Projets de recherche

Code: 22D06MT

Several cases of so-called sample swapping have been detected in recent years. During sample swapping an athlete substitutes the presumable positive doping control sample by another negative urine sample in order to circumvent an adverse analytical finding. For the exchange of urine, a sample from the same individual or from a different individual may be used. While the latter can result in a significantly different steroid profile, employing a urine sample from the same individual collected prior to administration of a doping agent will not show any differences in the steroid profile. Here it can be expected that the stored clean sample will be used several times and that this may result in steroid profiles with an atypical similarity. Two cases of this type of sample swapping have already been detected, but only by chance as the similar samples were analyzed in the same laboratory in a timely manner. 
The aim of this research project is the development of a screening tool based on machine learning and artificial intelligence which will enable to detect steroid profiles with an atypical similarity in all steroid profiles determined world-wide in different accredited doping control laboratories. The developed software will be able to search within the existing profiles for similar ones and will be trained to compared new entries to the dataset to existing ones in order to indentify highly similar patterns. Employing artificial intelligence will enable to train the software to become highly sensitive, selective and robust. In order to confirm potentially similar samples to belong to the same athlete, DNA analysis of these urine samples will be applied where applicable.

Code: 22C05MT

Higenamine is a non-selective β2-agonist, which has been described as a key component of a great variety of natural plants such as Nandina domestica, Tinospora crispa, and Annona squamosa. Although higenamine has never been approved as a drug by the US Food and Drug Administration (FDA), it plays an important role in traditional Chinese herbal medicine (it has been approved for clinical research by the China Food and Drug Administration - CFDA), and has been found to be an ingredient - often unlabeled - of different weight loss and sports supplements, which has been suspected or proven to have caused cases of unintentional anti-doping rule violations.
Within the scope of a previous elimination study aiming at the urinary quantification of higenamine after administration of tropical fruit plants from the Annonaceae family, it could be shown that single-dose administrations of these fruit species are rather unlikely to lead to AAFs in sports. The aim of this project is to conduct further investigation which complement the previous datasey. For that purpose, stability evaluation of componuds of interest, and specific contribution of the different higenamine conjugates will be performed. Besides, two different administrations studies with a total of 6 healthy volunteers  (3 x ♂, 3 x ♀) are planned. Each participant will eat 3 servings on Annona fruit puree with 24 h (8:00 am, 6:00 pm, and next day 8:00 am) and, at after washout, a single dose of two different higenamine-containing nutritional supplements. Urine samples will be collected up to three days following administration in both study phases, and comprehensive analyses are planned to support result management and decision-making processes in case of higenamine findings in sports drug testing programs, preferably by means of new potential biomarkers of plant-derived higenamine administrations.

Code: 22B06XZ

Upon our previous research, a variant in the EPO gene can encode a variant EPO (VAR-EPO) which has a similar molecule weight with recombinant EPO (rEPO), thus cannot be distinguished using the current method of rEPO analysis. Though WADA has established a detailed procedure for investigation and further analysis, it is a burden for both laboratories, Testing Authoity and WADA, nevertheless, rEPO abused by this variant carriers can be hardly detected. For this situation, we are going to develop an effective confirmation method that can discriminate the wild-type EPO, VAR-EPO and rEPO directly, by removing or eliminating the detection of VAR-EPO using the VAR-specific antibody. Two methods will be developed and validated in this project:: reverse-normal immunopurification and step-recognition western blotting. The rEPO administration study on individuals who carry this varient will also be conducted. The collected positive urine and blood samples will be used to evaluate the method. With out goals, the new method can not only protect the "clean" athlete with that variant but also effectively penalize the rEPO abused athelete whether he or she carries that variant. Another significant advantage, this method would be easily adopted by other WADA accredited anti-doping laboratories.

Code: 22B05PC

Erythropoietin receptor agonists (ERAs), e.g. darbepoetins (dEPO), erythropoietins (EPO), EPO-Fc were listed in WADA's Prohibited List under Class S2.1. Traditional polyacrylamide gel-electrophoretic analytic methods were applied by most of the WADA certified laboratories for ERAs detection for the last 20 years. In this project, a novel photoclick hydrogel electrophoretic and in situ immunoblotting method for the screening of ERAs in urine and blood samples of athletes will be developed with high sensitivity and high throughput. A tetrazole-functionalized photoclick hydrogel based chip will be designed first which can satisfy the processing of 15 samples simultaneously. Optimization of the parameters in SAR-PAGE and intensity immunoblotting assay will be carried out on this chip to realize the separation of ERAs, especially the distinguishment of endogenous EPO and recombinant human EPO )rhEPO). Along with the confocal microscope for image acquisition, the LOD of this method can be lower than 1 IU/L (plasma) and 0.1 IU/L (urine) with limited sample volumes. Meanwhile, the whole analysis time can be reduced within 6 hours for plasma samples. Finally, an intravenous administration of rhEPO drugs on healthy subjects will be carried out to verify the practicability of this new method.

Code: 22A10JP

There is a high demand for rapid screening methods using mass spectrometry (MS) that can decrease the turnaround time, cost, and limits of quantitation of existing methodologies. It is important to emphasize that appropriate sample preparation is required to perform proper sample cleanup and analyte enrichment. We are focused on using matrix-compatible SPME-based devices for direct immersion extraction of small molecules from biofluids and direct coupling to MS. In this context, matrix-compatible SPME-based devices have extraction phases comprised of high-capacity sorbent embedded in a polyacrylonitrile binder that ensures small molecules can be extracted while minimizing the co-extraction of macromolecules. This provides better performance and more sensitive analysis of a wide range of small molecules including many of those found on the 2022 Prohibited List. Futhermore, we are focused on couple SPME-based devices to MS via the microfluidic open interface (MOI) and coated blade spray (CBS) technology, as well as developing more efficient ways to separate isobaric/isomeric analytes in the gas phase before MS detection using ion mobility spectrometry (IMS). These developments are critical to decrease the detection limits and discern between permitted and prohibited substances while increasing the speed of analysis and the range of screened and quantified molecules. In this proposal, we build on this success and propose applying SPME technologies directly to MS which have been recently developed in our laboratory to further improve the speed of screening directly during sports events. In the long term, the goal of the team's research is to take advantage of ongoing advances to develop powerful new analytical technologies to assist in the ongoing fight against doping in sport, with an emphasis on lowering the detection limits to better distinguish between permitted from prohibited use, as well as improving the detection window of prohibited substances.

Code: 22A08XT

For substances produced endogenously and included in the WADA Prohibited List, its abuse is based on: 10 identification of the best biomarker of abuse to be applied at the ITP level and 2) confirmation by a procedure able to demonstrate the application of the drug or its origin. Usually, this is performed by establishing the population distribution of the biomarker in ITP and the application isotope ratio mass spectrometry (IRMS) as a confirmatory procedure. This has been applied successfully using the steroidal module of the ABP and GC-IRMS.
In addition to steroidal hormones, several other prohibited compounds may benefit from a similar approach. We intend to investigate the application of IRMS coupled to liquid chromatography to the confirmation of compounds that because of their physicochemical properties cannot be analyzed by GC and for which a definiticive confirmation procedure does not exist.
The analysis of AICAR by GC-C-IRMS has been described after silylation and its comparison with a steroid (pregnanediol) as an endogenous reference compound (ERC). The current limitations of this approach can be overcome by the use of LC-IRMS, allowing the underivatized compound analysis and the use of an ERC closer to its metabolic path.
The main 2-phenethylamine and octopamine target analytes are excreted as sulfates, whose hydrolysis is not obvious. The confirmation by GC-C-IRMS is impaired by the high interaction with GC systems. The direct analysis of their sulfated conjugates by LC-IRMS will unambiguously confirm their origin. The main objective is to prove the applicability for antidoping purposes of LC-IRMS in the analyses of intact sulfates.
For IGF-1 the main objective is the use of LC-IRMS for the analysis of recombinant preparations and preparations of human origin, to prove its applicability for anti-doping purposes. The protein will be analyzed intact or after trypsin digestion.

Code: 21B07CR

All detection methods for ERA-doping are currently based on electrophoretic separation and Western blotting and due to its high sensitivity apply the same primary antibody (clone AE7A5 anti-EPO

antibody). Upon initiative of the grant applicant, a biotinylated version of the antibody was generated in 2018, which led to further increase in sensitivity and simplification of the method. The antibody

also became part of WADA TD2021EPO. One drawback of clone AE7A5 antibody is that it non-specifically binds to some proteins, which are present in human urine and blood. The non-specific

binding is caused by the fact, that the peptide used for generating the antibody contained two errors in the amino acid sequence. Hence, all doping control samples have to be first immunoaffinity-purified before electrophoresis. For this purpose, a different EPO antibody has to be used. To ensure better selectivity both the ISL [11] and the TD2021EPO, even if not totally mandatory, recommend that the CP differs from the ITP. Since no alternative to clone AE7A5 antibody is known so far, laboratories have to establish two different immunoaffinity purification procedures for ITP and CP. On the other hand, there are ca. 150 anti-human EPO antibodies commercially available, which have largely not been compared with clone AE7A5 antibody regarding sensitivity and selectivity. If an alternative antibody could be found, this would not only simplify EPO testing but would also generate more flexibility for the ITP and CP. Aim of the proposed project is a large scale evaluation of commercial anti-human EPO antibodies. About 100 antibodies will be tested regarding their sensitivity and selectivity in comparison with clone AE7A5 EPO antibody.

Main findings

164 antibodies were tested regarding their sensitivity in comparison with clone AE7A5 EPO antibody. Out of 70 polyclonal and 94 monoclonal antibodies a clear favourite was identified. It surpassed clone AE7A5 not only by sensitivity but also by specificity. The antibody is a recombinant rabbit monoclonal antibody (clone HL1794) and is worldwide available from the manufacturer (Genetex) as well as large international distributors (e.g. Abcam, ThermoFisher Scientific, Fisher Scientific). The antibody was fully validated for urine using an HRP-labelled and cross-reactivity minimized secondary antibody. It allowed the detection of all ERAs (rEPO, NESP, EPO-Fc, CERA) down to at least 0.1 MRPL of TD2024EPO. Similar results were obtained serum samples. The antibody did also not bind to proteins known to be nonspecifically bound by clone AE7A5 (ZAG, TrxR, NSE, NNE, enolase). Conclusions: Anti-EPO antibody clone HL1794 outperformed the current detection antibody clone AE7A5 regarding sensitivity and specificity. Thus, it is a very promising antibody, which will further improve detection of ERA-doping.

Code: 21T07NN

Detection of blood doping remain a challenge, and novel detection methods are warranted. In the present project, the aim is to evaluate the potential of changes in red blood cell proteins for detection of recombinant human erythropoietin (rhEpo) misuse at sea level and at altitude in males and females. The red cell protein content is expected to change with rhEpo treatment, as the transcription of genes coding for red cell proteins change during blood doping practices. Indeed, changes in a few proteins of red blood cells are known to change with rhEpo misuse. However, whether the changes in these proteins are sensitive and specific to rhEpo misuse and whether more sensitive and more specific proteins exist remains unknown. Additionally, it remains unknown whether hypoxic exposure or gender are confounding factors to these changes. To answer these questions, the present project will investigate more than 1000 different red blood cell proteins using mass spectrometry and identify the most sensitive and specific ones. Further, the present project will develop and validate a model for rhEpo detection based on the detected proteins. Our goal is to develop a ready-to-use robust method that will allow precise quantification of our novel biomarkers within approximately 4 h, from receiving the sample to the result.

Code: 21C20VB

This project aims to determine whether new-generation ultra long-acting beta2-agonist (U-LABA) formulations containing vilanterol and indacaterol can enhance performance when inhaled at therapeutic or supratherapeutic doses in highly-trained males and females. The project will also examine whether U-LABA can induce muscle hypertrophy when used chronically.

Objectives: This project will investigate the potential performance-enhancing effects of vilanterol and indacaterol at therapeutic and supratherapeutic inhaled doses. THe formulations tested are Relvar® (22 µg/dose vilanterol+fluticasone 55 µg/dose) and Atectura® (indacaterol 125 µg/dose+mometasone 62 µg/dose) as these are U-LABA formulations of relevance for asthma treatment.

Code: 22E05MT

Gene doping has been classified as a prohibited method in the WADA prohibited list for many years. As gene therapeutic approaches improve and concerns regarding their safe clinical application decline, concerns about their illicit use in elite sports are rising sharply. The outlined project aims at the evaluation of a novel gene doping detection approach emplying high multiplex MALD-TOF mass spectrometry (MS). As preliminary work, a prototype panel able of the multiplex detection of transgenic expression of seven relevant gene doping targets (EPO, FST, GH-1, IGF-1 MSTN(-Propeptide), VEGF-A, VEGF-D) has been designed and pre-tested for its general specificity and applicability. Central aim of the proposed project is the in-depth validation of the prototype panel with regard to its specificity, sensitivity, and reproducibility. Futhermore, suitable reference material will be designed and tested taking advantage of the single-base extension and detection technique of the proposed chemistry and detection method. In a final step, the optimized and validated panel and reference material will be tested for its applicability to routine testine procedures with a first set of 1000 (doping control) specimens consented for research purposes. If successful, the described high multiplex gene doping detection panel and MALDI-TOF MS detection approach would allow a time- and cost-effective detection of multiple gene doping relevant targets at once and could thus expand the range of available tests for gene doping detection.