Abstract:

Objective To establish a qualitative and quantitative analytical method for the determination of 21 etomidate analogues and etomidate acid in hair and to investigate its application value in actual forensic cases. Methods Hair samples were extracted following cryogenic wet grinding. The supernatant was filtered through a 0.22 μm organic membrane filter. The Kinetex^® Biphenyl （100 mm×2.1 mm, 1.7 μm） column was used, and gradient elution was performed with methanol and 5 mmol/L ammonium acetate within 15 minutes. The data acquisition was acquired by UPLC-MS/MS with electrospray ionization in positive ion mode, followed by method verification. Results The 21 etomidate analogues and etomidate acid exhibited good chromatographic separation in hair samples. In particular, all eight compounds within three distinct groups of isomers, including propoxate and isopropoxate; butomidate, sec-butomidate, iso-butomidate and tert-butomidate; 1-（methoxycarbonyl）-2-methylpropan-2-yl 1-（1-phenylethyl）-1H-imidazole-5-carboxylate and 1-（methoxycarbonyl）-2-methylpropanyl 1-（1-phenylethyl）-1H-imidazole-5- carboxylate, were effectively separated. The method showed a good linear relationship in the quantitative linear range （r>0.999）， with the limits of detection （LOD） ranging from 0.005 to 0.020 ng/mg and the lower limit of quantification （LLOQ） of 0.05 ng/mg. The selectivity， accuracy and precision of the established method met the verification standards. The matrix effects and extraction recoveries showed good reproducibility at different concentration levels， and the quantitative results for processed hair samples remained stable after storage at 4 ℃ for 36 hours. Practical application results showed that etomidate, metomidate, isopropoxate, sec-butomidate, iso-butomidate, TF-etomidate, and ABP-700, and the metabolite etomidate acid could be detected in the hair of abusers, although the content of etomidate acid was relatively low. Conclusion The method established in this study is simple to perform and provides accurate quantification. It enables the simultaneous determination of etomidate acid, 13 scheduled etomidate analogues, and 8 unscheduled potential derivatives in hair. The analytical scope encompasses alkyl, alkoxy, alkoxycarbonyl, halogen, and sulfhydryl substitutions distributed across the ester side chain, benzene ring, and imidazole ring. It can provide technical support for combating the abuse of etomidate analogue substances.

Key words: forensic medicine, toxicological analysis, etomidate, etomidate acid, ultra-high performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）, hair