Projets de recherche

Code: 23C05MT

The two carbonic anhydrase inhibitors brinzolamide (BA) and dorzolamide (DA) are prohibited in sports after systemic administration (ophthalmic permitted) only, thus, the analytical evidence for the administration route represents a desirable tool in doping controls. The diuretic effects of BA and DA are responsible for the classification as masking agent, which are prohibited at all times (in- and out-of-competition). Preliminary results showed that the metabolism of BA and DA differs for the different application routes. Additionally, it is well known that these substances (together with their metabolites) are enriched in the red blood cells, with a resulting half-life of several weeks after administration. In the present study the in-vivo metabolism of BA and DA after ophthalmic (eye drop) and systemic (oral) administration to pigs will be evaluated with special focus on the characteristic application route derived metabolism. The metabolite pattern of BA and DA will be evaluated and compared to samples obtained from patients that therapeutically apply either BA or DA as ophthalmic preparations. The analysis of the parent drug as well as their metabolites are planned by means of liquid chromatography coupled to high resolution mass spectrometry.

Code: 23A17GM

While most anti-doping testing is conducted in urine, several analytes are better monitored in serum, including human growth hormone (hGH), certain erythropoiesis stimulating agents (ESAs), insulin-growth factor 1 (IGF1) and procollagen type-III N-terminal peptide (PIIINP). Additionally, more attention has recently been given to the longitudinal monitoring of the steroids testosterone (T) and androstenedione (A4) in serum. However, several factors currently make collecting anti-doping serum samples difficult and expensive, including the need for trained phlebotomists, the cold chain monitored shipping requirements, and the discomfort and difficulty of venipuncture blood draws, which may be uncomfortable and inconvenient for frequently tested athletes. In the last 5-10 years, there has been a push for increased testing using dried blood spots (DBS), obtainable using finger prick collections or specialized capillary draw devices. While innovative, not all analytes are easily measured in DBS, mainly due to sensitivity issues and lack of a controlled collection volumes. Recently, the Tasso+ capillary collection device has been adjusted and improved to now allow collection of whole blood instead of just DBS. Although this may not overcome all of the cold-chain shipping issues, collection of whole blood from a capillary device removes the phlebotomy requirement which can decrease collection costs, open up testing to more remote areas (more easily), and improve the athlete experience of providing blood samples. In 2022, our team highlighted the benefits of collecting whole blood using microcapillary Tasso+ K2EDTA devices, and how this method could serve as a replacement for Athlete Biological Passport (ABP) analysis from venous, whole blood samples. Now, the possibility exists to use this technology to also collect serum samples. We propose to use the newly designed Tasso+ SST devices to collect capillary serum from volunteers and compare analytical results to serum collected from a standard venipuncture method. Here, we will compare the capillary and venous serum for EPO detection following a single dose administration of recombinant EPO (identification and detection window), and measurement of the serum steroids T and A4 before and after a transdermal testosterone application. Additionally, we will compare the measurement of recombinant and pituitary HGH in matched capillary and venous samples, and finally assess the longitudinal stability of IGF-1 and P3NP in both types of serum.

Code: 23A15GG

Dried blood spot (DBS) applications get increasing attention in the field of anti-doping as indicated by the increasing number of scientific publications. The current comprehensive study is designed to investigate capabilities of DBS as complementary matrix to detect testosterone doping after oral, intramuscular and transdermal administration of testosterone undecanoate (TU) and/or testosterone gel in female and male populations. Analytical targets in DBS are consequently testosterone undecanoate itself as well as a panel of endogenous steroids, including testosterone, androstenedione and 5α-dihydrotestosterone. A sensitive, robust and already validated liquid chromatography mass spectrometry (LC-MS) methods will be applied, including derivatization with Girard P and hydroxylamine to significantly improve sensitivity and limits of detection. The results obtained are anticipated to provide a more detailed insight into the window of detection for TU and changes in endogenous steroid concentrations to uncover testosterone doping. The correlation between factors such as route of administration, gender and inter-individual variability as well as the detection of the TU as prodrug and endogenous steroid concentration changes will be established. The results of this study may provide important evidence for its suitability to be used for the steroid blood passport based on DBS as matrix.

Code: 23A08VN

Three commonly used stimulants (amphetamine, methylphenidate and modafinil) will be evaluated in the study. Therapeutic doses of each drug will be administered once a day to ten healthy participants (five males and five females) for five days. Paired collections of urine, dried blood spots and serum will be collected from each participant, once a day. Collections will be performed during the administration phase and for seven days after cessation of use. Concentrations of the primary target compound will be measured by LC/MS to determine the matrix that best reflects circulating levels. A time course of elimination will be determined and the possibility of compound specific thresholds will also be evaluated.

Code: DBS23SP02SR

Doping testing of small peptides (SPs) is mostly performed in urine using mixed-mode weak cation sorbents prior to LC-MS/MS. Complementary information can be obtained in a cost-effective way using dried blood spot (DBS) samples. However, many issues still need to be addressed before considering it reliable for routine use. A major issue is the demand for simpler and faster sample processing, which is mainly based on DBS treatment with boiling water or methanol followed by sample cleanup and eluate evaporation. On the other hand, there is the need for higher SP recoveries (e.g. most of them are below 30%), and lower LODs (e.g. although no MRPLs have been set for SPs in DBS, achieved LODs are above the requested MRPLs established for SPs in urine, i.e. 1-2 ng/mL). Other issues include the demand for more thorough studies about hematocrit effects, SP shipping and storage stability, and the timing and procedure for addition of internal standards. This project aims to develop an efficient and high throughput analytical platform for reliable testing and confirmation of SPs in DBS samples. The approach will be based on the combination of the capability of supramolecular solvents (SUPRASs) to integrate efficient compound extraction and sample clean-up with the power of liquid chromatography-mass spectrometry (LC-QTOF and LC-QQQ) for SP detection.

The analytical platform will be applied to the detection of 58 peptidic drugs and metabolites, most of them routinely analysed by the WADA accredited laboratory of the Health Institute Carlos III (ISCIII, Madrid) within the ITP for urinary SPs. They will include growth hormone releasing peptides and secretagogues, growth hormone fragments, antidiuretic hormones, gonadorelin releasing peptides and growth factor modulators) Some of the selected SPs such as GHRP-1, GHRP-2, GHRP-6, hexarelin, alexamorelin, triptorelin AOD9604, desmopressin, TB-500 are unstable in urine. The capability of SUPRASs for simultaneous SP extraction from DBS and sample cleanup will be investigated using SUPRASs made up of very different nanostructures (e.g. hexagonal inverted or ribbon-shaped aggregates, sponges, cubosomes, etc.) that offer mixed-interaction mechanisms, including dispersion, ionic, hydrogen bonding, dipole-dipole, CH-π and/or polar hydrophobicity. Five marketed collection devices based on cellulose and hydrophilic porous materials will be comparatively evaluated in terms of SP recoveries and matrix effects. Addition of internal standards on DBS and SUPRAS (before extraction) will be compared. Robustness will be evaluated in terms of hematocrit effect. The optimized SUPRAS-LC-MS platforms for SP detection will be validated according to WADA guidelines and an analytical workflow that includes the proposed MRPLs will be delivered. The proposed method will be validated by the ISCIII lab using LC-QTRAP-MS/MS. As a proof of concept, the SUPRAS-based method will be applied to the analysis of DBS samples obtained from the subcutaneous administration of a single dose of GHRP-2 and Ipamorelin to two intervention groups, each consisting of 10 elite athletes.

Code: 22C12MH

An emerging concern regarding the misuse of anabolic doping substances in sports is “muscle memory” – as skeletal muscle that has previously been exposed to an anabolic prohibited substance may adapt more readily to retraining. While this phenomenon has been demonstrated in rodents, the underlying mechanisms as well as whether this applies to humans remain unknown. The relevance of studying muscle memory induced by prohibited substances is clear when considering the current anti-doping regulations. One can imagine a scenario where an athlete is banned from competitive events for a few years, but upon return to competitive sport still enjoys an advantage in retraining potential because of anabolic compound-induced muscle memory.

This project will elucidate whether prior treatment of the two commonly misused prohibited anabolic compounds, clenbuterol or testosterone, induces muscle memory and thus improves the hypertrophic response to subsequent resistance training. To this end, two longitudinal randomized placebo-controlled trials will be conducted in healthy volunteers undergoing periods of resistance training. The project will utilize state-of-the-art methodologies to address aspects of myocellular addition, hypertrophy and myocellular reprogramming in muscle biopsies collected during the trials.

Code: 22D05KC

With the increasing participation of female athletes in international competitions, there is growing interest in the specificities of the elite female athlete. Indeed, the elite female athlete is subject to a menstrual cycle, with huge variations in estrogen and progesterone concentrations during the cycle. A relatively large number of elite female athletes suffers from menstrual disorders, often due to intensive training and eating disorders, with inhibition of their hypothalamic-pituitary-gonadal axis, resulting in very low estradiol and progesterone levels. Finally, approximately 1 in 2 high-level female athletes uses hormonal contraception, the estrogen-progestin pill (OOP) being the most frequently used. Female sex hormone levels therefore fluctuate greatly in elite female athletes, with likely a significant impact on blood and urine steroid profiles.

However, most of the time, there is only an approximate estimation of the cycle phase, without direct blood female sex hormones and precursors analysis and if any, on a punctual way. We therefore propose to dose these hormones on a longitudinal way in elite non-hyperandrogenic female athletes, taking into account the duration of the MC, menstrual disorders or the use of OOP, in order to assess their direct impact on the blood steroid profile, in particular on testosterone (TES) and androstenedione (4-AEN), the target compounds selected by WADA for the blood steroid profile. As the environment of the athlete directly modulates the secretion of these female sex hormones, and consequently the steroid profiles, we will in parallel quantify the training load and food intake thanks to questionnaires and investigate the blood level of TSH, T3, prolactin, leptin, DHEA and cortisol, as well as the body composition during the different hormonal status.

Code: 22D02RF

The goal of our research is to investigate the impact of altitude training on blood values in elite athletes preparing for a major competition, with special focus on the biomarkers considered in the framework of the Athlete Biological Passport (ABP). Testing the hypothesis that hypoxic training is related to hematological variations would allow the present ABP model to be refined, and its efficacy strengthened. Our study would challenge the complexity of confounding factors affecting the interpretation of the ABP in elite athletes. In addition, based on the current development of a multi-parametric model previously validated in elite cyclists to compare inferred and actual plasma volume (PV) variations, investigation of the potential confounding effects of altitude exposure, and pre-competition tapering phase, can provide additional experimental evidence towards future improvements of the ABP.

The main objectives of this study are the following: firstly, this study will allow to further investigate the hematological variables of the ABP before, during, after a 3-week altitude training camp and on days before and after a major competition (20 days after the return to see level); furthermore, the potential confounding effect of hypoxic exposure will be evaluated by measuring PV changes or inferring the latter changes from serum biomarkers that are sensitive to PV changes. Overall, our study aims at improving the ABP in its current form and at evaluating the possible implementation of additional biomarkers.

Code: T22M01RTRV - Part 2

12 hours starting from any dose”, which is considered as the cut-off between maximal therapeutic dosagesfor athletes benefiting from a therapeutic use exemption and doses at which a doping effect could begin to be expected from systemic exposure [8]. The WADA established a urinary concentration

Code: T22M01RTRV - Part 1

Salbutamolwas included in the prohibited list of the World Anti-Doping Agency (WADA) in 2004. While systemic salbutamol is banned, inhaled salbutamol is permitted but with dosage regimen restrictions. The dose should indeed not exceed “1600 µg over 24 hours in divided doses not to exceed 800 µg over.