Projets de recherche

Code: 17A20LM 

Urine samples collected worldwide for anti-doping testing are not always shipped in refrigerated conditions from collection sites to WADA accredited laboratories, in particular when several transport days are required. Commensal urethral microbiota, urinary pathogens and environmental bacteria may contaminate urine and the enzymatic activity by microbial contamination can cause severe modifications to the excreted compounds, in particular to doping-relevant peptides such as hormones and growth factors. Since delays are inevitable and sample refrigeration is not always possible and effective, sound methods for preserving and stabilizing urine samples are desirable.
This Project involves the use of Volumetric Absorptive Microsampling (VAMSTM) and Dried Urine Spot (DUS) strategies for the collection of dried microsamples processed by means of streamlined workflows to be developed ad hoc, for high-throughput LC-MS/MS analysis of several peptide hormones and growth factors (i.e. GHRP-1, GHRP-2, GHRP-6, hexarelin, alexamorelin, triptorelin, AOD9064, CJC-1293, desmopressin, TB-500, hCG and ACTH). DUS and VAMS approaches represent promising alternatives to the procedures currently performed by anti-doping laboratories, allowing analyte stabilization by water loss and the consequent broadening of their detection window. This innovative sampling produces logistics savings due to the small transported volume (by air shipments and through customs) and to the possibility to keep the specimens at room temperature, with significant implications on overall analysis cost. 
The ultimate goal of this project is to establish and validate feasible but reliable protocols for the collection of urine microvolumes, stably storable and shippable with no particular precautions. As a proof of concept, instrumental analytical methods will be developed, validated and applied for the stability assessment of several peptide hormones and grow factors in dried urine microsamples stored at different conditions. In parallel, stability will be assessed in classic fluid urine stored at temperature-controlled conditions and thorough comparisons with dried microsamples will be carried out.

Main Findings: 

•    Innovative microsampling and pretreatment procedures based on dried matrices (DUS and urine VAMS) were optimised for the application to urine specimens for anti-doping purposes. Important experimental parameters were studied and specifically optimised. 
•    Original MS, HRMS and FT-IR methods were used for peptide chemical identity confirmation; then LC-MS/MS and LC-HRMS methods were developed for the simultaneous analysis of the peptides of interest in dried urine microsamples. After suitable study of the experimental conditions, the final methods provided good, solid performance within relatively short run times.
•    The LC-MS/MS and LC-HRMS methods were validated according to current guidelines, with good results for all assays and all analytes, and with similar or better performances than those of comparable published methods.
•    The microsampling, pretreatment and analysis workflow was successfully applied to the study of peptide stability in dried matrices.
•    Mid-term stability assays were carried out, both on dried and fluid urine-based matrices, with outstanding results. In fact, DUS and urine VAMS were remarkably stable, even though they were kept at RT, with all studied peptides recovered in the 80-95% range at the end of the study period (3 months). The stability of the chosen peptides, which are known to be prone to degradation under common storage conditions, was greatly enhanced in comparison to fluid urine stored at freezing (-20°C) and ultra-freezing (-80°C) temperatures. Analyte loss in fluid urine at -80°C was regularly 5-10% larger at study end than the loss in dried matrices, with widely worse losses (up to 35% more) for fluid urine kept at -20°C.
The use of innovative microsampling media offers significantly interesting perspectives towards the development of engineered, highly reliable devices.

Code: ISF17D20AM

Our objectives are:
- to reproduce an ABT experiment similar to what athletes are susceptible to perform (small volume of blood: 200ml) and to compare in the same assay reinfusion of refrigerated blood (stored less than one month) or reinfusion of cryopreserved blood. A group of subjects that will donate blood but will not be reinfused will also be integrated as controls. 
- to focus on the characterization of microparticles before and after ABT:  to identify morphological and cell surface markers changes, as well as changes in protein content by proteomic analyses of microparticles. 

We will :
(i) determine if these techniques can efficiently identify an ABT of small volume. And if similar modifications are identified when blood was conserved refrigerated or frozen. 
(ii) determine the window of detection of an ABT of small volume with these tests..

Code: ISF17E07FB 

The rationale of our proposal is based on the hypothesis that gene transfer with AAVs will leave an immunological and genetic footprint in any human subject clearly distinguishable from a naturally occurring AAV infection with the wild-type virus. In the context of the detection of gene doping, the main objective of this project is to develop a two-step method for the detection of AAV-mediated gene doping based on the combination of immunological and molecular tools. This general objective can be divided in the following specific aims, describing the two-step approach proposed for the identification of subjects who underwent AAV-based gene doping. Specifically: 2.1. Profiling of the anti-AAV antibody responses. A sensitive, pan-AAV antibody assay will be developed, validated, and streamlined to distinguish humoral responses deriving from natural exposure to AAV from gene doping with AAV vectors. Thus, this assay will permit to establish a baseline to be used as reference value to discriminate between subjects positive or negative for AAV-gene doping. 2.2. Detection of AAV genomes in blood/PBMCs by means of target capture NGS. To this end, a panel will be designed containing at least 4 million capture probes specific for at least 100 genome elements, such as viral ITs, regulatory sequences and different doping genes involved in improvement of muscle strength and performance.

Main Findings

The main findings are not available due to the sensitivity of the information and results developed in this project.

Code: T17R03DC 

Athletes are required to provide urine samples, as part of an anti-doping programme, in a doping control station and the sample collection is witnessed by a Doping Control Officer. As part of the process, a Doping Control Form (example attached) is completed and the samples are transported to the Drug Control Centre (DCC) at King's College London. The part of the Doping Control Form received by the DCC contains no information about the athlete (e.g. name, address). It contains information about sample volume, specific gravity, event code, and any medication declared to have been used by the athlete. A box on the form indicates whether the athlete has consented for his/her samples to be used for the research purposes. Once the athlete’s sample arrives in the DCC, it is split and undergoes the sample preparation steps (e.g. solid
phase extraction) prior to analysis. The usual method for sample analysis is liquid chromatography-high resolution mass spectrometry (LC-HRMS) (please see Musenga and Cowan, Use of ultra-high pressure liquid chromatography coupled to high resolution mass spectrometry for fast screening in high throughput doping control. J. Chromatography A 2013;1288:82-95). In our research project, funded by the World Anti-Doping Agency (WADA), we would like to investigate the potential application of supercritical fluid chromatography-mass spectrometry (SFCMS) in the analysis of doping control samples. We aim to compare the performance of SFC-MS with LC-HRMS. In order to do our research, once the samples have been analysed by LC-HRMS, we will re-analyse them by SFCMS and compare two methods by results obtained. We plan to do our research with approximately 1000 samples and our research refers to secondary data analysis. The DCC is required by WADA to analyse all samples received by LC- HRMS no matter if the athlete had given research consent or not. However, we will only analyse samples by SFC-MS with the research consent provided by the athlete on the Doping Control Form. As a laboratory accredited by the WADA, we have to comply with strict criteria in the use of athlete samples for research. These include: waiting at least 3 months after the anti-doping analytical report has been issued; transference of the sample from the coded bottle to a separate container so that it is impossible for the identity of the athlete who provided the sample to be discoverable by anyone.

Main Findings:

This study clearly demonstrated the applicability of SFC-MS for routine antidoping analysis. We analysed approximately 3,000 samples (3 x 1,000) in total using SFC-MS equipment from three different vendors to investigate whether this technology is applicable for routine anti-doping analysis. Our work demonstrated SFC-MS to be a robust analytical technique. It was demonstrated that it may be considered as a complementary technique to LC-MS, which is readily available in anti-doping laboratories. Three SFC-MS instrument manufacturers (Agilent Technologies, Shimadzu UK Ltd and Waters Corporation) generously contributed to our study to investigate the routine applicability of SFC-MS in doping control. All three vendors dedicated instrument time and resource for the project. A large number of anti-doping samples and QCs were delivered to each manufacturer. We worked closely with each manufacturer in agreeing the method of operation and helped with the analysis of the samples, data review and undertook the independent statistical analysis of the data.
All three instruments appeared to meet the requirement for robustness needed for routine use. Most of our compounds showed excellent chromatography. There were only a few examples of co-elution due to interference with endogenous compounds that could benefit from modification of the chromatographic conditions. Retention times were generally remarkably stable over the analysis time. We demonstrated some software features that would facilitate data review especially with the software from one manufacturer. Despite using a “dilute and inject” approach, the SFC columns were found to be stable over more than 1,200 injections of samples, standards and quality controls on three different systems. In general, SFC-MS was found to be similar to LC-MS for routine use but with the advantage of complementarity providing additional information of the chromatographic properties of the analyte thereby further confirming the identity of a doping agent.

Code: T17R02TK

Ultra-high performance supercritical fluid chromatography-mass spectrometry (UHPSFC-MS) could represent in the near future an orthogonal technique to LC-MS and GC-MS for routine doping analysis. This technique now benefits from a broader recognition and interest, thanks to new technological improvements and the recent commercialization of new platforms.

The aim of this project is to evaluate the potential of UHPSFC-MS/MS for screening and confirmation purposes in routine anti-doping analysis. This aim will be achieved through a comprehensive robustness study (different columns chemistries, column batches, instruments and laboratories) by selecting representative compounds of major classes of prohibited substances (approx. 50 compounds) fortified in urine samples. Various aspects will be evaluated such as the stability of retention times and the inter-batch variability of SFC columns. Then, an inter-laboratory study as well as an inter-instrument study will be performed with other academic and/or industrial laboratories equipped with the same brand of SFC instrument (Waters Acquity UPC² system) or equipped with other brands of UHPSFC instruments, including Agilent and Shimadzu, to evaluate the ruggedness of the SFC-MS/MS method.

Main Findings

The aim of this study was to assess the interlaboratory reproducibility of ultra-high performance supercritical fluid chromatography coupled with tandem mass spectrometry method for routine antidoping analyses.To do so, a set of 21 doping agents, spiked in urine and analyzed after dilute and shoot treatment, was used to assess the variability of their retention times between four different laboratories, all equipped with the same chromatographic system and with the same ultra-high performance supercritical fluid chromatography stationary phase chemistry. The average relative standard deviations (RSD%) demonstrated a good reproducibility of the retention times for 19 out of 21 analytes, with RSD% values below 3.0%. Only for two substances, namely fenbutrazate and niketamide, the retention was not repeatable between laboratories, with RSD% of approximately 15% in both cases. This behaviour was associated with (a) the low organic modifier percentage (around 2-4%) in the mobile phase at the corresponding retention times, and (b) the influence of the system volume on poorly retained analytes. An analysis on seven “blind” urines was subsequently carried out in the same four laboratories. In these blind samples, either one, two, or none of the 21 doping agents previously analyzed were present at an unknown concentration. Each laboratory had to perform the identification of the compounds in the samples and estimate their concentrations. All laboratories assigned all target analytes correctly in all blind urine samples and provide a comparable estimation of their concentrations.

Code: 17D07MM

Azole compounds were introduced into the therapy of fungal infections of humans in the early 1970s. The primary target of azole compounds is the CYP51, the enzyme involved in the synthesis of ergosterol from lanosterol in the fungal cell membranes. However, azoles have a potential to interact also with other cytochrome P-450-dependent enzymes, leading to toxicologically relevant changes in the liver and endocrine system. Indeed, depending on their effects on specific enzymes inhibited they can cause (i) a reduced formation of either androgens or estrogens, and/or (ii) alterations in the elimination rate of xenobiotics. CYP19 (aromatase) is one of the cytochrome P450 enzyme inhibited by azoles. Several azole fungicides disrupt normal aromatase function leading to a decrease in estrogens formation, and for this reason these agents are not prescribed in pregnancy and are instead used in the management of advanced estrogen-responsive breast tumors in postmenopausal women. Reduction of estrogen levels by CYP19 inhibition is the working principle of anti-aromatase agents. These agents together with other anti-oestrogenic compounds, are included in the section S4 “Hormone and Metabolic Modulators” of the WADA prohibited List. Azole antifungals are instead included in the WADA TDEAAS as confounding factors. In a previous WADA-funded project, focused on the evaluation of the alterations provoked by the administration of azole antifungals on the basal levels of the parameters that are part of the steroid profile, we have reported that both miconazole and fluconazole is capable of altering the levels of the endogenous steroids in a different manner. In this project we aim to extend the study by increasing the number of measurements, including more individual, different ethnicities, route of administration, doses, range of age and by evaluating whether similar effects can be caused also by other azole antifungals.

Code: 17C02MP

Increasing numbers of adverse analytical findings were reported in the recent years due to the misuse of the anabolic androgenic steroid dehydrochloromethyltestosterone (DHCMT). Once developed in the former GDR for misuse in sports it regained enormous relevance especially in the samples from Bejing and London Olympic games. Several adverse analytical findings were reported after the samples had been retested for the newly reported long-term metabolite of DHCMT. At present, the use of post administration urines instead of purified reference material has been accepted in confirmatory analyses. Very recently the currently used metabolites have been questioned in the literature. Thus a controlled administration trial in humans will be performed to provide further evidence for tracing back the long term metabolites 20ξOH-NorTHCMT and 20βOH-NorDHCMT to a DHCMT administration. Furthermore, the utilisation of in-vitro experiments will further broaden the scientific insights into metabolic pathways that lead to the generation of these metabolites.

Main Findings

Dehydrochloromethyltestosterone (DHCMT) is an anabolic-androgenic steroid that was developed by Jenapharm in the 1960s and was marketed as Oral Turinabol®. It is prohibited in sports at all times. Even if discontinued as pharmaceutical in 1994, there are several adverse analytical findings by anti-doping laboratories every year. New long-term metabolites have been proposed in 2011/12, which resulted in adverse analytical findings in retests of the Olympic games of 2008 and 2012. However, no controlled administration trial monitoring these long-term metabolites was reported until now. In this study, a single oral dose of DHCMT (5 mg, p.o.) was administered to five healthy male volunteers and their urine samples were collected for a total of 60 days. The unconjugated and the glucuronidated fraction were analyzed separately by gas chromatography coupled to tandem mass spectrometry. The formation of the described long-term metabolites was verified, and their excretion monitored in detail. Due to interindividual differences there were several varieties in the excretion profiles among the volunteers. The metabolite M3, which has a fully reduced A-ring and modified D-ring structure, was identified by comparison with reference material as 4α-chloro-17β-hydroxymethyl-17α-methyl-18-nor-5α-androstan-13-en-3α-ol. It was found to be suitable as long-term marker for the intake of DHCMT in four of the volunteers. In one of the volunteers, it was detectable for 45 days after single oral dose administration. However, in two of the volunteers M5 (already published as long-term metabolite in the 1990s) showed longer detection windows. In one volunteer M3 was undetectable but another metabolite, M2, was found as the longest detectable metabolite. The last sample clearly identified as positive was collected between 9.9 and 44.9 days. Furthermore, the metabolite epiM4 (partially reduced A-ring and a modified D-ring structure which is epimerized in position 17 compared to M3) was identified in the urine of all volunteers with the help of chemically synthesized reference as 4-chloro-17α-hydroxymethyl-17β-methyl-18-nor-androsta-4,13-dien-3β-ol. It may serve as additional confirmatory metabolite. To improve tracing of cheating athletes, it is highly recommended to screen for all known metabolites in both fractions, glucuronidated and unconjugated. This study also offers some deeper insights into the metabolism of DHCMT and of 17α-methyl steroids in general.

Code: 17A24XD

The anabolic androgenic steroids (AAS) are prohibited in sports. They are included in the 2017 list of the World Anti-Doping Agency (WADA) as class S1. In the last years the anabolic agents accounted for most of the adverse analytical findings (AAF) in doping control (e.g. in 2015 50% of all ADAMS reported AAF). AAS undergo extensive metabolization, thus, urinary detection of a prohibited administration is mainly based on the detection of metabolites. As some steroids also occur naturally in the body, their uncoverage generally uses specific ratios, such as testosterone/epitestosterone (T/EpiT), androsterone/etiocholanolone (And/Etio), And/T, and 5α-/5β-androstane-3α,17β-diol (Adiol/Bdiol), that proved to be very stable in humans. Confirmation of the results generally require isotope ratio mass spectrometry. As confirmation is very elaborate and cost intense some minor metabolites came into the focus of anti-doping scientists to increase the efficiency of screening procedures. For the improved detection of an exogenous administration of androstenedione the usefulness of the A- or B-ring hydroxylated metabolites 4-hydroxy-androstenedione, 6z-hydroxy-androstenedione was reported. As already published 2β- and 15β-hydroxylation also occurs in testosterone metabolism with ~10% and 4% of the rate of the most dominant hepatic microsomal 6β-hydroxylation. Furthermore, it was demonstrated that the use of 2- and 4-hydroxyandrostenedione may serve as long term marker of an androstenedione administration. The metabolic generation could be confirmed by in-vitro experiments upon incubation with CYP1A2 and CYP1B1. No reports on the metabolic hydroxylation of androgens by CYP1A2 or by CYP1B1 are found in literature so far. The objective of the project is to further investigate the suitability of A-ring hydroxylation for long-term detection of endogenously occurring androgens and to extent the preliminary investigations to other prohibited steroids.

Main Findings

The hydroxylation pathway in vivo and/or in vitro studies for Testosterone (T), 4-androstenedione (AED), 19-norandrostenediol (NAD) and Methyltestosterone (MT) was investigated. Samples collected after the administration of T showed the presence of 6-OH-T according to an oral administration, but its concentration gave no additional information different to that already known (parameters of the steroid profile). On the other hand, the formation in vivo and in vitro of hydroxylated metabolites of AED, NAD and MT in position 4 allowed the formation of Formestane, Oxabolone and Oxymesterone respectively. Although the formation of these metabolites were significant (in some cases) and easily detectable, any of them showed a detection window longer that those metabolites known for AED (T/E ratio and others steroid profile parameters), NAD (19-norandrosterone, and 19-noretiocholanolone) and MT (5α and 5β reduced metabolites). Nevertheless, a special attention have to be paid when a sample with these compounds is reported, because under these circumstances a concomitant abuse of AED-formestane, NAD-oxabolone or MT-oxymetholone could be misinterpreted reported by the laboratory. Only for AED the 2-hydroxylation demonstrated an added value extending the suspicion of an AED administration based on an extended steroid profile.

Code: ISF17D04SI

The original idea of “athlete`s performance passport” or monitoring
individual performances for better informed decisions on doping
testing has been presented by Schumacher and Pottgiesser. The main objective of an “athlete`s performance passport” in sport is to distinguish between consistent and unexpectedly disproportionate performances. Excellent performance itself is not a proof of any wrongdoing or doping. However, through longitudinal monitoring, inconsistently excellent performance could be a warning sign that need further attention from anti-doping authorities.The purpose of this project is to establish framework for the longitudinal performance monitoring and identification criteria of athletes with outline performance in middle- and long distance
runners population.

Code: 17D09RV 

The detection of endogenous anabolic steroids (EAS) abuse is currently performed using the steroid profile. The steroid profile is composed of concentrations of testosterone and related endogenous metabolites excreted as glucurono-conjugates, and ratios between them, being Testosteone/Epitestosterone ratio the most important one. These parameters are monitored for each individual to define the individual basal ranges, and changes on them reveal the use of EAS. Suspicious samples are confirmed by carbon isotope ratio mass spectrometry to demonstrate the exogenous origin of testosterone and metabolites. The steroid profile is a powerful tool to detect EAS misuse, however improvements are needed to prolong detection windows. Testosterone and metabolites are also excreted as sulfates in urine. The sulfate fraction has not been comprehensively evaluated for the detection of EAS misuse. The objective of the project will be to evaluate the sulfate fraction of testosterone metabolites to look for new biomarkers to prolong the detectability of the misuse of EAS. First, a comprehensive methodology to quantify endogenous steroid sulfates based on their direct analysis by liquid chromatography-tandem mass spectrometry will be optimized and validated. Second, steroid sulfates will be quantified in urines obtained from healthy population to define normal population ranges. Finally, steroid sulfates will be quantified in urines collected after administration of testosterone to healthy volunteers by different routes. Evaluation of sulfate metabolites as markers of EAS administration will be performed by comparison of the excretion profiles of testosterone and metabolites excreted as sulfates with the excretion profiles of metabolites included in the conventional steroid profile. The successful outcome of the project will be directly applicable to sports drug testing by improving the detection of EAS misuse.

Main Findings: 

The objective of the project was a comprehensive evaluation of the sulfate fraction of testosterone (T) metabolites to look for new biomarkers to prolong the detectability of the misuse of endogenous anabolic steroids. First of all, an analytical method was developed and validated to quantify fourteen T related metabolites conjugated with sulfate, based on a mixed-mode solid pahse extraction and the direct measurement of sulfate metabolites by LC-MS/MS. The concentrations of sulfate metabolites in healthy volunteers, including Caucasion and Asian volunteers, were measured.

The usefulness of sulfate metabolites to detect oral T misuse was evaluated after administration of a single oral dose to five Caucasian male volunteers. Using individual threshold limits epiandrosterone sulfate (epiA-S)  improved the detection times (DTs) with respect to T/epitestosterone (E) ration in all five volunteers. Androsterone (A), etiocholanolen (Etio) and two androstanediol sulfates also improved DTs for some volunteers. The most promising results were obtained using ratios between sulfates of epiA, A or androstandediol 1 and E, and also sulfates of epiA or androstanediol 1 and dehydroandrosterone (DHA). These ratios prolonged the DT of oral T administration, in some cases several days after administration, and therefore significantly improving the retorspectivity compared to sulfate concentrations or to the conventionsl T/E ratio.

Sulfate metabolites were also evaluated after a single intramuscular (IM) injection of T to six Caucasian and six Asian helthy male volunteers. Principal component analysis (PCA) was used to obtain the most useful markers for discrimination between pre- and post-administration samples. For Caucasian volunteers, a separation between pre- and post-administration samples was observed in PCA, whereas for Asian no separation was obtained. Seventeen ratios between sulfate metabolites were selected and further considered. DTs of each ratio were evaluated using individual thresolds for each volunteer, and the best results were obtained using rations involving T and E sulfates in the denominator. The best marker was the ratio A-S/T-S which prolonged the DTs with respect to T/E ration in all Caucasian volunteers and in two Asian volunteers. Other ratios A-S or Etio-S and E-S, and, sulfates of Etio, DHA or epiA and T-S were also found adequate.

The data obtained in the project provide a comprehensive insight bout the usefulness of endogenous sulfate metabolites as biomarkers for the detection of oral and IM T misuse. They can drastically increase the DTs with respect to the conventional T/E ratio, especially after oral T administration and, according to our results, its inclusion in the steroid profile is strongly recommended.