LC–HRMS/MS Characterization of Hydrolytic and Photolytic Degradation Pathways of Ripretinib Following Forced Stress Studies

ABSTRACT Introduction Ripretinib is an innovative multitargeted kinase inhibitor introduced for the treatment of advanced gastrointestinal stromal tumors, was investigated to elucidate its degradation behavior and structural transformation pathways under forced stress conditions. Although degradation studies are essential for evaluating the intrinsic stability of pharmaceutical compounds, the degradation products of ripretinib have not been comprehensively characterized. The present work focuses on LC–HRMS/MS‐based structural elucidation of degradation products arising from photolytic and hydrolytic stress conditions. Methods In accordance with International Council for Harmonisation guidelines, ripretinib was subjected to acidic, alkaline, oxidative, thermal, and photolytic stress conditions. Liquid chromatography was employed to separate the degradation products, which were subsequently characterized by high‐resolution tandem mass spectrometry (LC–HRMS/MS) operated in positive electrospray ionization mode. Structural assignments were established using accurate mass measurements, isotopic pattern analysis, elemental composition determination, and collision‐induced dissociation pathways. Degradation kinetics under photolytic conditions were also evaluated. An in silico toxicity assessment of degradation products was performed using DEREK and SARAH prediction platforms. Results Ripretinib exhibited degradation predominantly under photolytic and hydrolytic stress conditions, leading to the formation of five degradation products. The protonated molecule of ripretinib at m/z 510 displayed a characteristic bromine isotopic distribution that facilitated differentiation between brominated and debrominated products. Hydrolytic degradation generated two products through cleavage of the urea linkage, whereas photolytic degradation produced three products involving intramolecular cyclization with concomitant debromination, hydroxylation, and N–C bond cleavage pathways. Collision‐induced dissociation of protonated molecules generated diagnostic product ions that enabled structural elucidation of all degradation products. Photodegradation followed apparent first‐order kinetics with a half‐life of 4.2 min. One hydrolytic degradation product containing a primary aromatic amine moiety was predicted to possess mutagenic potential. Conclusion This investigation elucidates the degradation pathways of ripretinib and highlights the utility of LC–HRMS/MS in the structural characterization of degradation products generated under stress conditions.