A Simple and Sensitive HPLC–MS/MS Method for the Simultaneous Quantitative Analysis and Pharmacokinetic Comparison of Four Imidazole‐Derived GABA Receptor Agonists: Etomidate, Metomidate, Propoxate, and Isopropoxate in Mouse Blood

Etomidate and its structural analogs-metomidate, propoxate, and isopropoxate-have emerged as prevalent substances of abuse in China due to their high addictive potential. This study developed and validated a sensitive, reliable, and high-throughput method using HPLC-MS/MS for simultaneous quantification of four imidazoline-derived new psychoactive substances and the metabolite etomidate acid and then applied it for the molecular pharmacokinetics of five analytes in murine blood. Method validation demonstrated excellent linearity (r2 ≥ 0.999) across calibration curves, with LLOQ ranging from 0.2 to 1 ng/mL and LOD ranging from 0.1 to 0.5 ng/mL. Intraday/interday precision reached 0.123%-11.2%, and accuracy was in the range of -9.68% to 9.86%, which met bioanalytical criteria. Recovery rates (89.3%-103%) and matrix effects (87.1%-105%) were within acceptable ranges. Male KM mice with a body weight of 25 ± 2 g were selected for pharmacokinetic evaluation. Pharmacokinetic analysis revealed significant dose-dependent relationships for maximum plasma concentration (Cmax), area under the concentration-time curve (AUC0-∞), and detection window (Twindow). Metabolic rates followed a descending order: propoxate > isopropoxate > etomidate > metomidate, likely attributed to their lipophilicity gradient. This study systematically elucidates the dose-metabolism kinetics and structure-activity relationships of etomidate analogs, addressing the knowledge gap in toxicokinetic data for propoxate and isopropoxate.