic separation was achieved on an Inertsil C18 column (150 mm × 4.6 mm, 5 μm) using a gradient mobile phase consisting of phosphate buffer (pH 3.5) and acetonitrile:methanol (50:50 v/v). The flow rate was maintained at 1.6 mL/min with UV detection at 260 nm and column temperature at 30°C. The retention times for Lamivudine, Tenofovir Disoproxil Fumarate, and Dolutegravir were 2.881, 7.813, and 8.633 minutes respectively. The method demonstrated linearity over concentration ranges of 75-225 μg/mL for Lamivudine and Tenofovir Disoproxil Fumarate, and 12-39 μg/mL for Dolutegravir with correlation coefficients >0.999. Method validation parameters including accuracy, precision, specificity, robustness, and system suitability met ICH guidelines. The limits of detection were 0.1, 0.1, and 0.18 μg/mL, while limits of quantification were 0.3, 0.3, and 0.55 μg/mL for Lamivudine, Tenofovir Disoproxil Fumarate, and Dolutegravir respectively. Recovery studies yielded mean recoveries of 100.30%, 100.17%, and 99.60% for the three drugs. The validated method was successfully applied for routine quality control analysis of pharmaceutical formulations containing these drugs in combination.