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 (C_max), area under the concentration–time curve (AUC_0–∞), and detection window (T_window). 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.