Prevention of Doping in Sport : Shortcomings and Challenges / Patrick Trabal. - Paris : United Nations Educational, Scientific and Cultural Organization (UNESCO), 2014. - Study of Social Science and Doping Group, CERSM, Université Paris Ouest Nanterre

Contents:

Part 1: Analysis
- Prevention: principles and values
- Describing the mechanisms
- Realities on the ground
- The problem of correlation
Part 2: Outlook
- Examining modes of public action
- Enlisting other paradigms
- Shifts
- Conclusions

Introduction
The World Anti-Doping Agency (WADA) promotes, coordinates and monitors the global fight against doping in sport. This review is the result of WADA’s identification of education and social science research as strategic priorities for developing evidence-based anti-doping education. To complete this commission we set out to identify evidence regarding the efficacy of prevention interventions across four social domains; bullying, alcohol, tobacco and social drug use. The main purpose of this review is to highlight the factors which have been determined, to date, as the most successful preventive approaches in these respective domains. Broad conclusions are drawn from the literature with a view to recommending ‘recipes of success’ which could be further refined and applied in the design of future anti-doping prevention programmes.

The Literature Review Methodology
The review process comprised two main stages. Stage one involved an examination and summation of tertiary and secondary level reviews (e.g., reviews of reviews metaanalyses, systematic reviews), published in the scientific literature or by government agencies between 2002 and November 2008. Stage two comprised the execution of a comprehensive search and review of primary studies based on the fact that the studies were (i) experimental or quasi-experimental, (ii) published from 2002 onward and (iii) not included (or excluded) in the reviews of stage one.

The Findings
Universal, school-based interventions are the most frequently studied prevention approach. This single setting offers the most systematic and efficient way of reaching the greatest number of young people each year. Although these interventions demonstrate immediate impact, their long-term effects are questionable. When school-based programmes are integrated into multi-level strategies involving school, family and community approaches, effectiveness is enhanced. However, community-based prevention alone appears to be ineffective in changing the behaviours considered. Based on the findings of research across the four domains, prevention programmes
should be:
- Targeted at young people and adolescents when attitudes and values are being formed.
- Tailored to fit the target population (e.g., risk factors, developmental).
- Interactive and emphasising of active participation (e.g., role-plays, discussions).
- Derived from social influence approaches and focused on developing core life skills (e.g., communication, decision-making, refusal skills) as knowledge dissemination alone is ineffective in changing behaviour.
- Monitored and delivered with high degrees of fidelity1, ensuring that programme implementation is as directed.
- Delivered by well trained individuals who, demonstrably, deliver the programme with high fidelity.
- Based on booster sessions delivered over a number of years. This reinforces and builds on intervention messages.

A number of questions still remain, even in those fields with a long history of research and evaluation. For example, intervention intensity appears to be an important determinant of intervention efficacy. However, it is unclear whether an ‘intense’ programme comprises (i) more sessions, or (ii) more content with fewer sessions. Similarly, the importance of training deliverers to ensure fidelity has been emphasised across the literature, but there is no consensus regarding who fits the role of ‘best’ deliverer.

Conclusion
This review has highlighted that, currently, there are no ‘magical ingredients’ to include in prevention programmes to ensure their effectiveness. However, there do seem to be some ‘recipes for success’ that should underpin any programme with primary prevention at its heart. Anti-doping education is a relatively young research field with few examples of best-practice. Therefore, anti-doping researchers, policy makers and practitioners are far from being able to rely on the level of evidence-based research that is currently available across the four domains we have considered in this review. It is also notable that even in these well established fields, more systematic research is needed to fully assess ideas across a variety of settings. Furthermore, researchers across each of these domains agree that little high quality information exists in developing countries in terms of prevention, evaluation and research. They also caution against assuming that research findings will readily transfer, and with equal impact, to prevent other undesirable/unhealthy behaviours.

On balance, this review has highlighted some of the lessons learned from research examining the prevention of bullying, alcohol, tobacco and social drug use. We hope the findings will assist active anti-doping educators in developing programmes from walled foundations rather than providing just bricks and mortar. The strategic goal of anti-doping education should be to develop an evidence-base that allows the ‘critical ingredients’ necessary for effective doping prevention education to be (i) discovered, (ii) applied and (iii) evaluated. In doing so, we will facilitate a long-term perspective which emphasises prevention, rather than detection, in the fight against doping in sport. Doping is a global issue and as such, requires ‘connected’ approaches, across countries and, most likely across the related organisations.

Preventive doping control analysis : liquid and gas chromatography time-of-flight mass spectrometry for detection of designer steroids / Costas G. Georgakopoulos, Ariadni Vonaparti, Marianna Stamou, Polyxeni Kiousi, Emmanouil Lyris, Yiannis S. Angelis, George Tsoupras, Bernhard Wuest, Michel W.F. Nielen, Irene Panderi, Michalis Koupparis. - (Rapid Communicaton in Mass Spectrometry 21 (2007) 15 (15 August); p. 2439-2446)

• PMID: 17610244
• DOI: 10.1002/rcm.3103

Abstract

A new combined doping control screening method for the analysis of anabolic steroids in human urine using liquid chromatography/electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (LCoaTOFMS) and gas chromatography/electron ionization orthogonal acceleration time-of-flight mass spectrometry (GCoaTOFMS) has been developed in order to acquire accurate full scan MS data to be used to detect designer steroids. The developed method allowed the detection of representative prohibited substances, in addition to steroids, at concentrations of 10 ng/mL for anabolic agents and metabolites, 30 ng/mL for corticosteroids, 500 ng/mL for stimulants and beta-blockers, 250 ng/mL for diuretics, and 200 ng/mL for narcotics. Sample preparation was based on liquid-liquid extraction of hydrolyzed human urine, and the final extract was analyzed as trimethylsilylated derivatives in GCoaTOFMS and underivatized in LCoaTOFMS in positive ion mode. The sensitivity, mass accuracy, advantages and limitations of the developed method are presented.

Preventive doping control screening analysis of prohibited substances in human urine using rapid-resolution liquid chromatography/high-resolution time-of-flight mass spectrometry / A. Vonaparti, E. Lyris, Y.S. Angelis, I. Panderi, M. Koupparis, A. Tsantili-Kakoulidou, R.J.B. Peters, M.W.F. Nielen, C. Georgakopoulos. - (Rapid Communicaton in Mass Spectrometry 24 (2010) 11 (15 June); p. 1595-1609)

• PMID: 20486255
• DOI: 10.1002/rcm.4554

Abstract

Unification of the screening protocols for a wide range of doping agents has become an important issue for doping control laboratories. This study presents the development and validation of a generic liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) screening method of 241 small molecule analytes from various categories of prohibited substances (stimulants, narcotics, diuretics, beta(2)-agonists, beta-blockers, hormone antagonists and modulators, glucocorticosteroids and anabolic agents). It is based on a single-step liquid-liquid extraction of hydrolyzed urine and the use of a rapid-resolution liquid chromatography/high-resolution time-of-flight mass spectrometric system acquiring continuous full scan data. Electrospray ionization in the positive mode was used. Validation parameters consisted of identification capability, limit of detection, specificity, ion suppression, extraction recovery, repeatability and mass accuracy. Detection criteria were established on the basis of retention time reproducibility and mass accuracy. The suitability of the methodology for doping control was demonstrated with positive urine samples. The preventive role of the method was proved by the case where full scan acquisition with accurate mass measurement allowed the retrospective reprocessing of acquired data from past doping control samples for the detection of a designer drug, the stimulant 4-methyl-2-hexanamine, which resulted in re-reporting a number of stored samples as positives for this particular substance, when, initially, they had been reported as negatives.

Procollagen type III amino-terminal propeptide and insulin-like growth factor I as biomarkers of growth hormone administration / David A. Cowan, Danielle A. Moncrieffe. - (Drug Testing and Analysis (2021) 21 August)

• PMID: 34418311
• DOI: 10.1002/dta.3155

Abstract

The acceptance in 2012 by the World Anti-Doping Agency (WADA) of the biomarker test for human growth hormone (hGH) based on procollagen type III amino-terminal propeptide (P-III-NP) and insulin-like growth factor I (IGF-I) was perhaps the first time that such a method has been used for forensic purposes. Developing a biomarker test to anti-doping standards, where the strict liability principle applies, is discussed. An alternative WADA-accepted approach is based on the measurement of different hGH isoforms, a method that suffers from the very short half-life of hGH limiting the detection period. Modification or withdrawal of the immunoassays, on which the biomarker measurements largely depend, has necessitated revalidation of the assays, remeasurement of samples and adjustment of the decision limits above which an athlete will be assumed to have administered hGH. When a liquid chromatography coupled mass spectrometry (LC-MS) method became a reality for the measurement of IGF-I, more consistency of results was assured. Measurement of P-III-NP is still dependent on immunoassays although work is underway to develop an LC-MS method. The promised long-term detection time for the biomarker assay does not appear to have been realised in practice, and this is perhaps partly the result of decision limits being set too high. Nevertheless, more robust assays are needed before a further adjustment of the decision limit is warranted. In the meantime, WADA is considering using P-III-NP and IGF-I as components of a biomarker passport system recording data from an individual athlete, rather than the population. Using this approach, smaller perturbations in the growth hormone (GH) score would mandate an investigation and possible action for hGH administration.

Profiling of 19-norandrosterone sulfate and glucuronide in human urine : implications in athlete's drug testing / Emmanuel Strahm, Norbert Baume, Patrice Mangin, Martial Saugy, Christiane Ayotte, Christophe Saudan. - (Steroids 74 (2009) 3 (March); p. 359-364)

• PMID: 19056413
• DOI: 10.1016/j.steroids.2008.11.005

Abstract

19-Norandrosterone (19-NA) as its glucuronide derivative is the target metabolite in anti-doping testing to reveal an abuse of nandrolone or nandrolone prohormone. To provide further evidence of a doping with these steroids, the sulfoconjugate form of 19-norandrosterone in human urine might be monitored as well. In the present study, the profiling of sulfate and glucuronide derivatives of 19-norandrosterone together with 19-noretiocholanolone (19-NE) were assessed in the spot urines of 8 male subjects, collected after administration of 19-nor-4-androstenedione (100mg). An LC/MS/MS assay was employed for the direct quantification of sulfoconjugates, whereas a standard GC/MS method was applied for the assessment of glucuroconjugates in urine specimens. Although the 19-NA glucuronide derivative was always the most prominent at the excretion peak, inter-individual variability of the excretion patterns was observed for both conjugate forms of 19-NA and 19-NE. The ratio between the glucuro- and sulfoconjugate derivatives of 19-NA and 19-NE could not discriminate the endogenous versus the exogenous origin of the parent compound. However, after ingestion of 100mg 19-nor-4-androstenedione, it was observed in the urine specimens that the sulfate conjugates of 19-NA was detectable over a longer period of time with respect to the other metabolites. These findings indicate that more interest shall be given to this type of conjugation to deter a potential doping with norsteroids.

Profiling of steroid metabolites after transdermal and oral administration of testosterone by ultra-high pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry / F. Badoud, J. Boccard, C. Schweizer, F. Pralong, M. Saugy, N. Baume. - (Journal of Steroid Biochemistry and Molecular Biology 138 (2013) November; p . 22-235)

• PMID: 23796409
• DOI: 10.1016/j.jsbmb.2013.05.018

Abstract

The screening of testosterone (T) misuse for doping control is based on the urinary steroid profile, including T, its precursors and metabolites. Modifications of individual levels and ratio between those metabolites are indicators of T misuse. In the context of screening analysis, the most discriminant criterion known to date is based on the T glucuronide (TG) to epitestosterone glucuronide (EG) ratio (TG/EG). Following the World Anti-Doping Agency (WADA) recommendations, there is suspicion of T misuse when the ratio reaches 4 or beyond. While this marker remains very sensitive and specific, it suffers from large inter-individual variability, with important influence of enzyme polymorphisms. Moreover, use of low dose or topical administration forms makes the screening of endogenous steroids difficult while the detection window no longer suits the doping habit. As reference limits are estimated on the basis of population studies, which encompass inter-individual and inter-ethnic variability, new strategies including individual threshold monitoring and alternative biomarkers were proposed to detect T misuse.

The purpose of this study was to evaluate the potential of ultra-high pressure liquid chromatography (UHPLC) coupled with a new generation high resolution quadrupole time-of-flight mass spectrometer (QTOF-MS) to investigate the steroid metabolism after transdermal and oral T administration. An approach was developed to quantify 12 targeted urinary steroids as direct glucuro- and sulfo-conjugated metabolites, allowing the conservation of the phase II metabolism information, reflecting genetic and environmental influences. The UHPLC-QTOF-MS(E) platform was applied to clinical study samples from 19 healthy male volunteers, having different genotypes for the UGT2B17 enzyme responsible for the glucuroconjugation of T. Based on reference population ranges, none of the traditional markers of T misuse could detect doping after topical administration of T, while the detection window was short after oral TU ingestion. The detection ability of the 12 targeted steroids was thus evaluated by using individual thresholds following both transdermal and oral administration. Other relevant biomarkers and minor metabolites were studied for complementary information to the steroid profile, including sulfoconjugated analytes and hydroxy forms of glucuroconjugated metabolites. While sulfoconjugated steroids may provide helpful screening information for individuals with homozygotous UGT2B17 deletion, hydroxy-glucuroconjugated analytes could enhance the detection window of oral T undecanoate (TU) doping.

Keywords: Anabolic androgenic steroids; Phase II metabolism; Quadrupole time-of-flight; Quantification; Testosterone; Ultra-high-pressure liquid chromatography.

Progress in the Detection of Erythropoietin in Blood, Urine, and Tissue / Yukiko Yasuoka, Yuichiro Izumi, Jeff M. Sands, Katsumasa Kawahara, Hiroshi Nonoguchi

• Molecules 28 (2023) 11 (June) 4446, p. 1-17
• PMID: 37298922
• PMCID: PMC10254663
• DOI: 10.3390/molecules28114446

Abstract

Detection of erythropoietin (Epo) was difficult until a method was developed by the World Anti-Doping Agency (WADA). WADA recommended the Western blot technique using isoelectric focusing (IEF)-PAGE to show that natural Epo and injected erythropoiesis-stimulating agents (ESAs) appear in different pH areas. Next, they used sodium N-lauroylsarcosinate (SAR)-PAGE for better differentiation of pegylated proteins, such as epoetin β pegol. Although WADA has recommended the use of pre-purification of samples, we developed a simple Western blotting method without pre-purification of samples. Instead of pre-purification, we used deglycosylation of samples before SDS-PAGE. The double detection of glycosylated and deglycosylated Epo bands increases the reliability of the detection of Epo protein. All of the endogenous Epo and exogenous ESAs shift to 22 kDa, except for Peg-bound epoetin β pegol. All endogenous Epo and exogenous ESAs were detected as 22 kDa deglycosylated Epo by liquid chromatography/mass spectrum (LC/MS) analysis. The most important factor for the detection of Epo is the selection of the antibody against Epo. WADA recommended clone AE7A5, and we used sc-9620. Both antibodies are useful for the detection of Epo protein by Western blotting.

Prohormone supplement 3β-hydroxy-5α-androst-1-en-17-one enhances resistance training gains but impairs user health / Jorge Granados, Trevor L. Gillum, Kevin M. Christmas, Matthew R. Kuennen

• Journal of Applied Physiology 116 (2014) 5 (March), p. 560-569
• PMID: 24381122
• DOI: 10.1152/japplphysiol.00616.2013

Abstract

Prohormone supplements (PS) are recognized not to impart anabolic or ergogenic effects in men, but the research supporting these conclusions is dated. The Anabolic Steroid Control Act was amended in 2004 to classify androstenedione and 17 additional anabolic compounds as controlled substances. The viability of PS that entered the market after that time have not been evaluated. Seventeen resistance-trained men (23 ± 1 yr; 13.1 ± 1.5% body fat) were randomly assigned to receive either 330 mg/day of 3β-hydroxy-5α-androst-1-en-17-one (Prohormone; n = 9) or sugar (Placebo; n = 8) per os and complete a 4-wk (16 session) structured resistance-training program. Body composition, muscular strength, circulating lipids, and markers of liver and kidney dysfunction were assessed at study onset and termination. Prohormone increased lean body mass by 6.3 ± 1.2%, decreased fat body mass by 24.6 ± 7.1%, and increased their back squat one repetition maximum and competition total by 14.3 ± 1.5 and 12.8 ± 1.1%, respectively. These improvements exceeded (P < 0.05) Placebo, which increased lean body mass by 0.5 ± 0.8%, reduced fat body mass by 9.5 ± 3.6%, and increased back squat one repetition maximum and competition total by 5.7 ± 1.7 and 5.9 ± 1.7%, respectively. Prohormone also experienced multiple adverse effects. These included a 38.7 ± 4.0% reduction in HDL (P < 0.01), a 32.8 ± 15.05% elevation in LDL (P < 0.01), and elevations of 120.0 ± 22.6 and 77.4 ± 12.0% in LDL-to-HDL and cholesterol-to-HDL ratios, respectively (both P < 0.01). Prohormone also exhibited elevations in serum creatinine (19.6 ± 4.3%; P < 0.01) and aspartate transaminase (113.8 ± 61.1%; P = 0.05), as well as reductions in serum albumin (5.1 ± 1.9%; P = 0.04), alkaline phosphatase (16.4 ± 4.7%; P = 0.04), and glomerular filtration rate (18.0 ± 3.3%; P = 0.04). None of these values changed (all P > 0.05) in Placebo. The oral PS 3β-hydroxy-5α-androst-1-en-17-one improves body composition and muscular strength. However, these changes come at a significant cost. Cardiovascular health and liver function are particularly compromised. Given these findings, we feel the harm associated with this particular PS outweighs any potential benefit.

Prohormones and sport / Frans T. Delbeke, Peter Van Eenoo, Wim Van Thuyne, Noël Desmet

• Journal of Steroid Biochemistry and Molecular Biology 83 (2002) 1-5 (December), p. 245-251
• PMID: 12650722
• DOI: 10.1016/s0960-0760(02)00274-1

Abstract

Several precursors of testosterone and nandrolone introduced on the nutritional supplement market as performance enhancing drugs are banned in sports. Until now they are legally sold without a prescription in the US. Results of excretion studies with related compounds including 7-keto-DHEA and 1-androstenediol are presented. The main metabolites of 7-keto-DHEA are 7-hydroxylated compounds. The commercial 1-androstenediol preparation was contaminated with several other anabolic steroids. Oxidation of 1-androstenediol to 1-androstenedione seems to be the major renal metabolic pathway. Additionally contaminated nutritional supplements containing banned substances not indicated on the label were administered. The results of the excretion studies indicate that after the intake of amounts substantially lower than the recommended dose athletes can fail a doping test for periods up to 120 h.