Application of testosterone to epitestosterone ratio to horse urine – a complementary approach to detect the administrations of testosterone and its pro-drugs in Thoroughbred geldings.
Abstract: Detection of testosterone and/or its pro-drugs in the gelding is currently regulated by the application of an international threshold for urinary testosterone of 20 ng/mL. The use of steroid ratios may provide a useful supplementary approach to aid in differentiating between the administration of these steroids and unusual physiological conditions that may result in atypically high testosterone concentrations. In the current study, an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed to quantify testosterone (T) and epitestosterone (E). The method was used to analyze 200 post-race urine samples from geldings in order to generate the ratios for the reference population. Following statistical analysis of the data, an upper limit of 5 for T:E ratio in geldings is proposed. Samples collected from 15 geldings with atypical urinary testosterone concentrations (>15 ng/mL) but otherwise normal steroid profile, had T:E ratios within those observed for the reference population. The applicability of an upper T:E ratio to detect an administration was demonstrated by the analysis of a selection of incurred samples from testosterone propionate, dehydroepiandrosterone (DHEA), and a mixture of DHEA and pregnenolone (Equi-Bolic®) administrations. These produced testosterone concentrations above the threshold of 20 ng/mL, but also T:E ratios above the proposed limit of 5. In conclusion, consideration of the T:E ratio appears to be a valuable complementary aid to evaluate whether an atypical testosterone concentration could be caused by a natural biological outlier as opposed to the administration of these steroids. Copyright © 2016 John Wiley & Sons, Ltd.
Copyright © 2016 John Wiley & Sons, Ltd.
Publication Date: 2016-11-11 PubMed ID: 27717154DOI: 10.1002/dta.2109Google Scholar: Lookup
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- Journal Article
- Analytical Methods
- Diagnosis
- Diagnostic Technique
- Disease Diagnosis
- Equine Diseases
- Equine Health
- Equine Research
- Equine Science
- Geldings
- High-performance Liquid Chromatography (HPLC)
- Horse Management
- Laboratory Methods
- Pharmacology
- Steroids
- Testosterone
- Urine Analysis
- Veterinary Care
- Veterinary Medicine
- Veterinary Research
Summary
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The researchers developed a technique to detect unnatural levels of testosterone and its pro-drugs in racing horses by using a ratio of testosterone to epitestosterone, facilitating differentiation between natural biological outliers and the doping of horses.
Technique Development
- The study first developed a method to quantify testosterone (T) and epitestosterone (E) in horse urine samples, using ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS).
- This technique was tested on 200 post-race urine samples from geldings to create reference T:E ratios.
- Statistical analysis of this data generated an upper limit of 5 for the T:E ratio in geldings.
Application Test
- The team then applied their method to urine samples collected from 15 geldings with higher than typical levels of urinary testosterone (>15 ng/mL) but otherwise typical steroid profiles.
- These yielded T:E ratios within the range observed for the control population, suggesting that the elevated testosterone levels were within normal physiological bounds rather than a result of testosterone administration.
Test of Additional Steroids
- Finally, the applicability of the method to detect steroid administration was tested using urine samples from horses given testosterone propionate, dehydroepiandrosterone (DHEA), and a mixture of DHEA and pregnenolone (Equi-Bolic®).
- Urine from these horses showed both testosterone concentrations above the existing threshold (20 ng/mL) and T:E ratios above the newly proposed limit of 5, suggesting steroid administration.
Conclusion
- The researchers conclude that examining the T:E ratio in horse urine samples could be an effective supplementary technique to determine whether elevated testosterone levels are due to natural variability or the administration of testosterone or its pro-drugs.
Cite This Article
APA
Viljanto M, Scarth J, Hincks P, Hillyer L, Cawley A, Suann C, Noble G, Walker CJ, Kicman AT, Parkin MC.
(2016).
Application of testosterone to epitestosterone ratio to horse urine – a complementary approach to detect the administrations of testosterone and its pro-drugs in Thoroughbred geldings.
Drug Test Anal, 9(9), 1328-1336.
https://doi.org/10.1002/dta.2109 Publication
Researcher Affiliations
- LGC, Newmarket Road, Fordham, Cambridgeshire, CB7 5WW, UK.
- Drug Control Centre, Analytical and Environmental Sciences Research Divisions, King's College London, 150 Stamford Street, London, SE1 9NH, UK.
- LGC, Newmarket Road, Fordham, Cambridgeshire, CB7 5WW, UK.
- LGC, Newmarket Road, Fordham, Cambridgeshire, CB7 5WW, UK.
- British Horseracing Authority, 75 High Holborn, London, WC1V 6LS, UK.
- Racing NSW, Level 11, 51 Druitt Street, Sydney, NSW, 2000, Australia.
- Racing NSW, Level 11, 51 Druitt Street, Sydney, NSW, 2000, Australia.
- School of Animal and Veterinary Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, NSW, 2678, Australia.
- Drug Control Centre, Analytical and Environmental Sciences Research Divisions, King's College London, 150 Stamford Street, London, SE1 9NH, UK.
- Drug Control Centre, Analytical and Environmental Sciences Research Divisions, King's College London, 150 Stamford Street, London, SE1 9NH, UK.
- Drug Control Centre, Analytical and Environmental Sciences Research Divisions, King's College London, 150 Stamford Street, London, SE1 9NH, UK.
MeSH Terms
- Animals
- Body Fluids / chemistry
- Chromatography, Liquid
- Dehydroepiandrosterone / analysis
- Dehydroepiandrosterone / urine
- Doping in Sports / statistics & numerical data
- Epitestosterone / analysis
- Epitestosterone / urine
- Horses
- Humans
- Prodrugs
- Steroids / analysis
- Steroids / urine
- Substance Abuse Detection
- Tandem Mass Spectrometry / methods
- Testosterone / analysis
- Testosterone / urine
Citations
This article has been cited 1 times.- Bhambri A, Srivastava M, Mahale VG, Mahale S, Karn SK. Mushrooms as Potential Sources of Active Metabolites and Medicines. Front Microbiol 2022;13:837266.
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