Antidoping programme and biological monitoring before and during the 2014 FIFA World Cup Brazil.

BACKGROUND: The FIFA has implemented an important antidoping programme for the 2014 FIFA World Cup. AIM: To perform the analyses before and during the World Cup with biological monitoring of blood and urine samples. METHODS: All qualified players from the 32 teams participating in the World Cup were tested out-of-competition. During the World Cup, 2-8 players per match were tested. Over 1000 samples were collected in total and analysed in the WADA accredited Laboratory of Lausanne. RESULTS: The quality of the analyses was at the required level as described in the WADA technical documents. The urinary steroid profiles of the players were stable and consistent with previously published papers on football players. During the competition, amphetamine was detected in a sample collected on a player who had a therapeutic use exemption for attention deficit hyperactivity disorder. The blood passport data showed no significant difference in haemoglobin values between out-of-competition and postmatch samples. CONCLUSIONS: Logistical issues linked to biological samples collection, and the overseas shipment during the World Cup did not impair the quality of the analyses, especially when used as the biological passport of football players.

A forensic perspective of the AFL investigation into peptides: an antidoping investigation case study.

BACKGROUND: The World Anti-Doping Agency (WADA) is introducing enhancements to doping investigations in its 2015 Code, which include improved sharing of information between antidoping organisations (including sporting bodies) and enhanced accountability of athlete support staff. These additions will improve the control of links between sports doping and organised crime. In February 2013 the Australian Crime Commission released a report that linked several professional sporting codes, professional athletes with links to organised crime, performance enhancing drugs and illicit substances. Following this report the Australian Football League (AFL) partnered the Australian national antidoping organisation to investigate peptide use in Australian football. METHODS: This review compared the model proposed by Marclay, a hypothetical model for anti-doping investigations that proposed a forensic intelligence and analysis approach, to use the forensic capabilities of the AFL investigation to test the model's relevance to an actual case. RESULTS: The investigation uncovered the use of peptides used to enhance athlete performance. The AFL investigation found a high risk of doping where athlete support staff existed in teams with weak corporate governance controls. A further finding included the need for the investigation to provide a timely response in professional team sports that were sensitive to the competition timing. In the case of the AFL the team was sanctioned prior to the finals as an interim outcome for allowing the risk of use of performance-enhancing substances. Doping violation charges are still being considered. DISCUSSION: Antidoping strategies should include the investigation of corporate officers in team doping circumstances, the mandatory recording of all athlete substance use during competition and training phases, the wider sharing of forensic intelligence with non-sporting bodies particularly law enforcement and collaboration between antidoping and sporting organisations in doping investigations. CONCLUSIONS: The AFL investigation illustrated the importance of the 2015 WADA Code changes and highlighted the need for a systematic use of broad forensic intelligence activities in the investigation of doping violations.

Time for change: a roadmap to guide the implementation of the World Anti-Doping Code 2015.

A medical and scientific multidisciplinary consensus meeting was held from 29 to 30 November 2013 on Anti-Doping in Sport at the Home of FIFA in Zurich, Switzerland, to create a roadmap for the implementation of the 2015 World Anti-Doping Code. The consensus statement and accompanying papers set out the priorities for the antidoping community in research, science and medicine. The participants achieved consensus on a strategy for the implementation of the 2015 World Anti-Doping Code. Key components of this strategy include: (1) sport-specific risk assessment, (2) prevalence measurement, (3) sport-specific test distribution plans, (4) storage and reanalysis, (5) analytical challenges, (6) forensic intelligence, (7) psychological approach to optimise the most deterrent effect, (8) the Athlete Biological Passport (ABP) and confounding factors, (9) data management system (Anti-Doping Administration & Management System (ADAMS), (10) education, (11) research needs and necessary advances, (12) inadvertent doping and (13) management and ethics: biological data. True implementation of the 2015 World Anti-Doping Code will depend largely on the ability to align thinking around these core concepts and strategies. FIFA, jointly with all other engaged International Federations of sports (Ifs), the International Olympic Committee (IOC) and World Anti-Doping Agency (WADA), are ideally placed to lead transformational change with the unwavering support of the wider antidoping community. The outcome of the consensus meeting was the creation of the ad hoc Working Group charged with the responsibility of moving this agenda forward.

Assessing tobacco dependence among cannabis users smoking cigarettes.

INTRODUCTION: This study examines the relationship between nicotine exposure and tobacco addiction among young smokers consuming either only tobacco or only tobacco and cannabis. METHODS: Data on tobacco and cannabis use were collected by a questionnaire among 313 adolescents and young adults in Western Switzerland between 2009 and 2010. In addition, a urine sample was used to determine urinary cotinine level. Nicotine addiction was measured using the Fagerström Test for Nicotine Dependence (FTND). In this study, we focused on a sample of 142 participants (mean age 19.54) that reported either smoking only tobacco cigarettes (CIG group, n = 70) or smoking both tobacco cigarettes and cannabis (CCS group, n = 72). RESULTS: The FTND did not differ significantly between CIG (1.96 ± 0.26) and CCS (2.66 ± 0.26) groups (p = 0.07). However, participants in the CCS group smoked more cigarettes (8.30 ± 0.79 vs. 5.78 ± 0.8, p = 0.03) and had a higher mean cotinine value (671.18 ± 67.6 vs. 404.32 ± 68.63, p = 0.008) than the CIG group. Further, the association between cotinine and FTND was much stronger among the CIG than among the CCS group (regression coefficient of 0.0031 vs. 0.00099, p < 0.0001). CONCLUSION: Adolescents smoking tobacco and cannabis cigarettes featured higher levels of cotinine than youth smoking only tobacco; however, there was no significant difference in the addiction score. The FTND score is intended to measure nicotine dependence from smoked tobacco cigarettes. Hence, to accurately determine nicotine exposure and the associated dependence among young smokers, it seems necessary to inquire about cannabis consumption.

To what extent does adding tobacco to cannabis expose young users to nicotine?

INTRODUCTION: To determine if mulling, the process of adding tobacco to cannabis for its consumption, exposes young cannabis users to significant levels of nicotine. METHODS: This observational study performed in 2009-2010 among Swiss youths aged 16-25 years involved the completion of a self-administrated questionnaire and the collection of a urine sample on the same day. Measures of urinary cotinine were blindly performed using liquid chromatography coupled-mass spectrometry. A total of 197 eligible participants were divided in 3 groups based on their consumption profile in the past 5 days: 70 abstainers (ABS) not having used cigarettes or cannabis, 57 cannabis users adding tobacco to the cannabis they smoke (MUL) but not having smoked cigarettes, and 70 cigarette smokers (CIG) not having smoked cannabis. RESULTS: Exposure to nicotine was at its lowest among ABS with a mean (SE) cotinine level of 3.2 (1.4) ng/ml compared, respectively, with 214.6 (43.8) and 397.9 (57.4) for MUL and CIG (p < .001). While consumption profile appeared as the only significant factor of influence when examining nicotine exposure from the ABS and MUL participants on multivariate analysis, it did not result in substantial differences among MUL and CIG groups. CONCLUSIONS: Urinary cotinine levels found among MUL are high enough to indicate a significant exposure to nicotine originating from the mulling process. In line with our results, health professionals should pay attention to mulling as it is likely to influence cannabis and cigarette use as well as the efficacy of cessation interventions.

Source inference of exogenous gamma-hydroxybutyric acid (GHB) administered to humans by means of carbon isotopic ratio analysis: novel perspectives regarding forensic investigation and intelligence issues.

γ-Hydroxybutyric acid (GHB) is an endogenous short-chain fatty acid popular as a recreational drug due to sedative and euphoric effects, but also often implicated in drug-facilitated sexual assaults owing to disinhibition and amnesic properties. Whilst discrimination between endogenous and exogenous GHB as required in intoxication cases may be achieved by the determination of the carbon isotope content, such information has not yet been exploited to answer source inference questions of forensic investigation and intelligence interests. However, potential isotopic fractionation effects occurring through the whole metabolism of GHB may be a major concern in this regard. Thus, urine specimens from six healthy male volunteers who ingested prescription GHB sodium salt, marketed as Xyrem(®), were analysed by means of gas chromatography/combustion/isotope ratio mass spectrometry to assess this particular topic. A very narrow range of δ(13)C values, spreading from -24.81‰ to -25.06‰, was observed, whilst mean δ(13)C value of Xyrem(®) corresponded to -24.99‰. Since urine samples and prescription drug could not be distinguished by means of statistical analysis, carbon isotopic effects and subsequent influence on δ(13)C values through GHB metabolism as a whole could be ruled out. Thus, a link between GHB as a raw matrix and found in a biological fluid may be established, bringing relevant information regarding source inference evaluation. Therefore, this study supports a diversified scope of exploitation for stable isotopes characterized in biological matrices from investigations on intoxication cases to drug intelligence programmes.

Integration of the Forensic Dimension into Anti-Doping Strategies.

Traditionally, research in anti-doping has been stimulated by the need for technological improvements to accommodate the expansion of the list of prohibited substances and methods. Nevertheless, in recent years, anti-doping found itself at a crossroads due to the increasing complexity and constant refinement of doping methods. As illustrated by the 2012 USADA (United States Anti-Doping Agency) versus Lance Armstrong case, a change in paradigm was necessary. The exploration of new scientific avenues to understand the mechanisms of doping and pinpoint its practice was most needed to allow designing more efficient preventive or disruptive strategies. In this context, and at the time of writing in 2017, transposing the concept of forensic intelligence to anti-doping was identified as a promising approach to address the different aspects of doping, from the individual athlete to organized doping and trafficking of substances in a proactive rather than a reactive way. Indeed, collection, structuring, and logical processing of multiple sources of information, and not strictly results of bioanalytical testing of urinary and blood samples, can bring additional value to detect and describe potential, emerging, or existing doping issues. This anti-doping intelligence can provide anti-doping authorities and relevant stakeholders with timely, accurate, and usable information for decision making to solve, reduce, and/or prevent doping-related activities. The integration of intelligence to complement other anti-doping approaches is a potentially major step forward in the development of more effective and robust anti-doping strategies.

Perspectives for Forensic Intelligence in anti-doping: thinking outside of the box.

Today's approach to anti-doping is mostly centered on the judicial process, despite pursuing a further goal in the detection, reduction, solving and/or prevention of doping. Similarly to decision-making in the area of law enforcement feeding on Forensic Intelligence, anti-doping might significantly benefit from a more extensive gathering of knowledge. Forensic Intelligence might bring a broader logical dimension to the interpretation of data on doping activities for a more future-oriented and comprehensive approach instead of the traditional case-based and reactive process. Information coming from a variety of sources related to doping, whether directly or potentially, would feed an organized memory to provide real time intelligence on the size, seriousness and evolution of the phenomenon. Due to the complexity of doping, integrating analytical chemical results and longitudinal monitoring of biomarkers with physiological, epidemiological, sociological or circumstantial information might provide a logical framework enabling fit for purpose decision-making. Therefore, Anti-Doping Intelligence might prove efficient at providing a more proactive response to any potential or emerging doping phenomenon or to address existing problems with innovative actions or/and policies. This approach might prove useful to detect, neutralize, disrupt and/or prevent organized doping or the trafficking of doping agents, as well as helping to refine the targeting of athletes or teams. In addition, such an intelligence-led methodology would serve to address doping offenses in the absence of adverse analytical chemical evidence.

A one-year monitoring of nicotine use in sport: frontier between potential performance enhancement and addiction issues.

Tobacco consumption is a global epidemic responsible for a vast burden of disease. With pharmacological properties sought-after by consumers and responsible for addiction issues, nicotine is the main reason of this phenomenon. Accordingly, smokeless tobacco products are of growing popularity in sport owing to potential performance enhancing properties and absence of adverse effects on the respiratory system. Nevertheless, nicotine does not appear on the 2011 World Anti-Doping Agency (WADA) Prohibited List or Monitoring Program by lack of a comprehensive large-scale prevalence survey. Thus, this work describes a one-year monitoring study on urine specimens from professional athletes of different disciplines covering 2010 and 2011. A method for the detection and quantification of nicotine, its major metabolites (cotinine, trans-3-hydroxycotinine, nicotine-N'-oxide and cotinine-N-oxide) and minor tobacco alkaloids (anabasine, anatabine and nornicotine) was developed, relying on ultra-high pressure liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-TQ-MS/MS). A simple and fast dilute-and-shoot sample treatment was performed, followed by hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) operated in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) data acquisition. After method validation, assessing the prevalence of nicotine consumption in sport involved analysis of 2185 urine samples, accounting for 43 different sports. Concentrations distribution of major nicotine metabolites, minor nicotine metabolites and tobacco alkaloids ranged from 10 (LLOQ) to 32,223, 6670 and 538 ng/mL, respectively. Compounds of interest were detected in trace levels in 23.0% of urine specimens, with concentration levels corresponding to an exposure within the last three days for 18.3% of samples. Likewise, hypothesizing conservative concentration limits for active nicotine consumption prior and/or during sport practice (50 ng/mL for nicotine, cotinine and trans-3-hydroxycotinine and 25 ng/mL for nicotine-N'-oxide, cotinine-N-oxide, anabasine, anatabine and nornicotine) revealed a prevalence of 15.3% amongst athletes. While this number may appear lower than the worldwide smoking prevalence of around 25%, focusing the study on selected sports highlighted more alarming findings. Indeed, active nicotine consumption in ice hockey, skiing, biathlon, bobsleigh, skating, football, basketball, volleyball, rugby, American football, wrestling and gymnastics was found to range between 19.0 and 55.6%. Therefore, considering the adverse effects of smoking on the respiratory tract and numerous health threats detrimental to sport practice at top level, likelihood of smokeless tobacco consumption for performance enhancement is greatly supported.

Use of forensic investigations in anti-doping.

The fight against doping is mainly focused on direct detection, using analytical methods for the detection of doping agents in biological samples. However, the World Anti-Doping Code also defines doping as possession, administration or attempted administration of prohibited substances or methods, trafficking or attempted trafficking in any prohibited substance or methods. As these issues correspond to criminal investigation, a forensic approach can help assessing potential violation of these rules. In the context of a rowing competition, genetic analyses were conducted on biological samples collected in infusion apparatus, bags and tubing in order to obtain DNA profiles. As no database of athletes' DNA profiles was available, the use of information from the location detection as well as contextual information were key to determine a population of suspected athletes and to obtain reference DNA profiles for comparison. Analysis of samples from infusion systems provided 8 different DNA profiles. The comparison between these profiles and 8 reference profiles from suspected athletes could not be distinguished. This case-study is one of the first where a forensic approach was applied for anti-doping purposes. Based on this investigation, the International Rowing Federation authorities decided to ban not only the incriminated athletes, but also the coaches and officials for 2 years.

Determination of nicotine and nicotine metabolites in urine by hydrophilic interaction chromatography-tandem mass spectrometry: Potential use of smokeless tobacco products by ice hockey players.

Consumption of nicotine in the form of smokeless tobacco (snus, snuff, chewing tobacco) or nicotine-containing medication (gum, patch) may benefit sport practice. Indeed, use of snus seems to be a growing trend and investigating nicotine consumption amongst professional athletes is of major interest to sport authorities. Thus, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection and quantification of nicotine and its principal metabolites cotinine, trans-3-hydroxycotinine, nicotine-N'-oxide and cotinine-N-oxide in urine was developed. Sample preparation was performed by liquid-liquid extraction followed by hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) operated in electrospray positive ionization (ESI) mode with selective reaction monitoring (SRM) data acquisition. The method was validated and calibration curves were linear over the selected concentration ranges of 10-10,000 ng/mL for nicotine, cotinine, trans-3-hydroxycotinine and 10-5000 ng/mL for nicotine-N'-oxide and cotinine-N-oxide, with calculated coefficients of determination (R(2)) greater than 0.95. The total extraction efficiency (%) was concentration dependent and ranged between 70.4 and 100.4%. The lower limit of quantification (LLOQ) for all analytes was 10 ng/mL. Repeatability and intermediate precision were ≤9.4 and ≤9.9%, respectively. In order to measure the prevalence of nicotine exposure during the 2009 Ice Hockey World Championships, 72 samples were collected and analyzed after the minimum of 3 months storage period and complete removal of identification means as required by the 2009 International Standards for Laboratories (ISL). Nicotine and/or metabolites were detected in every urine sample, while concentration measurements indicated an exposure within the last 3 days for eight specimens out of ten. Concentrations of nicotine, cotinine, trans-3-hydroxycotinine, nicotine-N'-oxide and cotinine-N-oxide were found to range between 11 and 19,750, 13 and 10,475, 10 and 8217, 11 and 3396, and 13 and 1640 ng/mL, respectively. When proposing conservative concentration limits for nicotine consumption prior and/or during the games (50 ng/mL for nicotine, cotinine and trans-3-hydroxycotinine and 25 ng/mL for nicotine-N'-oxide and cotinine-N-oxide), about half of the hockey players were qualified as consumers. These findings significantly support the likelihood of extensive smokeless nicotine consumption. However, since such conclusions can only be hypothesized, the potential use of smokeless tobacco as a doping agent in ice hockey requires further investigation.

Potential of IRMS technology for tracing gamma-butyrolactone (GBL).

Popularity of gamma-hydroxybutyric acid (GHB) is fairly stable among drug users, while the consumption of its chemical precursor, gamma-butyrolactone (GBL), is a growing phenomenon. Although conventional analytical methods allow to detect this substance in various matrices, linking a trace and a source is still a difficult challenge. However, as several synthesis pathways and chemical precursors exist for the production of GBL, its carbon isotopic signature may vary extensively. For that purpose, a method has been developed to determine the carbon isotopes content of GBL by means of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). The delta(13)C-values of 19 bulk samples purchased worldwide were in the range from -23.1 to -45.8 per thousand (SD<0.3 per thousand). Furthermore, testing on the purification of GBL by distillation has not been found to be consistent with such a large range of delta(13)C-values, which are likely to result from the isotopic composition of the organic precursors used to produce GBL together with the kinetic isotope effect associated with the synthesis routes. Finally, inter- and intra-variability measurements of the delta(13)C-values demonstrated the high potential of IRMS for discriminating between seizures of GBL and for source determination.

Validation and performance comparison of two carbon isotope ratio methods to control the misuse of androgens in humans.

Carbon isotope ratio of androgens in urine specimens is routinely determined to exclude an abuse of testosterone or testosterone prohormones by athletes. Increasing application of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) in the last years for target and systematic investigations on samples has resulted in the demand for rapid sample throughput as well as high selectivity in the extraction process particularly in the case of conspicuous samples. For that purpose, we present herein the complimentary use of an SPE-based assay and an HPLC fractionation method as a two-stage strategy for the isolation of testosterone metabolites and endogenous reference compounds prior to GC/C/IRMS analyses. Assays validation demonstrated acceptable performance in terms of intermediate precision (range: 0.1-0.4 per thousand) and Bland-Altman analyses revealed no significant bias (0.2 per thousand). For further validation of this two-stage analyses strategy, all the specimens (n=124) collected during a major sport event were processed.