A rapid, simple, and robust reverse-phase HPLC method was developed and validated for the quantification of related substances in dolutegravir 10 mg dispersible tablets. This study primarily aimed to establish a new RP-HPLC method to quantify impurity B (a degradation impurity) as a related substance, following USP guidelines. Chromatographic separation was performed using a phenyl-hexyl column (250 × 4.6 mm, 5µ) with a mobile phase consisting of 45% buffer (sodium dihydrogen phosphate dihydrate and EDTA), 49% methanol, and 6% acetonitrile, adjusted to a pH of 2.5 ± 0.05 with orthophosphoric acid. The method used isocratic elution at a flow rate of 1.2 mL/min and maintained a column temperature of 35 °C, with detection at 258 nm using a PDA detector. The method was evaluated for stability, specificity, ruggedness, precision, linearity, accuracy, and robustness, according to USP standards, and showing high resolution and short retention times. All system suitability and validation parameters met the required criteria. The method showed excellent sensitivity, with LOD and LOQ values confirming its capability. Linearity was confirmed for both dolutegravir and impurity B with a correlation coefficient greater than 0.997. The recovery of the impurity was consistent and ranged from 80 to 120%. Overall, the method was found to be accurate and reliable for the determination of related substances in dolutegravir 10 mg dispersible tablets.
