Biomarkers of alcohol abuse in racehorses by liquid chromatography/tandem mass spectrometry

A rapid and sensitive method was developed for the screening, quantification and confirmation of ethyl glucuronide (EG) and ethyl sulfate (ES) as biomarkers for alcohol administration to racehorses using liquid chromatography coupled on‐line with triple quadrupole tandem mass spectrometry. Urine sample aliquots (0.1 mL) were pre‐treated by protein precipitation. Separation of EG and ES was achieved on an Ultra PFP column. Isocratic elution with a flush step was performed using 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Analysis was performed by negative electrospray ionization in multiple reaction monitoring mode. The retention times for EG and ES were 1.7 ± 0.30 and 3.4 ± 0.30 min, respectively. The internal standard used was d5‐ethyl glucuronide with a retention time of 1.7 ± 0.30 min. The entire separation was completed in 0.995). Intra‐ and inter‐day accuracy and precision were less than 15%. The analytes were stable in urine for 24 h at room temperature, 10 days at 4°C and 21 days at −20°C and −70°C. Ion suppression or enhancement due to matrix effect was negligible. The measurement uncertainty was