Projets de recherche

Code: 11B10JP

All humans express antibodies against N-glycolyl-neuraminic acid (Neu5Gc), a sialic acidhumans cannot produce that is present in glycoproteins from animal sources. Small amounts ofNeu5Gc can metabolically be incorporated to human proteins resulting in the development of an immune response. 
Neu5Gc is present in recombinant glycoproteins (e.g. rEPO and analogues) since they are expressed in non-human cell lines. It has been proven that mice receiving injections of a recombinant glycoprotein, containing Neu5Gc, immediately produce antibodies against it. 
The hypothesis is that the concentration of antibodies against Neu5Gc must be much much higher in subjects treated with recombinant proteins (e.g. rEPO) than in the general population whre the exposure to Neu5Gc happens through a complex mechanism from the diet. 
The main objectives of the project are: 1- Developing a test to quantify antibodies against Neu5Gc circulating in human blood (serum or plasma) based on what has already been described in the literature.
2- Test human blood samples from healthy individuals and from patients being treated with rEPO.
3- Refine the analytical test in order to increase specificity and minimise background by testing different Neu5Gc containing antigens and approaches (inhibition).
4- Validate the test for anti-doping purposes establishing thresholds to ascertain the illegal exposure to recombinant glycoproteins as well as studying the pharmacokinetics of antibody production.

Main Findings: 

Humans do not possess the enzymatic capability of producing the sialic acid N-glycolyl-neuraminic acid (Neu5Gc). It has been described that, as Neu5Gc is present in animal products, small amounts of Neu5Gc can metabolically be incorporated into human proteins resulting in the development of an immune response. Anti-Neu5Gc antibodies (with different linkages to other monosaccharides in glycans) have been described as measurable in almost all human beings. We hypothesised that, as Neu5Gc is present in recombinant glycoproteins (e.g. rEPO and analogues), expressed in non-human cell lines, treatment with these substances should give rise to the development of a higher title of specific antibodies. Following the findings published in the literature, the overall aim of the project was the detection of circulating antibodies raised specifically against Neu5Gc, with the α2,3-linkage to Galactose found in recombinant EPO and analogues. 
Following the project plan, a specific antigen Neu5Gc-α2,3-Gal-β1,4-GlcNAc was synthesised linked to biotin (through an hexaethylenglycol bridge) or to KLH. 
Serum samples were obtained from patients under chronic NESP treatment as well as healthy untreated volunteers. Additionally samples were also obtained from volunteers participating in a clinical trial receiving rEPO treatment for 3 weeks. 
Detecting particular human antibodies in serum samples has proven to be very challenging as the concentration of total IgG is huge and non-specific binding is a major drawback. 
Different antigens were tested including Neu5Gc or Neu5Ac linked to a polyacrylamide (as described in the literature), the whole rEPO and our specific rEPO trisaccharide. 
After optimising all ELISA parameters, and being able to minimise background, small differences in anti-trisaccharide titre could be observed between patients treated with NESP and healthy volunteers. However those mean differences would not allow setting a cut-off to differentiate between them. However, when those tests were applied to samples from healthy volunteers submitted to rEPO treatment (e.g. resembling doping cases) there was no measurable increase in antibody titres. 
In conclusion, despite the many reports in the literature suggesting that circulating anti-Neu5Gc antibodies are ubiquitous, those measurements could not be consistently reproduced nor a clear titre of Anti-Neu5Gc antibodies could be detected either in patients using a rEPO analogue chronically nor in volunteers submitted to rEPO treatment. 

Code: 11D16GF

In late 2008, several synthetic cannabinoids were detected in herbal smoking mixtures, commonly called "spice". Today a great variety of such synthetic cannabinoid receptor agonists is known and it expected that further substances will appear. Many European countries have banned these substances, and the World Anti-Doping Agency (WADA) prohibited synthetic cannabinoids in-competition in 2010. To allow an adequat detection of synthetic cannabinoids in a urine sample, knowledge of metabolism is a key issue. Performing human clinical studies is however difficult, due to the lack of a toxicological profile. 
In vitro metabolism studies is an alternative to excretion studies in humans, and human hepatocytes are recognized to be a very close model to the human liver with intact cell membranes and the full complement of enzymes and cofactors. In the proposed project, an in vitro model using cryopreserved human hepatocytes will be used to investigate the metabolism of selected synthetic cannabinoids. The doping control laboratory in Seibersdorf is currently involved in a Wada-funded project testing and optimising the use of commercial available cryopreserved hepatocytes as an in vitro model for doping substances. The optimised model will be applied on the substances in the current study, and the main metabolites will be characterized by various mass spectrometric methods. A verification of the postulated metabolism from the in vitro studies is allowed by the investigation of in vivo urinary metabolic profiles in samples from drug drivers, supplied by the Vienna Police Department. 
Finally, the incubation extracts will be evaluated as a potential source of reference material in doping control, as access to such material is of major importance in an unequivocal identification of metabolites in a doping control sample. 

Main Findings:

Aim of the project was to use cryopreserved hepatocytes to study the metabolism and characterize metabolites of selected synthetic cannabinoids dominant on the market today. Due to the continuous emergence of new “spice” compounds on the marked, a rapid investigation of the metabolism is of major importance, especially since some synthetic cannabinoids are already reported not to be excreted unchanged in human urine [1]. 
Four of the most prevalent compounds in positive forensic samples are reported to be JWH-122, JWH-210, AM-2201 and JWH-081 [2]. Thus, these four compounds were selected for the studies on the human metabolism of cannabinoids, in addition to JWH-018 as a model compound. 
The results from the study suggest monohydroxylation as a major metabolic pathway for all five compounds investigated. Additionally, a major JWH 018 metabolite is carboxylated, while one of the major JWH-210 metabolites is dihydroxylated. Furthermore, additionally abundant metabolites for AM-2201 included one dihydroxylated, dihydrodiol, one desfluoropentylated, and defluorinated metabolites. Regarding JWH-081, a major dihydrodiol metabolite was identified, in addition to the abundant monohydroxylated compounds. Hence, even though similar metabolic pathways are observed for the five investigated compounds, differences are also present. Although varied conjugation rates were observed, the majority of the main metabolites were excreted as glucuronide conjugates. 
Incubations with hepatocytes seem to represent a very useful model for identifying and predicting potential major urinary metabolites. Looking at the model compound JWH-018, the metabolites generated in the incubation mixtures were in good correspondence with reported major metabolites excreted in urine. Furthermore, the JWH-210 hepatocyte extracts resembled the metabolic profile observed in a forensic urine sample. Altogether, taking information from our experiments and available literature into account, it seems that the metabolites generated in human hepatocyte incubation extracts reflect the prevailing human metabolism.

Code: 11A13CG

Anabolic androgenic steroids (AAS) are the most frequent abused substances in sports and are included in the World Antidoping Agency (WADA) List of prohibited substances. AAS are extensively metabolized in the liver and their target tissues. The metabolic pathways are divided into phase-I and phase-II reactions. Phase-I reactions involve oxidation, hydrolysis and reduction, which introduce new functional groups for the subsequent phase-II reactions (i.e. conjugation).

For the endogenous androgens and for exogenous AAS the main phase-II reactions are conjugation with glucuronic acid (glucuronidation) or with a sulfo-moiety (sulphatation). The screening methods used by the Antidoping Laboratories usually focus on those metabolites, that are excreted unconjugated or as glucuronides into the urine. Extraction of the gluco- deconjugated steroids from the matrix and concentration of the analytes is performed by liquid-liquid extraction (with diethylether or tertbutylmethylether) or solid phase extraction followed by mass spectrometric detection either by liquid chromatography mass spectrometry or gas chromatography mass spectrometry. By using this initial screening extraction protocol the Antidoping Laboratories substantially ignore the sulfo-conjugated part of AAS. Nevertheless there are AAS for which the longer detected metabolite is a sulfo-conjugate. Sulfo-conjugated AAS are relatively easy to be detected directly since the development of instrumentation providing interfacing of liquid chromatographic (LC) separation to mass spectrometric (MS) detection, especially via electrospray ionisation (ESI), has opened up broad possibilities for the direct analysis of sulfo-conjugated substances. Moreover, sulfo-conjugated substances can be extracted from urine using ethyl acetate, instead of diethylether, as extraction solvent. The objectives of this project will be:

a) To develop a screening method for the already known sulfo-conjugated metabolites of AAS, and

b) To investigate the existence of not yet reported sulfo-conjugated metabolites of AAS, that can improve detectability and identification in either initial screening protocol or confirmation methods.

Main Findings

Anabolic steroids such as oxandrolone, madol, formebolone, methenolone, 17- methylnandrolone and mesterolone were tested for the existence of sulfo-conjugated metabolites. Metabolic samples from long-term excretion studies were tested for any sulphate metabolite and where any sulphate metabolite was found, an evaluation of its retrospectivity was performed in comparison with their free and gluco-conugated metabolites used for their monitoring in GC/HRMS analysis. In most cases where new metabolites were found, a detailed characterization of their structures based on mass spectrometry techniques was also performed. Additionally, spotted metabolic samples for oxymetholone, drostanolone, norethandrolone, danazol, clostebol, methandriol, calusterone, furazabol, fluoxymesterone, oxymesterone, boldenone, mesterolone, methandienone, methyltestosterone, oral turinabol, methenolone, and tibolone that include the known, up to that time, anabolic steroids with sulphates metabolites, as well as other AASs with unknown sulphate metabolism were tested in order to develop a new screening method for sulphate metabolites. Samples from the above listed steroids were extracted and analyzed using a screening method based on alkaline extraction with ethylacetate and LC/QTOF analysis in a negative mode. Potential sulfate metabolites of these steroids were drawn and the molecular ions were calculated and extracted using the instrument software.

Conclusions

The investigation of sulfo-conjugated metabolites of methenolone and mesterolone led to the discovery of new metabolites of at least equal or better retrospectivity compared to the already known gluco-conjugated metabolites detected by GCMS. Furthermore, a sulfo-conjugated long-term metabolite of 17-methylnandrolone was discovered (unpublished results). The analysis of madol, formebolone and oxandrolone didn’t lead to any new (sulfo-) metabolite, at least to a concentration level that would be detectable by the technology used in this study. A screening method for sulfo-conjugated metabolites was developed for several anabolic steroids based on literature data, using accurate mass measurements for the extracted ion chromatograms and leading to a number of new sulfo-conjugated metabolites. Their structures, as well as their retrospectivity for the monitoring of their parent compounds abuse for doping control purposes were not evaluated in the framework of this project.

Code: 11D13MG

Performance-enhancing aids are not new to athletes. Owing to fame, honor and economic benefits arising from success in competitions, there is rather a long history of cheating in sports.  After the usage of erythropoietin could effectively been proven, lots of athletes turned to an older doping procedure: autologous blood doping to increase the number of circulating red blood cells (RBCs). This increases the oxygen delivery through the blood to the working muscle which profoundly enhances an athletes´ performance. The International Olympic Committee (IOC) forbade blood boosting after the 1984 Olympics, despite the fact that no methods had been devised for unequivocal detection. Valid and established methods for the detection of autologous blood doping are still missing but our recent findings indicate that the RBC flexibility represents a useful marker to detect blood doping. The deformability is an essential feature enabling RBCs to access and transit the capillary bed and to deliver oxygen to the muscle cells due to the fact that the vessel diameter of the capillary is smaller than the resting cell diameter. 
The aim of the presented study is the investigation of RBCs flexibility in male and female endurance athletes and male and female non-athletes during a one-year period. Thereby, a variety of parameters, known to influence RBC deformability like L-arginine and NO levels, are planned to be investigated. Also, the influence of the female hormone cycle on the RBC deformability should be investigated. Furthermore, the influence of competition and training periods and the influence of the ethnic background on RBCs flexibility will be evaluated. The findings obtained with this study will serve as a basis for further investigations and the development of a routinely applicable detection method to detect autologous blood doping.

Main Findings:

Autologous blood doping is the practice of boosting the number of red blood cells (RBC) in order to improve aerobic capacity and thus athletic performance and endurance. RBC deformability, the ability of RBC to change their shape under a given level of applied shear stress, is an important cell property to ensure oxygen and nutrient supply to the working tissue in the microcirculation. Thus, RBC deformability may also determine performance capacity. Hypothermic blood storage decreases RBC deformability, thus, monitoring RBC deformability of elite athletes during the course of the year may be suitable to detect for autologous blood doping.  
But first it was necessary to test for factors that possibly influence deformability in endurance athletes. Among these were age, ethnicity, gender, estrogen levels and training volume.  
The major results revealed increasing RBC deformability from pre-puberty to adulthood. Further, female athletes who used hormonal contraceptives and who thus showed reduced estrogen levels showed reduced RBC deformability compared to female athletes without hormonal contraception (-HC). An acute response of RBC deformability to increasing estrogen levels was not detected concluding a chronic impact of estrogen on RBC deformability. Both, male and female athletes (-HC) showed higher deformability values compared to respective non-athlete control groups, indicating a correlation of endurance exercise and RBC deformability. RBC deformability did not differ between male athletes and female athletes – HC but deformability values were significantly higher in male athletes compared to female athletes + HC. Female Asian and black athletes (all – HC) showed reduced RBC deformability compared to white female athletes – HC. Also, male Asian and black athletes showed reduced RBC deformability compared to white male athletes.  
The recent data indicated that RBC deformability is influenced by some variables such as gender or ethnicity. According to the recent data it is suggested to monitor individual RBC deformability values longitudinally to detect abnormalities possibly occurring after autologous blood doping.

Code: 11C9RS

Aim 1: Assay optimization

We will evaluate a combined assay format that is based on real-time PCR and ITC readout with a nested PCR pre-step to improve sensitivity. The current single-step qPCR assay has a sensitivity of 3 copies for the macaque Epo cDNA in the presence of 500ng - 1 OOOng macaque gDNA. A qPCR assay has been developed to the homologous locus in the human Epo cDNA with a sensitivity of 10 copies in 500ng human gDNA. A nested PCR assay has been developed for the human Epo cDNA with a sensitivity of 10 copies in 1250ng of human DNA. We will evaluate the enhanced sensitivity of nested PCR combined with the automation and rapid results of qPCR with the aim of reaching a sensitivity of less than 5 copies in 500ng genomic DNA. We will also examine the feasibility of utilizing the ITC assay format with the nested+qPCR approach. Once the assay is developed and tested using plasmids and virions spiked into gDNA and human blood, we will evaluate samples obtained directly from transduced macaques, and macaque PBLs spiked into na"fve human blood. The SV40 polyA site, which is not present in the human genome, but will be present in the rAAV vector used here, is an ideal target since there is no competition with endogenous sequences. The SV40 assay was previously developed [1Jfor the macaque studies and is considered to have the best sensitivity; it will serve as a reference for optimizing the hEPO Exon3-4 assay. Controls (ITC format) will be developed for each assay.

Aim 2: Assay validation

New or revised clinical diagnostic methods must be validated: defined as the process by which it is established, by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical application. Components of validation involve quality control (QC) and quality assurance (QA). We will perform analytical validation of the human Epo assay to ensure specificity, sensitivity, and ruggedness (multiple labs, multiple operators, repeats and replicates). This will involve drafting test records, along with assay specifications, reagent preparation logs, and a validation plan, and executing the assay(s) several times with several replicates in Florida and Mainz. Data will be captured and evaluated statistically to determine the assay performance criteria. For pre-clinical performance validation, we will evaluate spiked (using plasmid DNA) human blood samples with respect to: sex, age, race, medical history (if available), exercise status, general health, and prescription drugs (if known). The rate of false positives and false negatives will be established by testing a statistically significant number of samples. We will screen up to 100 blood samples (50 for each lab in Florida and Mainz) for false positives in order to get an idea of the specificity of our procedures. The Simon Lab will contribute 100 human samples (Germans), and if available, substitute some of the German samples with samples of other ethnicities.

The assay validation activities support the documentation for eventual regulatory submission and eventual manufacture of an in vitro diagnostic test (IVD) kit. Ultimately, data from the assay development, analytical validation, and pre-clinical performance validation studies will be documented along with a description of each kit (with all required components) in a form suitable for regulatory submission.

As for regulatory capability and expertise, in addition to his faculty appointment and research activities described in this proposal conducted in the Department of Molecular Genetics and Microbiology, Dr. Snyder is the Director of the Center of Excellence for Regenerative Health Biotechnology at University of Florida (UF CERHB, http://www.cerhb.ufl.edu/ ). Established in 2003, the UF CERHB is committed to translational research in many different biotechnological fields including viral vectors for gene therapy. UF CERHB's biopharmaceutical development operation, offers a wide range of cGMP-compliant biopharmaceutical manufacturing and testing services to the biotechnology industry and to biomedical research institutions. Furthermore, Dr. Moullier runs a similar operation in Nantes called ATLANTIC BIO GMP (ABG). Resources are available (on a fee-for-service basis) at UF CERHB and ABG, Nantes for anti-doping PCR assay kit manufacturing.

Main Findings

It is crucial to develop reliable test methods for the detection of gene doping, given that some members of the sports community are seeking for gene transfer technology to improve exercise performance. Based on previous work we established a ´nested real-time PCR´ assay. This is an assay based on the PCR technology which is used in criminology and well-known in the court of law for the identification of genetic material in samples from sites of crime. We adapted this technique to detect single erythropoietin (EPO) DNA molecules that had been introduced into the body by means of gene transfer. Due to its high sensitivity, our technique is principally able to detect genetic modifications of certain parts of the body in conventional blood samples. Here we aimed at providing evidence of the high sensitivity and specificity of our refined procedure that now enables quantification of the artificial DNA in the blood stream.

Through an inter-laboratory study between Nantes (France), Gainesville (Florida, USA), and Mainz (Germany) two nested real-time assays were validated and utilized for an in vitro blinded study. Both assays showed unique sensitivity and specificity in a large dynamic range. According to the nested assay protocol the detection of ~1 copy of circular transgene molecule in a background of 500 ng gDNA is possible reliably. In a blinded in-vitro study the reliability of the assays to detect between 1000 – 2 copies of transgene molecules and non-template control was proven.

To validate the suitability of the nested real-time assays for the detection of gene doping a non-human primate study was conducted. Two macaques were injected intramuscularly with a recombinant adeno-associated virus serotype 8 (rAAV8) vector harboring human EPO cDNA sequence. One macaque served as non-injected control. The vector was promoterless to avoid transgene expression. Following the injection of 2.5 x 1011 viral genomes/kg, the transgene molecules were detectable 8-14 weeks.

The high sensitivity of the nested qPCR assay along with the specificity for transgene detection is essential for gene doping surveillance. The assay can be adapted to other gene doping candidate genes.

Code: 11B1JB

Operational Technologies Corp. (OpTech) has already demonstrated and published (BrunoJ.G., et al. J. Biomolec. Techniques, In Press, 2011) the ability to develop aptamers (surrogate antibodies composed of DNA) which can discriminate synthetic (recombinant) from natural human growth hormone (hGH) with initial funding from WADA. The aptamers developed by OpTech detect minor amino acid modifications and differences added by E. coli host cells as noted by Hepner et al. (2005 and 2006) to research grade synthetic hGH. Under this follow on grant proposal, OpTech will develop a new set of discriminatory aptamers against one or more pharmaceutical grade recombinant hGH targets (Genotropin, Norditropin, etc.). OpTech has recently demonstrated ultrasensitive (sub-picogram/ml) detection of hGH with some of its original hGH aptamers in an aptamer-magnetic bead sandwich electrochemiluminescence (ECL) format. ECL is already used by Roche Diagnostics for ultrasensitive detection of numerous clinical analytes. Therefore, OpTech will develop several combinations of capture and reporter aptamers to detect pharmaceutical hGH at sub-pg levels. Finally, because ECL is a laboratory-bound technique, OpTech will investigate the potential for presumptive field testing for synthetic hGH in aptamer-based lateral flow test strips using blood or urine samples for preliminary screening of athletes. To enhance sensitivity, OpTech will explore the use of quantum dots (QDs) and fluorescent nanoparticles (FNPs) along with a UV penlight to enhance visual detection of synthetic hGH in lateral flow test strips. Disposable low pressure size-exclusion chromatography columns may be needed to remove creatinine and urea in serum and urine prior to aptamer-based assays. These substances appear to denature aptamer secondary and tertiary structures which are critical to proper folding and binding of aptamers to hGH and other targets. This is anticipated to be a two year project. 

Main Findings: 

Operational Technologies Corp. (OpTech) was unable to obtain Genotropin or Norditropin from Pfizer, Novo Nordisk, or other sources.  However, OpTech developed DNA aptamers against the oxidized peptide regions of Genotropin defined by Hepner et al. (2005 and 2006) and thought to be potential sites for discrimination of recombinant vs. natural hGH in ~ 2% of Genotropin molecules. OpTech screened these aptamers against research-grade rhGH and natural pituitary hGH and identified several candidate aptamers (e.g., Hep 5-6R, Hep 6-6R and Hep 6-12R) which may be able to discriminate Genotropin from natural hGH.  However, without access to authentic Genotropin, OpTech could not test this hypothesis fully.  Therefore, OpTech elected to use the remaining funds to explore new and potentially even better approaches to detecting hGH abuse in athletes with aptamer-based assays.  These new approaches were: 1) Development of aptamers to bind human IGF-1 and PIIINP which are both long-lived serum biomarkers of rhGh use.  This work resulted in development of a preliminary enzyme-linked IGF-1 aptamer-magnetic bead sandwich assay which functioned in pure human serum with a lower limit of detection of ~ 30 ng/mL and linear detection to > 1,000 ng/mL.  The top-ranked IGF-1 aptamer candidates (3F and 25R) may bind a region of IGF-1 that is accessible even when IGF-1 is bound by its binding proteins in serum.  Unfortunately, these IGF-1 aptamers also demonstrated significant cross-reactivity with Brain Natriuretic Peptide (BNP), bone collagen Helical Peptide (HP) and rhGH, but much lower affinity for C-telopeptide (CTx) or N-telopeptide (NTx) of bone collagen.  Preliminary data also indicated that development of a PIIINP aptamer assay with linear quantification in the high to mid-ng/mL range was possible using at least 3 different aptamer candidates identified during ELISA-like screening (ELASA).
2) Development of aptamers against specific peptide regions of the 20kD and 22kD isoforms of hGH to emulate the current ratiometric immunoassay.  OpTech developed aptamers against the 15-amino acid region (EEAYIPKEQKYSFLQ) of 22kD hGH which is not present in 20 kD hGH and also developed aptamers against the fused flanking regions (DTYQEFNPQTSL) resulting from alternative splicing to enable discrimination of unique regions on each isoform of hGH.  Two lead candidates designated “Splice 20-14R and Splice 22-5F” were identified for potential future ratiometric assay development.Both approaches appear promising and, if fully developed, would have the advantage of overcoming lot-to-lot variability associated with antibodies and immunoassays, because aptamer DNA synthesis from known sequences is a very high fidelity process.

Code: 11C12AB 

The anti-doping community is focusing efforts on developing methods to detect gene doping, a new threat to the world of sport. 
Methodology with potential to detect ‘doping’ genes in blood samples from athletes has recently been developed by us and other researchers. It involves PCR assays targeting intronless cDNA sequences that are present in doping genes but absent in intron-containing endogenous genes.  Acceptance of this methodology for routine gene doping testing requires reference materials as controls to ensure the method performs as intended. A positive control containing the cDNA sequence for the candidate doping gene is commonly used in method development. Inadvertent cross-contamination of a tested sample with this type of control would lead to a false positive test result. Hence, it is not suitable for routine testing because of legal implications. 
We propose to develop a DNA reference material for a positive control in gene doping testing that will overcome this problem. Using erythropoietin as a model doping gene, we will produce a reference material that will be detectable by the PCR assays with similar specificity and sensitivity as the doping gene. However, the reference material and gene doping products will differ, allowing easy discrimination between true positive and false positive test results. We will characterise this reference material for purity and quantity using latest technologies including digital PCR and next generation sequencing. In addition, using this reference material we will in vitro validate the complete erythropoietin gene doping detection method from sample processing to PCR detection. 
This model system will serve as a prototype for preparing reference materials for detecting doping with other candidate genes. 
The proposed research is crucial in the development of a routine method for direct detection of gene doping and for ensuring the method is reliable, reproducible and robust and will withstand legal scrutiny. 

Main Findings: 

The anti-doping community is focusing efforts on developing methods to detect gene doping, a new threat to the world of sport. Recently, methodology with potential to detect ‘doping’ genes in blood samples from athletes has been developed by us and other researchers. 
Acceptance and implementation of this methodology for routine gene doping testing requires Reference Material (RM) as a control to ensure the method performs as intended. Existing ‘in-house’ controls based on complementary DNA (cDNA) for targeted genes will not withstand legal scrutiny since any cross-contamination will generate ‘false positive’ test results. 
In this project, we generated and characterised a DNA RM that will overcome this problem. Using EPO as a model doping gene, we produced three forms of the RM. Each form is detectable by the PCR assays that target a doping EPO gene. However, the products generated in these assays from the RM and a doping gene differ, allowing easy discrimination between true positive and ‘false positive’ test results. We characterised these three forms of the RM for purity, quantity and stability, and studied their performance in gene doping detection assays. Finally, the three forms were in vitro validated in the complete EPO gene doping detection method from sample processing to PCR detection. 
This model system will serve as a prototype for preparing RM for detecting doping with other candidate genes. The produced RM is crucial in the development and acceptance of a routine test for direct detection of gene doping and for ensuring the method is reliable, reproducible and robust and will withstand legal scrutiny.

Code: 11A17PV

Anabolic steroids are the most frequently detected prohibited substances in sports. Analysis for these substances is routinely performed using gas chromatography-mass spectrometry (GCMS) and liquid chromatography mass spectrometry (LC-MS). Although the detection capabilities of LC-MS are limited for the detection of steroids with androstanediol as a back bone to their structure, LC-MS is often more sensitive for many other steroids. Moreover, recently several publications have shown that LC-MS can detect steroids (glucuronides) directly in urine without any sample preparation.

The advantages for such a direct urinalysis are multi-fold. It reduces costs, required volume of sample and analysis time, but also it allows for the direct detection of intact conjugated steroids rather than the liberated steroid molecules after hydrolysis.

In the past several small scale attempts have been made to detect these intact steroid conjugates in urine. These attempts were quite successful, but did not meet WADA’s minimum required performance limits. Recently however several scientific improvements have been made, including more sensitive instruments, optimized mobile phase compositions (e.g. methanol
instead of acetonitrile), which now allow for the detection of several steroids well below WADA’s MRPL. These preliminary results (e.g. 19-norandrosterone glucuronide) show that the direct urinalysis of steroid conjugates at concentrations at or below the MRPL is achievable.

Main Findings: 

The aim of this project was to study intact conjugated anabolic steroids in urine in comparison with the traditional methods where the aglycones are detected after enzymatic hydrolysis. This approach permitted the detection of anabolic steroids misuse by liquid chromatography mass spectrometer since the glucuronide and sulfate groups are easy to ionize by ESI. Consequently, steroids with a sulphated or glucuronidated androstane framework could be detected. Research resulted in the development of a multi-target analytical method for the screening of different exogenous anabolic steroids and also for the quantification of endogenous anabolic steroids.

The use of a dilute-and-shoot strategy allowed to minimize sample preparation yielding a short turnaround time. Additionally a small volume of urine can be used. This is a great advantage compared with traditional methodologies where liquid-liquid extraction or solid phase extraction after enzymatic hydrolysis and detection by LC-MS or GC-MS (after derivatisation step) is applied.

The use of excretion studies or synthesis in vitro are of great interest since the number of conjugated anabolic steroids commercially available is still very low. With this methodology new conjugated anabolic steroids were detected and included in the multitarget method.

Code: 11D7MD 

This project, which constitutes a follow-up from two previous works funded by WADA aims at widening our understanding of the effects of glucocorticoids (GC) on muscle function, exercise performance, postural control and health risks. Indeed, we demonstrated in our previous works that short-term but not acute GC administration improves performance during endurance exercise lasting at least 40 minutes, without any gender effect. However, no study, to our knowledge, has focused on the ergogenic effects of GC during repeated bouts of high-intensity exercises. In the same way, whether the numerous deleterious effects of chronic (>1 month) GC intake are well known, there is no consensus on health risks after short-term (1 week) systemic GC intake. 
We therefore propose to study whether short-term GC administration would improve performance during repeated bouts of high-intensity exercise. In parallel, we will investigate with complementary methods the repercussions of GC administration on biomechanical (especially the magnitude of the braking of the fall during the swing phase), and physiological parameters (cardio-respiratory, muscular and inflammatory) in order to assess both GC ergogenic effects and health risks induced by short-term high dosage treatment.

Main Findings: 

In the project “Effects of glucocorticoid during repeated bouts of high-intensity exercise” we tested the ergogenic effects and health risks of short-term GC administration. Physiological and biomechanical parameters were analyzed. Several results can be highlighted: 
- No ergogenic effect of short-term systemic GC (i.e. 60 mg prednisone/day/7 days) was found during repeated bouts of brief intense exercise. Muscle oxygenation parameters measured with the near infrared spectroscopy technique were not altered by short-term GC administration at rest and during repeated bouts of high-intensity exercise. These suggest that GC treatment had no effect on the blood oxygen transport capacity and on the muscle oxygen extraction during exercise. The unaltered oxygen availability at the muscle level seems to be in accordance with the lack of improvement in performance during the last sequence of hopping with the GC treatment.  
- Regarding hormone responses during exercise, while no change was found in saliva testosterone, GC intake induced changes in saliva cortisol and DHEA concentrations. In parallel, blood IL-6 and IL-10 were altered at rest and during the exercise performed. It is interesting to note that the anti-inflammatory effects of prednisone were maximal and stable from the beginning of treatment, in the resting and exercise conditions. However, hormonal concentrations continued to decline during short-term intake. Lastly, our study shows that short-term prednisone treatment affects the circadian pattern of saliva DHEA but not testosterone in our recreationally trained subjects.  
- Regarding baroreflex sensitivity (BRS), heart rate (HR) and systolic blood pressure (SBP) variability (HRV, SBPV) short-term GC intake did not change SBP. However, a low frequency (LF) SBPV increase associated with a LF-BRS decrease and a HR increase was observed, indicating a sympathetic cardiovascular stimulus.  
- Regarding EMG activities in the ankle extensors EMG during single leg hopping, and balance control during single stance of the first step in gait initiation, no GC effect was noted compared to placebo.

Code: 11A3JP 

Recent advances in liquid chromatography–mass spectrometry (LC-MS) have opened the possibility of converting many of the doping control analysis methods to LC-MS platforms. This would increase flexibility and selectivity for many analyses, while obviating the need for derivatization in most cases. A limitation to this switch is the established sample preparation strategies required for the numerous doping control methods. Developed for GC-MS analysis, they are time consuming and not always appropriate for LC-MS analysis. While the LC-MS technology holds the promise of providing a universal screening tool, the varied requirements for sample preparation for the numerous tests limit this potential. Professor Pawliszyn's group has long recognized the bottle-neck that sample preparation presents for the analytical laboratory, and have made significant strides in developing solid phase microextraction (SPME) technology to simplify sample preparation for both GC-MS and LC-MS, while maintaining sensitivity and enhancing options for automation.  
In this project we will evaluate automated SPME technology as a universal sample preparation strategy, coupled with LC-MS as a universal tool for identification and quantification of prohibited substances. We will develop validated methods for urine analysis for a range of prohibited substances from the S1, S3, S4, S5, S6, S7, S8, S9 and P2 categories. We will apply enzymatic deconjugation of phase II drug metabolites as required and investigate guidelines for confirmatory analysis. SPME technology is also readily applicable to plasma and whole blood analysis and has significant advantages relative to SPE. We will evaluate the performance of the system in these matrices as well. At the conclusion of the project we will have sufficient data to evaluate the suitability of automated SPME coupled with LC-MS as a universal tool in prohibited substance screening. If judged suitable, it will be immediately applicable for introduction to the screening program in WADA accredited facilities. 

Main Findings: 

• The potential of automated solid phase microextraction (SPME) in thin film geometry as simple and convenient sample preparation approach for urine, plasma and blood analysis was demonstrated.
• SPME conditions for each of the studied matrices were carefully optimized. The effect of experimental parameters such as pH, ionic strength, centrifugation and temperature was investigated.
• Protocols for the multiclass analysis of 110 and 25 prohibited substances in urine and plasma, respectively, were developed and validated.
• In the case of urine, 100 out of 110 compounds showed R2 above 0.991, intra and inter day precision was below 20 % in most cases, and in terms of accuracy only 6 compounds exhibited more than 20 % deviation from their nominal concentration value. Regarding LOQ, only 15 out of 110 compounds did not meet the MRPL values stipulated by WADA; however, as presented in the final report, by using a more sensitive mass spectrometry analyzer WADA requirements can be fulfilled.
• In the method developed for plasma analysis, satisfactory results in terms of linearity (R2> 0.99), inter and intra-day accuracy (85 – 130 %), precision (<20 %) and limits of quantitation (0.25 – 10 ng/mL for most compounds) were found.
• The suitability of the automated system to incorporate a hydrolysis step before SPME extraction was also verified.
• The most important advantages of SPME in doping controls can be listed as follows:
1. Minimum sample handling before SPME extraction. No need of centrifugation or protein precipitation (no clogging issues as in SPE).
2. High-throughput analysis. The automated 96 Concept system allows simultaneous preparation of 96 samples.
3. Efficient sample clean-up. Elimination or reduction of possible ionization suppression or enhancement effects.
4. No breakthrough volume issues. The open bed geometry of SPME allows simultaneous quantitation of a wide range of substances without concerning about extraction phase saturation.