Projets de recherche

Code: 14A18XD 

The detection of the exogenous administration of synthetic androgens (the so called “pseudo-endogenous” steroids) having the same chemical structure of the compounds produced endogenously (i.e testosterone, 5α-dihydrotestosterone or androstenedione) is primarily based on the alterations of the urinary endogenous steroid profiles. 
A Bayesian approach and adaptive model has been adopted by WADA for the management of the steroid profiles and all the parameters obtained by the Accredited Laboratories will be collected starting 1st January 2014 in a global database integrated in the Athletes Biological Passport (ABP), permitting to establish the individual reference ranges of every athlete. 
The detection capacity of the steroid profile can be improved by the inclusion of the urinary hydroxylated androgen metabolites excreted in lower amounts but whose diagnostic values become more significant after the administration of endogenous steroids. The additional inclusion of the isotope ratio mass spectrometric (IRMS) data has demonstrated to improve the statistical discrimination between basal samples and samples obtained after controlled administration of testosterone. 
Finally, the use of IRMS is a very powerful tool for the detection of pseudoendogenous steroids if the starting material for their synthesis has a 13C composition different from the endogenously produced molecule. The detection of formulations able to circumvent their detection by IRMS already occurred since the criteria are based on population data and not on athlete’s previous data. 
The main goal of this project is to establish the long term variability of the IRMS delta values of both target (TC) and endogenous reference compounds (ERC), and of the steroid profile parameters including hydroxylated androgens metabolites in order to improve the evaluation of the longitudinal profiles. This should permit to enlarge the detection capacity and to potentially detect the abuse of preparation.

Main Findings: 

There is a definite need of finding more efficient ways to detect the exogenous administration of pseudoendogenous steroids. Although the implementation of the ABP has been a clear step forward, there is still a gap between our capacity to suspect and to confirm the abuse of these substances. The use or more specific markers of the urinary steroid profile like some hydroxylated steroids has been investigated, although their implementation is not easy and would require big efforts to harmonize their detection and quantification as was done for the current markers of the ABP.
The potential inclusion of the IRMS data in the ABP as a direct evidence of doping by pseudoendogenous steroids has been evaluated. As for the ABP markers, instead of comparing the obtained data with reference population ranges (present approach as a confirmation strategy), we suggest to incorporate these data to the ABP and to evaluate the values to the reference data produced by every single athlete.

To reach this goal, the first step was to investigate the stability of the IRMS data in both healthy volunteers and athletes. 

The variability of the IRMS data in a short, medium and long term period in 8 males and 6 females volunteers when compared to data of athletes submitted to investigations by the respective NADO due to atypical steroid profiles, showed that the variability of the individuals’ absolute delta values of the parameters studied are at least one half lower than the population ones and  much lower than the markers of the steroid module of the ABP. The data obtained from real samples of athletes showing atypical steroid profiles, show a variability comparable to the non-athletes volunteers, demonstrating that sports practice and the use dietary supplements do not influence the delta values of the endogenously produce steroids. These IRMS values depend mainly on the individuals’ diet and metabolism. 

This would allow defining individual reference ranges much narrow than the currently applied ones. This would permit (1) to extend the detection window of the pseudoendogenous administration and (2) to potentially detect the use of steroids from pharmaceutical preparations showing delta values close to the endogenous values.

The results of the present project, suggest that the IRMS data obtained with the procedures already in place in the WADA accredited Laboratories, can be used in a more performing way. If the data instead of being only used during the confirmation evaluation of an atypical steroid profile would be evaluated longitudinally, the information obtained would permit to enlarge the detection window of pseudoendogenous steroids abuse and the potential detection of pharmaceutical preparations showing delta values in the endogenous region.
We are not proposing the implementation of a new method but the better exploitation of the data already obtained. By doing so, the gap between the ability of detecting suspicious steroid profiles and the capacity of confirming them by IRMS will be drastically reduced.
There is a definite need of finding more efficient ways to detect the exogenous administration of pseudoendogenous steroids. Although the implementation of the ABP has been a clear step forward, there is still a gap between our capacity to suspect and to confirm the abuse of these substances. The use or more specific markers of the urinary steroid profile like some hydroxylated steroids has been investigated, although their implementation is not easy and would require big efforts to harmonize their detection and quantification as was done for the current markers of the ABP.
The potential inclusion of the IRMS data in the ABP as a direct evidence of doping by pseudoendogenous steroids has been evaluated. As for the ABP markers, instead of comparing the obtained data with reference population ranges (present approach as a confirmation strategy), we suggest to incorporate these data to the ABP and to evaluate the values to the reference data produced by every single athlete.

To reach this goal, the first step was to investigate the stability of the IRMS data in both healthy volunteers and athletes. 

The variability of the IRMS data in a short, medium and long term period in 8 males and 6 females volunteers when compared to data of athletes submitted to investigations by the respective NADO due to atypical steroid profiles, showed that the variability of the individuals’ absolute delta values of the parameters studied are at least one half lower than the population ones and  much lower than the markers of the steroid module of the ABP. The data obtained from real samples of athletes showing atypical steroid profiles, show a variability comparable to the non-athletes volunteers, demonstrating that sports practice and the use dietary supplements do not influence the delta values of the endogenously produce steroids. These IRMS values depend mainly on the individuals’ diet and metabolism. 

This would allow defining individual reference ranges much narrow than the currently applied ones. This would permit (1) to extend the detection window of the pseudoendogenous administration and (2) to potentially detect the use of steroids from pharmaceutical preparations showing delta values close to the endogenous values.

The results of the present project, suggest that the IRMS data obtained with the procedures already in place in the WADA accredited Laboratories, can be used in a more performing way. If the data instead of being only used during the confirmation evaluation of an atypical steroid profile would be evaluated longitudinally, the information obtained would permit to enlarge the detection window of pseudoendogenous steroids abuse and the potential detection of pharmaceutical preparations showing delta values in the endogenous region.
We are not proposing the implementation of a new method but the better exploitation of the data already obtained. By doing so, the gap between the ability of detecting suspicious steroid profiles and the capacity of confirming them by IRMS will be drastically reduced.

Code: 14B14ZW

Detection of insulin or insulin analogue doping is still a challenge for doping analysis laboratories. Until now no screening method is available providing the fast and reliable detection of doping with insulin analogue. The insulin analogues are modified insulin preparations to show faster acting or long acting effects compared to regular insulin, which are therefore broadly used in clinical therapy for diabetes and might also be intensively misused in sport and body building. The insulin analogues have amino acid sequences different to regular insulin. Such difference could be recognized by specially selected monoclonal antibodies, which have their binding site or epitopes involved in the positions where amino acid sequences are altered in insulin analogues.  
In this project, we will at first generate and select two monoclonal antibodies which bind all insulin analogues significantly differently. Thereafter a differential immunoassay based on immune-PCR Imperacer Technologie from Chimera Biotech will be constructed with these 2 antibodies. The immune-PCR is the ultra sensitive immunoassay using antibody-DNA conjugate. The DNA fragment can specifically amplify millions fold and the signal will be directly read out in a real-time PCR machine.  An insulin analogue is detected if the insulin concentrations determined by the two assays are significantly differently. This ultrasensitive real-time immune-PCR duplex assay could directly use small amount of urine samples for doping control. Furthermore, efforts will be made to produce specific monoclonal antibodies to each insulin analogue. Each monoclonal antibody will only bind one insulin analogue but not regular insulin or other analogues. 
Differential immunoassays, especially multiplex immune-PCR constructed with these antibodies will be able to identify quickly and unequivocally which insulin analogues are misused. 

Main Findings: 

In this research project, high affinity anti-insulin monoclonal antibodies (mAbs) including several regular insulin specific mAbs and glargine specific mAbs were produced and they were carefully examined for their binding to insulin and insulin analogues. More than 30 best hybridomas were chosen to be cloned with limited dilution. The antibodies were produced in protein free medium, purified with affinity column and biotinylated. The mAb pairs for the sandwich immunoassays were identified through examining more than 900 antibody combinations. A permissive insulin sandwich assay using mAb 5E10 and a regular insulin preferential assay using mAb 7F3 were constructed. A glargine specific sandwich assay with mAb 6E8 was also established. These three assays are ultra-sensitive (limit of detection <1 pg/ml) and together they can identify any insulin analogue in urine and serum directly with only a few hundred microliters of samples. In a pilot study, these assays showed to be able to identify low amount of insulin analogues in the urine samples of the diabetic patients treated insulin analogues alone or combined with regular insulin preparations. 

Furthermore, a regular insulin specific sandwich assay was also constructed with mAb 1B7. This assay together with the permissive insulin assay can identify insulin analogues, bovine and porcine insulin. Due to its high specificity but moderate sensitivity, this assay would be more suitable for the confirmation assay. Additionally, a universal ultrasensitive Immuno-PCR assay format using real-time PCR cycler was constructed, which can easily be converted to the ultra-sensitive duplex assays

Code: 14A21RN 

Ultra-high performance supercritical fluid chromatography-mass spectrometry (UHPSFC-MS) could represent in the near future an orthogonal technique to LC-MS(/MS) and GC-MS(/MS) for routine doping analysis. 
UHPSFC is a normal phase separation technique that uses carbon dioxide as mobile phase, with the addition of small amounts of methanol as co-solvent. This technology is considered to be a green approach. Using appropriate stationary phase chemistries and dimensions, different pharmaceutical compounds (non-polar and polar) have already been analyzed. However, up to date, only few applications of this technique for the analysis of compounds in biological fluids (urine and blood) exist in scientific literature. Furthermore, no studies are available for the analysis of forbidden substances in anti-doping area. 
In this study, the performance of UHPSFC-MS(/MS) will be evaluated for the analysis of different classes of forbidden substances present in the prohibited list, such as exogenous and endogenous steroids, stimulants, diuretics, narcotics, corticoids, small peptides, etc.  Particular attention will be focused to endogenous and exogenous steroids in urine and blood samples as well as to the optimization of analytical conditions (separation and detection) for both screening and confirmation purposes. Results will be evaluated in terms of selectivity, matrix effect, sensitivity and limits of detection in both biological matrices. Benefits and drawbacks of this technique will be discussed and compared to conventional LC-MS(/MS) and GC-MS(/MS) analyses. An improvement of detection capabilities for different forbidden substances hardly detectable by conventional techniques could be expected.

Main Findings: 

Today, LC-MS(/MS) and GC-MS(/MS) remain the most widely used analytical strategies for doping control analysis. The goal of this project was to evaluate the potential of an alternative chromatographic strategy, namely SFC-MS/MS, for the determination of a wide range of doping agents present in the WADA prohibited list. For this purpose, a UHPSFC-MS/MS screening method was developed for 110 stimulants, diuretics and narcotics in urine samples. Then, the same analytical platform was used for the screening of about 100 analytes belonging to the difficult classes of anabolic agents, hormones and metabolic modulators, synthetic cannabinoids and glucocorticoids in urine samples. 
From these two large screening methods, we were able to demonstrate that SFC-MS/MS was extremely reliable and could be considered as an ideal alternative to the widely used LC-MS(/MS) and GC-MS(/MS) platforms for the determination of diverse substances in biological matrices. In this project, the following benefits were noticed in SFC-MS/MS: 1. The retention of SFC is mostly based on polar interactions with the stationary phases (i.e. dipole-dipole and hydrogen bonding), which is the opposite of RPLC, where the retention is driven by hydrophobic interactions. Then, some polar doping agents present in the WADA prohibited list (e.g. methylecgonine, octopamine, oxilofrine, synephrine, phenylephrine…) were sufficiently retained in SFC, while it was not the case in RPLC under generic screening conditions.
2. Because the retention mechanisms in SFC and RPLC are orthogonal, some problematic coelutions in RPLC can be “easily” resolved in SFC and vice-versa. This feature was particularly useful for the determination of anabolic agents. In this particular class of compounds, there were indeed a significant number of isobaric substances that deserve special chromatographic attention, since they cannot be resolved by MS/MS detection.
3. In SFC-MS/MS, and contrary to what happen for GC-MS(/MS), the urine samples can be directly analyzed, without the need for a chemical derivatization strategy. Therefore, the sample treatment was simplified and the analytical throughput was strongly enhanced in SFC vs. GC. In view of the workload undergone by a doping control analysis laboratory, such a rapid procedure could be of great value.
4. The instrumental sensitivities achieved in SFC-MS/MS were adequate for screening purposes. Indeed, the LOD achieved were systematically below the MRPLs fixed by the WADA. For the 100 anabolic agents, hormones and metabolic modulators, synthetic cannabinoids and glucocorticoids, there were only one metabolite of each of these substances (norethisterone, turinabol and norbolethone) that did not meet the MRPL criteria among the 100 tested compounds. Through this project, we demonstrated that the sensitivities achieved in SFCMS/MS were comparable and often improved with regard to RPLC-MS/MS and GC-MS/MS, due to the very limited amount of water in the SFC mobile phase.
5. The susceptibility of the developed SFC-MS/MS methods towards matrix effects was evaluated at different concentration levels and the obtained results were compared to that of LC-MS/MS and GC-MS/MS (in some cases). The matrix effects were very low in UHPSFCMS/ MS, and slightly higher in UHPLC-MS/MS, and unacceptable in GC-MS/MS, due to “matrix induced chromatographic response enhancement”, that could jeopardize the reliability of the method. hanks to this project, UHPSFC was for the first time proven to be applicable for the screening of doping agents in urine samples. The application range that can be managed in SFC-MS/MS was found to be similar and even larger than RPLC-MS/MS. Compared to GC-MS/MS, a much higher throughput can be achieved, as there was no need for chemical derivatization. The benefits reported in UHPSFC–MS/MS make this approach very attractive and promising for doping control analysis, as a screening strategy or possibly as a confirmatory approach.

Code: 14A03KP 

Since years high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) gained importance for the detection of various classes of doping relevant substances. In contrast to the classical GC-MS technique it allows for separation of analytes with different functional properties without derivatization. 
However some analytes are still challenging as HPLC-MS/MS shows limited resolution capabilities and highly polar analytes interact only insufficiently on the conventional analytical columns. Thus, especially the HPLC analysis of several steroidal doping substances and their metabolites but also the polar stimulants are dissatisfactory. Supercritical fluid chromatography (SFC) as orthogonal separation technique to HPLC may help to overcome these issues. 
During the project it will be tested for the analysis of stimulants. In this class some of the very polar compounds already proved to be nicely analyzed by SFC-MS/MS. A multi-analyte method will be developed and compared to the currently used method. Special focus is given to robustness, identification power and turn around times. 

Main Findings: 

High performance liquid chromatography (HPLC) is considered as method of choice for the separation of various classes of drugs. However, some analytes are still challenging as HPLC shows limited resolution capabilities for highly polar analytes as they interact only insufficiently on conventional reversed phase (RP) columns. Especially in combination with mass spectrometric detection limitations apply for alterations of mobile phase composition and thereby also stationary phases. Some highly polar sympathetic drugs and their metabolites showed almost no retention on different reversed phase columns. Even on phenylhexyl phases, that show different selectivity due to π-π-interaction their retention remains poor. 
Supercritical fluid chromatography (SFC) as orthogonal separation technique to HPLC may help to overcome these issues. Selected polar drugs and metabolites were analyzed utilizing SFC separation. In method development software assistance was used to create a robust separation. Thereby the response surface was generated as full central composite in quadratic design model focusing on the critical peak pairs, that show the same ion transitions in tandem mass spectrometry (MS/MS) and therefore require chromatographic separation for proper assignment. In the final SFC-MS/MS method, all compounds showed sharp peaks and good retention also for the very polar analytes, such as sulfoconjugates. Retention times and elution orders in SFC are different to both RP and HILIC separations due to the orthogonality. Furthermore, short cycle times could be realized. As temperature and pressure strongly influence the polarity of supercritical fluids, a precise temperature and backpressure regulation is required for the stability of the retention times. As CO2 is the main constituent of the mobile phase in SFC solvent consumption and solvent waste are considerably reduced.

Code: 14A08OM

For sports drug testing, the errorless discrimination of rEPOs from endogenous human EPO is important. The current gel electrophoretic methods are also labour-intensive for anti-doping laboratories. 
Mass spectrometry-based procedures provide strong identification power, robustness and reproducibility in sports drug testing. Recently, we have developed a high-sensitivity and high-throughput qualitative detection method for human urinary NESP using LC-MS/MS. This was the first report of successful practical application of the mass spectrometric identification of NESP in human urine. 
Following this outcome, the aims of this study are to optimize the NESP detecting method for a robust implementation in WADA accredited doping laboratories, and further to establish a novel detection method for other rEPOs, such as CERA in human matrices by LC-MS/MS for doping control purposes. 
CERA is a long-acting erythropoietin derivative with methoxy polyethylene glycol butanoic acid linking to the N-terminal amino group or the ε-amino group of any lysine, predominantly Lys45 and Lys52, in the protein molecule. The structure of CERA is different from that of human EPO and epoetins. After enzymatic digestion, the target Pegylated peptides will be searched by the LTQ Orbitrap high resolution hybrid mass spectrometer for discriminating between CERA and endogenous EPO. Furthermore mass spectrometric glycoform profiling of rEPOs (e.g. epoetins) will be performed by LC-ESI-MS/MS. After enzymatic digestion of core protein and glycan of rEPOs, the target digested glycosylated peptides will be identified by the LTQ Orbitrap high resolution hybrid mass spectrometer (Thermo) and SynaptG2 HDMS TOFMS (Waters) for discriminating between epoetins and endogenous EPO. We focus the differences of syalylation, N-glycosilation, O- acetylation of sialic acids and O-glycosilation between epoetins and human EPO, and we have an optimized CID technique for discriminating N-glycopeptides by LC-MS. 

Main Findings: 

The current analytical methods for recombinant human erythropoietin (rEPO) are mainly gell electrophoretic methods. Mass spectrometry is necessary for  the reliable identification of rEPOs in doping control. The aim of this research project is to develop the mass spectrometric detection method for human sports drug testing.  
The research comprises three topics, i.e., 'Mass spectrometric characterization of rEPOs and their biosimilars', 'Improvement of the LC-MS/MS method for detecting darbepoetin alfa in human urine', and 'Mass spectrometric approach for detecting CERA'. 
It has been recognized that EPO biosimilars typically have the same amino acid sequence as the innovator product, and that they have slight differences in overall chemical structure.  As expected, the biosimilars of darbepoietin alfa have different glycan distributions compared with the originator. Interestingly, the biosimilars of darbepoietin alfa and a biosimilar of epoetin alfa contain not only des-arginine product comprising 165 amino acids, but also a C-terminal arginine product comprising 166 amino acids, in contrast to the results in which the originator recombinant EPO products. darbepoetin alfa and CERA contain only des-arginine EPO comprising 165 amino acids. The presence of C-terminal arginine EPO in human matrices might be evidence of administration of EPO biosimilars.  
We established a high-quality and more robust UPLC-MS/MS method for detecting darbepoetin alfa using a deutrated internal standard. The lower detection limit was 1 pg/ml. The detection window in urine from patients treated with NESP was estimated to be up to 16 days after administration. The present method could be a useful tool for detecting the biosimilars of darbepoetin alfa for doping control testing because the method does not depend on the glycan profiles.  CERA and epoetin beta representing human EPO could be successfully discriminated using  LC-MS/MS with in-source CID. However, this mass spectrometry-based technology needs to be improved in terms of sensitivity for detecting CERA in human biological fluids. 

Code: T14M01GP 

Human chorionic gonadotropin (hCG) may be abused by male athletes in sports and is included in the banned substance list of the World Anti-Doping Agency. An ti - soping laboratories mainly use immunoassays to quantify hCG but cross-reactivity with the different forms of hCG can constitute a poblem for quantitative hCG determination especially in urine samples. Choriogonadotropin protein is a heterodimer composed of an alpha chain, whjich 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 ans 4O- glycosylations on the beta subunit and 2 N-glycosylations on the alpha subunit. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers analytical specificity superior to that of immunoassays and can be alternative method for wuantification of hCG in athletes biological samples. for optimal assay accuracy and reliability, a stable isotope labeled internal standard should be used. In a precious project, we developed a protpcol to produce an isotopically-labeled version of hCG (PSAQ standard). The choriogonadotrophin heretodimer was successfully expressed in mammalian cells and purified. Stable isotope incorporation was determined to be greater than 98%. The goal of this project is to produce 200 to 500μg of hCG PSAQ standard according to the protocol previously developed to deliver different anti-doping laboratories. 

Main Findings: 

Recombinant hCG protein was produced and labelled in HEK293 cells. The choriogonadotrophin heterodimer was successfully expressed and purified using Ni-IMAC resin. To obtain higher purity level, the sample was submitted to a second purification step using Ion Exchange Chromatography (IEX). The purity was estimated to be greater than 95%.

In this program, to increase the sequence coverage, a sample of hCG was treated with a  combination of five enzymes to remove N-linked glycans and many common O-linked glycans.  Following deglycosylation, hCG alpha and beta subunits sequences were verified by LC-MS/MS analysis after reduction/alkylation and in-gel trypsin digestion of the purified protein.  The sequence coverage of CGB is greatly improved after treatment with the Deglycosylation Mix compared to the previous feasibility study where the protein was only digested with PNGase F.  Stable isotope incorporation was estimated using MS data generated by LC-MS/MS analysis.  
hCG PSAQ™ standard will be delivered in aliquots of 20 μg.  We recommend to store at -80°C upon receipt.

Code: 14A30JM

The project has two main goals. Firstly, the project aims to evaluate the potential of 3α-glucuronide-6β-hydroxyandrosterone (6OH-A-3G) and 3α-glucuronide-6β-hydroxyetiocholanolone (6OH-Etio-3G) for the screening of endogenous anabolic androgenic steroid misuse. For this first purpose, a quantitative method validated during the project 12A13OP will be applied to several samples from excretion studies already available in our laboratory. Secondly, the project also aims to characterize, elucidate and synthesize the marker M170T298. For this purpose, a large range of analytical strategies will be applied e.g. different derivatization steps, an in-depth study of accurate mass and tandem mass spectra. Once elucidated, the marker will be synthesized and characterized by NMR. After the synthesis of the marker, it would be useful to develop a quantitative method and to check its potential for other administration routes of testosterone. It is intended to reach these goals in a follow-up project once the objectives of the present project are achieved.

Main Findings

[3:22 PM] Meirotti, Luciana

In previous WADA funded projects (11A9RV and 12A13OP), our research group revealed that two testosterone metabolites resistant to enzymatic hydrolysis: 3α-glucuronide-6β-hydroxyandrosterone (6OH-A-3G) and 3α-glucuronide-6β-hydroxyetiocholanolone (6OH-Etio-3G) were present in urine. We characterized them, synthesized them and validated a method for their quantitation. However, their potential usefulness for doping control analysis was not fully evaluated.
On the other hand, common investigations for the screening of testosterone misuse are focused on metabolites with a predicted structure. Thus, unpredicted markers remain not evaluated. In order to cover this gap, we performed a preliminary metabolomic study which showed the presence of a marker (code M170T298) which is a potential urinary marker for testosterone administration. In order to confirm this fact, the characterization, elucidation and synthesis of this marker are required.
Thus, the project has two main goals. Firstly, the project aims to evaluate the potential of 6OH-A-3G and 6OH-Etio-3G for the screening of EAAS misuse. Secondly, the project also aims to characterize, elucidate and synthesize the marker M170T298.
Regarding the first goal, the usefulness of 6OH-A-3G and 6OH-Etio-3G was tested in three scenarios: (i) oral administration, (ii) intramuscular administration and (iii) gel administration. After oral administration of testosterone undecanoate we found that four markers containing either 6OH-A-3G or 6OH-Etio-3G presented detection windows (DWs) larger for all volunteers than those obtained by T/E and comparable to those reported by using cysteinyl metabolites. After intramuscular administration, markers containing either 6OH-A-3G or 6OH-Etio-3G provided detection windows that were similar or longer than those obtained by markers currently included in the steroid profile and clearly higher than those obtained by cysteinyl markers. Finally, the administration of testosterone gel could be screened by markers containing either 6OH-A-3G or 6OH-Etio-3G reaching similar to those obtained by markers currently included in the Athlete Biological Passport.
Regarding the second goal, during this project, we have characterized, elucidated and synthesized the marker M170T298. Our research shows that this marker is 1-cyclopentenoyl glycine (1-CPG) which is probably coming from the metabolism of the cypionic acid released after the hydrolysis of testosterone cypionate. Therefore, it can be valuable to detect the misuse of testosterone cypionate. This fact has been confirmed by analysis of samples collected after testosterone cypionate administration. The determination of 1-CPG provided the longest DWs for these samples

Code: 14D11AR 

Our research program encompasses projects designed to investigate how low doses of topical testosterone interfere with the urinary steroid profile and Athletes Biological Passport (ABP). ABP is essential to detect doping with anabolic androgenic steroids, especially with the use of low-dose-testosterone being increasingly used by some athletes. 
In addition to genetic variation, there are many other factors that may contribute to the inter- and intra-individual variability in the steroid profile, i.e. concomitant drug use, diseases, menstrual cycle, and pregnancy. The latter two conditions have a marked influence on the metabolism and endocrinology of estrogens, progestagens, and peptide hormones (LH,FSH, hCG). However, very little is known about the natural androgen disposition in these situations. In view of the increasing use of LDD it is important to know the natural profile of androgens in the early post-conceptional phase when women still may not know, or know of the pregnancy. There is a dearth of information how pregnancy influences the androgen profile, but it is likely to confound the test interpretation. We will therefore study the androgen profile in females (no dose administration!) in the post-conceptional phase and first trimester.
It is of great importance that the athletes ABP will be able to compensate for all possible variabliity in longitudinal steroid profiles.  More knowledge is therefore needed about how drug use and pregnancy influence the ABP results and hence the outcome of doping tests. 

Main Findings: 

The low-dose androgen doping strategy practiced by certain individuals may be difficult to detect, particularly in women during the menstrual cycle and even more so in the early post-conceptional phase because of extensive hormonal changes. Even though doping puts the fetus at danger, highly motivated sports women may disregard this risk. Alternatively, they may be unaware of conception early in pregnancy. It is probable that exogenous androgens will interfere with the hormonal balance in pregnant women. For obvious reasons there is only sparse information on that in the literature.  
We have investigated the natural androgen excretion profile in women in different phases of pregnancy, and the potential effect on the testosterone/epitestosterone (T/E) ratio used in doping tests. First, pregnancy altered the excretion of glucuronide (G) and sulfate (S) conjugated androgen metabolites. Using an LC-MS/MS method we found that both epitestosterone-S (-S = sulfate conjugate) and epitestosterone-G (-G = glucuronic acid conjugate) were significantly increased throughout the trimesters, being normalized post-partum. Importantly, some of the urinary profiles of steroid ratios in the ABP were altered in pregnant women when compared to the same women after delivery, or with non-pregnant women. Two of the ratios were altered during pregnancy; T/E was lower, whereas A/Etio was higher in pregnant women. Prior information about pregnancy would make the interpretation of the test results safer since pregnancy has such an impact on the ABP steroid profile.  
The steroid profile was also studied in relation to ethnicity and genetic variation. The results show that pregnant women of Asian origin exert higher urinary concentrations of epitestosterone-G than Caucasians. The genotype – phenotype relation between UGT2B17 deletion polymorphism and T/E ratio was disrupted during pregnancy. 
19-Norandrosterone increases in pregnancy and, if above the Decision Limit (DL), analysis of hCG is performed. If the concentration is above 15ng/mL, an IRMS analysis needs to be done in order to confirm if 19-NA is of exogenous origin. In the first trimester 19-NA was below the DL in 55 % of the women. None of the pregnant women reached 15 ng/mL in the first trimester, but 45 % of them were above the DL. The median 19-NA concentrations were increased in the second and third trimester, and in the second and third trimester 56 and 71 %, respectively, of the pregnant women reached the individual DL.
Early pregnancy is a condition that perturbs the hormonal balance and affects the interpretation of common doping tests. Our results are informative about the critical points to be considered in relation to doping tests in fertile female athletes.

Code: T14M02AB 

Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles and is prohibited in males according to the World Anti-Doping Agency list of prohibited substances. Immunoassays are currently used by anti-doping laboratories to measure urinary hCG but results can vary widely among laboratories due to differences in cross-reactivity against different molecular forms of hCG (isoforms). We recently developed a sequential immunoextraction method with liquid chromatography tandem mass spectrometry (LC-MS/MS) detection for quantification of intact hCG, hCG free β-subunit and β-subunit core fragment in urine. Data from hCG excretion studies demonstrated that intact hCG should be monitored for detection of doping with hCG. In order to apply the LC-MS/MS method for confirmation of immunoassay screen positive hCG results a threshold concentration needs to be established. In preliminary studies we found that the current threshold concentration of 5 mIU/mL applied to immunoassays is not appropriate when measured by LC-MS/MS and needs to be significantly lower. 
In the present study we plan to determine the concentration of intact hCG in 600 non-doping male urine samples using the recently developed immunoextraction method with LC-MS/MS detection. Half of the urine samples will be from normal male volunteers and the other half from non-doping male athletes. The data from this study will be immediately used to determine the appropriate threshold concentration for confirming doping with hCG. 

Main Findings:

The concentrations of intact hCG in the male urine samples are provided in the attached file and are graphically displayed in Figure 1. Of the 570 male urine samples, 243 had undetectable intact hCG concentrations (<0.02 IU/L) not displayed on the graph. The remaining 327 urine samples had intact hCG concentrations ranging from 0.02 to 0.50 IU/L.  Of the 590 total samples, 542 (95.1%) had intact hCG concentrations <0.01 IU/L. Of the remaining 28 samples, 27 had intact hCG concentrations >0.01 and <0.26 IU/L. Only sample had an intact hCG concentration of 0.50 IU/L (27 year old, not shown on graph). Given these data, we recommend an extremely conservative intact hCG cutoff of 2 IU/L to be used during confirmation testing for detection of doping with hCG. At a concentration of 2.5 IU/L the imprecision of the method was 11.4%, when 3 separate urine samples were analyzed on 5 different days.

Code: 14A11CA 

The project that we propose implies the development of a novel two-dimensional HPLC purification method for the carbon isotopic analysis of two groups of molecules, corticoids and low concentrated steroids. Prednisolone is a direct metabolite of the synthetic corticoid prednisone but it can also be derived from the dehydrogenase bacterial enzymatic activity of cortisol. In the same manner cortisone, the parent molecule of cortisol, could produce considerable quantities of prednisone as well, yielding false positive cases. Additionally, numerous methods for the purification and C analysis of boldenone and nandrolone metabolites have been developed in the past years; however, to our knowledge, none yet has proven to produce measurements as precise and accurate as the testosterone metabolites, being unquestionably more concentrated. Furthermore two HPLC purifications are commonly required for their purification, from which testosterone metabolites are left aside due to the complexity of the separation even if the latter come necessary in the detection of multi-positives cases. To ascertain the exogenous origin of the corticoids, difficult to chromatographically purify due to their higher polarity, and to efficiently purify boldenone or nandrolone metabolites including testosterone metabolites, the development of a novel two dimensional HPLC method through the heart-cutting technique is sought for a rapid purification of those compounds from the urine matrix without any derivatization. The rationale behind this method development is (i) to save instrumental time for the purification of the compounds, (ii) obtain the same purification power as with two subsequent HPLC cleansing runs and (iii) to gain intensity, precision and accuracy owed to fewer manipulations and chemical reactions. Finally, we wish to validate the methods by confirming the absence of isotopic fractionation through the analysis of all the tested metabolites using the developed HPLC technique and to publish the methods used for routine δ13C measurements. 

Main Findings: 

The use of compound specific isotope analysis (CSIA) to investigate stable isotopic abundances of is still considered a niche discipline for well-trained specialists (Brand 2012) and indeed, the implementation of reliable and robust IRMS methods has proven to be quite a challenge for anti-doping labs. 
Possibly the most critical aspect of CSIA is achieving adequate purification of compounds prior to IRMS analysis since unlike most other mass spectrometric methods, the specificity of CSIA analysis hinges on achieving baseline separation of target GC peaks from all other carbon-containing molecules in the urinary matrix. Insufficient purification results in peak contamination and inconsistent or irreproducible δ13C values. The world anti-doping agency (WADA) recommends the use of high performance liquid chromatography (HPLC) for sample cleanup, however many WADA-accredited labs suggest more than just one purification step (Piper et al. 2008) or their methods require long HPLC columns and extended purification time (75 minutes, Ouellet, LeBerre and Ayotte, 2012). 
This WADA project provides a description of an automated two dimensional HPLC purification (2D-HPLC) method for urine extracts that has made possible the highest throughput CSIA purification of urine extracts described thus far by WADA-accredited labs, requiring only 35 minutes per sample or approximately 20-25 samples/day. In contrast to previously established CSIA methods, no sample manipulation is required between purification steps (e.g. Piper 2008). Six urinary steroids including testosterone/DHEA and 4 metabolites (5α-androstanediol, 5β-androstanediol, androsterone and etiocholanolone) as well as two endogenous reference compounds (pregnanediol and 3α-hydroxy-5α-androst-16-ene) were eluted and collected during HPLC purification. 
Comparative GC chromatograms are used to contrast the efficiency of 2D purification to a previously established 1D HPLC method (Ouellet et al 2014). While each sample requires less than half the time of 1D purification, sample purity is actually improved. Precision of δ13C for all analyzed compounds in negative and positive controls was 0.3‰ or better, which is comparable to the precision of pure compounds at similar concentrations. The appeal of 2D-HPLC is indeed that a properly configured method can easily surpass the efficiency of the highest performing 1D system – producing clean peaks in much shorter run times. No extra expenditure is incurred by the use of 2D-HPLC, aside from the initial instrument cost. The potential for a dramatic increase in the IRMS sample load offered by 2D-HPLC would be especially useful during international sporting events, during which a large number of tests are required over very short time delays. Increased sample throughput makes possible a larger number of IRMS tests, necessarily improves the likelihood of detection of the abuse of testosterone or testosterone-related steroids.