Projets de recherche

Code: 12A18GG

Testosterone is still regarded as the major contributor to steroid doping world-wide. Among the most common forms of application are injections of different sorts of esters. State-of-the-Art detection of Testosterone doping includes the quantification of the Testosterone /Epitestosterone – Ratio (T/E – ratio) as well as subsequent Isotope ratio mass spectrometry (IRMS). Previous published studies of our research group have demonstrated that a comparably high percentage of testosterone preparations do not significantly differ from endogenous values of testosterone and markers of testosterone doping. Consequently when such preparations with endogenous – like 13CVPDB values are applied, IRMS - technology fails to detect testosterone
doping. Direct detection of the testosterone ester leads to an unequivocal proof of doping with testosterone preparations, because such esters are not built endogenously.  Previous studies indicate that a direct detection of testosterone esters in both hair and plasma is possible.
Aim of the proposed project is the investigation and optimisation of the direct detection of testosterone esters in body fluids like serum, whole blood and stabilized blood with an already developed detection method using modern and sensitive technology. The project will gain information on diagnostic windows for detection of doping using testosterone esters and proper sampling conditions. Additional aim of the proposed project is to evaluate the suitability of already collected blood samples in doping control (e.g. samples collected for blood parameter measurement or growth hormone detection) for a possible reanalysis for testosterone esters.

Main Findings:

Injections of synthetic esters of testosterone are among the most common forms of testosterone application. In doping control, the detection of an intact ester of testosterone in blood gives an unequivocal proof of the administration of exogenous testosterone. The aim of the current project was to investigate the detection window for a number of testosterone esters in blood. Furthermore, the suitability of different types of blood collection devices was evaluated.
A clinical study with six participants was carried out, comprising a single injection of a testosterone ester preparation. The enrolled subjects were randomly assigned to receive either a single intramuscular injection of testosterone undecanoante 1000 mg (Nebido®) or a single intramuscular injection of a mixture of four testosterone esters: testosterone propionate 30 mg, testosterone phenylpropionate 60 mg, testosterone isocaproate 60 mg and testosterone decanoate 100 mg (Sustanon®). Three subjects were assigned to each study group and blood was collected throughout a testing period of 60 days. At each sampling, the blood was collected into three different blood collection tubes: Tube A: Vacuette 9 ml serum tube with clot activator and gel separator (Greiner bio-one Vacuette®, Kremsmünster, Austria). Tube B: Vacuette 6 ml plasma tube with sodium fluoride, potassium oxalate, and no gel separator (Greiner bio-one Vacuette®, Kremsmünster, Austria). Tube C: Vacutest 3.5 ml plasma tube with sodium fluoride, disodium ethylenediaminetetraacetic acid (Na2EDTA), and gel separator (Vacutest Kima, Arzergrande, Italy). Additionally, a study on the in vitro degradation of testosterone esters in blood was performed, using the same collection tubes as described above. The applied analytical method included liquid-liquid extraction and preparation of oxime derivatives, prior to TLX-sample clean-up and LC-MS/MS detection.
In the clinical study, the elimination half-lives and detection times depended on the type of testosterone ester administrated. The depot effect of an intramuscular testosterone ester preparation increases in proportion to the length of the ester side chain. This is because the half-life of the absorption increases with longer chains.
As expected, the shortest chained ester, testosterone propionate, showed the most rapid elimination and shortest half-life. Nevertheless, the ester could still be detected for 4-5 days in serum and plasma of all study participants receiving the drug. Testosterone phenylpropionate and testosterone isocaproate were detected for at least 8 days in serum and plasma, whereas testosterone decanoate showed a detection time of 18 days. Testosterone undecanoate was detectable in all post-administration blood samples collected during the whole study period (60 days), thereby giving the longest detection time of the esters investigated.
The stability studies showed that the shorter chained testosterone esters were hydrolysed more rapidly in blood collection tubes not stabilized with NaF (tube A) compared to stabilized tubes (tube B and C). The rate of hydrolysis seems to be dependent on storage temperature. In the clinical study, though, the testosterone ester detection window was not affected by the applied blood collection tube.

Code: 12B11GK

Recombinant human growth hormone (rhGH) has been on the list of forbidden substances since availability of its recombinant form in the early 1990s; however adequate routine doping tests are lacking. The project aims to develop a fast and highly sensitive drug test for detecting two or more hGH-dependent markers in the serum of elite adolescent athletes. In our approach proteomic markers for hGH action such as insulin-like growth factor 1 (IGF-1) and pro-collagen type III N-terminal peptide (P-III-P) will be identified in just a single immunoassay. Considering the desirable reduction of time, costs and workload using FCS instead of other currently available IGF-1 and P-III-NP assays the presented methodology will be an important contribution for a functional doping test for proper use of rhGH. 

Main Findings: 

Two approaches have been developed to detect recombinant hGH in blood in order to control its misuse with the intention of improving athletic performance. Adequate non-invasive tests for human growth hormone (hGH)-dependent markers such as insulin-like growth factor 1 (IGF-1) and pro-collagen type Ill N-terminal peptide (PIIINP) are still lacking. In this one-year project a fast and sensitive bead-based immunoassay for IGF-1 and PIIINP detection in serum was developed based on Fluorescence Correlation Spectroscopy (FCS). FCS was used as a reliable technology for measuring absolute concentrations in the nano-molar range. Three FCS immunoassays - a sandwich and a competitive immunoassay for IGF-1 as well as a competitive PIIINP assay - were established and validated against commercially available ELISA. We were able to detect molarities between 0.5 and lOnM of IGF-1 and between 0.5 and 2.5nM of PIIINP with high accuracy in serum samples.  
Considering the desirable reduction of time, costs and workload using FCS instead of other currently available IGF-1 and PIIINP assays the presented methodology might be an important contribution for proper use of rhGH in sports. 

Code: 12A23RC 

This proposal describes a detailed plan to improve the ability to analyze prohibited doping substances by the development of brand new, original methodologies in Analytical Chemistry.  The proposal teams up an advanced mass spectrometry laboratory at the University of Paris (VI) with France’s leading organization in the battle to ensure the absence of illegal performance enhancing substances in athletes, the “Agence Française de Lutte contre le Dopage” (AFLD). 
Using the original approach of regioselective anion attachment mass spectrometry that has been recently developed by the Cole research group, we seek to improve the limits of detection of prohibited steroids, especially those of limited polarity that exhibit poor responses by conventional mass spectrometric ionization approaches. In addition, we seek to expand the ability to unequivocally identify novel doping substances (those not on the current Prohibited list), while also enabling the direct analysis of conjugated steroids. The expected outcomes of this work are a substantial improvement in the ability to detect prohibited steroids at ultra-trace levels, and an enhancement of the specificity in structural analysis that is obtainable by tandem mass spectrometry.

Main Findings: 

Neutral anabolic steroids are doping agents that do not provide strong signals by electrospray ionization-mass spectrometry (ESI-MS), thus making their detection challenging when using this technique. While some steroids may be analyzed adequately with older more labor-intensive anti-doping analysis methods (such as GC-MS employing electron ionization (EI), often preceded by derivatization), there remains a need for the development of a new method that is capable of efficiently analyzing more difficult compounds, especially those that are less susceptible to ionization. We have investigated the addition of anions, in ammonium salt form, to anabolic steroid samples as ionization enhancers and confirmed that better signals and lower limits of detection are obtained using Anion Attachment Mass Spectrometry as compared to both positive and negative ESI in the absence of additives. Overall, the developed method allows for substantially improved detection of neutral steroids. For the neutral steroids targeted in this study, results show that the instrumental limit of detection (LOD) obtained using Anion Attachment Mass Spectrometry is always equal to, or better than, that obtained in either positive or negative ion ESI-MS in the absence of any additive. In the case of the most difficult compounds (calusterone-M, norbolethone-M), no signal was obtained in negative ion ESI-MS, but upon addition of NH4F, it becomes possible to detect these compounds at concentrations as low as 25 ng/mL, which represents a 100-fold improvement compared to the LODs obtained in positive ion ESI-MS. 
Atmospheric Pressure Photo-Ionization (APPI) Mass Spectrometry was also tested for its ability to produce mass spectrometric signals from trace level samples, and obtained results were compared to results acquired using Anion Attachment Mass Spectrometry. As a test case, the fluoxymesterone metabolite produces the cation radical ion M+. when analyzed using APPI, but a 50-fold loss in signal is observed from APPI when compared to the Anion Attachment approach. In summary, none of the tested "difficult to ionize" steroids exhibited better signal responses by APPI as compared to those obtained by Anion Attachment Mass Spectrometry. On the other hand, the formation of anion adducts does not appear to improve the sensitivity of conjugated steroids containing a carboxylic acid that is already susceptible to deprotonation and [M-H]- formation in conventional ESI-MS. 
Looking ahead to the implementation of this new approach to "real-world" antidoping samples, we have obtained preliminary Liquid Chromatography-Multiple Reaction Monitoring (LC-MRM) results that show trace level detection of the problematic fluoxymesterone metabolite down to the 5 ng/mL level. This improved sensitivity, relative to existing methods, will serve to reduce the number of false negatives in real sports doping analyses.

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Résumé

La présente étude vise à mettre en place une stratégie de prévention du dopage à partir d’une enquête sur les connaissances, attitudes et pratiques des élèves en rapport avec le dopage. Les résultats attendus permettront de faire progresser l’état des connaissances générales de la discipline, en ce qui concerne la situation du dopage en milieu scolaire ivoirien. En effet, en Côte d’Ivoire, les travaux réalisés sur le dopage concernent généralement les sportifs issus des clubs professionnels (football notamment), et ne prennent pas en compte le milieu scolaire.

Méthodologie

Il s’agit d’une recherche action fondée sur une enquête CAP diagnostique des connaissances, attitudes et pratiques des élèves en matière de produits dopants, dont les résultats serviront à concevoir des messages de communication. Ces messages seront utilisés d’une part pour une série de conférences publiques dans les écoles, et d’autre part pour une sensibilisation de proximité menée par des comités locaux (composés d’élèves des établissements scolaires concernés) mis en place par le projet, en vue d’un changement de comportement. En outre, une prise en charge psychosociale des cas de dépendance vis-à-vis des produits dopants, identifiés lors de l’enquête, sera réalisée par les assistants sociaux membres de l’équipe, en vue d’aider les élèves concernés à abandonner ces pratiques.

Résultats

• 20,6% des élèves enquêtés affirment connaître des élèves sportifs utilisant des produits pour améliorer leur performance dans le sport. Par ailleurs, 5,3% des élèves sportifs enquêtés affirment avoir eux‐mêmes déjà utilisé des produits pour améliorer leur performance. Recommandation: réduire la prévalence du dopage en milieu sportif scolaire.
• Le désir de vaincre la peur et le stress, ainsi que le besoin d’endurance/de vigueur constituent les principales raisons d’utilisation des produits dopants par les élèves sportifs enquêtés. Recommandation: Sensibiliser les élèves sportifs sur les méthodes recommandées pour vaincre la peur et le stress lors des compétitions et pour obtenir de l’endurance sur le plan physique.
• 10% des élèves sportifs affirment utiliser les multivitamines avant les compétitions ou pendant les entraînements, et 24,3% les utilise après. Recommandation: Sensibiliser les élèves sportifs en vue du non usage de produits multivitaminés avant et pendant les compétitions et les entrainements
• Les boissons alcoolisées constituent l’une des substances les plus utilisées par les élèves sportifs comme produit pour augmenter les performances dans la pratique du sport. Il est aussi fait cas de l’usage de drogue. Recommandation: sensibiliser les élèves sportifs à la non prise de boissons alcoolisées avant et pendant les compétitions et entrainements.
• 66,1% des élèves enquêtés ont déjà entendu parler de dopage. Soit 33,9% qui n’en ont pas entendu parler en dehors de l’enquête. Recommandation: informer les élèves sur le dopage, sa définition selon l’AMA et l’’OM.
• La télé (70,6%) est de loin le canal par lequel les élèves ont entendu parler du dopage. L’accès à cette information par le biais de l’école représente 15,1%. Recommandation: renforcer les capacités des écoles à diffuser des informations sur le dopage, notamment par la formation des membres de clubs de santé scolaire sur le dopage.
• 23,9% des élèves qui ont entendu parler de dopage ne sont pas en mesure de citer les produits interdits dans le sport, et certains parmi ceux‐qui disent connaître ces produits font parfois un amalgame avec des produits non nocifs. Recommandation : Informer les élèves sportifs sur la liste des produits interdits dans la pratique du sport.
• 92,2% des élèves admettent qu’il y a des conséquences négatives liées au dopage. Recommandation: informer plus largement les élèves sur les conséquences concrètes du dopage.

Ce document n'est actuellement disponible qu'en anglais.

Ce document n'est actuellement disponible qu'en anglais.

Ce document n'est actuellement disponible qu'en anglais.

Ce document n'est actuellement disponible qu'en anglais.

Ce document n'est actuellement disponible qu'en anglais.