Metabolic Profiling of Sitravatinib in Rat and Human Liver Microsomes Using LC-MS/MS and LC-Orbitrap-HRMS.

[RATIONALE] Sitravatinib is a receptor tyrosine kinase inhibitor that has been developed for the treatment of advanced nonsmall-cell lung cancer (NSCLC) and urothelial carcinoma. This study aimed at developing an integrated LC-MS/MS and LC-Orbitrap-HRMS platform for evaluating the metabolic stability and profiling the metabolites in rat and human liver microsomes.

[METHOD] Separation was achieved using a Waters ACQUITY BEH C column with a gradient of 0.1% formic acid in water and acetonitrile. Detection utilized positive electrospray ionization and multiple reaction monitoring of transitions m/z 630.2 → 555.2 for sitravatinib and m/z 502.5 → 323.2 for the internal standard. Metabolite identification was performed using LC-Orbitrap-HRMS through full-scan MS/dd-MS and parallel reaction monitoring. The structures of the metabolites were characterized via accurate mass measurement and MS fragmentation interpretation.

[RESULTS] The LC-MS/MS method showed excellent linearity over a concentration range of 1.0-2000 nM, suitable for high-throughput in vitro assays. Sitravatinib demonstrated poor metabolic stability (t = 17.47 ± 2.57 min) in rat liver microsomes, with seven metabolites identified. Among these, M3 and M5 were the major metabolites. In contrast, sitravatinib exhibited high metabolic stability in human liver microsomes (t = 96.06 ± 12.17 min), with only seven minor metabolites detected.

[CONCLUSIONS] Key metabolic pathways included O-demethylation, amide formation, N-dealkylation, and oxidative deamination. This study establishes the first integrated LC-MS/MS and HRMS strategy for in vitro metabolic profiling of sitravatinib.