Development and validation of a quick and sensitive UPLC-MS/MS method for measuring ensartinib in HLMs: investigation of structural alerts associated with metabolic lability and in silico toxicity

Abstract

Ensartinib (EST) is a new oral tyrosine kinase inhibitor authorized for the initial treatment of anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC). EST demonstrates efficacy against Met Proto-Oncogene (MET), Virus Oncogene Homolog 1 (ROS1), Receptor Tyrosine Kinase (AXL), Abelson Murine Leukemia (ABL), and v-Ros UR2 Sarcoma, and it is considered safer than existing ALK inhibitors. The current research involved the development of a rapid, green, and sensitive UPLC-MS/MS approach for detecting EST in human liver microsomes (HLMs). The approach was applied to determine the in vitro metabolic stability of EST. The UPLC-MS/MS method validation agreed with the US-FDA standards for bioanalytical approach validation. The StarDrop software package, which comprises the DEREK toxicity and WhichP450 modules, was used to identify potential toxicity alerts associated with the EST chemical structure and to estimate its in silico metabolic lability. EST and Encorafenib (ECB as the internal standard) were differentiated using an isocratic mobile phase system on a reversed stationary phase (Eclipse Plus C18) column. The EST calibration curve demonstrated linearity across the level range of 1 to 3000 ng mL⁻¹. This research determined the accuracy and precision of the UPLC-MS/MS procedure for intra- and inter-day valuations, spanning −5.22% to 9.67% and −5.22% to 10.67%, respectively. The in vitro half-life (t_1/2) was ascertained to be 19.29 min, while the intrinsic clearance (Cl_int) of EST was determined to be 42.03 mL min⁻¹ kg⁻¹. In silico data proposed that minor structural modifications to the dichlorophenyl moiety or the piperazine ring during drug design may enhance the safety profile and metabolic stability relative to the properties of EST. Evaluating the EST metabolic stability and in silico ADME characteristics is essential for advancing innovative therapeutic research focused on improving metabolic stability.