The project deal with the synthesis and analytical characterization of an epimeric long-term metabolite of the anabolic steroid Dehydrochloromethyltestosterone (4a-chloro-18-nor-17b-hydroxymethyl-17a-methyl-5a-androst-13-en-3a-ol)
Synthesis of 17-epi-Oxymesterone long term metabolite
By
Investigateur principal
G.
Gmeiner
Seibersdorf Labor GmbH
Autriche
―
2023
―
En vigueur
Sommaire
Code: T23A03GG
The project deal with the synthesis and analytical characterization of a long term metabolite of the anabolic steroid oxymesterone (17β-methyl-4,17α-dihydroxy-androst-4-en-3-one)
The project deal with the synthesis and analytical characterization of the glucuronide phase II metabolite of the stimulant Adrafinil (Diphenylmethyl)sulfinyl]-N-hydroxyacetamide)
The project deal with the synthesis and analytical characterization of the glucuronide phase II metabolite of the anabolic agent Andarine (2S)-3-(4-acetamidophenoxy)-2-hydroxy-2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]propanamide)
Detection of genetic polymorphisms of the human Erythropietin gene in urine, whole blood and dried blood spots
By
Investigateur principal
F.
Donati
Laboratorio Antidoping FMSI
Italie
―
2023
―
En vigueur
Sommaire
Code: 23E05FD
Several polymorphisms/variants of the human EPO gene could be the cause of confounding effects in the application of the analytical methods that are routinely applied for the detection of the abuse of recombinant erythropoietin (recEPO). The most known of them is the c.577del (rs369859204) variant, a single nucleotide deletion in the position 577 of the human EPO gene exon 5. The deletion causes a shifting of the sequence within the terminal exon 5 with consequent loss of the stop codon. At translational level, this results in the formation of a longer mature protein with a higher molecular weight of 3.3 kDa. In doping control, this results in the visualization of a “double band” during the application of the routinary SAR-Page method for the detection of the abuse of recEPO. The double band may be then erroneously interpreted as a false positive sample. As stated in the WADA technical document TDEPO2022, it is suggested to screen for the presence of the c.577del variant whenever a suspected double band is detected by the SAR-Page analysis, in the final aim to avoid the possibility of a false positive finding. Moreover, at least 12 other polymorphisms/variants have been described in the literature that are potentially capable of generating confounding effects in the interpretations of the antidoping analyzes. By following the input given by WADA TDEPO2022, this project involves the development and validation of a genetic test, based on the genomic DNA Sanger sequencing method, that is capable to identify the c.577del variant and all the other polymorphisms/variants present on the human EPO gene (namely: rs1554393458, rs1554393463, rs1419397684, rs773895305, rs763035217, rs1562901775, rs71517124, rs1562902091, rs893404064, rs752755372, rs951322017, rs369859204), and the study of their incidence in determining confounding effects in the interpretation of the routinary tests performed for the purpose of detecting the abuse of recombinant EPO by athletes.
Detection of rEPO administration using deep learning on blood smears
By
Investigateur principal
S.
Voss
IDAS Dresden
Allemagne
―
2023
―
En vigueur
Sommaire
Code: 23D05SV
The aim of this study is to use artificial intelligence (AI) to evaluate blood smears as a potential matrix to detect doping with recombinant EPO (rEPO). Previous publications reported changes in the Red blood Cell Morphology after rEPO administration (e.g. Macrocytes, Stomatocytes). During these times automated haematology analysers and manual microscopy were used for the estimation of these parameters. Recently, digital Morphology is a developing field in Haematology which enables the automated analysis of blood smears by artificial intelligence. With this technology it is possible to evaluate morphological changes with an increased precision, based on a higher number of cells and to discover even minor changes in cellular shapes. AI and deep learning is capable of revealing new insights which conventional approaches were lacking so far, like predicting molecular changes on cytomorphology. Therefore, the goal of this study is to identify relevant changes in cell morphology during rEPO administration which are not addressed using current state of the art techniques. In the long term, a blood smear-based athlete blood passport providing an individual erythrocyte signature might be a prospective application of monitoring athletes by using artificial intelligence based on this postulated deep learning model.
Investigations into the metabolism and elimination of flmodafinil and fladrafinil as well as their prevalence in elite sports
By
Investigateur principal
M.
Thevis
German Sport University
Allemagne
―
2023
―
En vigueur
Sommaire
Code: 23C03MT
Stimulants are substances with wake-promoting effects, whose misuse in sports is only prohibited in-competition. The high analytical sensitivity of anti-doping laboratories allows for comprehensive screening procedures, which cannot only reveal the misuse of stimulants, but also unintended doping due to the use of contaminated/adulterated nutritional supplements. Therefore, stimulants constantly account for more than 10% of all adverse analytical findings (AAF) reported by the World Anti-Doping Agency (WADA). Due to the easy detectability of prohibited stimulants, especially novel drugs such as flmodafinil and fladrafinil have an enormous potential for being misused in sports. These bisfluoro analogs of modafinil and its pro-drug adrafinil are supposed to be significantly more effective than the original compounds, but currently not explicitly mentioned in the WADA Prohibited List. Consequently, the aim of this research project is to investigate the urinary metabolism of flmodafinil and fladrafinil, as well as their elimination behavior in urine and blood. The acquired data will lay the foundation for the implementation of both drugs as well as their metabolites into doping control routine procedures. Moreover, a retrospective evaluation of in-competition routine samples will be conducted to determine the prevalence of these stimulants in sports.
Inhibitors of the activin receptor signaling pathways (IASPs) in sports drug testing
By
Investigateur principal
M.
Thevis
German Sport University
Allemagne
―
2023
―
En vigueur
Sommaire
Code: 23A13MT
Members of the transforming growth factor beta (TGF-β) superfamily such as myostatin (MSTN), activin A, and GDF-11, are dimeric cytokines signaling through activin receptors. They play important regulative roles in different biological processes as for example the formation of muscle and red blood cells. Therefore, inhibitors of the activin receptor signaling pathways (IASPs) can be considered as potential performance-enhancing agents in sports, which are included in sections S2 (“Peptide hormones, growth factors, related substances and mimetics”) and S4 (“Hormone and metabolic modulators”) of the WADA Prohibited List. They comprise targeted (e.g. anti-MSTN antibodies) as well as multi-targeting approaches (e.g. receptor competitors and anti-ActRII antibodies), and for several drug candidates, doping control detection methods based on electrophoretic techniques and Western blotting or LC-HRMS/MS have been proactively developed within the last years. This is of utmost importance, as, even without clinical approval, reference material for such emerging drugs is often available for research purposes and/or distributed on the black market. Therefore, the aim of this research project is to develop a combined mass spectrometric detection assay for the novel anti-activin A antibody Garetosmab and other IASPs such as Sotatercept, Domagrozumab, and Luspatercept. A combined extraction of the target analytes from plasma/serum can be achieved by using NHS magnetic sepharose beads coupled to different TGF-β cytokines such as activin A, MSTN, and GDF-11. Subsequent LC-HRMS/MS analysis will not only allow for an unambiguous identification of the target analytes based on the amino acid level, but also retrospective sample evaluation for the presence of novel IASPs binding to one of the TGF-β cytokines employed for affinity purification.
Investigations into the human metabolism of Trestolone (7α-methyl-19-nortestosterone, MENT)
By
Investigateur principal
M.
Thevis
German Sport University
Allemagne
―
2023
―
En vigueur
Sommaire
Code: 23A07MT
MENT (7α-methyl-19-nortestosterone) is not explicitly listed on the WADA´s Prohibited List, albeit it falls under section S1 Anabolic Agents. Developed in the 1960´s, its anabolic properties were found to be ten-times that of testosterone, the main endogenous anabolic androgenic hormone. Considering its fast-metabolic clearance rate, MENT seemed a suitable candidate for male hormonal contraception and hormonal replacement therapies. Despite promising results obtained in the 1990´s, MENT failed to receive full clinical approval due to ascertained side effects. Nevertheless, it is still easily available via internet-based providers and an interesting alternative for cheating athletes due to its described properties. Human urinary metabolites of MENT have only been described once in the context of sports drug testing and 3 metabolites were identified, detectable at high urinary concentrations for only 14 hours. Considering the human metabolism of steroids in general, it can be expected that metabolites are formed and excreted into urine for a longer time period, most probably at much lower concentrations. Aim of this research project will be the re-investigation of the human MENT metabolism employing an administration trial with deuterated MENT to enable the sensitive detection of all formed metabolites by hydrogen isotope ratio mass spectrometry. This method was already enrolled in several successful research projects on metabolite detection. All metabolites detected will be further investigated by high resolution/high accuracy mass spectrometry in order to potentially elucidate their chemical structural. In a second administration trial, samples will be collected from three volunteers after the administration of unlabeled MENT to confirm the findings of the deuterated excretion study and to enable consideration of inter-individual differences in steroid metabolism to identify those metabolites most suitable for drug testing. Promising metabolites will then be in-cooperated into existing sports drug testing methods.
Stability of Phase II long term sulfated steroid metabolites for their use in Quality Assessment exercises and routine doping control.
By
Investigateur principal
J.
Pascual
Institut Municipal d’Investigacio Medica (IMIM)
Espagne
―
2023
―
En vigueur
Sommaire
Code: 23A03JP
Sulfated phase II metabolites have gained great interest as long term metabolites of many anabolic steroids. The direct analysis of those sulfates using LC/MSMS has been extensively used as the research tool to finding new long-term metabolites. However, sulfates are known to be potentially unstable, and very particularly at acidic pH where catalysis occurs. Urine samples normally have acidic pH and the stability of those precious long-term metabolites in the biological matrix needs to be investigated. The preparation of reference urine samples to be used within the EQAS quality Assessment scheme has led us to obtaining shocking evidence on the instability of some of the analytes of interest in routine doping control. In particular, some long-term sulfated phase II steroid metabolites. As a good example, results obtained when assaying the stability of a excretion study of danazol, 7 days at different temperatures showed a huge increase in the concentration of the two metabolites (ethisterone and 2-OH-methyl-ethisterone). This increase is attributed to the hydrolysis of their sulfated phase II counterparts. The aim of the present project is the study of the stability of sulfated phase II metabolites using available excretion studies of danazol, mesterolone, clostebol and drostanolone, all having known sulfated long-term metabolites, as proof of concept. The idea is developing conditions to keep those sulfates stable for sample preparation and/or establish the conditions for a complete hydrolysis in order to target the analysis of the resulting compounds.