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

Shiqi Jiang,1,2,* Yan Chen,1,2,* Lu Cao,1 Haoxin Fu,1 Peiqi Wang,1 Chongmeng Yang31Department of Pharmacy, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China; 2Zhejiang Key Laboratory of Intelligent Cancer Biomarker Discovery and Translation, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China; 3Department of Ophthalmology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China*These authors contributed equally to this workCorrespondence: Chongmeng Yang, Email [email protected]: 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.Keywords: ensartinib, M465, pharmacokinetics, UPLC–MS/MS, rat