Detection of anabolic and androgenic steroids and/or their esters in horse hair using ultra-high performance liquid chromatography-high resolution mass spectrometry.
Abstract: Anabolic and androgenic steroids (AASs) are a class of prohibited substances banned in horseracing at all times. The common approach for controlling the misuse of AASs in equine sports is by detecting the presence of AASs and/or their metabolites in urine and blood samples using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). This approach, however, often falls short as the duration of effect for many AASs are longer than their detection time in both urine and blood. As a result, there is a high risk that such AASs could escape detection in their official race-day samples although they may have been used during the long period of training. Hair analysis, on the other hand, can afford significantly longer detection windows. In addition, the identification of synthetic ester derivatives of AASs in hair, particularly for the endogenous ones, can provide unequivocal proof of their exogenous origin. This paper describes the development of a sensitive method (at sub to low parts-per-billion or ppb levels) for detecting 48 AASs and/or their esters in horse hair using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). Decontaminated horse hair was pulverised and subjected to in-situ liquid-liquid extraction in a mixture of hexane - ethyl acetate (7:3, v/v) and phosphate buffer (0.1M, pH 9.5), followed by additional clean-up using mixed-mode solid-phase extraction. The final extract was analysed using UHPLC-HRMS in the positive electrospray ionisation (ESI) mode with both full scan and parallel reaction monitoring (PRM). This method was validated for qualitative identification purposes. Validation data, including method specificity, method sensitivity, extraction recovery, method precision and matrix effect are presented. Method applicability was demonstrated by the successful detection and confirmation of testosterone propionate in a referee hair sample. To our knowledge, this was the first report of a comprehensive screening method for detecting as many as 48 AASs and/or their esters in horse hair. Moreover, retrospective analysis of non-targeted AASs and/or their esters was made feasible by re-examining the full scan UHPLC-HRMS data acquired.
Copyright © 2017 Elsevier B.V. All rights reserved.
Publication Date: 2017-03-06 PubMed ID: 28314523DOI: 10.1016/j.chroma.2017.03.007Google Scholar: Lookup
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Summary
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The article is about a more effective technique to detect the use of banned anabolic and androgenic steroids in racehorses through analysis of horse hair using ultra-high performance liquid chromatography-high resolution mass spectrometry.
Introduction to Anabolic Androgenic Steroids
- Anabolic and androgenic steroids (AASs) are performance-enhancing drugs commonly misused in sports, including horse racing.
- Currently, the detection of these substances relies largely on the analysis of urine and blood samples using methods like gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS).
- However, because these substances can stay effective longer than they are detectable in these biological fluids, there’s a chance misconduct goes undetected, especially during the long periods of training leading up to races.
Approach via Hair Analysis
- The paper proposes a more reliable way of detecting AASs, by analyzing steroidal substances or their synthetic ester derivates present in horse hair.
- Hair tests can detect the presence of these substances over a longer period, thus circumventing the limitations inherent in blood and urine tests.
- Moreover, the detection of synthetic ester derivatives can provide indisputable evidence of the external origin of the drug, erasing any claim of its natural presence in the body.
“Ultra-high Performance Liquid Chromatography-high Resolution Mass Spectrometry” Method
- The new method involves the use of ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) to detect and identify up to 48 types of AASs or their esters in horse hair.
- To carry out the tests, the horse hair is first decontaminated, pulverized, and then subjected to ‘in situ’ liquid-liquid extraction before further clean-up via the solid-phase extraction process.
- The final extract is analyzed using UHPLC-HRMS in the positive electrospray ionization (ESI) mode, employing both full scan and parallel reaction monitoring (PRM).
- The method’s applicability was validated by successfully identifying testosterone propionate in a referee hair sample.
- This approach also allows for retrospective tests for detecting non-targeted AASs and/or their esters by reexamining the full scan UHPLC-HRMS data acquired.
Conclusion
- According to the researchers’ knowledge, this is the first report of a comprehensive screening method that can detect as many as 48 AASs and/or their esters in horse hair.
- The proposed method could represent a significant stride forward in the fight against doping in horse racing.
Cite This Article
APA
Kwok KY, Choi TLS, Kwok WH, Wong JKY, Wan TSM.
(2017).
Detection of anabolic and androgenic steroids and/or their esters in horse hair using ultra-high performance liquid chromatography-high resolution mass spectrometry.
J Chromatogr A, 1493, 76-86.
https://doi.org/10.1016/j.chroma.2017.03.007 Publication
Researcher Affiliations
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China. Electronic address: karen.y.kwok@hkjc.org.hk.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China. Electronic address: terence.sm.wan@hkjc.org.hk.
MeSH Terms
- Anabolic Agents / analysis
- Anabolic Agents / chemistry
- Androgens / analysis
- Androgens / chemistry
- Animals
- Chromatography, High Pressure Liquid
- Doping in Sports / prevention & control
- Esters / analysis
- Esters / chemistry
- Gas Chromatography-Mass Spectrometry
- Hair / chemistry
- Horses
- Liquid-Liquid Extraction
- Mass Spectrometry
- Reference Standards
- Retrospective Studies
- Solid Phase Extraction
- Steroids / analysis
- Steroids / chemistry
- Testosterone Propionate / analysis
Citations
This article has been cited 5 times.- Leise JM, Leatherwood JL, Paris BL, Walter KW, George JM, Martinez RE, Glass KP, Lo CP, Mays TP, Wickersham TA. Evaluation of an Oral Supplemental Cannabidiol Product for Acceptability and Performance in Mature Horses. Animals (Basel) 2023 Jan 10;13(2).
- Haynes AZ, Levine M. Detection of anabolic steroids via cyclodextrin-promoted fluorescence modulation. RSC Adv 2020 Jun 29;10(42):25108-25115.
- Chan WS, Wong GF, Hung CW, Wong YN, Fung KM, Lee WK, Dao KL, Leung CW, Lo KM, Lee WM, Cheung BK. Interpol review of toxicology 2016-2019. Forensic Sci Int Synerg 2020;2:563-607.
- Katahira T, Kanazawa A, Shinohara M, Koshibu M, Kaga H, Mita T, Tosaka Y, Komiya K, Miyatsuka T, Ikeda F, Azuma K, Takayanagi N, Ogihara T, Ohmura C, Miyachi A, Mieno E, Yamashita S, Watada H. Postprandial Plasma Glucagon Kinetics in Type 2 Diabetes Mellitus: Comparison of Immunoassay and Mass Spectrometry. J Endocr Soc 2019 Jan 1;3(1):42-51.
- Puscasiu D, Flangea C, Vlad D, Popescu R, Vlad CS, Barac S, Rata AL, Marina C, Cobec IM, Laitin SMD. Adulteration of Sports Supplements with Anabolic Steroids-From Innocent Athlete to Vicious Cheater. Nutrients 2025 Oct 1;17(19).
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