Biomarkers of alcohol abuse in racehorses by liquid chromatography/tandem mass spectrometry.
Abstract: 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 or =3) and 500 ng/mL, respectively. The limit of confirmations (LOC) for EG and ES were 500 ng/mL and 1.0 microg/mL, respectively. The assay was linear over a concentration range of 0.5-100 microg/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 degrees C and 21 days at -20 degrees C and -70 degrees 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.
Publication Date: 2007-11-02 PubMed ID: 17973234DOI: 10.1002/rcm.3282Google Scholar: Lookup
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Summary
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The research paper discusses a quick and accurate method developed to identify, quantify, and confirm the presence of alcohol use in racehorses. The method involves searching for ethyl glucuronide and ethyl sulfate biomarkers using liquid chromatography with mass spectrometry.
Recognizing Alcohol Biomarkers
- The study developed a new method to detect two alcohol biomass, ethyl glucuronide (EG) and ethyl sulfate (ES). Biomarkers help determine if alcohol has been given to racehorses.
- The process uses liquid chromatography, a technique that separates different components in a mixture. This separation is followed by mass spectrometry, a method used for measuring the characteristics of individual molecules.
Methodology
- The urine samples from racehorses were collected and pre-treated by protein precipitation.
- EG and ES from urine samples were separated using an Ultra PFP column. An isocratic elution with a flush step was carried out with 0.1% formic acid in water and 0.1% formic acid in acetonitrile.
- The analysis was performed under negative electrospray ionization in multiple reaction monitoring mode.
- The test identifies EG and ES, taking less than 5 minutes to provide results.
Efficacy of the Test
- The limit of detection (LOD) and quantification (LOQ) for both EG and ES were 100 ng/mL and 500 ng/mL respectively. The limit of confirmations (LOC) were also recognized at 500 ng/mL for EG and 1.0 μg/mL for ES.
- The test showed excellent accuracy with a linear result over the concentration range of 0.5-100 μg/mL.
- This method retained precision and accuracy both within the day of testing and between different days.
- The test also showed that EG and ES remain stable in urine samples under varying temperatures and conditions for up to 21 days.
Conclusion
- This method was successfully used on urine samples collected post-race from racehorses, providing quick and accurate results. The authors noted that the method could reliably reproduce results, making it a valuable tool for detecting alcohol intake in racehorses to ensure fair competition.
Cite This Article
APA
You Y, Uboh CE, Soma LR, Guan F, Li X, Rudy JA, Chen J.
(2007).
Biomarkers of alcohol abuse in racehorses by liquid chromatography/tandem mass spectrometry.
Rapid Commun Mass Spectrom, 21(23), 3785-3794.
https://doi.org/10.1002/rcm.3282 Publication
Researcher Affiliations
- University of Pennsylvania, School of Veterinary Medicine, Department of Clinical Studies, New Bolton Center Campus, Kennett Square, PA 19348, USA.
MeSH Terms
- Alcoholism / urine
- Animals
- Biomarkers / urine
- Chromatography, High Pressure Liquid / methods
- Chromatography, High Pressure Liquid / veterinary
- Glucuronates / urine
- Horses / blood
- Reproducibility of Results
- Sensitivity and Specificity
- Spectrometry, Mass, Electrospray Ionization / methods
- Spectrometry, Mass, Electrospray Ionization / veterinary
- Substance Abuse Detection / methods
- Substance Abuse Detection / veterinary
- Sulfuric Acid Esters / urine
- Urinalysis / methods
- Urinalysis / veterinary
Citations
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