Projets de recherche

Code: T11A03VB

Human chorionic gonadotropin (hCG) may be abused by male athletes in sports and 1s included in the banned substance list of the Wortd Anti-Doping Agency.The goal of this prej!;l.ct Is t9 develop an new analytical tool for the detection of hCG In eith!etes biological samples. In particular. we wm develop an isotopically-labeled version of hCG (PSAQ standard) to be used as qUi,,ritificaUon reference in mass spectrometry (MS)-based assays. ParUli:IJlarly, we w ii develop optimized protocols for expression, stable-Isotope-labelling and purification of hum?n choriogona9otropin (hCG).

hCG is an heterodfmer protein composed of an a chain (common to thyrotropin, lutropin and follitropin), and a ti chain. which confers its specific biplogical activity_ The a and (i subunM are non-covalently linked and carry several dtsulfide bonds (6 disulfide bonds per subunit). The 2 chains also harbour carbohydrate moieties. 2 N-glycosylations and 4 0-g!ycosylatlo s on the fi subunit and 2 N-glycosylatlp11s on the a subunit. For optimal assay accuracy and reliablUty. the PSAQ standard will be developed as an a? heterodimer. The PSAQ heterodimer will be purified and controlled for Isotope incorporation. MS-based analyses will also be perfomed to check the sequence, structure and post-translational modifications of the standard

Main findings

This program was a feasibility study aiming at developing expression, isotope-labelling and purification protocols to produce isotopically-labeled human choriogonadotropin PSAQ standard. Choriogonadotropin protein is a heterodimer composed of an alpha chain, which is also common to thyrotropin, lutropin, follitropin, and a beta chain, which confers its specific biological activity. The alpha and beta subunits are non-covalently linked and carry numerous disulfide bonds. The 2 chains also harbour carbohydrate moieties: 2 N-glycosylations and 4 O-glycosylations on the beta subunit and 2 Nglycosylations on the alpha subunit. The first step of evaluating the expression of a choriogonadotropin in human cell free expression system was failed. In a second step of the program, each subunit (alpha and beta) was cloned with its signal peptide into our dedicated mammalian expression vector. The DNA sequence coding for the poly-His purification tag was added at the C-terminal end of sequence coding for subunit alpha protein. Then both expression vectors coding for the alpha and beta chains were co-transfected in HEK 293 cells. The choriogonadotrophin heterodimer was successfully expressed and purified. The purity was estimated to be greater than 90% at this stage. The standard was treated with PNGase F which removes N-linked glycosylations. Alpha and beta subunits sequences were verified by LC-MS/MS analyses after reduction/alkylation and in-gel trypsin digestion. Sequences coverage was higher than 50% with 3 specific peptides detected for each subunit. Stable isotope incorporation was determined using MS data generated by LC-MS/MS analyses. This experiment confirmed that the isotope incorporation of arginine and lysine was greater than 98% with limited Arginine-to-Proline conversion.

Code: T11A02MT

Erythropoiesis-stimulating agents (ESAs) have been frequently detected in doping control samples in the past and are among the illicitly used drugs that are most often mentioned by confessing athletes. New ESA therapeutics represent new options for cheating athletes and new challenges for sports drug testing authorities as comprehensive detection assays are required. With the introduction of the recently approved EPO-mimetic drug Omontys (formerly referred to as Hematide and Peginesatide), a first peptidic ESA is principally available that necessitates complementary analytical methods as Omontys will not be detectable with conventional EPO analytical methodologies. First successful procedures for the analysis of Omontys from plasma, serum, and dried blood spots were recently reported; however, the most frequently available doping control samples are urine specimens. Since pharmacokinetic studies have demonstrated that a significant amount of Omontys is renally excreted, the transfer of the methodology to urine and the analysis of proof-of concept samples from human in-vivo studies are desirable.

In the present study, the completion of a detection assay for Omontys from urine samples by means of liquid chromatography-mass spectrometry is aimed. Pilot studies have demonstrated the capability of extracting, enzymatically hydrolysing and subsequently measuring a proteotypical peptide of Omontys from spiked urine specimens; in order to apply the methodology to authentic doping control samples, the method will be fully characterized and an administration study with the EPO-mimetic agent will be conducted with six human volunteers. The in-vivo derived samples will be measured with the developed assay to demonstrate its utility for the determination of the prohibited drug in authentic specimens.

Main findings

PEGylation of protein therapeutics by covalently attaching polyethylene glycol (PEG) polymers results in an increased molecular mass and, therefore, slower renal clearance, reduced proteolytic susceptibility, and decreased immunogenicity. Endogenously, PEGylated substances do not exist and, hence, the concept of targeting PEGylated compounds as an initial testing approach towards prohibited substances was considered. In this research project, different strategies including immunoaffinity purification & LC-MS/MS, commercial PEG-specific ELISAs, and SDS-PAGE in combination with iodine staining were tested to assess the capability of these approaches to allow for multiplexed detection of PEGylated proteins in doping control serum samples. In the course of the studies, ELISA kits and iodine staining were found to be of limited specificity. In addition, the diversity of PEGylations regarding size and composition resulted in limited recognition of PEGylated model compounds by anti-PEG antibodies used for immunoaffinity purification, suggesting that (to date) testing merely for the presence of PEG polymers in human serum does not facilitate sports drug testing approaches per se. However, Pegvisomant (i.e. PEGylated and sequence-modified human growth hormone) was successfully detected in serum samples with the employed methodologies, which demonstrates that in general mass spectrometric assays can be established if antibodies binding with appropriate efficiency to a broader spectrum of PEG conjugates are available.

Code: R11C01TF

We will analyze the effects of HGH and erythropoietin to search for genetic signatures of exposure to those doping agents. We have obtained results from several individual WADA-supported laboratories and will carry out extensive comparisons to identify genes whose expression is indicative of exposure to HGH or erythropoietin.

Main findings

These two related studies were designed to test the concept that exposure of humans to doping agents disturbs the normal expression of many of the 20,000 genes in human cells and whether those changes can be used as a rigorous proof or a “signature” of exposure to specific doping agents. These kinds of studies are made possible by modern genetic techniques that allow an estimate of the extent of expression of all human genes on a single square inch silicon chip. Our studies also were designed to determine if several studies carried out under different conditions in different laboratories could easily be analyzed even though they used slightly different techniques and methods of analysis. This method of analysis of multiple and slightly different sets of data from different studies is called “meta-analysis” and examples of meta-analysis have been successful in many other kinds of research. We therefore carried out meta-analysis of three independent studies of the effects on gene expression in blood samples from athletes of administration of human growth hormone (HGH) and three separate studies of the effects of erythropoietin (Epo) or hypoxia in the blood cells of human athletes and in mice.

As expected, we found that the genes that are expressed incorrectly after exposure to HGH and Epo are different from each other. However, we also discovered that the separate studies identified only a small number of genes that were disturbed in the same way in all three studies, leading to a conclusion that those genes are probably not specific “signatures” for exposure to HGH or Epo. We have concluded that these results could mean that these drugs do not cause significant changes in gene expression in blood cells. We prefer an explanation that the three separate HGH studies and the diverse Epo/hypoxia differed in many details that introduced too many slight differences in the timing of testing, dosages, methods of tissue preparation and methods of analysis that probably have hidden the gene expressions shared by the different studies. We are impressed that in other very recent studies supported by WADA in which one laboratory has carried out very careful studies of humans exposed to Epo, reproducible changes have been found that consistent with a genetic “signature” for exposure to Epo, demonstrating that extensive and well controlled single studies under some conditions can be an effective approach to identifying the genetic changes in blood samples or other tissues. We propose that a reasonable next step is to carry out a similar study with HGH exposure and compare results from such a study with those obtained in our meta-analysis. We are confident the overall concept of genetic signatures of doping is a correct and robust one and will add important new tools to the prevention and detection of doping.

Code: R11A01MT

The discovery and implementation of the long-term metabolite of metandienone, namely 17β-hydroxymethyl-17α-methyl-18-norandrost-1,4,13-trien-3-one to routine doping controls resulted in hundreds of adverse analytical findings for metandienone worldwide and impressively demonstrated that prolonged detection periods significantly increase the effectiveness of sports drug testing. For oxandrolone and other 17-methylated steroids, analogues to this metabolite have been described, but comprehensive characterization and pharmacokinetic data were still missing and dersirable. In this project, the synthesis of the two epimeric oxandrolone metabolites 17β-hydroxymethyl-17α-methyl-18-nor-2-oxa-5α–androsta-13-en-3-one and 17α-hydroxymethyl-17β-methyl-18-nor-2-oxa-5α–androsta-13-en-3-one using a fungus (Cunninghamella elegans) based protocol was conducted followed by full characterization of the obtained reference material by means of liquid chromatography-nuclear magnetic resonance spectroscopy and -high resolution/high accuracy mass spectrometry. To ensure a specific and sensitive detection in athlete’s urine samples, different analytical approaches were followed, such as LC-MS/MS (QqQ and Q-Orbitrap) and GC-MS/MS to detect and identify the new target analytes. The applied methods have demonstrated good specificity and no significant matrix interferences. Linearity (R2 > 0.99) was tested and precise results were obtained for the detection of the analytes (CV < 20%). Limits of detection (S/N) for confirmatory and screening analysis were estimated at one and two nanogram per milliliter of urine, respectively. The assay was applied to oxandrolone post-administration samples to obtain data on the excretion of the different oxandrolone metabolites. The studied specimens demonstrated significantly longer detection periods for the new oxandrolone metabolites compared to commonly targeted metabolites such as epioxandrolone or 18-nor-oxandrolone, presenting a promising approach to improve the fight against doping.

Main findings

According to the WADA statistics, anabolic androgenic steroids are still the most frequently detected substances in sports drug testing. Nevertheless, for some anabolic steroids as oxandrolone the number of positive findings is relativly low despite of very potent anabolic activity and only weak side effects making the steroid very attractive for cheating athletes. To improve sports drug testing and to develop comprehensive analytical strategies for an effective detection of an oxandrolone abuse, two isomeric oxandrolone metabolites Ox M1 and its epimer Ox M2 were successfully synthesized and fully characterized. Within an oxandrolone post administration study, this reference material was applied to obtain data on approximate detection windows. The first time, it has been demonstrated that Ox M1 and M2 provided significantly prolonged detection periods compared to commonly targeted metabolites such as epioxandrolone or 18-nor-oxandrolone. Based on these results, a very comprehensive, sensitive and reliable methodology based on LC-(ESI)-MS/MS and GC-MS/MS for screening and confirmatory analysis of the new long term metabolites Ox M1 and M2 was developed presenting a powerful tool in the fight against doping.

Code: 11A1JV 

When metabolites of nandrolone, boldenone or formestane are found at low concentrations in doping control tests, a further CIRMS-based analysis is compulsory in order to be sure that steroid misuse has occurred. For each analyte, the factors that could generate such findings are diffrent, but in all cases the urinary concentrations always range between 1 and 20 ng/mL. Due to the low concentration of the analytes and the small contribution of 13C to the analyte molecule, an important level of selectivity and sensitivity is necessary to characterize a 13C difference.  Therefore, the sample must be submitted to a long time-consuming sample preparation method. 
Nevertheless, to improve detection capabilities, some laboratories include a standalone clean-up step based on the use of high performance liquid chromatography (HPLC). The present project attempts to attain these objectives by means of on-line coupling of liquid chromatography and gas chromatography (LC-GC) using the TOTAD interface, developed and patented by the UCLM research group. This analytical methodology allows the complete LC fraction containing the analytes to be transferred to the GC and, moreover, provides a better signal/noise ratio because it provides cleaner extract. These aspects, will presumably improve sensitivity by one or two orders of magnitude. The project will focus on developing an analytical method in which the hydrolysed extract is derivatised (off-line) and analysed by LC-GC. Two different detectors will be used, leading to LC-GC-MS and/or LC-GC-C-IRMS coupling. Automation of the system by using an autosampler will also be attempted. The objective of the present project is to develop a sensitive, automatic, robust and reliable analytical method to be used for the analysis of the mentioned steroids in urine. The method should allow the detection limits required to be reached, thereby providing a powerful tool for the efficient control of the misuse of anabolic steroids. 

Main Findings: 

It has been developed a sensitive, automatic, robust and reliable method for analyzing steroid in a urine using on line coupled LC-GC-C-IRMS (liquid chromatography-gas chromatography-combustion isotope ratio mass spectrometry) with the TOTAD (Through iven Transfer Adsorption Desorption)  interface to discriminate between the endogenous or exogenous origin of the same, and by on-line coupled LC-GC-MS to confirm the purity and the identity of the steroid peaks. Urine samples spiked at 5 ng/mL or 10 ng/mL with the steroid to be analysed were used. Of each urine sample 20 mL was hydrolyzed, extracted, purified by a first RPLC step of un-derivatized steroids, derivatized to acetyl­steroids and, finally, submitted to on-line LC-GC with CIRMS or MS detection. In the coupled system a second RPLC cleaning of derivatized steroids is applied, and the l!...C fractions containing the analytes are completely transferred to GC-CIRMS or GC-MS. 
l!...C-GC-CIRMS methods were devefoped for the analysis of 19-Norandrosterone (19-NA) and for Bodenone (Bo) and its main metabolite (BoM). The method failed in the case of 19-NA due to an interfering compound. The derivatization of 
Formestane (F) did not work properly. so that the developed method is not applicable to F analysis. In the case of Bo and BoM the method was developed successfully. The volumes of the fractions transferred from LC to GC through a fraction collector were 1000 and 900 ftL, respectively. 138Cyp08 corrected values for Bo and BoM, as well as the /:J.8 in relation to the reference compound Pregnandiol (PD), clearly indicated the exogenous origin of the steroids. The Standard Deviations of the 138CVPoB values ranged from 0.85 to 1.11 for the entire method. A confirmatory on-line LC-GC-MS analytical method was developed for Bo and BoM. Relative Standard Deviations of the absolute peak areas were below 8%, except when Bo was spiked at 10 ng/L. Detection Limits were 0.5 ng/mL for Bo and 0.05 ng/mL for BoM in full scan. When 3 ions were selected, detection limits were 0.07 and 0.008 ng/mL respectively. 
For the first time, an analytical method involving the coupling of LC-GC with C­IRMS has been developed. The presented methods permit the origin of urinary Bo and BoM to be identified as endogenous or exogenous. The sensitivity is substantially improved compared with currently used methods, allowing the detection limits set by WADA to be attained.  The advantages of LC-CG coupling open uo the possibility of extending the methodology to other steroids and to other compounds currently analysed by GC. The high sensitivity achieved should permit the amount of the urine necessary for such analyses to be decreased. The LC-GC-C-IRMS method developed for the analysis of Boldenone has been published. Presumably, the LC-GC-MS method for the analysis of boldenone will be published shortly.

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Dr. Gertrud PFISTER, University of Copenhague, Danemark

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Dr. Haralambos TSORBATZOUDIS, University of Thessaloniki, Greece

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Dr. Fabio MANFREDINI, International Biathlon Union, in partnership with the University of Ferrara, Italy

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