Development and Validation of a UPLC–MS/MS Method for Simultaneous Quantification of Ensartinib and Its Major Metabolite M465 in Rat Plasma and Application to Pharmacokinetic Studies

Abstract

Introduction Ensartinib is a second-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor approved for the treatment of ALK-positive non-small cell lung cancer (NSCLC). However, its in vivo disposition and metabolic characteristics, particularly those of its major metabolite M465, remain incompletely understood. This study aimed to develop and validate a sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method for the simultaneous quantification of ensartinib and M465 in rat plasma and to apply the method to a pharmacokinetic study in rats. Methods Crizotinib was used as the internal standard (IS). Plasma samples were processed by one-step protein precipitation with acetonitrile. Chromatographic separation was achieved on a Waters Acquity BEH C18 column using gradient elution with 0.1% formic acid in water and acetonitrile. The method was validated in terms of selectivity, linearity, lower limit of quantification (LLOQ), precision, accuracy, recovery, matrix effect, stability, carry-over and dilution integrity. The validated method was subsequently applied to determine plasma concentrations of ensartinib and M465 after oral administration of ensartinib to rats. Results The method showed good linearity over the concentration ranges of 0.1–100 ng/mL for ensartinib and 0.05–10 ng/mL for M465, with LLOQs of 0.1 ng/mL and 0.05 ng/mL, respectively. The intra- and inter-day precision values ranged from 2.2% to 16.4%, and the accuracy ranged from −12.3% to 8.0%, meeting the acceptance criteria for bioanalytical method validation. Recovery, matrix effect, stability, carry-over and dilution integrity were all within acceptable limits. After oral administration of ensartinib to rats, the maximum plasma concentrations (Cmax) of ensartinib and M465 were approximately 64.7 ng/mL and 6.3 ng/mL, respectively. The AUC(0–t) values of ensartinib and M465 were 577.6 ng/mL*h and 32.5 ng/mL*h, respectively, indicating lower systemic exposure of M465 compared with the parent drug. Conclusion A sensitive, reliable, and rapid UPLC–MS/MS method was successfully developed and validated for the simultaneous determination of ensartinib and M465 in rat plasma. The method was successfully applied to a rat pharmacokinetic study and provides useful analytical support for further preclinical investigations of ensartinib and its major metabolite.