A new sensitive and stability indicating reverse phase high-performance liquid chromatography method has been developed for the quantitative determination of potential impurities of Ledipasvir, active pharmaceutical ingredient. Efficient chromatographic separation was achieved on waters × select column contains C18 stationary phase in gradient mode, quantitation at wavelength of 325 nm and mobile phase A is 50 mM ammonium formate pH 3.5 and mobile phase B is acetonitrile with flow rate 1.2 mL. The method was validated according to International Council for Hormonisation guideline, linearity studies has shown correlation coefficient value greater than 0.999 for Ledipasvir and all known impurities. Limit of detection and Limit of quantification were determined to be 0.01% and 0.03%, respectively, for all the impurities. The consistent recoveries were obtained for all impurities (95-105%). Various stress conditions were applied to study the degradation behavior of the drug substance. LC-MS was used to analyse the degraded samples and possible structural identifications were assigned based upon known reactivity of the drug. The method was found to be specific, selective and robust to the degradation products. Major degradation of the drug substance was found to occur under photolytic stress conditions resulting a novel impurity (impurity F). The formed degradant was identified and characterized by FT-IR, 1HNMR and LC-MS/MS. The stress samples quantified against qualified working standard and the mass balance found above 98%.

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