Chiral analysis of etomidate and metomidate enantiomers in hair by UHPLC-MS/MS: application to authentic human hair samples and enantiomeric fraction assessment.

Abstract

R-etomidate and metomidate, employed clinically as sedatives and hypnotics in human and veterinary medicine, respectively, have recently gained attention due to their escalating nonmedical use. Owing to their intoxicating effects and addictive potential, illicit abuse of these compounds has surged over the past few years. Understanding the enantiomer ratio of etomidate and metomidate in hair can help identify patterns of abuse and the source of the drugs. Here, a chiral separation and quantification method was developed to analyze etomidate and metomidate enantiomers in human hair. Approximately 20 mg of hair was extracted with acetonitrile by cryogenic grinding and then analyzed by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Chromatographic separation was achieved using a Lux 3 μm cellulose-3 chiral column (150 mm × 4.6 mm) under gradient elution. The linearity ranged from 10 to 1000 pg/mg for all compounds. The validated method was applied to 98 authentic hair samples. The enantiomeric ratios of etomidate and metomidate were quantified using the enantiomeric fraction (EF), defined as [R-enantiomer]/[R + S-enantiomers]. The observed EF values spanned 0.40-1.00 for etomidate and 0.49-1.00 for metomidate, indicating variable stereochemical compositions across samples. This study is the first to report the chiral separation and quantification of etomidate and metomidate enantiomers in human hair using UHPLC-MS/MS. Although only R-etomidate is clinically approved, forensic hair analysis identified S-etomidate coexisting with R-enantiomers in 8 samples, indicating nonmedical exposure to incompletely refined or racemic mixtures.