Gunagratinib

Gunagratinib is a potent fibroblast growth factor receptor (FGFR) inhibitor currently in clinical development for head and neck cancers and cholangiocarcinoma. However, information on its hepatic metabolism remains limited. In this study, we developed and validated an ultra-high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for quantifying gunagratinib in liver microsomes. Chromatographic separation used a Waters ACQUITY BEH C₁₈ column with a gradient elution system of 0.1 % formic acid in water and acetonitrile. Quantification employed positive electrospray ionization with selective reaction monitoring (SRM) for gunagratinib (m/z 424.2→407.3) and the internal standard (IS; m/z 419.2→296.1). The assay demonstrated excellent linearity over the concentration range of 2.0-2000 nM, suitable for in vitro high-throughput screening. Gunagratinib exhibited moderate projected hepatic extraction ratio in rat (E_H = 0.39), contrasting with high ER_H in monkey (E_H = 0.70) and human (E_H = 0.71). Metabolite characterization was performed using HPLC coupled with benchtop Orbitrap high-resolution mass spectrometry (HPLC-Orbitrap-HRMS) in full MS/dd-MS² scan mode, enabling accurate mass measurement, chemical formula assignment, and MS² fragmentation interpretation. Seven metabolites were structurally identified, with M7 (N-demethylation) being the most abundant. Demethylation and oxygenation were the primary metabolic pathways. Cytochrome P450 3A4 was the predominant enzyme responsible for gunagratinib metabolism based on recombinant human P450 enzyme analysis and chemical inhibition study. This study presents the first integrated analytical method based on HPLC-MS/MS and HPLC-Orbitrap-HRMS for the in vitro metabolic assessment of gunagratinib. We anticipate the clinical application of this method in future pharmacokinetic or metabolism studies.