Anabolic agents: recent strategies for their detection and protection from inadvertent doping

Hans Geyer, Wilhelm Schänzer, Mario Thevis, Hans Geyer, Wilhelm Schänzer, Mario Thevis

Abstract

According to the World Anti-Doping Agency (WADA) Prohibited List, anabolic agents consist of exogenous anabolic androgenic steroids (AAS), endogenous AAS and other anabolic agents such as clenbuterol and selective androgen receptor modulators (SARMs). Currently employed strategies for their improved detection include the prolongation of the detection windows for exogenous AAS, non-targeted and indirect analytical approaches for the detection of modified steroids (designer steroids), the athlete's biological passport and isotope ratio mass spectrometry for the detection of the misuse of endogenous AAS, as well as preventive doping research for the detection of SARMs. The recent use of these strategies led to 4-80-fold increases of adverse analytical findings for exogenous AAS, to the detection of the misuse of new designer steroids, to adverse analytical findings of different endogenous AAS and to the first adverse analytical findings of SARMs. The strategies of the antidoping research are not only focused on the development of methods to catch the cheating athlete but also to protect the clean athlete from inadvertent doping. Within the past few years several sources of inadvertent doping with anabolic agents have been identified. Among these are nutritional supplements adulterated with AAS, meat products contaminated with clenbuterol, mycotoxin (zearalenone) contamination leading to zeranol findings, and natural products containing endogenous AAS. The protection strategy consists of further investigations in case of reasonable suspicion of inadvertent doping, publication of the results, education of athletes and development of methods to differentiate between intentional and unintentional doping.

Keywords: Anabolic steroids; Doping; Drug control.

Figures

Figure 1
Figure 1
Adverse analytical findings and atypical findings of all World Anti-Doping Agency (WADA) accredited laboratories in 2012 reported via Anti-Doping Administration and Management System (ADAMS).
Figure 2
Figure 2
Adverse analytical findings for metandienone in the World Anti-Doping Agency (WADA) accredited laboratory Cologne before and after the prolongation of the detection window for metandienone by the implementation of a new long-term metabolite in the screening procedure in January 2006.
Figure 3
Figure 3
Adverse analytical findings for dehydrochloromethyltestosterone in the World Anti-Doping Agency (WADA) accredited laboratory Cologne before and after the prolongation of the detection window for dehydrochloromethyltestosterone by the implementation of three new long-term metabolites in December 2012 in the screening procedure (status from 1 December 2013).
Figure 4
Figure 4
Adverse analytical findings for stanozolol in the World Anti-Doping Agency (WADA) accredited laboratory Cologne before and after the prolongation of the detection window for stanozolol by the use of high-resolution mass spectrometry in combination with the implementation of old and new stanozolol metabolites and a new long-term metabolite in December 2012 (status from 1 December 2013).
Figure 5
Figure 5
Non-targeted analysis (precursor ion scan): screening for a common fragment of different steroids (m/z 241).
Figure 6
Figure 6
Chemical structures of selected selective androgen receptor modulator drug candidates: andarine (A), ostarine (B), BMS-564929 (C), 4-(7-hydroxy-1,3-dioxo-tetrahydro-pyrrolo[1,2-c]imidazol-2-yl)-naphthalene-1-carbonitrile (D), LGD-121071 (E), LGD-2226 (F), S-40503 (G), 2-methyl-2-(8-nitro-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]chinolin-4-yl)propan-1-ol (H), RAD140 (I), and ACP-105 (J).
Figure 7
Figure 7
Steroids detected in a musk pod sample.

References

    1. WADA. 2012 Anti-Doping Testing Figures Report. World Anti-Doping Agency, 2013
    1. WADA. The 2014 prohibited list. Montreal: World Anti-Doping Agency, 2014
    1. Schänzer W, Delahaut P, Geyer H, et al. Long-term detection and identification of metandienone and stanozolol abuse in athletes by gas chromatography-high-resolution mass spectrometry. J Chromatogr B 1996;687:93–108
    1. Schänzer W, Horning S, Opfermann G, et al. Gas chromatography/mass spectrometry identification of long-term excreted metabolites of the anabolic steroid 4-chloro-1,2-dehydro-17alpha-methyltestosterone in humans. J Steroid Biochem Mol Biol 1996;57:363–76
    1. Fußhöller G, Mareck U, Schmechel A, et al. Long-term detection of metandienone abuse by means of the new metabolite 17ß-hydroxymethyl-17α-methyl-18-norandrost-1,4,13-trien-3-one. In: Schänzer W, Geyer H, Gotzmann A, Mareck U.eds Recent advances in doping analysis. Cologne: Sportverlag Strauss, 2007:15:393–6
    1. Sobolevsky T, Rodchenkov G. Detection and mass spectrometric characterization of novel long-term dehydrochloromethyltestosterone metabolites in human urine. J Steroid Biochem Molec Biol 2012;128:121–7
    1. Sobolevsky T, Rodchenkov G. Mass spectrometric description of novel oxymetholone and desoxymethyltestosterone metabolites identified in human urine and their importance for doping control. Drug Test Anal 2012;4:682–91
    1. Lootens L, Meuleman P, Pozo OJ, et al. uPA+/+-SCID mouse with humanized liver as a model for in vivo metabolism of exogenous steroids: methandienone as a case study. Clin Chem 2009;55:1783–93
    1. Lootens L, Van Eenoo P, Meuleman P, et al. Steroid metabolism in chimeric mice with humanized liver. Drug Test Anal 2009;1:531–7
    1. Thevis M, Piper T, Horning S, et al. Hydrogen isotope ratio mass spectrometry and high-resolution/high-accuracy mass spectrometry in metabolite identification studies: detecting target compounds for sports drug testing. Rapid Commun Mass Spectrom 2013;27:1904–12
    1. Delbeke FT, Van Eenoo P, Van Thuyne W, et al. Prohormones and sport. J Steroid Biochem Molec Biol 2002;83:245–51
    1. Catlin DH, Sekera MH, Ahrens BD, et al. Tetrahydrogestrinone: discovery, synthesis, and detection in urine. Rapid Commun Mass Spectrom 2004;18:1245–9
    1. Geyer H, Parr MK, Koehler K, et al. Nutritional supplements cross-contaminated and faked with doping substances. J Mass Spectrom 2008;43:892–902
    1. Thevis M, Geyer H, Mareck U, et al. Screening for unknown synthetic steroids in human urine by liquid chromatography-tandem mass spectrometry. J Mass Spectrom 2005;40:955–62
    1. Thevis M, Opfermann G, Bommerich U, et al. Characterization of chemically modified steroids for doping control purposes by electrospray ionization tandem mass spectrometry. J Mass Spectrom 2005;40:494–502
    1. Williams TM, Kind AJ, Houghton E, et al. Electrospray collision-induced dissociation of testosterone and testosterone hydroxy analogs. J Mass Spectrom 1999;34:206–16
    1. Pozo OJ, Van Eenoo P, Deventer K, et al. Collision-induced dissociation of 3-keto anabolic steroids and related compounds after electrospray ionization. Considerations for structural elucidation. Rapid Commun Mass Spectrom 2008;22:409–24
    1. Thevis M, Beuck S, Höppner S, et al. Structure elucidation of the diagnostic product ion at m/z 97 derived from androst-4-en-3-one-based steroids by ESI-CID and IRMPD spectroscopy. J Am Soc Mass Spectrom 2012;23:537–46
    1. Thevis M, Guddat S, Schänzer W. Doping control analysis of trenbolone and related compounds using liquid chromatography-tandem mass spectrometry. Steroids 2009;74:315–21
    1. Thevis M, Schänzer W. Mass spectrometric analysis of androstan-17beta-ol-3-one and androstadiene-17beta-ol-3-one isomers. J Am Soc Mass Spectrom 2005;16:1660–9
    1. Thevis M, Schänzer W. Mass spectrometry in sports drug testing: structure characterization and analytical assays. Mass Spectrom Rev 2007;26:79–107
    1. AAP. Greek weightlifters banned from Beijing Olympics. Perth now. Perth: Australian Associated Press, 2008
    1. Pozo OJ, Deventer K, Eenoo PV, et al. Efficient approach for the comprehensive detection of unknown anabolic steroids and metabolites in human urine by liquid chromatography-electrospray-tandem mass spectrometry. Anal Chem 2008;80:1709–20
    1. Schänzer W, Guddat S, Thomas A, et al. Expanding analytical possibilities concerning the detection of stanozolol misuse by means of high resolution/high accuracy mass spectrometric detection of stanozolol glucuronides in human sports drug testing. Drug Test Anal 2013;5:810–18
    1. Donike M, Geyer H, Schänzer W, et al. Die Suppression der endogenen Androgenproduktion durch Metandienon. In: Franz I-W, Mellerowicz H, Noack W.eds Training und Sport zur Prävention und Rehabilitation in der technisierten Umwelt. Berlin, Heiderlberg, New York: Springer Verlag, 1985:508
    1. Donike M, Geyer H, Kraft M, et al. Longterm influence of anabolic steroid misuse on the steroid profile, in Official Proceedings of the IInd I.A.F. Belotti P, Benzi G, Ljungqvist A.eds World symposium on doping in sport. Monte Carlo: International Athletic Foundation, 1989:107–16
    1. Catlin DH, Ahrens BD, Kucherova Y. Detection of norbolethone, an anabolic steroid never marketed, in athleteś urine. Rapid Commun Mass Spectrom 2002;16:1273–5
    1. Donike M, Bärwald KR, Klostermann K, et al. The detection of exogenous testosterone. In: Heck H, Hollmann W, Liesen H.eds Leistung und Gesundheit, Kongressbd, Dtsch. Sportärztekongress. Köln: Deutscher Ärtze-Verlag, 1983:293–8
    1. Donike M, Zimmermann J, Bärwald KR, et al. Routinebestimmung von Anabolika in Harn. Dtsch Z Sportmed 1984;35:14–24
    1. Donike M, Rauth S, Mareck-Engelke U, et al. Evaluation of longitudinal studies: the determination of subject-based reference ranges of the testosterone/epitestosterone ratio. In: Geyer H, Gotzmann A, Mareck-Engelke U, Rauth S.eds Recent advances in doping analysis. Cologne: Sport&Buch Strauss, 1993:33–9
    1. Geyer H, Mareck U, Schänzer W, et al. The Cologne protocol to follow up high testosterone/epitestosterone ratios. In: Schänzer W, Geyer H, Gotzmann A, Mareck U.eds Recent advances in doping analysis. Cologne: Sport und Buch Strauss, 1997;4:107–26
    1. Donike M, Ueki M, Kuroda Y, et al. Detection of dihydrotestosterone (DHT) doping: alterations in the steroid profile and reference ranges for DHT and its 5 alpha-metabolites. J Sports Med Phys Fitness 1995;35:235–50
    1. Sottas PE, Saugy M, Saudan C. Endogenous steroid profiling in the athlete biological passport. Endocrinol Metab Clinics North Am 2010;39:59–73, viii-ix
    1. Sottas PE, Baume N, Saudan C, et al. Bayesian detection of abnormal values in longitudinal biomarkers with an application to T/E ratio. Biostatistics 2007;8:285–96
    1. Mareck U, Geyer H, Opfermann G, et al. Factors influencing the steroid profile in doping control analysis. J Mass Spectrom 2008;43:877–91
    1. Van Renterghem P, Van Eenoo P, Van Thuyne W, et al. Validation of an extended method for the detection of the misuse of endogenous steroids in sports, including new hydroxylated metabolites. J Chromatogr B 2008;876:225–35
    1. Van Renterghem P, Van Eenoo P, Geyer H, et al. Reference ranges for urinary concentrations and ratios of endogenous steroids, which can be used as markers for steroid misuse, in a Caucasian population of athletes. Steroids 2010;75:154–63
    1. Piper T, Emery C, Saugy M. Recent developments in the use of isotope ratio mass spectrometry in sports drug testing. Anal Bioanal Chem 2011;401:433–47
    1. Becchi M, Aguilera R, Farizon Y, et al. Gas chromatography/combustion/isotope-ratio mass spectrometry analysis of urinary steroids to detect misuse of testosterone in sport. Rapid Commun Mass Spectrom 1994;8:304–8
    1. Dalton JT, Mukherjee A, Zhu Z, et al. Discovery of nonsteroidal androgens. Biochem Biophys Res Commun 1998;244:1–4
    1. Dalton JT, Taylor RP, Mohler ML, et al. Selective androgen receptor modulators for the prevention and treatment of muscle wasting associated with cancer. Curr Opin Support Palliat Care 2013;7:345–51
    1. Thevis M, Kamber M, Schänzer W. Screening for metabolically stable aryl-propionamide-derived selective androgen receptor modulators for doping control purposes. Rapid Commun Mass Spectrom 2006;20:870–6
    1. Thevis M, Schänzer W. Mass spectrometry of selective androgen receptor modulators. J Mass Spectrom 2008;43:865–76
    1. Thevis M, Piper T, Beuck S, et al. Expanding sports drug testing assays: mass spectrometric characterization of the selective androgen receptor modulator drug candidates RAD140 and ACP-105. Rapid Commun Mass Spectrom 2013;27:1173–82
    1. Thevis M, Gerace E, Thomas A, et al. Characterization of in vitro generated metabolites of the selective androgen receptor modulators S-22 and S-23 and in vivo comparison to post-administration canine urine specimens. Drug Test Anal 2010;2:589–98
    1. Thevis M, Geyer H, Kamber M, et al. Detection of the arylpropionamide-derived selective androgen receptor modulator (SARM) S-4 (Andarine) in a black-market product. Drug Test Anal 2009;1:387–92
    1. Thevis M, Geyer H, Thomas A, et al. Trafficking of drug candidates relevant for sports drug testing: detection of non-approved therapeutics categorized as anabolic and gene doping agents in products distributed via the Internet. Drug Test Anal 2011;3:331–6
    1. Grata E, Perrenoud L, Saugy M, et al. SARM-S4 and metabolites detection in sports drug testing: a case report. Forensic Sci Int 2011;213:104–8
    1. Starcevic B, Ahrens BD, Butch AW. Detection of the selective androgen receptor modulator S-4 (Andarine) in a doping control sample. Drug Test Anal 2013;5:377–9
    1. Geyer H, Braun H, Burke LM, et al. Inadvertent doping. In: A-Z of nutritional supplements: dietary supplements, sports nutrition foods and ergogenic aids for health and performance—part 22. Br J Sports Med 2011;45:752–4
    1. Vernec A, Stear SJ, Burke LM, et al. A-Z of nutritional supplements: dietary supplements, sports nutrition foods and ergogenic aids for health and performance: part 48. Br J Sports Med 2013;47:998–1000
    1. Guddat S, Fusshöller G, Geyer H, et al. Clenbuterol—regional food contamination a possible source for inadvertent doping in sports. Drug Test Anal 2012;4:534–8
    1. Thevis M, Geyer L, Geyer H, et al. Adverse analytical findings with clenbuterol among U-17 soccer players attributed to food contamination issues. Drug Test Anal 2013;5:372–6
    1. Nicoli R, Petrou M, Badoud F, et al. Quantification of clenbuterol at trace level in human urine by ultra-high pressure liquid chromatography-tandem mass spectrometry. J Chromatogr A 2013;1292:142–50
    1. Thevis M, Thomas A, Beuck S, et al. Does the analysis of the enantiomeric composition of clenbuterol in human urine enable the differentiation of illicit clenbuterol administration from food contamination in sports drug testing? Rapid Commun Mass Spectrom 2013;27:507–12
    1. Segura J. Is anti-doping analysis so far from clinical, legal or forensic targets?: The added value of close relationships between related disciplines. Drug Test Anal 2009;1:479–84
    1. Thevis M, Fusshöller G, Schänzer W. Zeranol: doping offence or mycotoxin? A case-related study. Drug Test Anal 2011;3:777–83
    1. Thevis M, Schänzer W, Geyer H, et al. Traditional Chinese medicine and sports drug testing: identification of natural steroid administration in doping control urine samples resulting from musk (pod) extracts. Br J Sports Med 2013;47:109–14
    1. Schänzer W, Geyer H, Fusshöller G, et al. Mass spectrometric identification and characterization of a new long-term metabolite of metandienone in human urine. Rapid Comm Mass Spectrom 2006;20:2252–8
    1. Gomez C, Pozo OJ, Garrostas L, et al. A new sulphate metabolite as a long-term marker of metandienone misuse. Steroids 2013;78:1245–53
    1. Guddat S, Fusshöller G, Beuck S, et al. Synthesis, characterization, and detection of new oxandrolone metabolites as long-term markers in sports drug testing. Anal Bioanal Chem 2013;405:8285–94

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