Development and validation of HPLC-UV and HPLC-MS/MS methods for ML-210 quantification

ML-210 is a GPX-4 inhibitor with potential therapeutic relevance in cancer therapies. However, no validated (bio)analytical methods for its quantification have been reported, limiting its pharmacokinetic and stability investigations. This study describes the development and validation of complementary analytical and bioanalytical methods for the quantification of ML-210 in formulation and biological matrices. An HPLC-UV method was developed for the quantification of ML-210 in intravenous formulations, while a HPLC-MS/MS method was established for its analysis in DMEM culture medium and mouse plasma. Methods were validated according to ICH Q2(R1) and FDA bioanalytical guidelines. The HPLC-UV method demonstrated high specificity, confirmed by forced degradation studies, with accuracy ranging from 98.4% to 108.4% and precision within acceptable limits (intra-day RSD ≤ 0.9%, inter-day RSD ≤ 3.3%). The HPLC-MS/MS method met regulatory criteria for selectivity, sensitivity, carryover, accuracy (89.6-106.7%), precision (RSD ≤ 9.6%), and linearity (R² ≥ 0.9994) in both matrices. Matrix effects and recoveries showed RSD values below 15%. ML-210 remained stable for at least 6 months at -20°C in both matrices and for approximately 2 h in mouse plasma in vitro at 37°C. The validated HPLC-MS/MS method was applied to an in vivo pharmacokinetic study following intravenous administration in mice (5 mg/kg), revealing a rapid decline in plasma concentration and an elimination half-life of approximately 300 min. These validated methods provide a reliable analytical platform for the quantification of ML-210 in formulation and biological studies, supporting future pharmacokinetic and preclinical investigations.