STABILITY INDICATING HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF EMTRICITABINE, DOLUTEGRAVIR AND TENOFOVIR ALAFENAMIDE IN THEIR COMBINED PHARMACEUTICAL DOSAGE FORM

Abstract

A simple, rapid, precise and accurate reversed phase HPLC stability indicating method was developed for simultaneous determination of emtricitabine (ECT), dolutegravir (DOL) and tenofovir alafenamide (TEN) in their combined dosage marketed formulation. The chromatographic separation was achieved on Hypersil BDS C18 (250  4.6 mm, 5.0 µ) column using 0.5 M potassium dihydrogen phosphate buffer: acetonitrile (80: 20, v/v), pH 3.5 ± 0.1 maintained by 0.1 % o-phosphoric acid as mobile phase at a flow rate of 1.0 mL/min and monitored at 260 nm. All three analytes were resolved at retention times of 5.777, 6.783, 8.393 min for ECT, DOL, and TEN respectively. The assay performance of the present developed method was validated for various parameters like system suitability, linearity, accuracy, precision, robustness, limit of detection and quantification. The calibration curves were linear over concentration ranges of 12.5-75.0, 50.0-300, and 6.25-37.5 μg/ml for DOL, ECT, and TEN, respectively with correlation coefficients  0.9995. All three analytes were subjected to various stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found in alkaline condition. The present method demonstrated to be effective stability-indicating by resolution of the analytes from their forced-degradation products. The validated method was successfully applied for the simultaneous estimation three drugs in their combined pharmaceutical dosage form Spegra.