Simultaneous determination of bosutinib and its metabolite in rat plasma by UPLC-MS/MS: Development, validation and application to pharmacokinetic and metabolic stability studies.

Bosutinib is a second-generation tyrosine kinase inhibitor (TKI) primarily used for the treatment of chronic myeloid leukemia (CML). The aim of this study was to develop and validate a rapid and accurate ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of bosutinib and its major metabolite M5. Chromatographic separation was performed on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) using a gradient elution with acetonitrile and 0.1% formic acid as the mobile phase, and gefitinib was used as the internal standard (IS). Biological analysis showed that both bosutinib (2-400 ng/mL) and its metabolite M5 (1-60 ng/mL) exhibited good linearity in their respective concentration ranges, with correlation coefficients (r2) of 0.996 and 0.993, respectively. Both of them exhibited intra- and inter-day precision (RSD%) below 15% and accuracy (RE%) within ± 15%. Recoveries were all within the range of 85.3-98.0%, and matrix effects were all within the range of 91.6-101.6%. Ultimately, human liver microsomes (HLM) was used to study in vitro metabolic stability, while in vivo pharmacokinetics were evaluated in rats. In vivo pharmacokinetic studies confirmed that this method could be successfully used for the analysis of bosutinib after oral administration of 50 mg/kg in rats. In vitro results showed that bosutinib had the intrinsic clearance (CLint) value of 0.03 mL/min/mg and the half-life (t1/2) value of 79.94 min. This study provides a potential reference for the clinical investigation of bosutinib.