Projets de recherche

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.

This study aims to investigate and compare the concentration changes of GCs in DBSs and urine during the washout period following GC administration. A rapid and sensitive UPLC-MS/MS-based method has been developed for the simultaneous quantitation of six common GCs in DBSs, with the extraction procedure optimised to ensure favourable recoveries. DBSs and urine samples are then collected from subjects receiving oral prednisolone, intramuscular dexamethasone, and locally injected triamcinolone acetonide. The pharmacokinetics and detection windows of each GC in DBSs are established, and reference ranges are suggested. The DBS data are compared with those from urine. The developed analytical method and the resulting elimination datasets are readily deliverable to anti-doping labs and authorities, enhancing the applicability of DBS for GC testing.

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.

Code: 241C03JFN

The REV-ERB alpha and beta agonist SR9011 was initially synthesized to explore the relations between sleep disorders and circadian rhythm in rodent models. In parallel, studies highlighted that SR9011 ameliorates muscular growth and affects mitochondrial composition, making this compound attractive to athletes who desire to improve their sports performance. Consequently, SR9011 was placed on the WADA prohibited substance list in 2018. However, only the compound was included because of poorly understood and characterized metabolism. Since January 2024, some metabolites of SR9011 have been included in the WADA list of banned substances. So far, two publications have suggested structures some metabolites, although their structure must be re-examined. The first objective of this project is to adapt our previous protocol using human subcellular liver fractions to spheroid cell culture to corroborate the presence of oxidative metabolites. By this, we aimed to refine our previously suggested metabolites to reinforce those susceptible to be detected in urine samples. The spheroid which are terminally differentiated hepatic cells derived from a human hepatic progenitor cell line will be used to obtain metabolites. To achieve metabolites generation, spheroid and SR9011 will be incubated in a cell culture medium. In parallel, considering the phase II metabolism of SR9011 has not been addressed to date, the investigation of glucuronide conjugates on SR9011 will constitute the second objective. Finally, since WADA has included four oxidative metabolites in the list of banned substances and considering that no reference materials are currently available, the third objective of this proposal consists of adapting small-scale synthesis routes to large-scale production of those metabolites in readiness to provide enough reference material to worldwide anti-doping laboratories.

Code: 242E03KC

Gene doping, which is banned by WADA, is the alteration of an athlete’s DNA in order to obtain athletic advantage. Rapid advances in genome editing technologies pose a significant future threat to fair competition and athlete safety. This project aims to address this challenge by focusing on detecting performance-enhancing genome modifications.

This research will begin with animal models to study how effectively genome editing can be achieved in tissues relevant for doping and then be detected non-invasively. From there, the research will work towards developing accurate, affordable, and scalable methods to detect genome editing in humans in the context of performance enhancement.

The project consists of three main objectives:

1. Animal studies. Using state-of-the-art genomic technologies, we will perform genome editing experiments in mice. This will help us assess the efficiency of genome editing across different tissues and identify non-invasive detection methods.
2. Human genome editing detection assay. We will create a targeted deep sequencing tool to detect edits in 20-30 human genes linked to doping. We will develop software to differentiate genuine edits from experimental errors and naturally occurring variations.
3. Validation. We will validate the assay by applying it to lab-edited primary human hematopoietic stem cells and employing a blinded computational analysis to identify genome editing events.

This pioneering research will provide crucial insights into how new genomic technologies could be used in doping and on the factors that affect editing detectability. Our goal is to create novel, cost-effective methods for detecting genome editing in humans, supporting anti-doping efforts worldwide.

Code: 242B05SV

ERA analysis has been repeatedly legally challenged. One of the criticised points is potential protein overload related to exercise induced proteinuria. Although exercise induced proteinuria has been investigated for IEF and SDS-PAGE and samples are immunopurified, no standardized data are available for the currently used SAR-PAGE method.

Although both methods apply the same measurement principle, which is basically separation by migration behaviour based on molecular weight and posttranslational modifications such as glycosylation or pegylation of the molecule, the detergent is different. In addition, the analysis method evolved with the introduction of the biotinylated version of the AE7A5 Anti-EPO antibody being now used for the analysis, and no data exist which can exclude the possibility that contents of exercise urines might cross-react with, for example, the Streptavidin-HRP complex which is used for the visualization by chemiluminescence.

Further, it has not been demonstrated whether high intensity exercise in a hypoxic environment could further affect the ERA analysis, for example by a higher excretion of erythropoietin. Therefore, it is planned to investigate urine samples from 30 athletes who are subjected to an exercise protocol on a treadmill or cycling ergometer known to induce proteinuria. In addition, urine samples from 10 athletes following an acute hypoxic exercise protocol will be collected and examined.

Code: 241E07CS

The detection of gene and cell doping in athletes is crucial to maintain fair competition and prevent the use of performance-enhancing substances that can be harmful to an athlete's health. In this project, we propose the development of all-in-one gene doping detection chip paper based on Multiplexed Recombinase Polymerase Amplification (RPA) and CRISPR/Cas12a system with on-chip target gene amplification and detection functions. This chip will facilitate the sequential execution of multiple isothermal nucleic acid amplification and CRISPR/Cas12a sensing reactions directly on small blood samples, eliminating the need for separate pre-processing steps. Our proposed all in one gene doping chip paper features three key components: a sample pad, four conjugation pads, and a test membrane. The sample pad, preloaded with dried reaction buffer, allows for simple heat treatment of whole blood, releasing transgene DNA directly onto the chip without pre-preparation step. Each conjugation pad, linked to a specific target gene, combines on-chip RPA with a Cas12a reaction. In just 15 minutes at 37°C, RPA amplifies the target gene using specific primers. Next, Cas12a cleaves a reporter molecule (biotin-FAM) in the presence of the target exogenous DNA, but leaves it intact in its absence. Finally, the test membrane, pre-coated with streptavidin and anti-anti-FAM lines, captures the reporter depending on the target presence.