Purpose: Following the regulation of etomidate, novel etomidate analogues have continuously emerged, posing increasing challenges to forensic toxicology. This study was prompted by the identification of a novel etomidate analogue, DF-etomidate, in an e-cigarette liquid and aimed to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of DF-etomidate and etomidate acid in human blood, urine, and hair samples.

Methods: DF-etomidate was identified by liquid chromatography-high resolution mass spectrometry (LC-HRMS) and confirmed by gas chromatography-mass spectrometry (GC-MS). A sensitive UPLC-MS/MS method was developed and validated for the simultaneous quantification of DF-etomidate and etomidate acid in blood, urine, and hair, and subsequently applied to authentic forensic specimens.

Results: The method showed excellent linearity (r > 0.999), satisfactory selectivity, precision, accuracy, extraction recovery, matrix effect, and sensitivity in all three biological matrices. The lower limits of quantification were 0.1 ng/mL for DF-etomidate and 2 ng/mL for etomidate acid in blood and urine, and 0.005 ng/mg and 0.1 ng/mg, respectively, in hair. DF-etomidate was detected in blood, urine, and hair, whereas etomidate acid was detected only in urine.

Conclusions: This study presents the first validated UPLC-MS/MS method for the simultaneous determination of DF-etomidate and etomidate acid in human blood, urine, and hair. Its application to authentic forensic specimens provides initial analytical evidence of DF-etomidate exposure across multiple biological matrices and supports further investigations of this emerging analogue.