An Effective and Systematic Strategy for Metabolic Profiling of Golvatinib In Vitro by Combining UHPLC‐MS/MS and UHPLC‐Q‐Orbitrap‐HRMS

Golvatinib is a tyrosine kinase inhibitor in development for treating solid tumors such as advanced hepatocellular carcinoma. The present study aimed to clarify the metabolic fates of golvatinib using human and rat liver microsomes. For this purpose, a comprehensive and systematic workflow was established for investigating the in vitro metabolic fates. Separation was performed on a Waters ACQUITY BEH C 18 column with a mobile phase consisting of 0.1% formic acid in water and acetonitrile. Detection was carried out by multiple reaction monitoring mode using the transitions m/z 634.3→184.2 for golvatinib and m/z 650.3→200.2 for golvatinib N ‐oxide. This method exhibited excellent linearity across a concentration range of 1.0–2000 nM ( r > 0.995). The validated assay was subsequently applied to evaluate the metabolic stability of golvatinib. The results revealed that golvatinib underwent rapid metabolism in rat liver microsomes ( t 1/2 = 13.76 ± 0.79 min), while its turnover was considerably slower in human liver microsomes ( t 1/2 = 56.93 ± 3.17 min). Furthermore, twelve metabolites were identified using Orbitrap high‐resolution mass spectrometry (HRMS) via accurate mass determination and fragmentation analysis. Major metabolic pathways of golvatinib encompass oxygenation, N ‐demethylation, N ‐oxidation, N ‐hydroxylation, dealkylation, oxidative deamination, and oxidative defluorination. Further study demonstrated that CYP3A4 was the principal enzyme involved in metabolizing golvatinib. To the best of our knowledge, this is the first report combining ultra‐high‐performance liquid chromatography‐tandem MS (UHPLC‐MS/MS) with UHPLC‐Quadrupole‐Orbitrap‐HRMS for profiling golvatinib metabolism in vitro, thereby laying a foundation for subsequent pharmacokinetic study.