The present study describes the development and validation of a simple, rapid, precise, and economical reversed-phase high-performance liquid chromatographic (RP-HPLC) method for simultaneous estimation of dolutegravir and lamivudine in bulk and pharmaceutical dosage forms. The chromatographic separation was achieved on Waters XBridge C18 column (250 mm × 4.6 mm, 5 μm) using isocratic elution with mobile phase consisting of acetonitrile and 10 mM phosphate buffer pH 4.5 (40:60 v/v) at a flow rate of 1.0 mL/min. Detection was performed at 254 nm with column temperature maintained at 30°C. The retention times were 3.18 minutes for lamivudine and 6.82 minutes for dolutegravir with excellent resolution (9.18). The method was validated according to ICH Q2(R1) guidelines demonstrating excellent linearity (r = 0.9999), accuracy (recovery 99.89-99.94%), precision (RSD <0.2%), sensitivity (LOQ: 0.25 μg/mL for lamivudine, 0.15 μg/mL for dolutegravir), and robustness. Comprehensive forced degradation studies established the stability-indicating capability of the method, with successful separation of degradation products formed under acidic, basic, oxidative, thermal, humidity, and photolytic stress conditions. The validated method was successfully applied for assay of commercial formulations, demonstrating its suitability for routine quality control applications.

Dolutegravir, Lamivudine, RP-HPLC, Method Validation, Stability-Indicating, Simultaneous Estimation