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.

The effect of a period of intense exercise on the marker approach to detect growth hormone doping in sports.

The major objective of this study was to investigate the effects of several days of intense exercise on the growth hormone marker approach to detect doping with human growth hormone (hGH). In addition we investigated the effect of changes in plasma volume on the test. Fifteen male athletes performed a simulated nine-day cycling stage race. Blood samples were collected twice daily over a period of 15 days (stage race + three days before and after). Plasma volumes were estimated by the optimized CO Rebreathing method. IGF-1 and P-III-NP were analyzed by Siemens Immulite and Cisbio Assays, respectively. All measured GH 2000 scores were far below the published decision limits for an adverse analytical finding. The period of exercise did not increase the GH-scores; however the accompanying effect of the increase in Plasma Volume yielded in essentially lower GH-scores. We could demonstrate that a period of heavy, long-term exercise with changes in plasma volume does not interfere with the decision limits for an adverse analytical finding. Copyright © 2014 John Wiley & Sons, Ltd.

Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry I. Screening analysis.

The general strategy to perform anti-doping analyses of urine samples starts with the screening for a wide range of compounds. This step should be fast, generic and able to detect any sample that may contain a prohibited substance while avoiding false negatives and reducing false positive results. The experiments presented in this work were based on ultra-high-pressure liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry. Thanks to the high sensitivity of the method, urine samples could be diluted 2-fold prior to injection. One hundred and three forbidden substances from various classes (such as stimulants, diuretics, narcotics, anti-estrogens) were analysed on a C(18) reversed-phase column in two gradients of 9min (including two 3min equilibration periods) for positive and negative electrospray ionisation and detected in the MS full scan mode. The automatic identification of analytes was based on retention time and mass accuracy, with an automated tool for peak picking. The method was validated according to the International Standard for Laboratories described in the World Anti-Doping Code and was selective enough to comply with the World Anti-Doping Agency recommendations. In addition, the matrix effect on MS response was measured on all investigated analytes spiked in urine samples. The limits of detection ranged from 1 to 500ng/mL, allowing the identification of all tested compounds in urine. When a sample was reported positive during the screening, a fast additional pre-confirmatory step was performed to reduce the number of confirmatory analyses.

The effect of a period of intensive exercise on the isoform test to detect growth hormone doping in sports.

OBJECTIVE: The major objective of this study was to investigate the effects of several days of intense exercise on growth hormone (hGH) testing using the World Anti-Doping Agencies hGH isoform differential immunoassays. Additionally the effects of circadian variation and exercise type on the isoform ratios were also investigated. STUDY DESIGN: 15 male athletes performed a simulated nine day cycling stage race. Blood samples were collected twice daily over a period of 15days (stage race+three days before and after). hGH isoforms were analysed by the official WADA immunoassays (CMZ Assay GmbH). RESULTS: All measured isoform ratios were far below the WADA decision limits for an adverse analytical finding. Changes in the isoform ratios could not be clearly connected to circadian variation, exercise duration or intensity. CONCLUSIONS: The present study demonstrates that the hGH isoform ratios are not significantly affected by exercise or circadian variation. We demonstrated that heavy, long term exercise does not interfere with the decision limits for an adverse analytical finding.

Challenges and threats to implementing the fight against doping in sport

Prominent doping cases in certain sports have recently raised public awareness of doping and reinforced the perception that doping is widespread. Efforts to deal with doping in sport have intensified in recent years, yet the general public believes that the 'cheaters' are ahead of the testers. Therefore, there is an urgent need to change the antidoping strategy. For example, the increase in the number of individual drug tests conducted between 2005 and 2012 was approximately 90 000 and equivalent to an increase of about 50%, yet the number of adverse analytical findings remained broadly the same. There is also a strikingly different prevalence of doping substances and methods in sports such as a 0.03% prevalence of anabolic steroids in football compared to 0.4% in the overall WADA statistics. Future efforts in the fight against doping should therefore be more heavily based on preventative strategies such as education and on the analysis of data and forensic intelligence and also on the experiences of relevant stakeholders such as the national antidoping organisations, the laboratories, athletes or team physicians and related biomedical support staff. This strategy is essential to instigate the change needed to more effectively fight doping in sport.

Detection in urine of 4-methyl-2-hexaneamine, a doping agent.

Stimulants are banned in-competition for all categories of sports by the World Anti-Doping Agency. A simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay employing electrospray ionisation in positive mode was developed in that work for the quantification in urine specimens of 4-methyl-2-hexaneamine, a primary amine exhibiting sympathomimetic properties. Following a simple pretreatment procedure, the analyte was separated using a gradient mobile phase on reverse phase C8 column. Selected reaction monitoring m/z 116.2-->57.3 was specific for detection of 4-methyl-2-hexaneamine and the assay exhibited a linear dynamic range of 50-700 ng/mL. The validated method has been successfully applied to analyze the target compound in food supplements as well as in urine specimens. The administered drug (40 mg) was detected at the level of 350 ng/mL in the urine up to 4 days.

An alternative approach to the prevention of doping in cycling

The article proposes an alternative approach to policies for preventing doping in cycling, based on in-depth analysis of the functioning of nine of the 40 world professional teams and the careers of the 2,351 riders who were or have been professionals since 2005. The first part shows that the instruments of prevention have been based on a questionable understanding of doping as an individual moral fault, and have not produced the expected results. The second part proposes to analyse the ways in which teams and riders produce their achievments, so as to put forward an alternative to the anti-doping policies used hitherto, which have little impact on riders. The study shows that it is more pertinent to examine the forms of employment and the business models, because these have important effects on cycling professionals' conditions of work. It makes it possible to identify three dimensions of the risk of doping on which organisations can act in their antidoping policies: team organisation, riders' preparation and workload, and the precarity of employment.

Circulating microRNAs: The Future of Biomarkers in Anti-doping Field.

microRNAs (miRNAs) are small non-coding RNAs that regulate various biological processes. Cell-free miRNAs have been proposed as biomarkers of disease, including diagnosis, prognosis, and monitoring of treatment responses. These circulating miRNAs are highly stable in several body fluids, including plasma and serum; hence, in view of their potential use as novel, non-invasive biomarkers, the profiles of circulating miRNAs have been explored in the field of anti-doping. This chapter describes the enormous potential of circulating miRNAs as a new class of biomarkers for the detection of doping substances, and highlights the advantages of measuring these stable species over other methods that have already been implemented in anti-doping regimes. Incorporating longitudinal measurements of circulating miRNAs into the Athlete Biological Passport is proposed as an efficient strategy for the implementation of these new biomarkers. Furthermore, potential challenges related to the transition of measurements of circulating miRNAs from research settings to practical anti-doping applications are presented.

Substance use among sportive adolescents in the French-speaking part of Switzerland

[Contents] Introduction. Objectives. Methodology. Results. Characteristics of the sample. Substance use (Psychoactive substances, Performance-enhancing substances). Profile of sportive adolescents using substances. Mixed substance use. Other factors related to substance use. Inactivity. Conclusions. References. Annexes. Annex 1. Questionnaire. Annex 2. Sample weighting procedure. Annex 3. Sports type.

Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. II: Analysis of biological samples.

The potential and applicability of UHPSFC-MS/MS for anti-doping screening in urine samples were tested for the first time. For this purpose, a group of 110 doping agents with diverse physicochemical properties was analyzed using two separation techniques, namely UHPLC-MS/MS and UHPSFC-MS/MS in both ESI+ and ESI- modes. The two approaches were compared in terms of selectivity, sensitivity, linearity and matrix effects. As expected, very diverse retentions and selectivities were obtained in UHPLC and UHPSFC, proving a good complementarity of these analytical strategies. In both conditions, acceptable peak shapes and MS detection capabilities were obtained within 7min analysis time, enabling the application of these two methods for screening purposes. Method sensitivity was found comparable for 46% of tested compounds, while higher sensitivity was observed for 21% of tested compounds in UHPLC-MS/MS and for 32% in UHPSFC-MS/MS. The latter demonstrated a lower susceptibility to matrix effects, which were mostly observed as signal suppression. In the case of UHPLC-MS/MS, more serious matrix effects were observed, leading typically to signal enhancement and the matrix effect was also concentration dependent, i.e., more significant matrix effects occurred at the lowest concentrations.

Anti-doping testing at the 2008 European football championship.

Big sports events like the 2008 European Football Championship are a challenge for anti-doping activities, particularly when the sports event is hosted by two different countries and there are two laboratories accredited by the World Anti-Doping Agency. This challenges the logistics of sample collection as well as the chemical analyses, which must be carried out timeously. The following paper discusses the handling of whereabouts information for each athlete and the therapeutic use exemption system, experiences in sample collection and transportation of blood and urine samples, and the results of the chemical analysis in two different accredited laboratories. An overview of the analytical results of blood profiling and growth hormone testing in comparison with the distribution of the normal population is also presented.

Quantification of clenbuterol at trace level in human urine by ultra-high pressure liquid chromatography-tandem mass spectrometry.

Clenbuterol is a β2 agonist agent with anabolic properties given by the increase in the muscular mass in parallel to the decrease of the body fat. For this reason, the use of clenbuterol is forbidden by the World Anti-Doping Agency (WADA) in the practice of sport. This compound is of particular interest for anti-doping authorities and WADA-accredited laboratories due to the recent reporting of risk of unintentional doping following the eating of meat contaminated with traces of clenbuterol in some countries. In this work, the development and the validation of an ultra-high pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the quantification of clenbuterol in human urine is described. The analyte was extracted from urine samples by liquid-liquid extraction (LLE) in basic conditions using tert butyl-methyl ether (TBME) and analyzed by UHPLC-MS/MS with a linear gradient of acetonitrile in 9min only. The simple and rapid method presented here was validated in compliance with authority guidelines and showed a limit of quantification at 5pg/mL and a linearity range from 5pg/mL to 300pg/mL. Good trueness (85.8-105%), repeatability (5.7-10.6% RSD) and intermediate precision (5.9-14.9% RSD) results were obtained. The method was then applied to real samples from eighteen volunteers collecting urines after single oral doses administration (1, 5 and 10μg) of clenbuterol-enriched yogurts.

Endogenous steroid profiling in the athlete biological passport.

The Athlete Biological Passport (ABP) is an individual electronic document that collects data regarding a specific athlete that is useful in differentiating between natural physiologic variations of selected biomarkers and deviations caused by artificial manipulations. A subsidiary of the endocrine module of the ABP, that which here is called Athlete Steroidal Passport (ASP), collects data on markers of an altered metabolism of endogenous steroidal hormones measured in urine samples. The ASP aims to identify not only doping with anabolic-androgenic steroids, but also most indirect steroid doping strategies such as doping with estrogen receptor antagonists and aromatase inhibitors. Development of specific markers of steroid doping, use of the athlete's previous measurements to define individual limits, with the athlete becoming his or her own reference, the inclusion of heterogeneous factors such as the UDPglucuronosyltransferase B17 genotype of the athlete, the knowledge of potentially confounding effects such as heavy alcohol consumption, the development of an external quality control system to control analytical uncertainty, and finally the use of Bayesian inferential methods to evaluate the value of indirect evidence have made the ASP a valuable alternative to deter steroid doping in elite sports. The ASP can be used to target athletes for gas chromatography/combustion/ isotope ratio mass spectrometry (GC/C/IRMS) testing, to withdraw temporarily the athlete from competing when an abnormality has been detected, and ultimately to lead to an antidoping infraction if that abnormality cannot be explained by a medical condition. Although the ASP has been developed primarily to ensure fairness in elite sports, its application in endocrinology for clinical purposes is straightforward in an evidence-based medicine paradigm.

Fast screening and confirmation of doping agents by UHPLC-QTOF-MS/MS

Introduction: The general strategy to perform anti-doping analysis starts with a screening followed by a confirmatory step when a sample is suspected to be positive. The screening step should be fast, generic and able to highlight any sample that may contain a prohibited substance by avoiding false negative and reducing false positive results. The confirmatory step is a dedicated procedure comprising a selective sample preparation and detection mode. Aim: The purpose of the study is to develop rapid screening and selective confirmatory strategies to detect and identify 103 doping agents in urine. Methods: For the screening, urine samples were simply diluted by a factor 2 with ultra-pure water and directly injected ("dilute and shoot") in the ultrahigh- pressure liquid chromatography (UHPLC). The UHPLC separation was performed in two gradients (ESI positive and negative) from 5/95 to 95/5% of MeCN/Water containing 0.1% formic acid. The gradient analysis time is 9 min including 3 min reequilibration. Analytes detection was performed in full scan mode on a quadrupole time-of-flight (QTOF) mass spectrometer by acquiring the exact mass of the protonated (ESI positive) or deprotonated (ESI negative) molecular ion. For the confirmatory analysis, urine samples were extracted on SPE 96-well plate with mixed-mode cation (MCX) for basic and neutral compounds or anion exchange (MAX) sorbents for acidic molecules. The analytes were eluted in 3 min (including 1.5 min reequilibration) with a S1-25 Ann Toxicol Anal. 2009; 21(S1) Abstracts gradient from 5/95 to 95/5% of MeCN/Water containing 0.1% formic acid. Analytes confirmation was performed in MS and MS/MS mode on a QTOF mass spectrometer. Results: In the screening and confirmatory analysis, basic and neutral analytes were analysed in the positive ESI mode, whereas acidic compounds were analysed in the negative mode. The analyte identification was based on retention time (tR) and exact mass measurement. "Dilute and shoot" was used as a generic sample treatment in the screening procedure, but matrix effect (e.g., ion suppression) cannot be avoided. However, the sensitivity was sufficient for all analytes to reach the minimal required performance limit (MRPL) required by the World Anti Doping Agency (WADA). To avoid time-consuming confirmatory analysis of false positive samples, a pre-confirmatory step was added. It consists of the sample re-injection, the acquisition of MS/MS spectra and the comparison to reference material. For the confirmatory analysis, urine samples were extracted by SPE allowing a pre-concentration of the analyte. A fast chromatographic separation was developed as a single analyte has to be confirmed. A dedicated QTOF-MS and MS/MS acquisition was performed to acquire within the same run a parallel scanning of two functions. Low collision energy was applied in the first channel to obtain the protonated molecular ion (QTOF-MS), while dedicated collision energy was set in the second channel to obtain fragmented ions (QTOF-MS/MS). Enough identification points were obtained to compare the spectra with reference material and negative urine sample. Finally, the entire process was validated and matrix effects quantified. Conclusion: Thanks to the coupling of UHPLC with the QTOF mass spectrometer, high tR repeatability, sensitivity, mass accuracy and mass resolution over a broad mass range were obtained. The method was sensitive, robust and reliable enough to detect and identify doping agents in urine. Keywords: screening, confirmatory analysis, UHPLC, QTOF, doping agents

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.