Development of a UPLC-MS/MS method and its application for the pharmacokinetic analysis of regorafenib in rats

Regorafenib (REG) is a multikinase inhibitor commonly used in the management of hepatocellular carcinoma, gastrointestinal stromal tumors, and colorectal cancer. Nevertheless, adverse effects or insufficient efficacy appear frequently due to individual variability of plasma concentration of this drug. This work was designed to develop a validated bioanalytical method enabling the accurate quantification of regorafenib (REG) and its metabolites, regorafenib N-oxide (M-2) and N-desmethyl regorafenib N-oxide (M-5), in plasma, and to assess the impact of trametinib (TRA) on their pharmacokinetic profiles. A quantitative detection method for REG, M-2 and M-5 in rat plasma were developed using UPLC-MS/MS, with regorafenib D3 as an internal standard. This method was subsequently applied to pharmacokinetic and drug-drug interaction studies in rats. The method demonstrated good linearity within the range of 50-8000 ng/mL, 25-2500 ng/mL and 25-175 ng/mL, for REG, M-2 and M-5, respectively. Both intra-day and inter-day precisions and accuracy (CV% and bias%) were less than 15%, and the recovery, matrix effect, and stability met EMA guideline. The method demonstrated high effectiveness in the quantitative determination of REG, M-2, and M-5 in rat plasma. Results showed that after administration of TRA, the C_max and AUC_0-∞ for REG increased by 375% and 553%, respectively (p < 0.05), and for M-5: by 779% (p < 0.05) and 482% (p > 0.05). For M-2, there was a respective decrease of 52% and 44% (p < 0.05). This study successfully established a reliable UPLC-MS/MS method for detecting REG, M-2 and M-5 plasma concentrations in rats. The study demonstrated that co-administration of TRA significantly enhances the systemic exposure of REG.