Method development and validation of reverse phase high-performance liquid chromatography for Teriflunomide with comprehensive forced degradation analysis and impurity profiling using high resolution mass spectroscopy

The current project describes the development and validation of a simple, precise RP-HPLC method for Teriflunomide (TER), accompanied by forced degradation studies and impurity profiling using HRMS. The separation was achieved on a Dionex HPLC system with a PDA detector and an Altima C18 column (250 × 4.6 mm; 5 µm), using a mobile phase of Water:Acetonitrile (60:40 v/v). Method Validation followed ICH guidelines and demonstrated excellent linearity (R2 = 0.999), precision with %RSD < 0.3%, accuracy with mean recovery between 102.88 and 103.00%, robustness under varied chromatographic conditions, and specificity with no interference from excipients or degradation products at the TER retention time. TER was exposed to forced degradation under certain conditions according to the ICH guidelines Q1A (R2). The degraded samples were analyzed by HRMS, which identified eight impurities (Impurity A–Impurity H), including a known genotoxic impurity, benzimidamide. The developed method was accurate, precise, and robust for routine analysis of TER and profiling its impurities.