Projets de recherche

Code. 12A21RV 

Anabolic androgenic steroids (AAS) are the most important group of forbidden substances detected in antidoping analyses, and improvement of the detection capabilities of their misuse is a priority in antidoping control. Studies on steroid metabolism have been traditionally performed using GC-MS methods. Phase II metabolic reactions of AAS have been normally studied by using hydrolysis with -glucuronidase enzymes, thus mainly conjugates with glucuronic acid have been systematically studied using both GC-MS and LC-MS/MS approaches. The study of sulfates has been limited by the difficulties of their efficient hydrolysis to phase I metabolites. LC-MS/MS offers the possibility to directly detect sulfate conjugates without previous hydrolysis to phase I metabolites. The application of LC-MS/MS has enabled the detection and characterization of unreported sulfate metabolites for boldenone and methyltestosterone, useful to improve detection capabilities of these AAS. Sulfated metabolites of boldenone can be used as markers of exogenous administration of boldenone. For methyltestosterone, long-term sulfated metabolites have been identified that allow for an increase between two and three times the retrospectivity of the detection. These results indicate that sulfatation is an important metabolic pathway for AAS and deserves to be comprehensively studied for all AAS using adequate technology (LC-MS/MS).
The objective of the present proposal is to continue the systematic study of sulfate conjugated metabolites of other AAS that could be used to improve detection capabilities of their misuse. A methodology based on the direct analysis of the sulfates by LC-MS/MS will be applied. Detected sulfate metabolites will be characterized and their excretion profiles will be compared with those of the steroid metabolites targeted in conventional screening procedures, in order to evaluate their interest as long-term markers of steroid misuse. Finally, a methodology addressed to the reliable detection and confirmation of new sulfate metabolites in routine antidoping analysis will be developed.

Main Findings: 

The objective of the study was to evaluate sulfate conjugated metabolites of different anabolic androgenic steroids (AAS). Based on the common ionization and fragmentation behaviour of steroid sulfates, precursor and neutral loss scan methods, and selected reaction monitoring methods including theoretical transitions of potential sulfate metabolites were used to detect new sulfate metabolites in samples obtained after administration of different AAS, including 4-chloro-metnadienone, stanozolol and clostebol.
For 4-chloro-metandienone, four conjugates with sulfate were detected, and two of them (identified as isomers of 4-chloro-18-nor-17€-hydroxymethyl-17€-methylandrost-1,4,13- triene-3-one conjugated with sulfate) were detected in urine up to the last samples collected in the excretion studies available, showing for these studies the same detection window as the recently reported long-term metabolites excreted in the glucuronide fraction.
For stanozolol, eleven sulfate metabolites were detected, including 16Å-hydroxy-stanozolol 16Å-sulfate and other metabolites characterized based on mass spectrometric data: two isomers of 16Å-hydroxy-stanozolol 16Å-sulfate; two isomers of stanozolol N-sulfate; three isomers of hydroxy-stanozolol N-sulfate; 16-oxo-stanozolol N-sulfate; and 4Å-hydroxy-16- oxo-stanozolol N-sulfate. Detection times up to 10 days were obtained after oral administration and, therefore, the sulfate metabolites did not improve the retrospectivity obtained with the recently described epistanozolol N-glucuronide, resulting from the initial formation of a sulfate conjugate.
For clostebol, sixteen sulfate metabolites were detected. One of them, characterized as 4€- chloro-5‚-androst-3Å-ol-17-one 3Å-sulfate, was detected up to the last sample collected in the excretion studies evaluated (31 days) and, therefore, significantly improves the detection time of clostebol misuse with respect to the commonly monitored metabolites, excreted in the glucuronide fraction.
The results of the project demonstrate the importance of sulfatation as a phase II metabolic pathway of AAS and the interest to study this metabolic fraction to look for new long-term metabolites, and the need to incorporate these metabolites in the initial testing procedures for AAS in all antidoping laboratories.

Code: 12C17MM 

The metabolic steps that lead to drug clearance in the human body are divided into two distinct parts: chemical modifications of the parent compound (phase I); and conjugation of either the parent drug and/or the phase I metabolite(s) with endogenous molecules (phase II).  Generally, cytochrome P450 systems are most important for phase I, while UDP glycosyltransferases (UGTs) for phase II reactions. UGTs catalyze the conjugation of the glucuronic acid moiety from UDP-glucuronic acid to many xenobiotics, including drugs/metabolites banned by the WADA. Conjugation facilitates the excretion of the compounds into urine. The kinetics of elimination of a compound could be influenced by genetic variability and/or by the co-administration of drugs that interact with UGTs. In this context, previous studies demonstrated that non steroidal anti-inflammatory drugs, like diclofenac and ibuprofen, decrease the enzymatic activity of the most important UGTs involved in the phase II metabolism of testosterone, whereas benzodiazepines and antifungals alter the glucuronidation kinetics of morphine and codeine.

The preliminary results of an ongoing project, also funded by the WADA, focused on the study of CYP450s inhibitors as potential “masking agents”, proved that in the presence of CYP450s inhibitors the in vitro phase I metabolic profiles of several classes of banned substances were extensively modified. In this project we plan to follow an analogous approach to preliminarily investigate whether the co-administration of doping agents with other xenobiotics that are substrate of the UGTs could be used by athletes as a strategy to evade anti-doping testing.  Specifically, the in vitro phase II metabolic profile of selected banned compounds, with especially emphasis on threshold substances (i.e 19-norandrosterone, morphine and tetrahydrocannabinol) will be assessed individually and in the presence of selected medicaments commonly used by athletes (non steroidal anti-inflammatory agents, antifungal agents and benzodiazepines).

Main Findings: 

This research project focused on the evaluation of, the inhibitory effect of  on-prohibited drugs on the phase II metabolism of prohibited drugs. More specifically, we have considered the effect, on the kinetics of the glucuronation reaction catalyzed by uridinediphosphateglucuronosyltransferases (UGT) of 19-norandrosterone, testosterone, epitestosterone and morphine, of non-prohibited drugs belonging to the classes of antifungals (fluconazole, itraconazole, ketoconazole and miconazole), benzodiazepines (alprazolam, bromazepam, diazepam and triazolam) and non-steroidal anti-inflammatory drugs (diclofenac, ibuprofen, ketoprofen and nimesulide).

In the first part of this project, the in vitro metabolism protocol using either pooled human liver microsomes or recombinant human UGT isoenzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A10, UGT2B4, UGT2B7, UGT2B10, UGT2B15 and UGT2B17) was optimized and validated in order to obtain a good correlation with the metabolism reported in humans. The optimized in vitro protocol was subsequently used to perform the inhibition studies in the presence of non banned medicaments commonly used by athletes (primarily among them antifungals, benzodiazepines and non-steroidal anti-inflammatory drugs).

19-norandrosterone, testosterone, epitestosterone and morphine undergo extensive glucuronidation confirming that the in vitro model developed and optimized in this study provides a good representation of the metabolic glucuronidation reactions in humans, being these data in accordance with information reported by other investigators.  The UGT isoforms principally involved in the phase II metabolism are the UGT2B7 and UGT2B17 for 19-
norandrosterone, the UGT2B15 and UGT2B17 for testosterone, the UGT2B4 and UGT2B7 for epitestosterone and UGT2B7 for morphine.

Concerning the inhibition study, the results obtained showed that the enzymatic activity of the UGT isoforms involved in the glucuronidation of 19-norandrosterone, testosterone, epitestosterone and morphine are extensively altered in the presence of the antifungals ketoconazole and miconazole. Moderate variations were, instead, registered in the presence of itraconazole, triazolam, diclofenac and ibuprofen and no significant modifications were measured in the presence of the others agents studies. The inhibitory effect of diclofenac, ketoconazole, ibuprofen, itraconazole, miconazole and triazolam on the 19-norandrosterone, epitestosterone, testosterone and morphine glucuronide formation is not an irreversible process, but involves competitive and miхed type mechanisms.

These evidences impose that drug-drug interaction has to be taken into account in doping control analysis, to reduce the risk of reporting uncorrected results.

Code: 12D18TM

In 2011 a study reported that of 2185 urine samples from professional athletes (spanning 43 different sport disciplines), ‘active’ consumption (not passive environmental exposure) of nicotine and/or tobacco-related alkaloids were detected in 15% immediately prior to and during sport practice. That same year WADA added nicotine to its Monitoring Program. Taken together, this highlights the extensive and alarming use of nicotine in professional sport.

Inclusion on the Monitoring Program implies nicotine satisfied three criteria of potentially i) enhancing performance, ii) representing a health threat for athletes and, iii) altering the spirit of sport. However, whilst evidence for the latter two criteria features in abundance the former criterion suffers from a lack of evidence or examination. To date, only two studies have investigated the effects of nicotine on sporting/exercise performance, with one showing an endurance improvement of 17% whilst the other found no effect.

However, evidence from similarly acting drugs such as bupropion and methylphenidate suggests that any performance improvements only occur at warmer ambient temperatures, when their action as central stimulants becomes more obvious. These substances also cause body temperature and heart rate to be pushed closer to the limit of safety.

Many competitive sporting events, especially endurance, take place during the summer period or in warm climates. Nicotine is known to reduce skin blood flow. When combined with exercise this raises a serious concern over the safety of its use during exercise/sport in a warm environment, where increasing skin blood flow and sweating are the primary routes of heat loss, possibly placing an athlete at greater risk of developing a heat illness.

This project will extend the limited data on whether nicotine does improve endurance performance, and determine whether at higher ambient temperatures nicotine may lead to developing heat illness, increasing an athlete’s health risk.

Main Findings

Nicotine was added to the 2012 WADA Monitoring Program after it was found that 15% of professional athletes actively consume it. Previous research has demonstrated that transdermal nicotine administration improves cycling time to exhaustion but does not influence 1h cycling time-trial performance in a moderate (~20°C) ambient temperature. However, evidence from similarly acting drugs suggests that any performance improvements only occur at warmer ambient temperatures, when they also cause body temperature and cardiovascular function to be pushed closer to the limit of safety.

Many competitive sporting events take place during the summer period or in warm climates. Nicotine is known to reduce skin blood flow and when combined with exercise this raises a serious concern over the safety of its use during exercise in a warm environment, where increasing skin blood flow and sweating are the primary routes of heat loss, possibly placing an athlete at greater risk of developing a heat illness.

Therefore, this project extended the limited data on whether nicotine does improve endurance performance, and whether at higher ambient temperatures it may lead to developing heat illness, increasing an athlete’s health risk. A final aim was to determine whether measurement of nicotine’s major metabolite, cotinine, should be performed using blood (serum) or urine. Ten trained, male cyclists cycled for 60 min at 55% of their maximum power output followed by a work-dependent time-trial at 75% of their maximum power output (~30 min) on four occasions. Twice trials were conducted in 18°C and twice in 30°C and in each environment they were administered placebo and nicotine via transdermal patch (7 mg·24hr-1) the evening before.

Ambient temperature affected time-trial performance, such that both time to complete the set work was faster (4±1min) and mean power output was higher (24±8W) in 18°C than 30°C. Forearm blood flow (venous occlusion plethysmography) values increased from 18°C to 30°C (6±1ml·100ml·min-1) and decreased from placebo to nicotine (3±1ml·100ml·min-1). Mean weighted (12-site) skin temperature values were lower in 18°C than 30°C (3.7±0.3°C) and higher with nicotine than placebo (0.5±0.2°C). Core (gastro-intestinal) temperature values were lower in 18°C than 30°C (0.4 ± 0.1°C) and higher with nicotine than placebo (0.2±0.1°C). Total sweat loss and % body weight loss were lower in 18°C than 30°C (0.6±0.1L and 0.7±0.2%). Heart rate was lower in 18°C than 30°C (8±1 beats·min-1). Cotinine concentrations immediately before exercise were higher in urine (140 ± 127 ng/ml-1) than serum (31 ± 29 ng/ml-1).

On the basis of these results, i) under these conditions, nicotine is neither ergogenic nor ergolytic, ii) nicotine does increase the risk for heat illness when administered during exercise coupled with heat stress, and iii) anti-doping efforts should concentrate on athlete urine sampling.

Code: 12C18PS

The detectability of erythropoiesis stimulating agent abuse is a major challenge on the way to a doping free sport. Despite existing tests, the detection of ESA abuse has proven to be very difficult. According to the large number of novel ESAs that are marketed around the world, or are in preclinical or clinical trials it seems to be virtually impossible to develop direct tests for each substance. To face this problem, indirect detection strategies have been suggested. During the last decade, molecular biological techniques enabled screening for the human transcriptome.

However, the ´transcriptomic´ approach is still facing a number of problems before robust biomarker can be detected. The problems include biological- and technical bias that are accompanied with the choice of target RNA, sample type and sample processing. Here, we suggest for the first time to utilize micro RNA (miRNA) extracted from mature red blood cells.

Since erythrocytes do not contain nuclei, gene – environment interactions that typically hamper establishing gene signatures for doping are excluded and will not affect miRNA levels. This stability against physiological bias or other factors along with their long life-span makes the erythrocytes the ultimate cell type for addressing the question of whether gene signatures for ESA doping can be found. With this pilot study we want to evaluate the feasibility of erythropoiesis specific miRNA profiling for ESA doping detection in red blood cells.

Main findings

This study intended to examine recombinant human EPO related effects on microRNA (miRNA) profile of red blood cells (RBCs), to discover markers which are indicative for altered erythropoiesis in induced by rhEPO. RBCs, as a homogenous sample material, were chosen to avoid typical bias that can hamper reliable miRNA marker detection, such as shifts in blood cell composition and cell lysis.

A double blind placebo controlled cross over study, including 14 healthy male participants, who received microdoses of recombinant human erythropoietin was conducted at the University of Glasgow, supervised by Prof. Pitsiladis. To discover potential novel biomarkers, which are indicative for rhEPO doping with microdoses, highly purified red blood cell samples from 7 subjects before and after 6 weeks of rhEPO administration were analyzed using Next Generation Sequencing. As a result of 6 weeks rhEPO administration mean haematocrit and haemoglobin concentration values increased significantly (p<0.01) from baseline values (42,57 ± 1,71 to 47,41 ± 2,97 (%) and 14.77 ± 0.61 to 16.36 ± 1,12 (g/dl), respectively). Concomitantly, the percentage of reticulocyte remained unchanged.

Using the Illumina NextSeq500 sequencing system -Illumina, San Diego, CA, USA- for small RNAs, RBC miRNA profiles of 7 subjects were discovered before and after 6 weeks of microdoses rhEPO administration. 13 out of 1,202 discovered mature miRNAs were significantly dysregulated (P<0.05, FDR<0.05). The most dysregulated miRNA, has-miR-150-5p is known to be down-regulated in RBCs and erythropoietic progenitors cells of polycythemia vera patients who suffer from disordered erythropoiesis. This information is concordant with our findings that has-miR-150-5p is down-regulated in healthy subjects who received rhEPO. Presumably, microdoses of rhEPO dysregulate the tightly controlled process of erythropoiesis and can be reflected by screening the RBCs miRNA transcriptome. This makes RBC specific miRNAs valuable biomarker candidates, which could be used in an additive way to generate a predictive marker for rhEPO doping, independent of changes in reticulocyte counts.

Code: 12B3CH

The Harrison lab has been developing a capillary electrophoretic method for the detection of autologous blood transfusions. The method is capable of identifying transfused red blood cells by the difference in their mobilities as compared to non-transfused red blood cells. The development of the test for autologous blood transfusions has been conducted simulating transfusions by mixing aged and fresh blood samples in vials, this study aims to examine the capabilities of the test in detecting autologous transfusions that have taken place in human subjects. This will examine how long after a transfusion the detection can reliably be made. In addition, we seek to determine how extended periods of exercise may impact the reliability of the analysis.

Main Findings: 

The work conducted by the Harrison research group at San Diego State University built on a prior WADA funded grant (09A23CH) to identify the efficacy of capillary electrophoresis (CE) at identifying autologous blood transfusions in endurance athletes. The process takes advantage of the changes in the physical characteristics of the erythrocytes while in storage, a change which is dominated by a reduction in the mean size of the erythrocytes. This change is important as endurance athletes have been shown to have a larger mean size for their erythrocytes than the average population, due to the higher rate of turnover of the cells, and the inherently larger size of new erythrocytes (reticulocytes). 
The CE separation is based on the difference in mobilities of analytes in a buffer solution in a uniform electric field. The mobilities of the analytes, in this case the different erythrocytes, are a function of their charge to size ratio. Consequently, erythrocytes that have been stored will have a different mobility than those that have not been removed from the athlete’s body. The CE separation had been previously shown to be able to identify mixtures of fresh and stored erythrocytes from endurance athletes, when mixed in a vial. The current project aimed to examine how effectively this mixed population could be identified when an actual autologous blood transfusion had taken place. 
In the current study a small group of endurance athletes (cyclists) were recruited to evaluated both the impact of a prolonged period of exercise on the results of the erythrocyte separation, and to evaluate the efficacy of the test to identify actual instances of autologous blood doping.  To this end the tests revealed that prolonged exercise does not negatively influence the outcome of the analysis, specifically it does not result in false positive results for the test. The athlete volunteers for autologous blood transfusions were tested before and after the transfusions. These test results demonstrated variations in the results for each individual, yet each individual’s blood profile varied between the pre and post transfusion periods, indicating that detection of an autologous blood transfusion could be possible. 

Code: 12A16PV

Steroid profiling and analysis of the sterome provide valuable information on the homeostasis of the androgenic system in a doping control setting. New biomarkers based upon minor steroid metabolites showed to provide a substantial advantage to detect misuse with endogenous steroids. Within the framework of the biological passport for steroids, these metabolites have been included in an adaptive model as applied for the T/E ratio resulting in improved detection accuracy. Additionally, a lateral discrimination model was developed that could simplify the evaluation of a steroid profile greatly by condensing it to a single score. The input of this steroidomic model consists of traditional steroid metabolites complemented with new minor steroid metabolites found as good biomarkers. It was proved that the model could successfully differentiate with great sensitivity (more than twice better compared to the mere T/E) between normal and altered steroid profiles which originate from administration studies performed with various endogenous steroids.

However, the steroid profile is not only altered after the administration of endogenous steroids. Multiple confounding factors such as alcohol and medication also contribute to the variation in steroid profiles and might lead to misinterpretations of these new biomarkers and model. Therefore, it is necessary to study the new biomarkers and steroidomic model under circumstances where steroid profiles are not changed by intake of endogenous steroids. In this project, the effect of ethanol, corticosteroids, 5-reducatse inhibitors, hormonal contraceptives, therapeutic exogenous steroids and ACTH on an extended steroid profile will be investigated.

Main Findings

The combination of extended steroid profiling using hydroxylated metabolites and longitudinal following from the biological passport has the potential to very sensitively detect alterations of the excreted androgens and metabolites. These more sensitive individual thresholds calculated by the Bayesian software can also be exceeded whenever confounding factors alter the steroid profile causing false negative atypical findings. Knowledge on these confounders is important to correctly interpret the steroid passport and inform its custodian in the best possible way.

EtG is the preferred alcohol markers to find alterations in primarily T/E, T/Andro, 5α/βAdiol and 7β-OH-DHEA/E. It is proposed to lower that EtG threshold to 20μg/ml for men and 10 μg/ml for women based on changes in sensitive steroid profile marker T/Andro.

Female’s E and PD levels in the luteal phase are highly suppressed by oral contraceptives. The T/E ratio becomes more stable with use of oral contraceptives whereas Andro/Etio slightly increases. The ratios of DHEA (metabolites) over E increase mid-cycle while using OC whereas this they tend to decreased without contraceptives.

After ACTH use, adrenal androgens DHEA and Adion in females increased and E maximally increased to 10 times normal concentrations. The T/E ratio showed a significant reduction in women and a small reduction in men. Andro/Etio first slightly increased before showing a 50% reduction. Both parameters might be altered sufficiently in females to trigger atypical results with the adaptive model.

After use of corticosteroids, E concentrations were only suppressed after multiple high doses which also cause prolonged suppression of the endogenous corticosteroids. In steroid profiles, Andro values showed a decline affecting Andro/Etio similarly. DHEA and its metabolites presented relatively large decreasing trends to 60h after intake. With respect to the steroidomic model, no steroid profile changes were sufficient to alter abnormal steroid profile scores to exceed their threshold.

Mesterolone, as a model for exogenous anabolic steroids was used to investigate the general influence of a small amounts of exogenous steroids onto the steroid profile. For single small doses of non-endogenous steroids, it can be concluded that these do not trigger any alteration in the steroid profile, steroid passport or steroidomic model.

5α-reductase inhibitors like finasteride has a big impact on the steroid profile. Although the T/E ratio is unchanged, the effect on steroid profiles occurs downstream the metabolic pathway where 5α/β-Adiol and Andro/Etio show larger suppressions to 5-20% of their original values. Changing Andro concentrations also result in altered degradation parameters with 5α-Aadion/Andro almost reaching the WADA threshold for invalid steroid profiles.

Code: 12A3JW 

Testosterone, an endogenous steroid banned by the World Anti-Doping Agency, is still one of the most widely used performance-enhancing drugs in the athletic community. Isotope ratio mass spectrometry (IRMS) is a commendatory technology used to detect urinary endogenous steroids by determination of the carbon isotope ratio (13C value). Recent investigation revealed that 13C labeled testosterone standard could be used to regulate the 13C value of testosterone products and lead to the false negative results in doping test. With increasing availability of the manipulated testosterone, significant efforts are needed by anti-doping authorities to develop methods to detect its abuse. 
The aim of this study is to establish method for the detection of testosterone manipulated with 13C labeled standards.  The key difference in structure between manipulated testosterone and non-manipulated testosterone is that certain amount of C-3 or/and C-4 are labeled by 13C in manipulated product which makes significant positive contribution to 13C value. In this study, chemical reactions will be explored to remove C-3 or C-4. For the manipulated testosterone, the 13C value of resultant of the reaction would be more negative than that of the reactant. By comparing the 13C values of reactant and resultant, the manipulated T could be detected. 

Main Findings: 

13C labeled standards can be used to control the δ13C value and produce manipulated testosterone (T) which cannot be detected by the current isotope ratio mass spectrometry (IRMS) method. The aim of this study is to establish a method for the detection of T manipulated with 13C labeled standards. A method was explored to remove the 13C labeled atom at C-3 in androsterone (Andro), the metabolite of T in urine, to produce the resultant (A-nor-5α-androstane-2,17-dione, ANAD). The difference in δ13C values between Andro and ANAD (∆δ13CAndro-ANAD, ‰) would change significantly in case manipulated T is abused. After administration of T manipulated with 13C labeled standards, urine samples were collected from volunteers and analyzed with the established method. Based on the population reference, the cut-off value of ∆δ13CAndro-ANAD for positive result was assessed. The developed method could be used to detect T manipulated with 3-13C labeled standards. The study results have been published in Analytica Chimica Acta (DOI: 10.1016/j.aca.2014.09.007).

Code: 12A13OP 

Availability of reference materials is crucial in doping control since they are needed to confirm adverse analytical findings and to develop quantitative methods.  Most of the analytes included in doping control routine methods are phase I metabolites released to urine after enzymatic hydrolysis. However, the direct detection of phase II metabolites by LCMS/ MS is a common trend in doping control analysis. Therefore, the synthesis of phase II metabolites will become more important for the field in the next years. 
In previous WADA funded projects, our research group has identified several phase II metabolites which are useful for the detection of the misuse of several anabolic steroids. Among them, preliminary results indicate that 6β-hydroxyandrosterone glucuronide and 6βhydroxyetiocholanolone glucuronide can improve the detection of testosterone misuse. Several sulphate metabolites like epiboldenone sulphate or 17β-methyl-5β-androstane-3α,17α-diol 3αsulphate can help in the detection of boldenone and methyltestosterone misuse. The structure of these metabolites has been confirmed by their synthesis at small scale. This synthesis at small scale was necessary because none of the detected metabolites is commercially available. 
Larger amounts of the metabolites are needed for their complete characterization by NMR techniques. Besides, the availability of these larger amounts would also allow for the development of analytical approaches able to detect and confirm their presence in urine samples. 
Therefore, the goals of this project are (i) to synthesize at a larger scale several phase II metabolites previously identified as useful for the doping control field (6β-hydroxyandrosterone glucuronide, 6β-hydroxyetiocholanolone glucuronide, epiboldenone sulphate, 17α-methyl-5βandrostane-3α,17β-diol 3α-sulphate, 17β-methyl-5β-androstane-3α,17α-diol 3α-sulphate and 17βmethyl-5α-androstane-3α,17α-diol 3α-sulphate), (ii) to fully characterize them by well established techniques (NMR spectroscopy, mass spectrometry), (iii) to study their stability and (iv) to develop analytical approaches to quantify conjugated metabolites of testosterone (6βhydroxyandrosterone glucuronide, 6β-hydroxyetiocholanolone glucuronide). The synthetized standards will be distributed among WADA accredited laboratories. 

Main Findings: 

Availability of reference materials is crucial in doping control since they are needed to confirm adverse analytical findings and to develop quantitative methods. Most of the analytes included in doping control routine methods are phase I metabolites released to urine after enzymatic hydrolysis. However, the direct detection of phase II metabolites by LC-MS/MS is a common trend in doping control analysis. Therefore, the synthesis of phase II metabolites will become more important for the field in the next years.In previous WADA funded projects, our research group has identified several phase II metabolites which are useful for the detection of the misuse of several anabolic steroids. Among them, preliminary results indicate that 6β-hydroxyandrosterone glucuronide and 6β-hydroxyetiocholanolone glucuronide can improve the detection of testosterone misuse. Several sulfate metabolites like epiboldenone sulfate or 17β-methyl-5β-androstane- 3α,17α-diol 3α-sulfate can help in the detection of boldenone and methyltestosterone misuse. The structure of these metabolites has been confirmed by their synthesis at small scale since none of the detected metabolites is commercially available.

This project aimed to (i) synthesize selected phase II metabolites at large scale, (ii) characterize these metabolites (iii) develop analytical methodology for the quantitation of conjugated metabolites of testosterone by LC-MS/MS and (iv) distribute of the synthesized material among WADA accredited laboratories.

The main goals of this project have been reached. Firstly, we have synthesized at a larger scale 6β-hydroxyandrosterone glucuronide, 6β-hydroxyetiocholanolone glucuronide, epiboldenone sulfate, 17α-methyl-5βandrostane-3α,17β diol 3α-sulfate, 17β-methyl-5α-androstane-3α,17α-diol 3α-sulfate and 17βα-methyl-5α-androstane-3α,17α-diol 3α-sulfate. Additionally, we have fully characterized these metabolites by well established techniques like NMR spectroscopy and mass spectrometry. We have developed and validated an analytical method based on LC-MS/MS for the quantification of glucuronide metabolites of testosterone. Finally, we have distributed the synthesized sulfates among WADA accredited laboratories.

Code: 12D11VB 

No studies have examined the influence of factors such as exercise and dehydration on the pharmacokinetic profile of asthmatic drugs (beta2-agonists). Exercise may modify the metabolism and excretion of drugs, and sweat loss during exercise can result in dehydration, both affecting the urine concentration of the drug. Furthermore, most current pharmacokinetic studies of beta2-agonists have been performed in non-athlete European subjects, and are therefore not representative for neither athletes nor for the most of the human population not being Europeans. 
During the recent years WADA have changed the restriction towards beta2-agonists with the introduction of urinary thresholds instead of “therapeutic use exceptions” for the commonly used asthmatic drugs salbutamol, salmeterol and formoterol. A large number of studies have investigated the pharmacokinetic profile of these substances, providing support for the urinary threshold of 1000 ng/mL for salbutamol and of 30 ng/mL for formoterol. However, the background evidence for supporting these thresholds is to some degree still inadequate. Humans from various ethnical backgrounds respond different to drugs. 
The genetic variations and impact of exercise and fluid intake are of crucial relevance when it comes to determination of urinary thresholds on the prohibited list. Today, little are known of the pharmacokinetic and pharmacodynamic profile of asthmatic drugs in response to exercise or of genetic variations. It is not unreasonable to assume that therapeutic use of asthmatic medication could result in urine concentrations above the urinary threshold limit of salbutamol, and hence a false positive doping test. If this is the case, the current threshold on the prohibited list may be too low and should be changed. 
The purpose of this study is to examine the pharmacokinetic and pharmacodynamic profile of inhaled beta2-agonists in athletes’ blood and urine during exercise and dehydration, as well as the variations between ethnical groups.

Main Findings: 

The purpose of the research proposal was to investigate the influence of exercise and deshydration on the pharmacokinetics of therapeutic inhaled salbutamol and terbutaline in healthy well-trained men. In study I, we investigated pharmacokinetics of salbutamol after inhalation of the maximal WADA-permitted dose (8×200 μg) during three conditions: exercise (EX), exercise+dehydration (EXD) and rest (R). Exercise consisted of 75 min cycling at 60% of VO2max and a 20-km time-trial. Fluid intake was 2300, 270, and 1100 mL during EX, EXD, and R, respectively. The 2016 WADA decision limit (1200 ng/mL) for salbutamol was exceeded in 23, 31, and 10% of the urine samples during EX, EXD and R, respectively, when unadjusted for USG. When adjusted for USG, the corresponding percentages fell to 21, 15, and 8%. During EXD, mean urine concentrations of salbutamol exceeded (1325±599 ng/mL) the decision limit 4 h after administration when unadjusted for USG. Our data from study I demonstrated a high risk of AAFs in urine samples of salbutamol the first 6 hours after inhalation during EXD and EX with 28 and 20% samples being above the threshold limit for salbutamol, respectively. Adjustment of samples to a USG of 1.02 g/mL reduced the amount of false positive AFFs. Nine of 13 subjects had urine samples that exceeded the decision limit, hence being false positive. In conclusion, exercise and dehydration affect urine concentrations of salbutamol and increase the risk of Adverse Analytical Findings in samples collected after inhalation of that maximal permitted (1600 μg) for salbutamol. This should be taken into account when evaluating doping cases of salbutamol. 
In study II, we investigated pharmacokinetics of terbutaline after inhalation of 8×500 μg as a single dose during three conditions: Exercise in hot ambient conditions (30-35°C)(EXH), exercise in normal ambient conditions (20-25°C)(EX), and rest (20-25°C)(R). Exercise consisted of 130 min at various intensities. When unadjusted for USG, urine concentrations of terbutaline after 4 h were different in the order EXH>EX>R (P≤0.01). When unadjusted for USG, urine concentrations of terbutaline were 299±151 ng/mL higher (P≤0.001) after 4 h compared to adjusted concentrations in EXH. In conclusion, exercise in hot ambient conditions results in higher urine concentrations of terbutaline. This should be taken into account when evaluating doping cases of terbutaline. 
Our observations from study I & II clearly indicate that dehydration due to insufficient fluid intake or exercise in hot ambient conditions increases urine concentrations of beta2-agonists in urine samples that are unadjusted for USG. While adjustment of urine samples for USG also has some limitations, the present findings suggest that it may be relevant to consider USG of beta2-agonist urine samples in doping control.

Code: 12A5CG

The number of peptide hormones, growth hormones and related substances which are required to be tested for are consistently increasing. The main catalyst for this fact is the marketing of products which have had limited data published in medical journals and are then produced and sold via internet websites. This makes them easily accessible and a concern for fair play in sport as well as the health of the athlete as the products may not have therapeutic use approval in humans and can be of poor quality.

In general the analysis of peptide hormones has been addressed one by one with many of the techniques requiring large volumes of urine and complicated extraction protocols. Recently several publications have multiplex methods to include a selection of peptides of interest to antidoping laboratories. These methods have involved either a direct analysis of urine samples for compounds expected at high concentrations or used antibodies for purification to enable lower detection limits.

Particular areas have been identified as playing an important role for the development of a multi-residue analysis technique; extraction/concentration including the use of two dimensional (2D) liquid chromatography, availability of standards and metabolites of peptide hormones. The extraction/concentration of the samples will be examined include solid phase extraction (cartridges/pipette tips), magnetic beads with antibodies, molecular weight cut-off filtration and 2D liquid chromatography. Many of the peptides which are sold over the internet are not available from suppliers of standard materials which makes it more difficult for laboratories to provide testing methodologies. This project includes making available standards suitable for laboratories to develop method with for testing procedures. An in vitro enzyme assay will also be setup with kidney microsomes to be applied to new peptide based drugs to determine the metabolites that are likely to be found in urine.

Main Findings

For over a decade the Australian Sports Drug Testing Laboratory (ASDTL) has been analyzing black market seizures for the Australian Border Force. Many different peptide hormone products have been identified and include Gonadotropin Releasing Hormone (GnRH) and analogues (eg. triptorelin), Growth Hormone Releasing Hormone (GHRH) and analogues (eg. CJC-1295), Growth Hormone Secretagogues (GHS) and mimetics (eg. ipamorelin) and Growth Hormone Releasing Peptides (eg. GHRP-6). Many of these substances continue to be confiscated at the border which means the risk of abuse by athletes in Australia, as well as other in countries around the world remains. The detection in bodily matrices is important, not only for ensuring fairness in sport but also ensuring the health and wellbeing of athletes.

As instrument sensitivity improved, publications were emerging outlining ‘dilute and shoot’ methods for small molecules. Given the difficulties encountered using SPE with the increasing list of peptides being analysed, a ‘dilute and shoot’ method was developed and validated where diluted samples were passed through Agilent Captiva ND Lipid plate to remove interferences. This methodology was superseded with the implementation of ionKey/MS systems and SPE sample preparation which can analyse approximately 50 small peptides, metabolites and peptide mimetics. Limits of detection for the majority of sample range between 0.1-0.5 ng/mL with recoveries between 20-99%. The workflow for the small peptide should also allow the addition of conjugated small molecules and the development will be included in future planned work.

The most significant impact of this project has been the production of reference material and the availability of these to WADA Laboratories. Some of the materials are available from commercial providers but the identification and production of new materials in particular metabolites can be considerably more responsive when initiated by a WADA laboratory than a commercial provider. The ASDTL now has available 60 parent and metabolites peptides.