Chromatographic Profiling of Encorafenib Impurities Using HPLC and LC-MS/MS Structural Characterization and In Silico Toxicity Evaluation of Degradation Products

This study systematically evaluated the impurities of Encorafenib along with structural elucidation, and in silico toxicological potential of its degradation products (DPs). The HPLC resolution of Encorafenib and its impurities was achieved on a Waters Symmetry C18 column using an isocratic mobile phase of methanol and 0.1 M ammonium acetate (pH 4.1, 60:40 v/v) at a flow rate of 0.85 mL/min with detection at 231 nm. The method effectively resolves Encorafenib and its three known impurities (Rs > 2) with acceptable ICH validation criteria such as linearity ( r ² > 0.999), sensitivity (LOD: 0.075 µg/mL), precision (RSD < 2%), robustness, and specificity. The base hydrolysis induces 16.31% degradation of Encorafenib with the formation of three new DPs, whereas acid stress produces 8.28% degradation. The LC-MS/MS analysis with fragmentation pattern was utilized for structural elucidation of major DPs. The in silico toxicity assessment proves that acid DP 2 and base DPs 3 and 4 display moderate toxicity (Class 4; LD 50 : 1000–1300 mg/kg), whereas base DP 1 shows high toxicity (Class 3; LD 50 : 155 mg/kg). The ADMET prediction using the pkCSM tool indicates favorable absorption characteristics and absence of predicted mutagenicity. Hepatotoxicity was predicted for all DPs; DP3 shows hERG II inhibition, and DP4 displays a broad toxicity profile. The developed method and comprehensive characterization of DPs provide a solid foundation for routine analysis and stability studies of Encorafenib.