LC–MS/MS Method Development and Validation for Quantification of Repotrectinib in Rat Plasma and its Application to Pharmacokinetic Study

Abstract

Objective: Repotrectinib is a next-generation tyrosine kinase inhibitor (TKI) recommended for adults with ROS1-positive, locally advanced or metastatic non-small cell lung cancer (NSCLC). The present study aimed to develop and validate a sensitive LC-MS/MS method for quantifying repotrectinib in rat plasma. Methods: Analysis was carried out using a SCIEX QTRAP 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode with SCIEX Analyst software. Separation of the analyte was achieved using a reversed-phase Luna C18 column with a mobile phase of acetonitrile and 0.1% formic acid at a 30:70 (v/v) ratio, a total run time of 5 min, and a flow rate of 1.0 ml/min. Optimized multiple reaction monitoring transitions were observed at m/z 356.37 → 295.45 for repotrectinib and m/z 533.57 → 335.60 for ponatinib (internal standard). Results: The method exhibited strong linearity over the concentration range of 1–120 ng/ml (R² = 0.9992). The method was found to be precise (%CV ≤ 5) and accurate, with a range of 99.99% to 101.00%. Recovery was consistent and reproducible, ranging from 98.8% to 99.97% (%CV<3). Stability studies confirmed that repotrectinib remained stable under bench-top, autosampler and freeze–thaw conditions. The method exhibited reliability and suitability for pharmacokinetic evaluation, as evidenced by a Cmax of 52.30±0.79 ng/ml at Tmax of 5 h in rats. Conclusion: The developed LC-MS/MS method metal validation requirements and is suitable to support future preclinical and, potentially, clinical studies.