Projets de recherche

Code: 25T04MT

A major concern both for athletes and result management authorities is the possibility of nutritional supplement/dietary product contamination with minute amounts of anabolic agents leading to adverse-analytical findings (AAFs) in doping controls. The analytical sensitivity of anti-doping laboratories has been optimized to allow for utmost retrospectivity in sports drug testing, but the problem arising from such a performance is that also trace amounts introduced into an athlete’s organism by contaminated supplements or food are detected.

To support the process of fair and comprehensive case management, in-depth investigations into the elimination profiles and metabolite ratios of micro-dosed LGD-4033 were conducted in 2020/21. For that purpose, single- and multi-micro-dose excretion studies at 1, 10 and 50 μg LGD-4033 were conducted, and the collected samples analyzed by enzymatic hydrolysis, solid-phase extraction (SPE) and LC-HRMS/MS. A total of 15 phase I metabolites was detected and the ratios of LGD-4033 and the epimer M1 as well as the metabolites M5-a and M5-b were found to shift over time. The combined evaluation of the LGD-4033/metabolite concentrations and ratios can be utilized to estimate whether a minute amount of the drug was recently ingested or a larger, pharmacologically relevant amount of the doping agent was administered several days/weeks ago and thus evaluate the plausibility of potential contamination scenarios.

Within this follow-up research project, the available micro-dose administration study samples will be re-analyzed to quantify the main long-term metabolite of LGD-4033 (M5-a) by using meanwhile available certified reference material.

Code: 25T02AM

Despite suspected high prevalence of Growth Hormone (GH) doping, detection of GH doping is complex. The indirect method based on two biomarkers increased by GH (growth factor IGF-I and procollagen propeptide P-III-NP) has recently been integrated in the endocrine module from the Athlete biological passport (ABP) to better identify doping with GH using a P-III-NP assay for Centaur Analyzer and IGF-I by top-down mass spectrometry.

However Centaur analyzer and its P-III-NP assay are to be ended soon (2027) and need to be replaced. Siemens proposes a new Atellica analyzer with a test for P-III-NP that is described as highly similar to Centaur results in the normal to pathological range, but this need further confirmation in the athletes population range The aims of the project are to evaluate and confirm this new technical option for P-III-NP quantification in the coming years.

1- Comparison of P-III-NP assays performed on two Siemens analyzers and impact of storage : 200 serum samples previously analyzed for endocrine passport will be reanalyzed for P-III-NP in parallel on Advia Centaur and on the new Siemens analyzer Atellica in order to evaluate the similarity of results in an athletes population.

This will also allow a direct comparison to initial values to confirm storage conditions (-80°C) are appropriate for reanalysis;

2- Impact of various degrees of hemolysis on P-III-NP and IGF-I by adding free hemoglobin to clear sample and checking the quantification of both markers.

These data will help to confirm that the new technical options for P-III-NP detection are fit-for-purpose and will increase knowledge on how variations in quality of serum impact endocrine passport.

Code: 25T01RV

Glucocorticoids (GC) are forbidden in sports competitions when used by injectable, oral or rectal routes. They are allowed by other routes for therapeutic purposes. Since most GCs are marketed in different administration forms, the distinction between routes of administration is needed to ensure safe treatments by allowed routes of administration and to detect the use for doping purposes during competitions. Based on scientific data available, compound-specific reporting levels were established for some GC and washout periods after some administration routes.

There is no data in literature regarding concentrations of GCs in urine after intravenous administration. The objective of the present project will be to perform administration studies with dexamethasone (DEX) and methylprednisolone (MP) using intra-venous (IV) administration, to evaluate the suitability of the reporting levels already defined for DEX and MP to detect IV administration and to define the washout periods after IV administration to avoid fase adverse analytical findings due to treatments close to competitions.

Code: 241C05MP

Recently, the success of the integration of new long-term markers for anabolic steroids such as metandienone greatly improved the detectability of their misuse in sports. Tetrahydromethyltestosterone-sulfate (THMTS) was proposed as a promising long-term marker for methyltestosterone administration, however, reference material for direct analysis of the intact conjugates is not available for all diastereomers. Reportedly, the longest detectable diastereomer was tentatively assigned as 3α5α17epiTHMTS after solvolysis using GCMS and literature reported Kovac indices for diastereomer allocation. Our preliminary data from intact sulfoconjugate analysis suggest 3α5βTHMTS instead. It remains open until now weather this difference is due to potential epimerization by solvolysis, sample pre-treatment or interindividual variability. Additionally, A-Ring reduced 17β-hydroxymethyl-13-ene (20OHNorTHMT) metabolites have not yet been consequently investigated after methyltestosterone administration. Analogous metabolites resulted in drastic extension of the detection window after Oral Turinabol administration.

Within the project the analysis of tetrahydromethyltestosterone (THMT) diastereomers as classically monitored metabolites of methyltestosterone, A-Ring reduced 17β-hydroxymethyl-13-ene metabolites (20OHNorTHMT), as well as intact sulfoconjugates, will be included in the analysis to evaluate the best suited metabolites after an administration of methyltestosterone.

Therefore, methods will be optimized for sensitive and selective detection using the various diastereomeric sets of target analytes. Furthermore, the suitability of different possibilities for sample preparation will be evaluated specially focusing on the sulfoconjugates. Their diastereomeric structure will unambiguously be confirmed by direct analysis of the intact conjugates and comparison with references of all diastereomers. Full characterization of the relevant THMTS diastereomers with LC-HRMS and NMR will be provided after chemical synthesis and purification. Including postadministration samples from six volunteers will help for investigation of interindividual variabilities of metabolite excretions.

Code: 242A12JFN

7-keto-DHEA is a performance enhancing anabolic androgenic steroid on the WADA prohibited list and abuse by athletes can be detected unambiguously using carbon stable isotope analysis after urine sample purification and isotope ratio mass spectrometric analysis. Stable isotope confirmation is required as 7-keto-DHEA and its primary metabolites can be formed through microbial transformation of naturally produced endogenous steroids. As the 13C content of steroids is conserved during their metabolism, the 13C content of the metabolites and parent compound excreted in urine will reflect that of the source of the compounds (either endogenous through in situ microbial reworking or exogenous through doping). Existing methods for detecting 7-keto-DHEA doping target multiple metabolites found principally as free form or glucuronidated metabolites. Due to method artifacts, it is possible that the proportion of sulfoconjugated metabolites of 7-keto-DHEA is currently being under reported. During initial scouting investigations, a significant increase in recovery of the analytes of interest has been observed using an acidic hydrolysis involving tetrahydrofuran and methanol as solvents. We aim to develop and validate a GC/C/IRMS confirmation method targeting the principal metabolites of 7-keto-DHEA in both the sulfoconjugated and glucruonidated steroid fractions. We will analyze a series of anonymized athlete urine samples which contain high concentrations of either 7α-OH-DHEA and 7β-OH-DHEA during our routine initial testing procedure over the last 5 years. This will serve as a reference population for North American athletes and will be of use in the context of determining positivity thresholds for inclusion in future WADA technical documents.

Code: 242D07FP

The longitudinal monitoring of Testosterone and Androstenedione blood concentrations was introduced in the Steroidal Module of Athlete Biological Passport in 2023 as a complementary tool to the urinary module. Although this approach has proven to increase the detection capability of endogenous anabolic androgenic steroids doping, limited information is currently available regarding confounding factors of blood steroid profiling. In this context, alcohol consumption is one of the exogenous confounding factors in urinary steroid analysis, but its influence on blood steroid profile has not yet been investigated in detail. The aim of this project is therefore to fill this gap by firstly focusing on the development of a novel LC-MS method for the simultaneous quantification of Testosterone, Androstenedione, and two markers of alcohol consumption, Ethyl glucuronide and Ethyl sulfate. Mass spectrometric parameters will be fine-tuned to achieve maximal sensitivity, and chromatographic conditions will be optimized to find the best compromise between resolution and analysis time. Once the developed method is validated according to WADA's requirements and transferred to a WADA-accredited laboratory, it will be employed in both laboratories to analyze real serum samples collected during a dedicated clinical study, allowing for inter-laboratory comparison. The clinical study will involve 20 healthy volunteers, who will be administered a moderate dose of alcohol. For each participant, both alcohol and placebo will be administered in two separate sessions. Serum and urine samples will be collected before the intervention and at 5 consecutive time points during the day of administration, with an additional sample collected the morning after the intervention. The results obtained from serum samples analyzed with the novel method and from urine samples analyzed using the routine method of WADA-accredited laboratory will then be investigated to characterize the effect of alcohol consumption on both parts of the Steroidal Module.

Code: 242C06DK

Understanding the metabolism of a drug is essential for the development of analytical assays, as it can reveal metabolic markers that can significantly increase the sensitivity of detection and extend detection times. 19-Nordehydroepiandrosterone (19-nor-DHEA) is an unlicensed steroid currently available for online purchase and is a combined derivative of the androgen/anabolic steroid nandrolone (19-nortestosterone) and the androgen prohormone dehydroepiandrosterone (DHEA), both of which are on the WADA Prohibited List. 19-Nor-DHEA is not on the WADA Prohibited List and its metabolism has never been reported in humans. It can be categorized as a nandrolone prohormone where, according to TD2021NA, the target metabolite to detect its abuse is 19-norandrosterone (19-NA) in its glucuronide conjugate, which is currently monitored by routine analytical methods. However, quantities of 19-nor steroid metabolites are also excreted in the sulphate fraction and their detection may help to improve long-term detection of their misuse. This study aims to elucidate the metabolic fate of 19-nor-DHEA, with a particular interest in obtaining the most abundant metabolites and those that can be excreted over a longer period. The study will focus on the detection, in addition to 19-NA and 19-NE, of metabolites expected from the metabolism of its 19-methyl analogue, DHEA, in both the glucuronide and sulphate fractions. Long-term markers for the 19-methyl counterpart, DHEA, have been reported in the sulphate fraction and their detection and identification on 19-nor-DHEA is the main objective of this study. The characterization of these metabolites is also a critical point of this study.

Code: 242C05PB

Beta2-agonists are commonly used by elite endurance athletes to counteract asthma and exercise-induced bronchoconstriction, whereby more than 50% of athletes experience lower airways dysfunction. Inhaling long acting beta2-agonists (such as formoterol) is an efficient administration route that achieves high systemic concentrations and has the potential to be ergogenic to exercise performance. Concerningly, recent reports suggest that Olympic athletes who use beta2-agonists outperform their non-user competitors. Therefore, given the potential performance-enhancing and health-related adverse effects of formoterol, WADA has further restricted formoterol intake allowance (2025 WADA Prohibited List) in an attempt to limit the ergogenic effects. Nonetheless, compared to other beta2-agonists, such as salbutamol and terbutaline, the ergogenic potential of formoterol has been less studied. Furthermore, most of the research has focused on short duration, strength and sprint-type exercise, providing minimal data assessing the ergogenic potential of formoterol on endurance events. As such, more research is required to investigate the performance effects of different doses of formoterol that reflect the new 12-hourly dosing intervals and different combinations of divided doses over a 24-hr period. This line of research can confirm whether the 2025 Prohibited List Guidelines are appropriate such that the symptoms of asthma and exercise-induced bronchoconstriction can be minimized, without providing a substantial ergogenic effect on exercise performance. There is also a need to better understand the mechanisms underpinning the performance-induced changes of formoterol administration. Therefore, the aims of this project are to investigate the ergogenic potential (prolonged endurance, and sprint ability) and characterize the resultant blood, muscle and urine metabolite changes induced by different single and divided doses of formoterol administration, compared to a placebo device.

Code: 242C03WCWC

Glucocorticoids (GCs) are widely used in medicine among athletes and are prohibited in competition only when administered orally, rectally, or by injection. However, an in-competition urine sample may show GC levels above the established laboratory reporting levels even if the administration occurred out-of-competition. Urinary washout periods have been established to mitigate this risk. Since blood levels correlate better with substance exposure than urine, dried blood spots (DBSs) collected through microsampling may provide valuable support in determining the timing of GC administration.

Code: 242B02CR

Chapter S2 of WADA’s Prohibited List 2024 (“Peptide hormones, growth factors, related substances, and mimetics”) lists under sub-chapters 2 and 3 “Peptide hormones and their releasing factors” and “Growth factors and growth factor modulators” as prohibited substances. Chapter S4 “Hormone and metabolic modulators”, sub-chapter 3 (“Agents preventing activin repetor IIB activation) additionally mentions “Activin receptor IIB competitors” (e.g. ACE-031) and “Myostatin inhibitors” (e.g. follistatin, myostatin propeptide) as prohibited proteins. Most of these compounds have not been pharmaceutically approved but are available on the black market. In the past, only studies on injectable black market peptides and proteins were performed. They led to the identification of unknown compounds with doping relevance and revealed the presence of “tags”, which made them clearly distinguishable from the body's own proteins (e.g. follistatin with His-tags, myostatin propeptide with GST-tags). Recently, an increasing amount of nasal sprays containing growth promoting peptides and proteins (e.g. AOD-9694, ACE-031, CJC-1295, Follistatin 344, GDF-8) appeared on the black market. So far, no comprehensive study on these products has been performed. Hence, it is unclear whether the compounds contained in nasal sprays are detectable with the current methods or not. For example, some might contain “tag-free” proteins that pass through mucous membranes more easily but are undetectable using existing methods. Within the scope of this project, ca. 50 peptide/protein-based nasal sprays will be purchased on the black market and analyzed with high resolution mass spectrometry and gel-electrophoresis. Subsequently, their detectability with the currently applied methods will be evaluated. In case of unknown compounds, top-down and bottom-up proteomics will be applied for identification. For compounds which cannot be detected with the current methods, alternative strategies will be proposed.