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High-throughput UHPLC-MS/MS method for the detection, quantification and identification of fifty-five anabolic and androgenic steroids in equine plasma.
Abstract: Anabolic and androgenic steroids (AASs) are synthetic substances related to the primary male sex hormone, testosterone. AASs can be abused in both human and equine sports and, thus, are banned by the International Olympic Committee and the Association of Racing Commissioners International (ARCI). Enforcement of the ban on the use of AASs in racehorses during competition requires a defensible and robust method of analysis. To address this requirement, a high-throughput ultra high-performance liquid chromatography-mass spectrometric (UHPLC-MS) method was developed for the detection, quantification and confirmation of 55 AASs in equine plasma. AASs were recovered from equine plasma samples by liquid-liquid extraction with methyl tert-butyl ether (MTBE). Analytes were chromatographically separated on a sub-2 µm particle size C(18) column with a mobile phase gradient elution and detected by selected-reaction monitoring (SRM) on a triple quadrupole mass spectrometer. AASs with isobaric precursor ions were either chromatographically resolved or mass spectrometrically differentiated by unique precursor-to-product ion transitions. A few of them that could not be resolved by both approaches were differentiated by intensity ratios of three major product ions. All the epimer pairs, testosterone and epitestosterone, boldenone and epiboldenone, nandrolone and epinandrolone, were chromatographically base-line separated. The limit of detection and that of quantification was 50 pg/ml for most of the AASs, and the limit of confirmation was 100-500 pg/ml. Full product ion spectra of AASs at concentrations as low as 100-500 pg/ml in equine plasma were obtained using the triple quadrupole instrument, to provide complementary evidentiary data for confirmation. The method is sensitive and selective for the detection, quantification and confirmation of multiple AASs in a single analysis and will be useful in the fight against doping of racehorses with AASs.
Copyright © 2010 John Wiley & Sons, Ltd.
Publication Date: 2010-09-25 PubMed ID: 20872903DOI: 10.1002/jms.1816Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The study introduces a precise method using ultra high-performance liquid chromatography-mass spectrometry (UHPLC-MS) for detecting, measuring, and confirming the presence of 55 varieties of anabolic and androgenic steroids in horse blood. These steroids are synthetics of the male hormone testosterone, typically exploited in human and equine sports. The research contributes to enforcing restrictions on the use of these steroids in horse-racing, through providing a detailed process of analysis.
Anabolic and Androgenic Steroids in Equine Sports
- This research focuses on the detection of Anabolic and Androgenic Steroids (AASs), synthetic substances associated with testosterone, the primary male sex hormone.
- These steroids can be misused in human performances and horse racing and are strictly prohibited by the International Olympic Committee and the Association of Racing Commissioners International (ARCI).
- A legitimate way of enforcing the prohibition of AASs in horse racing is to formulate a robust method to detect these substances efficiently.
The UHPLC-MS/MS Method
- A high-throughput ultra high-performance liquid chromatography-mass spectrometric (UHPLC-MS/MS) technique was developed to detect, quantify, and confirm 55 AASs in horse blood plasma.
- The detection of these steroids was via a chosen-reaction tracking approach on a triple quadrupole mass spectrometer.
- AASs with matching precursor ions were either differentiated chromatographically or distinguished by unique precursor-to-product ion transitions. Some were identified by the intensity ratios of three major product ions.
Chromatographic Separation and Differentiation
- All the pairs of AAS’s mentioned, for example, testosterone and epitestosterone, were completely separated by chromatography.
- The detection and quantification limit was 50 pg/ml for most AASs. The limit of validation ranged from 100-500 pg/ml.
- The method developed is both sensitive and selective, allowing for the identification, quantification, and validation of multiple AASs in a single analysis.
- This study’s findings could significantly aid in combating the illegal administration of AASs in horse racing.
Cite This Article
APA
Guan F, Uboh CE, Soma LR, You Y, Liu Y, Li X.
(2010).
High-throughput UHPLC-MS/MS method for the detection, quantification and identification of fifty-five anabolic and androgenic steroids in equine plasma.
J Mass Spectrom, 45(11), 1270-1279.
https://doi.org/10.1002/jms.1816 Publication
Researcher Affiliations
- School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, 382 West Street Road, Kennett Square, PA 19348, USA.
MeSH Terms
- Anabolic Agents / blood
- Anabolic Agents / chemistry
- Animals
- Chromatography, High Pressure Liquid / methods
- Doping in Sports
- High-Throughput Screening Assays / methods
- Horses / blood
- Isomerism
- Reproducibility of Results
- Sensitivity and Specificity
- Steroids / blood
- Steroids / chemistry
- Tandem Mass Spectrometry / methods
Citations
This article has been cited 4 times.- Behairy A, Mohamed WAM, Ebraheim LLM, Soliman MM, Abd-Elhakim YM, El-Sharkawy NI, Saber TM, El Deib MM. Boldenone Undecylenate-Mediated Hepatorenal Impairment by Oxidative Damage and Dysregulation of Heat Shock Protein 90 and Androgen Receptors Expressions: Vitamin C Preventive Role. Front Pharmacol 2021;12:651497.
- Dornelles GL, Bueno A, de Oliveira JS, da Silva AS, França RT, da Silva CB, Machado MS, Petry LD, Abdalla FH, Lhamas CL, de Andrade CM. Biochemical and oxidative stress markers in the liver and kidneys of rats submitted to different protocols of anabolic steroids. Mol Cell Biochem 2017 Jan;425(1-2):181-189.
- Musharraf SG, Arfeen QU, Mazhar W, Kanwal N. A validated stability-indicating TLC-densitometric method for the determination of stanozolol in pharmaceutical formulations. Chem Cent J 2013 Aug 27;7(1):142.
- Matraszek-Żuchowska I, Kłopot A, Grzelak J, Zdonek P. Determination of clostebol residues in the urine of slaughter animals using liquid chromatography-tandem mass spectrometry. J Vet Res 2024 Dec;68(4):611-621.
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