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Published on: December 15, 2011
Metabolic Profiling of Sitravatinib in Rat and Human Liver Microsomes Using LC-MS/MS and LC-Orbitrap-HRMS
Shan Xu1, Ying Zhu1, Wenxia Liu1
1Department of Pharmacy, Xuzhou Central Hospital, Xuzhou, China.
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 C18 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-MS2 and parallel reaction monitoring. The structures of the metabolites were characterized via accurate mass measurement and MS2 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 (t1/2 = 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 (t1/2 = 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.
Insights
Sitravatinib showed poor metabolic stability in rat liver microsomes but high stability in human liver microsomes. This study developed an integrated LC-MS/MS and HRMS platform for sitravatinib metabolite profiling.
Area of Science:
- Pharmacokinetics and Drug Metabolism
- Analytical Chemistry
- Mass Spectrometry
Background:
- Sitravatinib is a receptor tyrosine kinase inhibitor for advanced non-small-cell lung cancer (NSCLC) and urothelial carcinoma.
- Understanding sitravatinib's metabolism is crucial for its clinical development.
Purpose of the Study:
- To develop an integrated liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-high-resolution mass spectrometry (LC-Orbitrap-HRMS) platform.
- To evaluate the metabolic stability and profile metabolites of sitravatinib in rat and human liver microsomes.
Main Methods:
- Utilized a Waters ACQUITY BEH C18 column with a formic acid/acetonitrile gradient.
- Employed positive electrospray ionization and multiple reaction monitoring for sitravatinib quantification.
- Performed metabolite identification using LC-Orbitrap-HRMS with full-scan MS/dd-MS2 and parallel reaction monitoring.
Main Results:
- The LC-MS/MS method demonstrated excellent linearity (1.0-2000 nM) for high-throughput in vitro assays.
- Sitravatinib exhibited poor metabolic stability in rat liver microsomes (t1/2 = 17.47 ± 2.57 min) with seven identified metabolites (M3, M5 major).
- Sitravatinib showed high metabolic stability in human liver microsomes (t1/2 = 96.06 ± 12.17 min) with seven minor metabolites detected.
Conclusions:
- Key metabolic pathways identified include O-demethylation, amide formation, N-dealkylation, and oxidative deamination.
- This study presents the first integrated LC-MS/MS and HRMS strategy for in vitro metabolic profiling of sitravatinib.
- The developed platform enables efficient metabolic stability assessment and metabolite identification for sitravatinib.
