An Effective and Systematic Strategy for Metabolic Profiling of Golvatinib In Vitro by Combining UHPLC-MS/MS and UHPLC-Q-Orbitrap-HRMS
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Published on: March 28, 2017
Lvhao Cao1, Tongyuan Zhang1, Yunfeng Zhu1
1Department of Emergency Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, China.
Abstract:
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 C18 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 (t1/2 = 13.76 ± 0.79 min), while its turnover was considerably slower in human liver microsomes (t1/2 = 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.
Insights
Golvatinib, a cancer drug, is metabolized faster in rats than humans. This study identified twelve metabolites and the key enzyme CYP3A4, aiding future pharmacokinetic research.
Area of Science:
- Pharmacology
- Drug Metabolism
- Analytical Chemistry
Background:
- Golvatinib is an investigational tyrosine kinase inhibitor for solid tumors, including advanced hepatocellular carcinoma.
- Understanding its metabolic fate is crucial for clinical development and pharmacokinetic profiling.
Purpose of the Study:
- To elucidate the in vitro metabolic pathways and stability of golvatinib in human and rat liver microsomes.
- To establish and validate a sensitive analytical method for golvatinib and its metabolites.
Main Methods:
- Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was employed for separation and detection.
- Orbitrap high-resolution mass spectrometry (HRMS) was utilized for metabolite identification.
- Metabolic stability was assessed by determining half-life (t1/2) in liver microsomes.
Main Results:
- Golvatinib exhibited significantly faster metabolism in rat liver microsomes (t1/2 = 13.76 min) compared to human liver microsomes (t1/2 = 56.93 min).
- Twelve metabolites were identified, revealing major pathways including oxygenation, N-demethylation, and oxidative defluorination.
- CYP3A4 was identified as the primary enzyme responsible for golvatinib metabolism.
Conclusions:
- This study provides a comprehensive profile of golvatinib's in vitro metabolism, highlighting species-specific differences.
- The developed analytical method and identified metabolic pathways lay the groundwork for future pharmacokinetic studies of golvatinib.
