Comparative study of alkaline digestion and solvent‐assisted grinding methods for the quantitative extraction of etomidate and etomidate acid based on the hair from etomidate users

Hair can trace drug use history, but prevailing extraction methods often struggle to fully extract analytes because of tight encapsulation by hair keratin. Since etomidate (ET) was controlled as a Category II psychotropic drug in China in 2023, developing an analytical method to accurately quantify ET and its metabolite etomidate acid (ETA) in hair is essential for establishing cutoff values in hair testing. This study compared extraction efficiencies of alkaline digestion and four solvent-assisted grinding methods (methanol, ammonium formate buffers, 10% formic acid) employing deuterated internal standards (ISs). The optimal method was validated by UPLC-MS/MS and applied to 53 authentic hair samples (11 also analyzed by alkaline digestion for relative extraction efficiency). The ammonium formate-formic acid buffer (pH 3.6) grinding method was proven to be optimal, providing high efficiency while preserving ET stability. It exhibited good linearity (r² >0.999) in a range of 0.005-5 ng/mg for ET and 0.002-10 ng/mg for ETA. Limits of detection (LOD) for ET and ETA were 0.001 ng/mg, with lower limits of quantification (LLOQ) being 0.005 and 0.002 ng/mg, respectively. All 53 samples had detectable ET (0.013-219.36 ng/mg), while ETA was quantifiable in only 32 samples (0.002-1.873 ng/mg), with ET/ETA ratios of 2.2-615.4. This study establishes a simple, reliable, and validated method for simultaneous quantification of ET and ETA in human hair, demonstrating high practicality for forensic and drug control applications.