Projets de recherche

Code: T19M02MT

Stimulants have been banned as performance-enhancing drugs in sport since first anti-doping rules were introduced, and they are still frequently detected in doping controls.[1-3] Most of the stimulants on the World Anti-Doping Agency’s (WADA’s) Prohibited List, such as amphetamine, cocaine or ephedrine are either synthetic drugs or compounds of plant-origin. In addition, some (semi)synthetically derived substances are also naturally produced in the human body and involved in processes of the central nervous system.[4,5] This includes 2-phenylethylamine (PEA), which represents the core structure of several stimulants and psychoactive drugs and belongs to the so-called “trace amines”, present only at trace levels in the mammalian central nervous system.[6,7]. While these structurally related biogenic amines, also including tyramine and octopamine, have long been considered as simple metabolic by-products, the characterization of mammalian trace amine associated receptors (TAAR) has led to the understanding that trace amines act as potent neuromodulators, modifying the activity of coexisting neurotransmitters.[8-11] In physiological concentrations, PEA mainly affects dopamine-mediated neurotransmission, with elevated levels of PEA resulting in increased responsiveness to dopamine.[12,13] At high concentrations, PEA has welldocumented “amphetamine-like” effects on the release and reuptake of noradrenaline, dopamine and serotonin, thereby affecting appetite, mood and mental alertness.[5,14,15] Endogenous PEA is formed by enzymatic decarboxylation of phenylalanine. With an elimination half-life of only 0.4 minutes it is rapidly metabolized, mainly by monoamine oxidase (MAO) B, to phenylacetic acid.[11,16] Accordingly, urinary levels of PEA can be influenced by various factors, including nutrition, pharmacological interventions and mental diseases.[8,17-20] Being involved in the monoaminergic neurotransmission, PEA has been associated with major human disorders, including schizophrenia, Parkinson’s disease or depression.[21-23] In a study by Szabo et al. in 2001, it has further been linked to the antidepressant effects of sport, by showing substantial increases of phenylacetic acid in urine after aerobic exercise.[24]

Two different studies were conducted to examine the excretion pattern of 2-phenylethylamine in urine.
a) Single dose (SD) administration study (100 mg): A single dose of 100 mg of PEA was administered to 14 healthy volunteers (7 male, 7 female). Urine samples were collected both before and up to 72 h following administration. Within the first 24 h, every urine sample was collected, afterwards only two urine samples approximately every 12 h.
b) Multiple dose (MD) administration study (5x100 mg): Five doses of 100 mg PEA each were administered on five consecutive days to 14 healthy volunteers (7 male, 7 female). Before and during drug administration, morning urine specimens were collected. Within 24 h after the last dose, every urine sample was collected, afterwards only two urine samples approximately every 12 h.
Dosing was facilitated by encapsulating an amount of the nutritional supplement corresponding to 100 mg of PEA. The collected urine specimens were frozen at -20 °C until analysis. The administration studies were approved by the local ethics committee (#107/2019) and all participants provided written consent.

Main Findings: 

Offering analytical strategies to detect the misuse of substances naturally produced in the human body remains crucial in the fight against doping in sport. Investigating the metabolism of prohibited substances in sport drug testing is a kay element, in order to find efficient markers for their detection, prolong detection windows or even enable the discrimination between the abuse of substances from the consumption of contaminated food or their natural occurences in hte human body. Witrhin this study, new insights could be provided concerning the renal elimination of 2-phenylethylamine and two of its respective metabolites on the basis of human administration studies. Given the rapied metabolism and thereby low impact of PEA ingestion on its urinary concentrations, threshold levels for PEA in doping control deemed inadequate. Evaluating the concentration ratio of M1 and PEA to detect potential misuse seemed promising at first, but respecting the large inter-individual variation within the calculated ratios, the establishment of a cut-off level for this marker proved difficult. However, assessing the urinary levels of M1 and a second major metabolity, PAG, combined by means of ligistic regression analysis presents a novel approach to distinguish endogenous PEA from a potential misuse. In regard to the natural presence of both metabolites in the human body and the large variability observed within urinary levels, further research into potential confirmation methods, i.e. isotope ratio mass spectrometry, seems advisable in order to clarify potential suspicions of PEA administration.

Code: 19D04IH

The aim of this study is to gain more knowledge about the longitutidanl variation in the concentrations and ratios of "new" steroid markers not yet included in the steroid module of the Athlete Biological Passport (ABP). This will include variations of sulphate metabolites, as well as other testosterone metabolites from the glucuronide fraction which are not included in the ABP today. In addition, we will investigate non-analyticl factors that may influence the new markers, as well as markers of the established steroid module. When combining these factors, we expect to improve the understanding of the observed biological variation in the steroidal passport, and with this increase the sensitivity of the ABP.

We will conduct a longitudinal study of steroid profiles from 30 individual athletes, over a time period of one year. The confounding factors included in routine doping analysis will be analysed and evaluated. Samples will be collected in and out of season, and from different sports. In addition, participants will be asked to provide information on stress level; both physical and psychological at the time of sampling (by self-reporting). Information about these factors is normally not given during routine doping controls. Knowledge of the possible influence of these factors on the steroid markers is important in the evaluation of the passports.

The samples will be analysed using routine methods in doping analysis, using gas and liquid chromatography coupled to mass spectrometry. Both present and possibly new markers for the ABP will be evaluated.

Code: 19D07XD 

The detection of the abuse of pseudo-endogenous steroid doping is based on the longitudinal monitoring of six urinary steroidal markers and their relative ratios, as descrived in the steroidal module of the Athlete Biological Passport (ABP) by the application of a Bayesian adaptive model that is able to predict the maximum variability for each marker based on the previous data and to outline atypical results. Even if the introduction of the longitudinal evaluation of the steroidal markers of the ABP improved the detection of the pseudo-endogenous steroid doping, it does not allow to gather any information on the occurrence of atypical profiles due to the presence of endogenous and/or exogenous confounding factors that could influence the urinary excretion of the markers of the steroid profile. To overcome this drawback, the evaluation of a parallel "blood steroid profile" has been proposed.

While the analysis of blood samples have become more widespread in doping control and are essential for the detection of human growth hormone (hGH) and erythropoiesis stimulating agents (ESAs), its collection necessitates venipuncture and reliable conditions for transportation and storage.

For the above mentioned reasons the aim of the current project is to develop and validate a liquid chromatography - tandem mass spectrometry method for the analysis of a wide panel of potenital steroid biomarkers in blood using dried blood spots (DBS) as a sampling and transport devices. The sampling of DBS is less invasive, easy to perform and cost-reduced compated to the collection of blood samples. This will certainly facilitate the widespread collection of blood samples in a larger population of athetes. In addition, analytes in DBS are usually more stable than in whole blood. The research of testosterone esters in blood that is contemplated int he WADA TD2018EEAS would be facilitated in DBS.

Code: 19D03RV 

The detection of EAS abuse is currently performed using the steroid profile, however improvements are needed to prolong detection times (DTs). Results of a previous WADA (17D09RV) demonstrated that ratios between some sulfate metabolites significantly prolonged the DTs of oral T administration in Caucasian volunteers with respoec to T/E ratio. Some of the ratios were elevated up to the lsat sample collected several days later. Steroid sulfates have not been evaluated comprehensively in Asian population after oral T administration. And dermal administration has not been evaluation in Caucasian or Asian populations.

The objective of the present project is to continue the evaluation of sulfate metabolites to prolong the detectability of T misuse. The research will be focused in the following specific objectives:
- To verify the usefulness of sulfate markers in urine in Asian population after oral T administration.
- To verify the usefulness of sulfate markers in urine in Asian and Caucasian populations after dermal T administration.
- To identify androstanediol sulfate 1, which was one of the most useful markers identified in the previous study.
- To develop an initial testing procedure to quantify the relevant steroid sulfates in urine on a routine basis.

Code: 19A11MM 

The 5a-reductase enzyme system is involved in the metabolism of endogenous and exogenous steroids. The substrates are the C19/C21 steroids with a keto group at the carbon 3 and a double bond between carbons 4 and 5. The reaction mechanism is complex and involves the binding of a reducted pyridine nucleotide cofactor to the enzyme followed by the substrate.

The modulation of the 5α-reductase activity by 4-azasteroids is used in therapy for the treatment of benign prostatic hyperplasia or androgenic alopecia. In the anti-doping field, the possibility to use the 4-aza-steroids (finasteride and dutasteride) to manipulate the steroid excretion profiles and, consequently, to mask the abuse of both pseudoendogenous and exogenous steroids was demonstrated. From 2005 to 2009, this class of compounds was included in the WADA list in the section S5 “Diuretics and other Masking agents”; whereas since 2014 the 5α-reductase inhibitors are included in the Technical Document TDEAAS “Endogenous Anabolic Androgenic Steroids Measurement and Reporting” as confounding factor. Indeed, the administration of these agents leads to a decrease of the 5α-steroids with a consequent alteration of the ratios between androsterone and etiocholanolone; 5α-androstane-3α,17β-diol and 5β-androstane-3α,17β-diol; and androsterone and testosterone.

Different analytical procedures are reported in literature to determine finasteride in biological fluids, whereas for dutasteride only determinations in blood samples are reported due to its pharmacokinetics properties.

The aim of this project is to select the most appropriate analytical strategy and matrix to detect dutasteride during doping control test in order to avoid the risk to give uncorrected results.

Code: 19C03AM 

Tramadol is potent narcotic analgesic that acts on the opioid system. Data from the WADA Monitoring Programme and from athlete testimonies suggest that this drug is used across multiple sports in order to reduce exertional pain and allow the athlete to work even harder. In doing so, it is likely that tramadol is being used to provide the athlete with a performance advantage. However, there is currently no convincing research evidence to support or reject whether tramadol is performance enhancing in highly trained, healthy athletes. This project will employ an experimental design that focuses purely on the question of whether tramadol allows athletes to work harder by reducing pain, and thus allow for a better performance. Therefore, the main outcome of this project will be to provide robust experimental evidence to inform whether the use of tramadol in competition should be regulated. Thirty highly trained road racing cyclists will be recruited for this study, as the efficacy of performance enhancing interventions vary according to athletic ability. In a randomized, controlled, double-blinded crossover design, these cyclists will complete a laboratory cycling task that replicates the time/intensity demands of professional road cycling following the ingestion of tramadol or a placebo. The cycling task will involve fixed intensity and self-paced time trial cycling, amounting to approximately 1.5 hours of hard cycling. The intensity/time of the task is critical, as the ergogenic effects of tramadol are likely to be reduced in shorter duration exercise that induces less fatigue, and is therefore not representative of the context in which it is purportedly taken. Exercise performance (completion time) and perceptual responses (perceived pain and effort) will be compared between conditions, with results used to inform consultation with WADA regarding the S7 Narcotics category of the Prohibited List.

Code: 19B06MM 

Recently a wide variety of peptide hormones were included in the World Anti-Doping Agency List of prohibited substances and methods. The analytical approach of choice to detect these substances in biological fluids is most of the times linked to (i) the matrix selected, (ii) the concentration of both the analyte of interest and the interferences, (iii) the natural occurrence in human body and finally (iv) the molecular weight of the compound under investigation. Concerning, specifically, those peptide hormones with a molecular weight in the interval of 2000-10000 Da (i.e. insulins, GHRHs, synacthen or IGF-1 and its analogues), they are excreted in urine, either as parent compounds and/or as metabolites/degradation products, in very low levels.

Different analytical approaches have already been developed and published for the detection of these agents in doping control specimens (plasma/serum or urine samples) employing immunoafinity purification and liquid chromatographic-mass spectrometric-based techniques. The sample pre-treatment protocols proposed in literature are mainly based on ultrafiltration followed by the use of immunoaffinity purification: magnetic beads, immune-affinity chromatography or antibody pre-coated ELISA plates. Although better recoveries were achieved by using immune-affinity chromatography (>70%), the strategies currently adopted by the anti-doping laboratories are based on the use of magnetic beads (<30%) or of antibody pre-coated ELISA plate, mainly due to their simpler protocols and to the possibility to fully automate.

This project is focused on the evaluation of alternative off-line immunocapture technqiues, such as for example, pre-coated pipette tips or spin trap column with the aim to improve the efficacy, repeatability and robustness of the protocols currently used by the anti-doping laboratories to extract large peptides from biological fluids.

Code: 19A10MP

In the fight against doping the laboratories are confronted with an increasing number of substanced to screen on. Thus, a comprehensive screening for different classes of substanced using dilute-and-inject methods in anti-doping screening is desirable. As lots of xenobiotics are excreted as conjugates a detection of the intact conjugates is performed by this approach. While chemical synthesis of sulfoconjugates works efficiently for compounds having only one potential conjugation site, several analogous compounds could not be chemically synthesized effectively, due to their more complex chemical structure. For the synthesis of the phase-II metabolites (glucuronides and sulfates) of these compounds a biotechnological production will be implemented.

In part 1 of the project fission yeast strains, that enable the biotechnological production of glucuronies and sulfates that cannot be synthesized efficiently via classical chemical synthesis were generated. In part 2 they will used to produce the relevant human conjugates of prohibited substances. It is planned to address the sulfoconjugation of some challenging compounds such as salbutamol-sulfate, salbutamol-glucoronide, fenoterol-sulfate and 4-hydroxy-DHEA-sulfate within the project.

The produced reference material can be used for method set-up for direct detection. If laboratories still rely on hydrolysis of the conjugates, these reference compounds may serve as control for hydrolysis efficiency and quality assurance. Furthermore, artifact generation during conjugate cleavage can be evaluated by the help of this newly generated reference compounds. The relevance of the generated reference substances in doping control analysis will be demonstrated by comparison with authentic samples from doping control analysis.

Code: ISF19E03AM

Blood doping and in particular transfusions were used by athletes for decades, as an easy way to increase red blood cells, oxygen transportation to muscles and endurance. Homologous transfusion (blood from a compatible donor) was the simplest method used.

However, in 2004 a method based on flow cytometry was implemented in anti-doping laboratory to identify homologous transfusion. While being efficient to identify around 2% stranger blood in a sample, it requires the use of mnay antibodies and still could lead to false negative results. Nowadays, only few anti-doping laboratories are still running flow cytometry and athletes could feel free to come back to homologous transfusion.

With the development of forensic science, DNA has proven to be a reliable source of identification of the presence of two different DNA in a single blood sample. The power of amplification of DNA based techniques is sufficient to start from very small volumes and Dried Blood Spots (DBS) is an interesting matrix for the future of doping controls. The aim of the project is to evaluate the interest of implementing a new test to detect homologous transfusion by using a forensic DNA-based protocol on DBS for anti-doping purpose.

The objectives of the project are:

1. To validate the conditions to propose a robust protocol for detection of DNA mixture from Dried Blood Spots.

2. To estimate the window of detection after transfusion of healthy volunteers by analyzing DBS spotted from venous blood and to compare these results with those of the current flow cytometry method for detection of homologous blood transfusion.

Main findings

The protocol was successfully developed and validated. With 100μL of dried blood collected on a card treated to protect nucleic acids. A mix of two DNA was robustly identified when the minor blood proportion was 2% or higher in vitro when two whole bloods were mixed. Following one month of storage of the cards, at room temperature, the sensitivity of detection was not altered.

A blinded in vitro study with samples containing mixed compatible, bloods in different proportions as well as negative samples (one DNA) was conducted. For DNA analysis of DBS the limit of detection was confirmed at 2% donor blood. while the flow cytometry method performed on fresh blood was slightly more sensitive and could sometimes detect 1% donor blood in a sample.

However, the in vivo evaluation of this method with transfusion of 150 mL of a red blood cells concentrate (RBCC) using the forensic DNA approach gave disappointing results as no analyzed DBS showed any presence of a second DNA even one day following transfusion.

This result is most likely due to a lack of a sufficient number of white cells (the only blood cells containing DNA) from the donor in the RBCC and consequently in the DBS. On the contrary the flow cytometry method was very efficient and the transfusion could still be detected for more than 40 days. The classic flow cytometry method was performed with a tube by tube analysis of 11 antigens (C, c, E, e, Jka, Jkb, S, K, Fya, Fyb and s). A new simplified protocol for sample analysis by flow cytometry was also developed and evaluated using 96 well plates with 9 antigens detected (C, c,E, Jka, Jkb, S, Fya, Fyb and s). In each well two antibodies against two different RBC antigens can be mixed if one is an Immunoglobulin subtype G (IgG) and the other an immunoglobulin subtype M (IgM). Two secondary antibodies with two different fluorophores are added in each well to identify presence/absence or mixed expression of each antigen. Both flow cytometry methods detected well the double populations for several antigens after transfusion and were sufficiently sensitive to confirm at least two antigens with a double population at day 42 and day 50 post-transfusion.

In conclusion, analysis of DBS with the validated forensic DNA method to detect DNA mixes did not demonstrate enough performance to be considered as an alternative to the flow cytometry method for detection of HBT. In particular, it completely failed to detect the transfusion of the RBCC while the flow cytometry method was very efficient with a window of detection of 1.5 months. To simplify the laboratory work needed to analyse blood by flow cytometry, a technical evolution of the method was evaluated with success and the new protocol will soon be fully validated for a future application to doping control samples.

Code: 19A08LM 

Microsampling provides a wide range of applications that may offer advantages over traditional fluid samples on logistics and bioanalytical workflow. Among microsampling methods, dried matrix spots represent a feasible method for the microsampling of biological matrices to obtain for example dried urine spots (DUS). Moreover, volumetric absorptive microsampling (VAMS) has been recently introduced for the sampling of small, accurate biological fluid volumes. Dried microsamples can usually be stored under ambient conditions, although comprehensive analyte stability assessments are still under research for many compounds. Following the promising results previously obtained from the definition of mid-term stability of some doping-relevant peptides (e.g. GnRH analogues) in urine collected as DUS and VAMS, aim of this research is to carry out a systematic study on the stability in a wider time frame of such compounds in urine sampled as DMS and VAMS and to expand the study to additional peptides and to relevant compounds included into the Athlete Biological Passport (ABP) steroidal module. All variables involved in the sampling process will be assessed: humidity, temperature, light exposure will be evaluated to determine the optimal sampling, storage and transport conditions, and to evaluate results obtained from microsamples. In addition, possible scenarios will be simulated, representing the life cycle of an anti-doping sample: from collection to shipment, storage and handling before being subjected to pretreatment procedures and LC-MS/MS and LC-HRMS analysis. The project goal is to establish feasible and reliable workflows for microsample collection, stably storable and shippable with minimum precautions. These procedures could then be proposed as effective anti-doping strategies to be compared to conventional fluids. 

Main Findings: 

Two different dried microsampling and analysis workflows, based on dried urine spots (DUS)-LC-MS/MS and volumetric absorptive microsampling (VAMS)-LC-MS/MS respectively, were developed and validated for the testing of S2 peptides and of steroids included in the ABP. Validation included sampling time and drying time assays. Moreover, different microsampling volumes (10, 20, 30 μL) were tested to provide better versatility; 30 μL always granted the best sensitivity/selectivity compromise. Eleven-months stability assays showed that both microsampling matrices, stored at 25°C, provided good stability for all analytes at all time points, with end-point stability in the 52-66% range for peptides and in the 79-85% range for steroids. Similarly, both DUS and VAMS at 25°C produced better stability than fluid urine frozen at either -18°C or -80°C, with large differences (28-32.5%) for peptides and marginal ones (2-12%) for peptides. VAMS produced slightly better results than DUS: Stability in the former was 1-8% higher than in in the latter for peptides and 1-3% higher for steroids. Urine microsample storage for 11 months in sub-optimal conditions (up to 35°C, up to 40% HR, 12 h light exposure) caused larger analyte degradation, but again with better performance for VAMS over DUS (-9% vs. -18%, respectively, in comparison to optimal conditions). 11-months stability studies were completed on doping-relevant peptides and ABP-listed steroids, providing positive, interesting results. In particular, innovative microsampling and pretreatment procedures based on dried matrices (DUS and urine VAMS) were optimised for application to urine specimens for anti-doping purposes. Analytical platforms based on LC coupled to different MS detectors were exploited to develop two sets of validated analytical methods, one for the analysis of peptides and another for the analysis of steroids, in dried urine microsamples. Solid, satisfactory performance and good throughput were obtained for each analyte group. After 11 months, peptide stability in dried micro-matrices was above 51% and steroid stability was above 78%. Regarding peptides, stability in the micromatrices at RT was vastly superior to that of frozen or ultra-frozen urine. Results for steroids were less starkly defined, with both microsampling matrices providing marginal advantages in comparison to urine. In all cases, there was no need for any refrigerated or frozen microsample storage. Among the two microsampling strategies, VAMS granted significantly superior performance over DUS for peptides, with much smaller differences for steroids. However, VAMS generally gave better precision, extraction yield and matrix effect in the analytical workflow than DUS. In conclusion, dried urine micromatrices are a very promising tool for enhancing the stability of peptides and steroids in an anti-doping testing perspective. They may also provide reduced storage and shipping requirements and expenses, and simpler pretreatment procedures.