Preprint / Version 1

Doping Agents In Sports

##article.authors##

  • ZhongYu Richard Xun Kristin School

DOI:

https://doi.org/10.58445/rars.1553

Keywords:

Chemistry, Analytical Chemistry Doping, Drug Detection, Anabolic Androgenic Steroids

Abstract

Even in 2024, doping in sports remains a significant concern, posing challenges regarding health, ethics, and equity. This paper provides an exploration of doping agents, with a focus on their chemistry, mechanisms of action and methods of their detection. The structures and modes-of-action of testosterone derivates are discussed, and the analytical methods of gas chromatography coupled with mass spectrometry, gas chromatography coupled with tandem mass spectrometry, gas chromatography/combustion/isotope ratio mass spectrometry, and liquid chromatography are introduced. The paper concludes with a discussion of ways in which users of doping agents attempt to avoid detection and introduces the emerging field of gene doping.

References

(1) Te Kahu Raunui. The truth about steroids. Sport Integrity Comission. https://sportintegrity.nz/news/steroids (accessed 2024-08-20).

(2) Chrysopoulos, P. Doping in Ancient Greece. October 21, 2022. https://greekreporter.com/2022/10/21/doping-sports-ancient-greece/ (accessed 2024-08-20).

(3) Dotson, J. L.; Brown, R. T. The History of the Development of Anabolic-Androgenic Steroids. Pediatr. Clin. North Am. 2007, 54 (4), 761–769. https://doi.org/10.1016/j.pcl.2007.04.003.

(4) Hoberman, J. M.; Yesalis, C. E. The History of Synthetic Testosterone. Sci. Am. 1995, 272 (2), 76–81. https://doi.org/10.1038/scientificamerican0295-76.

(5) Baum, Z. The science behind performance-enhancing drugs. CAS. https://www.cas.org/fr/resources/cas-insights/science-behind-performance-enhancing-drugs (accessed 2024-07-14).

(6) Trenton, A. J.; Currier, G. W. Behavioural Manifestations of Anabolic Steroid Use: CNS Drugs 2005, 19 (7), 571–595. https://doi.org/10.2165/00023210-200519070-00002.

(7) Blue, J. G.; Lombardo, J. A. STEROIDS AND STEROID-LIKE COMPOUNDS. Clin. Sports Med. 1999, 18 (3), 667–689. https://doi.org/10.1016/S0278-5919(05)70175-7.

(8) WADA. Anti-Doping Process. WADA. https://www.wada-ama.org/en/athletes-support-personnel/anti-doping-process (accessed 2024-07-25).

(9) American Addition Centers Editorial Staff. Effects of Steroids | Short & Long-Term Effects of Steroid Use. DrugAbuse.com. https://drugabuse.com/stimulants/steroids/effects-use/ (accessed 2024-05-01).

(10) Bond, P.; Smit, D. L.; de Ronde, W. Anabolic–Androgenic Steroids: How Do They Work and What Are the Risks? Front. Endocrinol. 2022, 13. https://doi.org/10.3389/fendo.2022.1059473.

(11) Handelsman, D. J. Androgen Physiology, Pharmacology, Use and Misuse. In Endotext; Feingold, K. R., Anawalt, B., Blackman, M. R., Boyce, A., Chrousos, G., Corpas, E., de Herder, W. W., Dhatariya, K., Dungan, K., Hofland, J., Kalra, S., Kaltsas, G., Kapoor, N., Koch, C., Kopp, P., Korbonits, M., Kovacs, C. S., Kuohung, W., Laferrère, B., Levy, M., McGee, E. A., McLachlan, R., New, M., Purnell, J., Sahay, R., Shah, A. S., Singer, F., Sperling, M. A., Stratakis, C. A., Trence, D. L., Wilson, D. P., Eds.; MDText.com, Inc.: South Dartmouth (MA), 2000.

(12) Tauchen, J.; Jurášek, M.; Huml, L.; Rimpelová, S. Medicinal Use of Testosterone and Related Steroids Revisited. Molecules 2021, 26 (4), 1032. https://doi.org/10.3390/molecules26041032.

(13) Davey, R. A.; Grossmann, M. Androgen Receptor Structure, Function and Biology: From Bench to Bedside. Clin. Biochem. Rev. 2016, 37 (1), 3–15.

(14) Bond, P.; Llewellyn, W.; Van Mol, P. Anabolic Androgenic Steroid-Induced Hepatotoxicity. Med. Hypotheses 2016, 93, 150–153. https://doi.org/10.1016/j.mehy.2016.06.004.

(15) Tan, M. E.; Li, J.; Xu, H. E.; Melcher, K.; Yong, E. Androgen Receptor: Structure, Role in Prostate Cancer and Drug Discovery. Acta Pharmacol. Sin. 2015, 36 (1), 3–23. https://doi.org/10.1038/aps.2014.18.

(16) Coffey, K.; Robson, C. N. Regulation of the Androgen Receptor by Post-Translational Modifications. J. Endocrinol. 2012, 215 (2), 221–237. https://doi.org/10.1530/JOE-12-0238.

(17) Pozo, O. J.; Eenoo, P. V.; Deventer, K.; Delbeke, F. T. Detection and Characterization of Anabolic Steroids in Doping Analysis by LC-MS. TrAC Trends Anal. Chem. 2008, 27 (8), 657–671. https://doi.org/10.1016/j.trac.2008.06.003.

(18) DNALegal. A Guide to Anabolic Steroids Drug Testing. DNALegal Helping you find the truth. https://www.dnalegal.com/anabolic-steroids-drug-testing (accessed 2024-07-18).

(19) Urine vs Saliva Drug Testing: Pros and Cons - Australia Drug Testing. https://www.australiadrugtesting.com/urine-vs-saliva-drug-testing/ (accessed 2024-08-23).

(20) Lewis, J. G. Steroid Analysis in Saliva: An Overview. Clin. Biochem. Rev. 2006, 27 (3), 139–146.

(21) Alquraini, H.; Auchus, R. J. Strategies That Athletes Use to Avoid Detection of Androgenic-Anabolic Steroid Doping and Sanctions. Mol. Cell. Endocrinol. 2018, 464, 28–33. https://doi.org/10.1016/j.mce.2017.01.028.

(22) Hübener, H. J. Steroid Alcohols in Urine. In Methods of Enzymatic Analysis; Elsevier, 1965; pp 485–490. https://doi.org/10.1016/B978-0-12-395630-9.50100-6.

(23) Trout, G. J.; Kazlauskas, R. Sports Drug Testing ? An Analyst’s Perspective. Chem. Soc. Rev. 2004, 33 (1), 1. https://doi.org/10.1039/b201476a.

(24) Krumm, B.; Botrè, F.; Saugy, J. J.; Faiss, R. Future Opportunities for the Athlete Biological Passport. Front. Sports Act. Living 2022, 4, 986875. https://doi.org/10.3389/fspor.2022.986875.

(25) Goldschmidt, H. Demystifying the Athlete Biological Passport. Squire Patton Boggs. https://www.sports.legal/2024/02/demystifying-the-athlete-biological-passport/?__cf_chl_tk=YXGj.ZlMdytoJR3hoEJl83BNPDd0M1PkXmpf6MjvBM0-1721347094-0.0.1.1-3966 (accessed 2024-07-19).

(26) De La Torre, X.; Iannone, M.; Botrè, F. Improving the Detection of Anabolic Steroid Esters in Human Serum by LC–MS. J. Pharm. Biomed. Anal. 2021, 194, 113807. https://doi.org/10.1016/j.jpba.2020.113807.

(27) Krieger, J. Intended. Underrated. Disputed. The IOC Medical Commission’s “Subcommission on Doping and Biochemistry in Sport” between 1980 and 1988. Perform. Enhanc. Health 2016, 4 (3–4), 88–93. https://doi.org/10.1016/j.peh.2016.03.002.

(28) UCLA. Gas Chromatography Theory. UCLA CHEM EDU. https://www.chem.ucla.edu/~bacher/General/30BL/gc/theory.html#:~:text=The%20mobile%20phase%20(%3Dcarrier,phase%20(%3Dhigh%20boiling%20polymer). (accessed 2024-07-21).

(29) Arruda, T. R.; Leite Junior, B. R. D. C.; Marques, C. S.; Bernardes, P. C.; Magalhães, C. G.; Pinheiro, P. F. Emerging Techniques for Extraction and Characterization of Natural Compounds. In Green Products in Food Safety; Elsevier, 2023; pp 29–79. https://doi.org/10.1016/B978-0-323-95590-4.00009-6.

(30) Tyagi, R.; Kumar, P.; Sharma, U. Metabolomics Techniques: A Brief Update. In Epigenetics and Metabolomics; Elsevier, 2021; pp 1–29. https://doi.org/10.1016/B978-0-323-85652-2.00007-5.

(31) Snow, N. Flying High with Sensitivity and Selectivity: GC–MS to GC–MS/MS. LCGC N. Am. 2021, 39 (2). https://doi.org/10.56530/lcgc.na.yn3065q6.

(32) Danaceau, J. P.; Scott Morrison, M.; Slawson, M. H. Quantitative Confirmation of Testosterone and Epitestosterone in Human Urine by LC/Q‐ToF Mass Spectrometry for Doping Control. J. Mass Spectrom. 2008, 43 (7), 993–1000. https://doi.org/10.1002/jms.1443.

(33) Fernández-Peralbo, M. A.; Luque De Castro, M. D. Preparation of Urine Samples Prior to Targeted or Untargeted Metabolomics Mass-Spectrometry Analysis. TrAC Trends Anal. Chem. 2012, 41, 75–85. https://doi.org/10.1016/j.trac.2012.08.011.

(34) Mischak, H.; Kolch, W.; Aivaliotis, M.; Bouyssié, D.; Court, M.; Dihazi, H.; Dihazi, G. H.; Franke, J.; Garin, J.; De Peredo, A. G.; Iphöfer, A.; Jänsch, L.; Lacroix, C.; Makridakis, M.; Masselon, C.; Metzger, J.; Monsarrat, B.; Mrug, M.; Norling, M.; Novak, J.; Pich, A.; Pitt, A.; Bongcam‐Rudloff, E.; Siwy, J.; Suzuki, H.; Thongboonkerd, V.; Wang, L.; Zoidakis, J.; Zürbig, P.; Schanstra, J. P.; Vlahou, A. Comprehensive Human Urine Standards for Comparability and Standardization in Clinical Proteome Analysis. PROTEOMICS – Clin. Appl. 2010, 4 (4), 464–478. https://doi.org/10.1002/prca.200900189.

(35) SeparationScience. Internal Standards: How Does It Work? https://learning.sepscience.com/hubfs/Technical%20Blogs/HPLC_134_v2.pdf (accessed 2024-08-22).

(36) Hewavitharana, A. K.; P. Nicholas Shaw; H.D.C. Smyth; L.P. Samaranayake; H.M.H.N. Bandara. The Importance of Complete Overlapping of Analyte and Internal Standard Peaks in Eliminating Matrix Effects with Liquid Chromatography–Mass Spectrometry (LC–MS). Intellisphere LLC 2021, 39 (7).

(37) Allende, F.; Solari, S.; Campino, C.; Carvajal, C. A.; Lagos, C. F.; Vecchiola, A.; Valdivia, C.; Baudrand, R.; Owen, G. I.; Fardella, C. E. LC–MS/MS Method for the Simultaneous Determination of Free Urinary Steroids. Chromatographia 2014, 77 (7–8), 637–642. https://doi.org/10.1007/s10337-014-2638-4.

(38) Skov, K.; Johansen, S. S.; Linnet, K.; Rasmussen, B. S.; Nielsen, M. K. K. Exploring Enzymatic Hydrolysis of Urine Samples for Investigation of Drugs Associated with Drug-Facilitated Sexual Assault. Pharmaceuticals 2023, 17 (1), 13. https://doi.org/10.3390/ph17010013.

(39) Stevenson, D. IMMUNOAFFINITY EXTRACTION.

(40) Solid Phase Extraction SPE; 2021. https://www.youtube.com/watch?v=yCD_z-q_6iA (accessed 2024-07-23).

(41) Mandal, S.; Poi, R.; Hazra, D. K.; Ansary, I.; Bhattacharyya, S.; Karmakar, R. Review of Extraction and Detection Techniques for the Analysis of Pesticide Residues in Fruits to Evaluate Food Safety and Make Legislative Decisions: Challenges and Anticipations. J. Chromatogr. B 2023, 1215, 123587. https://doi.org/10.1016/j.jchromb.2022.123587.

(42) Lord, H. L.; Pfannkoch, E. A. Sample Preparation Automation for GC Injection. In Comprehensive Sampling and Sample Preparation; Elsevier, 2012; pp 597–612. https://doi.org/10.1016/B978-0-12-381373-2.00061-2.

(43) Lin, D.-L.; Wang, S.-M.; Wu, C.-H.; Chen, B.-G.; Liu, R. H. Chemical Derivatization for the Analysis of Drugs by GC-MS - A Conceptual Review. J. Food Drug Anal. 2020, 16 (1). https://doi.org/10.38212/2224-6614.2373.

(44) Sigma-Aldrich Co. Guide to Derivatization Reagents for GC., 1997. https://gcms.cz/labrulez-bucket-strapi-h3hsga3/application::paper.paper/t196909.pdf (accessed 2024-08-20).

(45) Bogos, L.-G.; Pralea, I.-E.; Moldovan, R.-C.; Iuga, C.-A. Indirect Enantioseparations: Recent Advances in Chiral Metabolomics for Biomedical Research. Int. J. Mol. Sci. 2022, 23 (13), 7428. https://doi.org/10.3390/ijms23137428.

(46) Turner, D. GC-MS Principle, Instrument and Analyses and GC-MS/MS. Analysis & Separations from Technology Networks. http://www.technologynetworks.com/analysis/articles/gc-ms-principle-instrument-and-analyses-and-gc-msms-362513 (accessed 2024-08-22).

(47) Van Renterghem, P.; Polet, M.; Brooker, L.; Van Gansbeke, W.; Van Eenoo, P. Development of a GC/C/IRMS Method – Confirmation of a Novel Steroid Profiling Approach in Doping Control. Steroids 2012, 77 (11), 1050–1060. https://doi.org/10.1016/j.steroids.2012.05.009.

(48) Hackney, A. C. Athlete Testing, Analytical Procedures, and Adverse Analytical Findings. In Doping, Performance Enhancing Drugs, and Hormones in Sport; Elsevier, 2018; pp 113–127. https://doi.org/10.1016/B978-0-12-813442-9.00010-9.

(49) De La Torre, X.; González, J. C.; Pichini, S.; Pascual, J. A.; Segura, J. 13C/12C Isotope Ratio MS Analysis of Testosterone, in Chemicals and Pharmaceutical Preparations. J. Pharm. Biomed. Anal. 2001, 24 (4), 645–650. https://doi.org/10.1016/S0731-7085(00)00452-0.

(50) Cheriyedath, S. Liquid Chromatography versus Gas Chromatography. New Medical Life Sciences. https://www.news-medical.net/life-sciences/Liquid-Chromatography-versus-Gas-Chromatography.aspx (accessed 2024-03-08).

(51) A. Schug, K. The LCGC Blog: Forensic Drug Analysis: GC–MS versus LC–MS. https://www.chromatographyonline.com/view/lcgc-blog-forensic-drug-analysis-gc-ms-versus-lc-ms (accessed 2024-08-12).

(52) Cadwallader, A. B.; de la Torre, X.; Tieri, A.; Botrè, F. The Abuse of Diuretics as Performance-Enhancing Drugs and Masking Agents in Sport Doping: Pharmacology, Toxicology and Analysis. Br. J. Pharmacol. 2010, 161 (1), 1–16. https://doi.org/10.1111/j.1476-5381.2010.00789.x.

(53) Hemmersbach, P. The Probenecid-Story - A Success in the Fight against Doping through out-of-Competition Testing. Drug Test. Anal. 2020, 12 (5), 589–594. https://doi.org/10.1002/dta.2727.

(54) Botrè, F. Are Liposomes Masking Agents? An in-Progress Study. 2009.

(55) Schänzer, W.; Geyer, H.; Gotzmann, A.; Mareck, U. Proceedings of the Manfred-Donike-Workshop, 29th Cologne Workshop on Dope Analysis 13th to 18th February 2011; Sportverl. Strauß: Köln, 2012.

(56) Kazlauskas, R. Designer Steroids. In Doping in Sports; Thieme, D., Hemmersbach, P., Eds.; Handbook of Experimental Pharmacology; Springer Berlin Heidelberg: Berlin, Heidelberg, 2009; Vol. 195, pp 155–185. https://doi.org/10.1007/978-3-540-79088-4_7.

(57) Brodwin, E. Olympians may be taking cues from Silicon Valley’s favorite way to do drugs. BusinessInsider. https://www.businessinsider.com/doping-cheating-winter-olympics-microdosing-testosterone-steroids-2018-2 (accessed 2024-09-11).

(58) Chayen, J.; Bitensky, L. BIOASSAYS | Overview. In Encyclopedia of Analytical Science; Elsevier, 2005; pp 259–265. https://doi.org/10.1016/B0-12-369397-7/00041-8.

(59) Lund, R. A.; Cooper, E. R.; Wang, H.; Ashley, Z.; Cawley, A. T.; Heather, A. K. Nontargeted Detection of Designer Androgens: Underestimated Role of in Vitro Bioassays. Drug Test. Anal. 2021, 13 (5), 894–902. https://doi.org/10.1002/dta.3049.

(60) Martín-Escudero, P.; Muñoz-Guerra, J. A.; García-Tenorio, S. V.; Serrano-Garde, E.; Soldevilla-Navarro, A. B.; Cortes-Carrillo, N.; Galindo-Canales, M.; Del Prado, N.; Fuentes-Ferrer, M.; Fernández-Pérez, C.; Behnisch, P. A.; Brouwer, A. Bioanalytical Detection of Steroid Abuse in Sports Based on the Androgenic Activity Measurement. Chemosensors 2021, 9 (4), 62. https://doi.org/10.3390/chemosensors9040062.

(61) Sanders, K. L.; Edwards, J. L. Nano-Liquid Chromatography-Mass Spectrometry and Recent Applications in Omics Investigations. Anal. Methods 2020, 12 (36), 4404–4417. https://doi.org/10.1039/D0AY01194K.

(62) Thevis, M. The 41 st Manfred Donike Workshop on Doping Analysis. Drug Test. Anal. 2023, 15 (11–12), 1310–1311. https://doi.org/10.1002/dta.3601.

(63) Li, R.; Su, P.; Shi, Y.; Shi, H.; Ding, S.; Su, X.; Chen, P.; Wu, D. Gene Doping Detection in the Era of Genomics. Drug Test. Anal. 2024, dta.3664. https://doi.org/10.1002/dta.3664.

(64) Proszenko, A. Why Gene Doping Is the next Great Olympic Threat. July 20, 2024. https://www.smh.com.au/sport/why-gene-doping-is-the-next-great-olympic-threat-20240720-p5jv5w.html (accessed 2024-08-20).

(65) Lippi, G.; Longo, U. G.; Maffulli, N. Genetics and Sports. Br. Med. Bull. 2010, 93 (1), 27–47. https://doi.org/10.1093/bmb/ldp007.

(66) Varillas-Delgado, D.; Del Coso, J.; Gutiérrez-Hellín, J.; Aguilar-Navarro, M.; Muñoz, A.; Maestro, A.; Morencos, E. Genetics and Sports Performance: The Present and Future in the Identification of Talent for Sports Based on DNA Testing. Eur. J. Appl. Physiol. 2022, 122 (8), 1811–1830. https://doi.org/10.1007/s00421-022-04945-z.

(67) Alvarez‐Romero, J.; Laguette, M. N.; Seale, K.; Jacques, M.; Voisin, S.; Hiam, D.; Feller, J. A.; Tirosh, O.; Miyamoto‐Mikami, E.; Kumagai, H.; Kikuchi, N.; Kamiya, N.; Fuku, N.; Collins, M.; September, A. V.; Eynon, N. Genetic Variants within the COL5A1 Gene Are Associated with Ligament Injuries in Physically Active Populations from Australia, South Africa, and Japan. Eur. J. Sport Sci. 2023, 23 (2), 284–293. https://doi.org/10.1080/17461391.2021.2011426.

(68) Posthumus, M.; Schwellnus, M. P.; Collins, M. The COL5A1 Gene: A Novel Marker of Endurance Running Performance. Med. Sci. Sports Exerc. 2011, 43 (4), 584–589. https://doi.org/10.1249/MSS.0b013e3181f34f4d.

Downloads

Posted

2024-08-31