2. Academic Validation
  3. Metabolic stability assessment and metabolite profiling of gunagratinib, a novel FGFR inhibitor, in rat, monkey and human liver microsomes by an integrated analysis method based on HPLC-MS/MS and HPLC-Orbitrap-HRMS

Metabolic stability assessment and metabolite profiling of gunagratinib, a novel FGFR inhibitor, in rat, monkey and human liver microsomes by an integrated analysis method based on HPLC-MS/MS and HPLC-Orbitrap-HRMS

  • J Pharm Biomed Anal. 2026 Feb 15:269:117253. doi: 10.1016/j.jpba.2025.117253.
Lei Zhang 1,  Jiabiao Ji 1,  Zhen Hu 2,  Shuai Qu 3,  Yayun Zhou 4,  Hongjian Zhang 5,  Jianming Yang 6
Affiliations

Affiliations

  • 1 Department of Otorhinolaryngology, Head and Neck Surgery, the Second Affiliated Hospital of Anhui Medical University, Hefei 230601, China.
  • 2 Department of Radiology, the Second Affiliated Hospital of Anhui Medical University, Hefei 230601, China.
  • 3 Department of Otorhinolaryngology, the Second People's Hospital of Hefei, Hefei 230601, China.
  • 4 Department of Thoracic Surgery, the Second Affiliated Hospital of Anhui Medical University, Hefei 230601, China.
  • 5 Department of Head and Neck Oncology, Hefei Center Hospital, Chinese Academy of Science, Hefei 230000, China. Electronic address: [email protected].
  • 6 Department of Otorhinolaryngology, Head and Neck Surgery, the Second Affiliated Hospital of Anhui Medical University, Hefei 230601, China. Electronic address: [email protected].
PMID: 41241972 DOI: 10.1016/j.jpba.2025.117253
Abstract

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 (EH = 0.39), contrasting with high ERH in monkey (EH = 0.70) and human (EH = 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.

Keywords

CYP3A4; Fibroblast growth factor receptor; Gunagratinib; High-resolution mass spectrometry; Metabolic stability; Metabolite characterization.

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