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Biomarkers of alcohol abuse in racehorses by liquid chromatography/tandem mass spectrometry
Author(s) -
You Youwen,
Uboh Cornelius E.,
Soma Lawrence R.,
Guan Fuyu,
Li Xiaoqing,
Rudy Jeffrey A.,
Chen Jinwen
Publication year - 2007
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.3282
Subject(s) - chemistry , chromatography , ethyl glucuronide , detection limit , formic acid , protein precipitation , selected reaction monitoring , electrospray ionization , mass spectrometry , tandem mass spectrometry , analyte , liquid chromatography–mass spectrometry , triple quadrupole mass spectrometer , elution , alcohol , biochemistry , alcohol consumption
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 d 5 ‐ethyl glucuronide with a retention time of 1.7 ± 0.30 min. The entire separation was completed in <5 min. The limit of detection (LOD) and of quantification (LOQ) for both analytes were 100 ng/mL (S/N ≥3) and 500 ng/mL, respectively. The limit of confirmations (LOC) for EG and ES were 500 ng/mL and 1.0 µg/mL, respectively. The assay was linear over a concentration range of 0.5–100 µg/mL (r 2 > 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 <14% for EG and <25% for ES. This method was successfully used for the quantification of EG and ES in urine samples following alcohol administration to research horses and for screening and confirmation of EG and ES in urine samples obtained from racehorses post‐competition. The method is simple, rapid, inexpensive, and reliably reproducible. Copyright © 2007 John Wiley & Sons, Ltd.