Projets de recherche

Code: 15A22MT 

The number of samples and target analytes is constantly increasing, necessitating all analytical methods to be optimized with regards to speed and costs effectiveness to follow the challenges of the modern anti-doping fight. Low molecular mass peptidic drugs (< 2kDa) have become more and more prominent on the Internet and in black markets, and their misuse in elite sport has been proven in the past.

The analysis of these new compounds is possible and methods are installed in the accredited anti-doping laboratories worldwide.  Unfortunately, these methods are largely designed to cover small peptidic drugs only in exclusive categories or after dedicated sample preparation as shown for instance for growth hormone releasing peptides, gonadorelins, desmopressin, TB-500, AOD-9604 and ARA-290. This is mainly due to the required sensitivity of these methods that enables the detection of the drugs in very low concentrations (sub-ng/mL). Within this study, a generic sample preparation strategy (dilute and inject) which is valid for several classes of compounds (diuretics, stimulants, anabolic agents etc.), will be evaluated for the detection of low molecular mass peptide hormones in urine even in the sub-ng/mL-levels. Therefore, several types of mass spectrometers and liquid chromatography methodologies will be assessed to find the best option for the aimed combination. This will enable a comprehensive multi-target screening within one analysis.

Main Findings: 

The number of small peptides prohibited in sports is steadily increasing, as well as the number of samples that have to be analyzed in sports drug testing programs. Consequently, fast, comprehensive, sensitive, and reliable new methods are needed to accordingly cover all these challenges. “Dilute-and-inject” approaches for urine analysis by liquid chromatography/mass spectrometry represent the most simplified way with a minimum of sample preparation workload. But due to the missing pre-concentration and purification steps, sophisticated chromatographic and mass spectrometric conditions are required to enable sensitive initial testing procedures. All of these aspects were taken into consideration when developing this new screening procedure by direct urine injection with online pre-concentration using a trapping column, liquid chromatographic separation, and subsequent detection by means of ion mobility coupled to high resolution mass spectrometry. 
Additionally, the in-vitro metabolism of selected model peptides such as ARA-290 and GHRP-3 was evaluated and new metabolites were identified by using different strategies as for example incubation with plasma, serum, human liver microsomes, and specific enzymes (e.g. amidases).

Code: 14A29OP

The project has two main goals. Firstly, the project aims to evaluate the potential of 3α-glucuronide-6β-hydroxyandrosterone (6OH-A-3G) and 3α-glucuronide-6β-hydroxyetiocholanolone (6OH-Etio-3G) for the screening of endogenous anabolic androgenic steroid misuse. For this first purpose, a quantitative method validated during the project 12A13OP will be applied to several samples from excretion studies already available in our laboratory. Secondly, the project also aims to characterize, elucidate and synthesize the marker M170T298. For this purpose, a large range of analytical strategies will be applied e.g. different derivatization steps, an in-depth study of accurate mass and tandem mass spectra. Once elucidated, the marker will be synthesized and characterized by NMR. After the synthesis of the marker, it would be useful to develop a quantitative method and to check its potential for other administration routes of testosterone. It is intended to reach these goals in a follow-up project once the objectives of the present project are achieved.

Main findings

In previous WADA funded projects (11A9RV and 12A13OP), our research group revealed that two testosterone metabolites resistant to enzymatic hydrolysis: 3α-glucuronide-6β-hydroxyandrosterone (6OH-A-3G) and 3α-glucuronide-6β-hydroxyetiocholanolone (6OH-Etio-3G) were present in urine. We characterized them, synthesized them and validated a method for their quantitation. However, their potential usefulness for doping control analysis was not fully evaluated.

On the other hand, common investigations for the screening of testosterone misuse are focused on metabolites with a predicted structure. Thus, unpredicted markers remain not evaluated. In order to cover this gap, we performed a preliminary metabolomic study which showed the presence of a marker (code M170T298) which is a potential urinary marker for testosterone administration. In order to confirm this fact, the characterization, elucidation and synthesis of this marker are required.

Thus, the project has two main goals. Firstly, the project aims to evaluate the potential of 6OH-A-3G and 6OH-Etio-3G for the screening of EAAS misuse. Secondly, the project also aims to characterize, elucidate and synthesize the marker M170T298.

Regarding the first goal, the usefulness of 6OH-A-3G and 6OH-Etio-3G was tested in three scenarios: (i) oral administration, (ii) intramuscular administration and (iii) gel administration. After oral administration of testosterone undecanoate we found that four markers containing either 6OH-A-3G or 6OH-Etio-3G presented detection windows (DWs) larger for all volunteers than those obtained by T/E and comparable to those reported by using cysteinyl metabolites. After intramuscular administration, markers containing either 6OH-A-3G or 6OH-Etio-3G provided detection windows that were similar or longer than those obtained by markers currently included in the steroid profile and clearly higher than those obtained by cysteinyl markers. Finally, the administration of testosterone gel could be screened by markers containing either 6OH-A-3G or 6OH-Etio-3G reaching similar to those obtained by markers currently included in the Athlete Biological Passport.

Regarding the second goal, during this project, we have characterized, elucidated and synthesized the marker M170T298. Our research shows that this marker is 1-cyclopentenoyl glycine (1-CPG) which is probably coming from the metabolism of the cypionic acid released after the hydrolysis of testosterone cypionate. Therefore, it can be valuable to detect the misuse of testosterone cypionate. This fact has been confirmed by analysis of samples collected after testosterone cypionate administration. The determination of 1-CPG provided the longest DWs for these samples

The WADA (World Anti-Doping Agency) Education Committee is seeking to target its Social Science Research Program to areas that would have greatest return in terms of enhancing the effectiveness of anti-doping education interventions. The objective of this project was to use the Sport Drug Control Model framework (SDCM; Donovan et al., 2002) to establish priorities for future social science research by reviewing what research has been completed in each of the model’s domains and identifying where more research is needed. The primary required outcome was the list of recommendations rather than the review per se.

Recognising the pivotal role that coaches play in clean sport, the World Anti-Doping Agency
(WADA) commissioned the International Council for Coaching Excellence (ICCE) to conduct a
research project to explore the potential of High Performance Training Centres (HPCs) to
become anti-doping training providers. This report provides guidance for the development of
a sustainable, cooperative and international anti-doping training programme for high
performance coaches.

The “One Resource Kit for Teachers” project was promoted in 2014 by a group of international Organizations composed by the World Anti‐Doping Agency (WADA), the United Nations Educational, Scientific and Cultural Organization (UNESCO), the International Olympic Committee (IOC), the International Paralympic Committee (IPC), the International Council for Sport Science and Physical Education (ICSSPE) and the International Fair Play Committee (IFPC). The main goals reported in the call were to map curriculum requirements for values education across the globe and to develop and pilot a contemporary resource kit for teachers that supports the delivery of their existing curriculum requirements for values-based education through sport.

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Jiri Mudrak, République Tchèque

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