Green Ultra‐Fast UPLC–MS/MS Method for Enasidenib Quantification in HLMs Matrix With the Metabolic Stability Assessment: In Silico Study for Structural Alerts, ADME Characteristics and Metaboslic Lability

Enasidenib (ESD), an oral medication, was used for the treatment of patients with refractory or relapsed Acute Myeloid Leukemia. No prior UPLC-MS/MS method with combined green metrics and in silico metabolism analysis for ESD was reported, so the research sought to establish an ultrafast UPLC-MS/MS method for estimating ESD in the HLMs matrix. The MS/MS analysis was applied applying positive ESI ionization mode, and chromatography using an Eclipse Plus C8 column (reversed phase) within one minute. The UPLC-MS/MS method exhibited a good degree of greenness as approved by the MoGAPI score (68.0) and the AGREE score (0.66). The linearity range was form 1 ng/mL (LOQ) to 4000 ng/mL. The precision and accuracy for intraday and interday measurements varied from -3.11% to 10.67% and -7.22% to 12.33%, respectively. The in vitro t_1/2 and the intrinsic clearance (Cl_int) were determined to be 82.52 min (long) and 9.83 mL/min/kg (low), respectively that is considered a low clearance drug. Slight structural modifications to the 2-methyl propan-2-ol moiety and pyridine ring in drug design could upsurge the ESD metabolic stability. The combined in vitro/in silico method delivers a resource-efficient strategy for early-stage metabolic screening and progressing innovative drug research focused on improving metabolic stability.