Ultra-performance liquid chromatography/tandem mass spectrometry in high-throughput detection, quantification and confirmation of anabolic steroids in equine plasma.
Abstract: An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method for fast-throughput analysis of eight anabolic and androgenic steroids (AAS) in equine plasma is reported. Analytes were recovered by liquid-liquid extraction using methyl tert-butyl ether, separated on a 1.9 microm C(18) reversed-phase column, and analyzed in positive electrospray ionization mode on a triple quadrupole mass spectrometer with selected reaction monitoring (SRM) and full product ion scans. Two SRM ion transitions were monitored for each AAS during screening to obtain highly selective screening results. Full product ion spectra of excellent quality for AAS, at 100 pg/0.5 mL in plasma, devoid of interfering spectra from impurities in plasma, were obtained. To our knowledge, this is the first report on the acquisition of full product ion spectra at such a low analyte concentration and plasma volume using a triple quadrupole instrument. In addition to product ion intensity ratios obtained from three SRM scans for identifying AAS in equine plasma, full product ion spectra were used as supporting evidence for confirmation. For quantification, deuterium-labeled testosterone and stanozolol were used as internal standards (ISs). The limits of detection, quantification and confirmation were 6.25-12.5 pg/0.5 mL, 25 pg/0.5 mL and 50-100 pg/0.5 mL, respectively. There was no significant matrix effect on the analysis of all eight AAS. Intra-day precision and accuracy were 2-15% and 91-107%, respectively. Inter-day precision and accuracy were 1-21% and 94-110%, respectively. Total analysis time was 5 min. To date, the method has been successfully used in the analysis of >12,000 samples for AAS in plasma samples from racehorses competing in the State of Pennsylvania. The method is fast, selective, reproducible, and reliable.
Copyright (c) 2009 John Wiley & Sons, Ltd.
Publication Date: 2009-06-09 PubMed ID: 19504479DOI: 10.1002/rcm.4114Google Scholar: Lookup
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Summary
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The research presents a method for quick and reliable detection of anabolic steroids in horse plasma using ultra-performance liquid chromatography/tandem mass spectrometry.
Methodology
- An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) technique was utilized to analyze eight anabolic and androgenic steroids (AAS) in equine plasma.
- The substances were recovered using a liquid-liquid extraction method with methyl tert-butyl ether.
- The analytes were separated on a 1.9 microm C(18) reversed-phase column and subjected to analysis in positive electrospray ionization mode on a triple quadrupole mass spectrometer.
- The method monitored two SRM ion transitions for each AAS to ensure highly selective screening results. Full product ion spectra were also acquired, free from interference by impurities in the plasma.
Findings
- This study is the first to report the acquisition of full product ion spectra at such low analyte concentration and plasma volume using a triple quadrupole instrument.
- The limits of detection, quantification, and confirmation were determined as 6.25-12.5 pg/0.5 mL, 25 pg/0.5 mL, and 50-100 pg/0.5 mL respectively.
- No significant effect of the matrix was found on the analysis of all eight AAS.
- The intra-day precision and accuracy values were 2-15% and 91-107% respectively. For inter-day values, precision ranged from 1-21% while accuracy fell between 94-110%.
- The total time taken for analysis was relatively short, only 5 minutes.
- All these emphasizes that the method is quick, selective, reproducible, and reliable.
Real-world Application
- This technique has been successfully utilized in the analysis of more than 12,000 plasma samples from racehorses in Pennsylvania, proving its practical effectiveness.
- It offers promises for regulation and oversight bodies to quickly and effectively detect potential instances of performance-enhancing drug use in racehorses.
Cite This Article
APA
You Y, Uboh CE, Soma LR, Guan F, Li X, Rudy JA, Liu Y, Chen J.
(2009).
Ultra-performance liquid chromatography/tandem mass spectrometry in high-throughput detection, quantification and confirmation of anabolic steroids in equine plasma.
Rapid Commun Mass Spectrom, 23(13), 2035-2044.
https://doi.org/10.1002/rcm.4114 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
- Anabolic Agents / blood
- Anabolic Agents / chemistry
- Animals
- Chromatography, High Pressure Liquid / methods
- Horses
- Male
- Steroids / blood
- Steroids / chemistry
- Tandem Mass Spectrometry / methods
Citations
This article has been cited 2 times.- Matraszek-Zuchowska I, Wozniak B, Posyniak A. Comparison of the Multiple Reaction Monitoring and Enhanced Product Ion Scan Modes for Confirmation of Stilbenes in Bovine Urine Samples Using LC-MS/MS QTRAP(®) System.. Chromatographia 2016;79:1003-1012.
- Chen JW, Uboh CE, Soma LR, You Y, Jiang Z, Li X, Guan F, Liu Y. Identification of sample donor by 24-plex short tandem repeat in a post-race equine plasma containing dexamethasone.. Springerplus 2014;3:94.
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