Investigations into the feasibility of routine ultra high performance liquid chromatography-tandem mass spectrometry analysis of equine hair samples for detecting the misuse of anabolic steroids, anabolic steroid esters and related compounds.
Abstract: The detection of the abuse of anabolic steroids in equine sport is complicated by the endogenous nature of some of the abused steroids, such as testosterone and nandrolone. These steroids are commonly administered as intramuscular injections of esterified forms of the steroid, which prolongs their effects and improves bioavailability over oral dosing. The successful detection of an intact anabolic steroid ester therefore provides unequivocal proof of an illegal administration, as esterified forms are not found endogenously. Detection of intact anabolic steroid esters is possible in plasma samples but not, to date, in the traditional doping control matrix of urine. The analysis of equine mane hair for the detection of anabolic steroid esters has the potential to greatly extend the time period over which detection of abuse can be monitored. Equine mane hair samples were incubated in 0.1M phosphate buffer (pH 9.5) before anabolic steroids (testosterone, nandrolone, boldenone, trenbolone and stanozolol), anabolic steroid esters (esters of testosterone, nandrolone, boldenone and trenbolone) and associated compounds (fluticasone propionate and esters of hydroxyprogesterone) were extracted by liquid-liquid extraction with a mix of hexane and ethyl acetate (7:3, v:v). Further sample clean up by solid phase extraction was followed by derivatisation with methoxylamine HCL and analysis by UHPLC-MS/MS. Initial method development was performed on a representative suite of four testosterone esters (propionate, phenylpropionate, isocaproate and decanoate) and the method was later extended to include a further 18 compounds. The applicability of the method was demonstrated by the analysis of mane hair samples collected following the intramuscular administration of 500 mg of Durateston(®) (mixed testosterone esters) to a Thoroughbred mare (560 kg). The method was subsequently used to successfully detect boldenone undecylenate and stanozolol in hair samples collected following suspicious screening findings from post-race urine samples. The use of segmental analysis to potentially provide additional information on the timing of administration was also investigated.
Copyright © 2013 Elsevier B.V. All rights reserved.
Publication Date: 2013-06-10 PubMed ID: 23830435DOI: 10.1016/j.aca.2013.05.058Google 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
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
This research primarily focuses on assembling an effective method to detect the illegal administration of anabolic steroids and their esters in equine sports, using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to analyze horse mane hair samples.
Background and Research Objective
- The research addresses the challenge of detecting illicit use of anabolic steroids in horse racing; this is complicated by some steroids like testosterone and nandrolone, being naturally occurring or endogenous.
- Anabolic steroids are typically injected in an esterified form which enhances their effects and bioavailability compared to oral intake.
- The detection of an intact anabolic steroid ester in the system implies an illegal administration since these esterified forms do not occur naturally.
- While steroid esters can be detected in plasma samples, conventional methods using urine samples have not been successful.
- The key objective of this research was to analyze horse mane hair to detect the presence of anabolic steroid esters, potentially extending the window of detection for steroid abuse.
Methodology
- Equine mane hair samples were soaked in a particular buffer and the steroids, their esters, and related compounds were extracted through liquid-liquid extraction with a mix of hexane and ethyl acetate.
- Sample purification was conducted through solid phase extraction, followed by derivatisation with methoxylamine HCL for UHPLC-MS/MS analysis.
- This process was initially undertaken for four derivatives of testosterone and was later extended for an additional eighteen compounds.
Application and Findings
- The method’s effectiveness was tested by analyzing mane hair samples from a Thoroughbred mare given a 500mg dose of a mixed testosterone ester.
- Through this method, researchers successfully detected boldenone undecylenate and stanozolol in samples collected from suspicious screening results of post-race urine samples.
- The research also considered the use of segmental analysis for additional information about the timing of the steroid’s administration.
The study has successfully demonstrated the potential of using ultra high performance liquid chromatography-tandem mass spectrometry for analysing hair samples, as a feasible approach for detecting misuse of anabolic steroids, anabolic steroid esters and related compounds in equine sports.
Cite This Article
APA
Gray BP, Viljanto M, Bright J, Pearce C, Maynard S.
(2013).
Investigations into the feasibility of routine ultra high performance liquid chromatography-tandem mass spectrometry analysis of equine hair samples for detecting the misuse of anabolic steroids, anabolic steroid esters and related compounds.
Anal Chim Acta, 787, 163-172.
https://doi.org/10.1016/j.aca.2013.05.058 Publication
Researcher Affiliations
- HFL Sport Science, LGC, Fordham, Cambridgeshire, UK. bgray@hfl.co.uk
MeSH Terms
- Anabolic Agents / analysis
- Animals
- Chromatography, High Pressure Liquid / methods
- Doping in Sports
- Esters
- Feasibility Studies
- Female
- Hair / chemistry
- Horses
- Male
- Steroids / analysis
- Tandem Mass Spectrometry / methods
- Testosterone / analysis
Citations
This article has been cited 5 times.- Koutsoumanis K, Allende A, Alvarez-Ordóñez A, Bolton D, Bover-Cid S, Chemaly M, Davies R, De Cesare A, Herman L, Lindqvist R, Nauta M, Peixe L, Ru G, Simmons M, Skandamis P, Suffredini E, Sánchez JÁ, Blagojevic B, Fürst P, Garin-Bastuji B, Jensen HE, Paulsen P, Baert K, Barrucci F, Broglia A, Georgiadis M, Hempen M, Hilbert F. Evaluation of public and animal health risks in case of a delayed post-mortem inspection in ungulates.. EFSA J 2020 Dec;18(12):e06307.
- Matraszek-Żuchowska I, Woźniak B, Sielska K, Posyniak A. Determination of Steroid Esters in Hair of Slaughter Animals by Liquid Chromatography with Tandem Mass Spectrometry.. J Vet Res 2019 Dec;63(4):561-572.
- Fabresse N, Grassin-Delyle S, Etting I, Alvarez JC. Detection and quantification of 12 anabolic steroids and analogs in human whole blood and 20 in hair using LC-HRMS/MS: application to real cases.. Int J Legal Med 2017 Jul;131(4):989-999.
- Moon JY, Choi MH, Kim J. Metabolic profiling of cholesterol and sex steroid hormones to monitor urological diseases.. Endocr Relat Cancer 2016 Oct;23(10):R455-67.
- Madry MM, Spycher BS, Kupper J, Fuerst A, Baumgartner MR, Kraemer T, Naegeli H. Long-term monitoring of opioid, sedative and anti-inflammatory drugs in horse hair using a selective and sensitive LC-MS/MS procedure.. BMC Vet Res 2016 Jun 1;12:84.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
