Traceability of etomidate and its analogs in biological samples using “specific” metabolites

Abstract

With the regulation of etomidate (ETO), criminals continue to use chemical modifications to derive various ETO analogues as substitutes to enter the illegal market in order to evade punishment, and the phenomenon of substitution abuse is becoming increasingly serious. After these substitutes are ingested by the human body, the prototype drug is almost undetectable in the biological samples. It is necessary to search for “characteristic” metabolite traceability prototype drugs to effectively prevent misjudgment of cases. The fragmentation patterns and metabolites of seven ETO analogs were analyzed by ultra high-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS) and liquid chromatography tandem triple quadrupole mass spectrometry (HPLC-MS/MS) using an in vitro metabolic model of human liver microsomes. These seven compounds undergo I-phase metabolites, including imidazole ring disconnection from alkyl groups, benzene ring and alkyl group interactions, dealkylation, and so on. The parent compounds propoxate and sec-butomidate were not detected in the actual 83 hair and 4 urine samples tested; ETO and isopropoxate were the two main drugs detected in hair samples; and CF3-etomidate, a novel alternative, was detected in hair at a higher rate than metomidate. In addition, 47 hair samples showed a single parent substance, and 35 samples contained two or more drugs, indicating the possibility of mixed use of ETO and its analogues. The biomarkers of metomidate in both urine and hair are hydroxylated metabolites on the benzene ring; the biomarkers of isopropoxate undergo a hydroxylation reaction on the benzene ring (hair) and cleavage of the C–N bond between the imidazole ring and alkyl group (urine); the metabolic product of the hydroxylation reaction on alkyl groups is a biomarker of butomidate. In this study, the metabolite differences of seven ETO analogs were compared for the first time in order to infer the parent compounds based on biomarkers. Meanwhile, based on the existing knowledge of the metabolism of the seven known substances in this experiment, it is possible to make a preliminary prediction of emerging ETO analogs in order to improve the understanding of early warning systems and the timely updating of monitoring techniques.