Detection of Etomidate and Etomidate Acid in Urine Using HPLC-MS/MS Method.

Abstract

Objectives: To establish a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the detection of etomidate and etomidate acid in urine samples.

Methods: Protein in the urine samples was precipitated by adding acetonitrile, and the supernatant was obtained after centrifugation and filtered. The supernatant was separated on a C₁₈ column with a mobile phase consisting of 0.1% formic acid solution and acetonitrile at a flow rate of 0.4 mL/min. The detection was performed in positive electrospray ionization (ESI) and multiple reaction monitoring (MRM) modes. The method was validated for selectivity, linearity and limit of detection (LOD), and applied to a case of etomidate poisoning death.

Results: The LOD of etomidate and etomidate acid were 0.2 and 0.5 ng/mL, respectively, and the limit of quantitation (LOQ) were 0.5 and 1.0 ng/mL, respectively. Good linear relationship was observed within the linear range (r>0.995 0). At three concentration levels (0.5, 5, 50 ng/mL for etomidate and 1, 10, 100 ng/mL for etomidate acid), the matrix effect was within the range of 5.42% to 18.47%, the extraction recovery rate was greater than 84.25% and the stability was greater than 88.23%. The accuracy, precision and dilution reliability all met the experimental requirements. Etomidate and etomidate acid were successfully detected with the concentrations of 8.82 and 27.88 μg/mL in the urine of a deceased individual who had consumed excessive etomidate.

Conclusions: The method has simple pretreatment, high sensitivity and wide linear range, which can be applied to the detection of etomidate and etomidate acid in urine samples in forensic science.