Structural and In silico safety evaluation of asciminib degradation products with a validated stability indicating HPLC method for genotoxic impurity determination

This study aimed to develop and validate an efficient and robust analytical HPLC method for the resolution and quantification of asciminib and its genotoxic impurities along with isolation and characterization of stress degradation products (DPs). Various method optimization studies were performed for development of analytical method for the analysis of asciminib and its genotoxic impurities. The DPs were characterized through LC-MS/MS and NMR spectroscopy. Further, an in-silico software assisted tools were utilized for the assessment pharmacokinetic and toxicological profile of DPs. The optimization proved Kinetex C18 (250 mm × 4.6 mm, 5 μm) column was ideal for the resolution of analytes with 0.7 mL/min flow of 0.01% formic acid buffer (pH 3.9) and acetonitrile and 276 nm wavelength. The forced degradation studies show significant degradation occurred under acidic (12.36%), basic (3.54%), oxidative (10.63%), and UV (7.28%) stress. This stress study leads to the formation of 5 DPs in acid, one DP in base, three DPs in peroxide, one DP in UV light stress study. The formed DPs were isolated through preparative HPLC and structural elucidation of DPs was conducted using LC-MS/MS and NMR spectroscopy. The toxicity and ADME profiles of the identified DPs reveal that some DPs exhibit potential hepatotoxicity, nephrotoxicity, and neurotoxicity with significant variations in toxicological and pharmacokinetic properties. The results underscore the importance of understanding the stability, toxicity, and pharmacokinetics of asciminib and its DPs for regulatory and pharmaceutical applications.