Introduction: Recently, numerous abuse and intoxication cases related to imidazole-derived γ-aminobutyric acid type A (GABA _A ) agonists, such as etomidate and its analogs, have been reported in China. In this study, we encountered a novel etomidate analog, iso-butomidate, in human biological samples from suspected abuse cases.

Methods: The metabolic profile of iso-butomidate was investigated using mice and human liver microsomes (HLMs) incubated with the compound, followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis to identify metabolites. We have established a simultaneous and reliable analytical method for etomidate acid and iso-butomidate in the human urine and blood by liquid chromatography−tandem mass spectrometry (LC-MS/MS), and the method could be applied to authentic cases. Sample preparation involved protein precipitation with acetonitrile after addition of internal standard, followed by centrifugation and analysis of the supernatant.

Results: Eight metabolites of iso-butomidate were identified in both mice and HLMs models, with metabolic pathways including monohydroxylation, dihydroxylation, O-dealkylation, and O-glucuronidation. Among these, etomidate acid was considered a suitable biomarker for monitoring exposure to iso-butomidate. The developed LC-MS/MS method was successfully applied to authentic human urine and blood samples, demonstrating its reliability for forensic toxicological investigations.

Conclusions: This is the first report to present the metabolic profiles of the novel imidazole-derived GABA _A agonist iso-butomidate. The established analytical method for simultaneous quantification of iso-butomidate and etomidate acid in human biological samples provides a valuable tool for monitoring exposure in abuse cases and further toxicological studies.