Projets de recherche

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Code: DBS20GC01RV

Increasing interest has been dedicated to DBS in sports drug testing, due to the advantages regarding sample collection and transportation.

Few data is published in the literature regarding analysis of GCs in DBS and concentrations of GCs after administrations of GCs, and it deserve to be studied.

The objective of the project will be to study the detection of oral administration of GCs in DBS. The research will be focused on the following specific objectives:

- To collect DBS samples in excretion studies of DEX, MP and DEF with administration of the compounds in one single oral dose. For DEX, multiple oral doses will be also studied.

- To develop and validate methods to detect DEX, MP and DEF metabolite in DBS.

- To measure concentrations of the compounds in the DBS samples collected after oral administration of GC.

- To evaluate correlation between DBS and plasma concentrations

Main findings

Dried Blood Spots (DBS) have increasing interest in sports drug testing due to easier sample collection compared to urine and blood, and advantages regarding sample transportation and storage. The objective of the project was to study the detection of oral administration of glucocorticoids (GCs) in DBS samples. The concentrations of dexamethasone (DEX), methylprednisolone (MP) and deflazacort (DEF) and respective metabolites were evaluated after single-dose oral treatments or multiple-dose oral treatments (for DEX). For that purpose, analytical methods for the quantitation of these GCs and metabolites, and cortisol (CORT) in DBS samples were developed and validated.

After single oral dose, GCs were detected in all DBS samples collected during the first 8 h post-administration, showing maximum concentrations in the samples collected 1 to 2 h post-administration, and decreasing over time. In the multiple-dose oral treatment, both DEX and 6βOH-DEX showed an increase after each dose followed by a decrease over time. For DEF, only the metabolites 21DES and 6βOH-21DES were detected. The suppression of CORT due to the administration of the GCs was observed in all studies.

The correlation between DBS and plasma was good and was established by calculating the DBS-to-plasma ratios for each compound. Based on the data of the study minimum reporting levels in DBS samples were proposed for DEX, MP and 21DES.

Dried Plasma Spots (DPS) were also collected in the administration studies and methods to quantify DEX, 21DES and CORT in these samples were also developed and validated. The methods showed higher variabilities compared to DBS analysis. They were applied to the analysis of DPS samples collected after the administration of DEX or DEF. The correlation between DPS and plasma samples was evaluated and no significant differences were obtained, in general, for the analytes evaluated.

Compared to plasma, both DPS and DBS present similar advantages regarding sample collection, stability, transportation and storage, however an important limitation is the larger volumes of blood required to collect DPS samples. As a general conclusion, DBS analysis is preferred to DPS for antidoping testing.

WADA has repeatedly encouraged social science research projects to take a regional perspective. The Caribbean is one region where systematic research on doping has been absent, despite calls for action (Acevedo et al., 2011). The paucity of doping research on Caribbean countries is surprising given the region’s strong sporting tradition and history. Sport is part of the Caribbean’s regional identity (CARICOM Commission on Youth Development, 2010), as witnessed by their success internationally. For instance, at the 2016 Olympic Games, 5% of Gold medals were won by Caribbean countries (Jordens, 2016).

Description coming soon.

Code: T20M03RV

Recent research has demonstrated that the criterion of discrimination between allowed and prohibited administrations of GCs needs to be compound specific. For some GCs, largely detected in doping controls, no sufficient data is available, and the objective of this project is to perform excretion studies with these GCs to generate the data needed to define the reporting levels and the washout periods.

The project will be focused on dexamethasone (DEX), methylprednisolone (MP) and deflazacort (DEF).

The research will be focused on the following specific objectives:

• To perform excretion studies of DEX, MP and DEF with administration of one single oral dose. For DEX, multiple oral doses will be also studied.
• To measure concentrations in urine of the parent compounds and the main metabolite in case of DEF, to evaluate the reporting lebels and washout periods.
• To measure concentrations of the compounds and cortisol in plasma, to evalulate the concordance of the urinary reporting levels and washout periods proposed with plasma concentrations of the active drug and the systemic effect

Main findings

Glucocorticoids (GCs) are prohibited in-competition by all injectable routes (including intraarticular and periarticular routes) in addition to oral and rectal administrations. In out-ofcompetition periods, there is no restriction of use. Since most GCs are marketed in different administration forms, the distinction between administration routes is needed to ensure safe treatments by allowed administration routes and to detect the use of prohibited administration routes during competitions. The regulations of GCs in sports were revisited in 2022, and new criteria were established for some of the compounds. In the present study, the discrimination criteria was evaluated for three GCs: dexamethasone (DEX), methylprednisolone (MP) and deflazacort (DEF).

Five clinical studies which involved oral administration of GCs to healthy volunteers were performed: single oral dose of DEX (4 mg, n=8), multiple oral dose of DEX (2mg/12h/5 days, n=8), single oral dose of MP (12 mg, n=8), multiple oral dose of MP (12 mg/3 days, n=8) and single oral dose of DEF (30 mg, n=8). Urine and plasma samples were collected before, during and after the treatments, and both the urinary and plasmatic excretion profiles of each GCs and their metabolites were evaluated using liquid chromatography-tandem mass spectrometry. Overall, the results of the project demonstrate that the minimum reporting levels (MRL) of 60 ng/mL for DEX, 30 ng/mL of MP and 30 ng/mL of desacetyldeflazacort (DEF metabolite) are suitable to detect oral administration of the compounds and the washout-period of 3 days for oral administration is also adequate for these GCs. Results obtained in this project provide additional data that supports that criteria based on substance-specific MRL improve the discrimination between prohibited and permitted use of GCS in sports.

Code: T20M02DM

The differential immunoassays to detect doping with recombinant human growth hormone (hGH) have been in use at WADA accredited laboratories for more than 10 years now. From the beginning, the assays were produced in the coated tube (CT) format. In the past, this format, where the specific antibodies are coated to plastic tubes, and the final assay signal is determined by a tube luminometer, was very popular for immunoassays used in laboratory medicine and research applications. However, it meanwhile has been largely abandoned, and the respective assays were switched to the so-called microtiter plate (MTP) format. Here, the antibodies are coated to 96 wells of a plastic plate, and the final assay signal is read by a plate luminometer. The microplate format has advantages in terms of production, but also in terms of workflow in the laboratory. As a consequence of the grossly reduced use of CT assays in laboratories worldwide, production capacities for such CT assays become rare, and might become unavailable in the near future. To ensure continued production and availability of the differential immunoassays it is suggested to convert these assays to the MTP format now. While assay ingredients (and particularly the monoclonal antibodies involved) remain unaffected by the conversion to the MTP format, and it has been demonstrated for many assays used in clinical routine that key parameters such as assay specificity, linearity, reproducibility etc. remain unaffected by the change, the MTP format nevertheless has to be optimized and validated for each individual assay. The current project aims to establish the MTP format for the differential immunoassays.

Code: R20M01MS

Primarily, the objective of this project is to conduct an exploratory study to monitor the blood steroid profile of women subjects over two menstrual cycles to compare the intra- and inter-individual variability of the blood steroid profile of women subjects during a menstrual cycle. Secondly, transdermal testosterone gel (Androgel 1%, AbbVie, North Chicago, IL) will be administered to all volunteers once. The study will enable the pharmacokinetic study of transdermal testosterone gel in women in addition to its detection and will allow to determine putative differences of steroid metabolism between men and women. Simultaneously, the urinary steroid profile will be monitored to allow comparison of the steroid profile between both matrices. This study should help to develop further the blood steroidal of the ABP and to refine the identification of doping behaviours in females with adequate investigations allowing to define reference values for specific female athletic populations. This study will also allow to lay the foundations for a further untargeted metabolomics project for the discovery of new steroidomic biomarkers for steroid detection in female athletes. The design of the study was modified and extended to three menstrual cycles and the treatment regimen was modified. Instead of a single application, testosterone gel (Tostran 20mg/g, 0.5g applied corresponding to 10mg of testosterone) is applied daily for a menstrual cycle (corresponding to 28 days).

Main Findings

In women, hormonal fluctuations related to menstrual cycle may impose a great source of variability for some urinary biomarkers of testosterone (T) administration, which can ultimately disrupt the sensitivity of their longitudinal monitoring. Additional biomarkers and alternative matrices need therefore to be investigated to improve the detection capability for doping practices with T, especially in female athletes. The aim of this study was therefore to investigate the impact of menstrual cycle combined with T gel administration on the biomarkers of the ABP (steroidal and haematological module) and on serum steroid biomarkers in females. It allowed to directly compare the sensitivity of T gel detection between urinary and blood steroid profiling either for targeting samples for IRMS or for longitudinal evaluation. To achieve this, a clinical trial involving fourteen healthy women subjects was conducted over three consecutive menstrual cycles with the second cycle combined with a daily administration of T gel for 28 days. The sensitivity of the current urinary and haematological markers of the Athlete Biological Passport (ABP), as well as serum steroid biomarkers was investigated for the monitoring of the T gel treatment. Additionally, endogenous fluctuation of these parameters were monitored within the menstrual cycle.

Code: 20D09OP

In previous WADA funded projects (11A9RV, 12A13OP, 14A29OP) our research group discovered two endogenous steroid (EAAS) metabolites, namely, 6β-hydroxyandrosterone-3-glucuronide (6bOHA-3G), and 6β-hydroxyetiocholanolone-3-glucuronide (6bOHEtio-3G) particularly selective markers of the administration of testosterone. Small quantities of the compounds were synthesized and fully characterized. They showed to be resistant to enzymatic hydrolysis, a reason for its late discovery.

An analytical method has already been developed, fully validated and published for the direct analysis of those substances together with other regular steroid profile glucuronide markers. One way to improve the the application of the method would be to have access to labelled internal standards which will decrease the variability of the basal values.

These two new markers have proven to be sensitive to testosterone administration (oral, intramuscular and even transdermal, improving its detection window, as compared to the current T/E ratio. We are  currently testing their specificity to differentiate testosterone administration from ethanol consumption or the combination (WADA project ISF18D13OP). Preliminary results of this on-going project show that while TG/EG increases both with T and EtOH administration, the new markers increase specifically after T administration while they do not increase after EtOH administration. This differential behavior adds specificity and improves the steroid profile. 

The current project (inter-ALMA) aims at finally incorporating these new markers into a routine screening by performing an interlaboratory validation incorporating 5 different laboratories. For that purpose, analytical methodology, standards (already synthesize in the frame of the 2019 PCC project entitled “Essential reference materials to advance the long term detection of testosterone abuse”) and their deuterated analogues (synthesized in the frame of iner-ALMA) will be provided to the laboratories. The outcome after using common or comparable methodologies to analyze selected samples will be compared.