Chiral Separation and Determination of Imidazole Compounds in Biological Matrices Using UHPLC-MS/MS

Objective: To establish a robust ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of imidazole compounds and their enantiomers in blood, urine, and hair. Materials and Methods: Blood and urine samples were subjected to protein precipitation with acetonitrile, whereas hair samples were pulverized after washing and extracted with methanol, followed by ultrasonication and centrifugation. The supernatant was filtered through a 0.22 μm membrane for analysis. Enantiomeric separation was achieved on a Phenomenex Lux ® Cellulose-3 chiral column using an isocratic mobile phase of 5 mmol/L ammonium acetate with 0.1% formic acid (60%) and acetonitrile (40%) at a flow rate of 0.5 mL/min. Detection was performed in positive electrospray ionization mode with multiple reaction monitoring. The method was validated according to international forensic toxicology standards and applied to authentic case samples. Results: The method demonstrated excellent selectivity and linearity ( R ²>0.996). Calibration ranges were 5–3000 ng/mL for blood and urine and 0.05–30 ng/mg for hair. Limits of detection were 1 ng/mL for blood and urine and 0.02 ng/mg for hair, with corresponding limits of quantification (LLOQ) of 5 ng/mL and 0.05 ng/mg. Accuracy ranged from 86.00% to 114.58%, recoveries exceeded 86.75%, and matrix effects were within 0.01%–14.82%, all meeting forensic validation criteria. Application to 22 authentic hair samples revealed widespread exposure to etomidate, with the R-isomers of metomidate, propoxate, and isopropoxate detected more frequently and at higher concentrations than their S-counterparts. Conclusion: The established UHPLC-MS/MS method provides high sensitivity, selectivity, and reproducibility, with a wide linear range and straightforward sample preparation. Its successful application to multiple biological matrices underscores its potential as a powerful tool for the screening, quantification, and source tracing of imidazole compounds and their enantiomers in forensic toxicology and drug abuse monitoring.