Projets de recherche

Code: 11C14NL 

MicroRNAs (miRNAs) are small (19 to 25 nucleotides) noncoding transcripts involved in many cellular and physiological mechanisms such as erythropoiesis. Recently, a new class of miRNAs was found in cell-free body fluids such as serum and plasma. These new class of miRNAs are called "circulating miRNAs". Circulating miRNAs have been found as very stable, specific and sensitive biomarkers. Therefore, they could be altered in a specific manner in doping interventions such as erythropoiesis-stimulating agents (ESA). 
First promising results have been obtained from our laboratory related to the utilization of circulating miRNAs to detect ESA abuse. 
After microarray profiling and RT-qPCR analysis, an increase of miR-144 was observed up to 27 days after ESA injection. Interestingly, miR-144 has been reported to be essential in erythropoiesis in human and other organisms. 
In this project, we plan to investigate more in details the pre-analytical and analytical characterization of the use of miR-144 as long-term biomarker for the detection of ESA. Indeed, we plan to test different biological matrix, extraction methods and the stability in order to facilitate the future utilization of miR-144 as indirect biomarker. Moreover, negative population will be tested to study, more in details, background noise and inter-individual variability of miR-144 concentration in plasma to define threshold limit.

Main Findings: 

In this project, different pre-analytical tests have been performed to characterize two selected miRNA. These two miRNAs were potential biomarkers for the detection of ESAs abuse and autologous blood transfusion, respectively. 
Extraction protocol was demonstrated to be the most important step in miRNAs analysis. Standard extraction protocol is based on phenol/chloroform extraction followed by silica columns purification. Although very efficient, the use of these toxic solvents should be handled carefully. To prevent the use of phenol/chloroform and to decrease the cost of analysis, different extraction procedures were tested. Since circulating miRNAs are very stable, extraction protocol based on boiling was possible. Comparison with standard method demonstrated that heat-and-shoot protocol was efficient only for minute amount of plasma. In contrast, with a volume of 100 µl standard method is close to 10 fold times better regarding qPCR signal. Plasma matrix is known to contain reverse transcription and PCR enzyme reaction. Since more inhibitors were present in 100 µl of plasma than 12 µl could explain this observation. Thus, boiling is sufficient to counteract inhibitors of enzyme reaction only in small volume.  
Minimally invasive test such as fingertip prick test possess some advantages. We observed that heat-and-shoot and standard method could be used to extract circulating miRNAs from digital blood. Digital blood collections are beneficial in clinics but this utility in anti-doping is questioned as some athletes preferred standard blood collection to fingertip prick test because of the pressure applied on fingers when they compete or train. In addition, it is difficult to analyse several variable in small amount of plasma and thus, combination of biomarkers are not possible. 
Circulating miRNAs have been demonstrated to be very stable. From our experiment, one of the miRNAs was unstable after heating sample at 60°C with the 3 pool of plasma tested. In contrast, the other one showed a good stability even in extreme conditions. This observation supported the use of the circulating miRNA as an efficient biomarker to detect autologous blood transfusion.

Code: 11A23JM 

The objective of this project is to further strengthen the ability of anti-doping laboratories to unambiguously identify mis-use of the anabolic steroid nandrolone for athletic performance enhancement. This will be achieved through preparation of a reference material that has an accurately known value for the ratio of stable isotopes of carbon in the chemical residue found 
in urine after nandrolone is metabolised by the body. The presence in a urine sample of 19- norandrosterone glucuronide, the main urinary metabolite of nandrolone, is indicative of the use of this prohibited drug. Under certain circumstances 19-NA may be present at low concentrations in samples of human urine for reasons unconnected with doping. To confirm Adverse Analytical Findings (AAFs) for samples containing 19-NA at concentrations between 2 
ng/mL and 10 ng/mL, WADA Technical document TD2010NA requires that carbon isotope ratio analysis be performed to demonstrate that the metabolite has not been produced naturally in the urine. Provided that other criteria are fulfilled, samples with 19-NA concentrations in this range are only 
to be reported as an AAF if the carbon isotope ratio of endogenous androsterone in the sample is greater than 3 per mille (‰) different to that 
of the 19-NA detected. 
In this project a stable solution of 19-NA glucuronide with an accurately assigned value for the carbon isotope ratio of the steroid will be prepared. This can then be added by laboratories to a urine CRM free of 19-NA but containing the endogenous reference compound for this analysis 
(androsterone). The freeze-dried urine CRM MX005, prepared to validate detection of testosterone abuse, has already been assigned a carbon isotope ratio value for androsterone in a previous WADA-funded project and could be used for this purpose. 

Main Findings: 

A certified reference material (CRM) has been prepared in the form of a solution with a metrologically-traceable reference value for the carbon isotope delta value 
(δ13CVPDB) of 19-norandrosterone (19–NA) in its glucuronide conjugate (19–NAG). The CRM has been packaged in 800 amber glass ampoules containing 1 mL of the solution and is now available to WADA-accredited laboratories. It consists of 19-NAG in water containing 20% methanol at a concentration equivalent to 201  ng/mL 19-NA (as the unconjugated steroid).  The certification of the isotope ratio of the 19-NA component of 19-NAG was complicated by the requirement to hydrolyse and remove the glucuronide moiety prior to measuring the isotope ratio of the steroid. The hydrolysis can introduce impurities that must be fully separated from the 19-NA prior to isotope ratio measurement. This was achieved by solvent extraction prior to gas chromatography coupled with combustion isotope ratio mass spectrometry  (GC/C/IRMS). Calibration of the GC/C/IRMS was performed using the isotope reference material CU/USADA 34-1, permitting metrological traceability to the international carbon isotope ratio reference standard VPDB. Potential sources of bias in the reference method including isotopic fractionation during extraction, internal standard selection and reproducibility of measurement were investigated. The homogeneity and stability of the CRM was verified by analysis of randomly selected vials after storage at -80 °C, -20 °C and +40 °C for periods up to 10 months. 
The certified property value (with uncertainty at the 95% level of confidence) for 19-NA in the CRM is provided in the following table: 

The CRM is intended for fortification by end users into urine material known to be free of 19-NA in which the δ13CVPDB value for an endogenous reference compound such as androsterone has been well characterised. It is designed to assist laboratories in validation and quality control of methods for determination of the carbon isotope ratio of 19–NA in urine at concentrations in the range 2 – 15 ng/mL as required by WADA technical documents TD2010DL and TD2014NA. The use of the glucuronide conjugate for fortification into urine will permit full validation of the sample workup in analytical methods for confirmation of the presence of exogenous 19-NA in its most abundant metabolised form, 19-NAG

Code: 11A12CG

Detection of all performance enhancing drugs is essential to help maintain an environment in which drug free athletes can compete fairly. The modern athlete however is becoming more and more advanced in their efforts to avoid detection by anti-doping laboratories. This became evident in the BALCO conspiracy where compounds were made specifically to circumvent detection by laboratories for the athletes to gain an unfair competitive advantage. The current methods available to WADA laboratories are largely based on the detection of known compounds. A method which laboratories can use to detect new and previously unknown compounds - clandestine compounds is required. The project will apply advanced mass spectrometry and computing technology to develop a database for comparison to athlete samples. The comparison of an athlete’s sample to the database will enable the detection of clandestine compounds such as new “designer” steroids. These clandestine compounds which are detected will undergo further analysis for identification purposes and thus will be able to be classified as either a prohibited substance or not. The developed techniques will be made available to all WADA laboratories.

Main Findings

• The software package Expressionist from Genedata is capable of reliably detecting new compounds in a sample by comparing the peaks detected in the sample with all those in a library created from a set of blank samples. Of course if the new substance has the same exact mass (within ±0.005 amu) and lies within the retention time window (± 0.1 minutes) of a compound already in the library then it will not be detected.

• Optimisation of the parameters used for compound detection is a long and slow process owing to the multiple processes involved with each process typically having several adjustable parameters.

• Once optimised, the use of the software is relatively simple. Automatic processing of a single sample takes less than two minutes. Manual checking to determine if the non-annotated peaks detected are indeed not in typical blank samples may take another five minutes.

• The software is capable of processing the large data sets (hundreds of samples) needed to create blank libraries in less than a day.

• The sensitivity of new compound detection depends primarily on the signal strength obtained in the mass spectrometer. Detection levels down to 1 ng/mL have been achieved for some compounds.

• There is a trade-off between the ability to detect new compounds and the desire to have few if any false positives. The more peaks there are in the library the less likely it is that a new compound will be detected but fewer false positives from new blank samples will be produced. The best way of improving this would be if the retention time alignment could be improved to reduce the variability from ± 0.1 minutes to ± 0.05 minutes.

• The ideal of detecting all new compounds has not been achieved due to the desire of not having too many false positives in new blank samples. Having more than a few peaks to manually check in each sample will make use of the software impractical.

• It is unlikely that a laboratory would use the software to check all samples owing to the time involved. It is more likely that it would be used to check suspect or high risk samples. Once a new peak has been detected then further work using other techniques such as MSMS would be needed to assist in identification of the compound responsible for the peak.

Code: 11A8CG

The World Anti-Doping Agency Prohibited List precludes the use of anabolic steroids including nandrolone and boldenone. The metabolites of nandrolone and boldenone can on occasion be found naturally at low concentration in some individuals. Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS) is the preferred method of confirming doping when a substance can both have been produced naturally by the body or through the ingestion of a prohibited substance. The use of GC-C-IRMS is recommended for samples containing metabolites of nandrolone and boldenone in the 2 ng/mL to 10 ng/mL range, as they could have been produced endogenously. GC-C-IRMS relies on the fact that synthetic versions of administered steroids are depleted in carbon-13 compared to those produced de novo. There is concern, however, that custom synthesis may produce carbon-13 enriched substrates likely to circumvent GC-C-IRMS confirmations. Analysis of commercially marketed and illegally derived (“black market”) materials will provide intelligence concerning trends in steroid manufacture. 
Furthermore, hydrogen is an element for which characteristic isotopic signatures may also be determined for steroids of endogenous and synthetic origin. The project proposes to measure the carbon and hydrogen isotope ratios of seized samples of nandrolone and boldenone over the course of a 12-month period to reveal any differences with values derived from legitimate pharmaceutical preparations that are known to be depleted in carbon-13. This project will build on the successful profiling of testosterone preparations (Cawley et al. 2010) by targeting an international sample population provided by WADA-accredited and law enforcement laboratories. GC-Thermal Conversion (TC)-IRMS analysis will be applied here to investigate the potential of hydrogen isotope ratios values to provide improved detection of administered nandrolone or boldenone that would otherwise be considered to be endogenous.

Main Findings: 

Previous studies have reported the use of chemical hydrolysis for the cleavage of testosterone esters. It was proposed that a similar reaction mechanism could be implemented for nandrolone and boldenone, however, the results revealed that a side reaction had occurred resulting in various rearrangement products. The current study presents a novel application of cholesterol esterase for the hydrolysis of steroid esters as an alternative to chemical hydrolysis. 
Additional purification procedures were implemented to prepare legitimate and seized samples for CIR profiling. The validity of the complete method was thoroughly evaluated to ensure that the analysis procedures did not hinder the measurement of δ13C values of the samples. The method presented herein has demonstrated its capacity to determine the δ13C values of nandrolone, testosterone and boldenone esters (n=62) and is an improvement to former chemical methods. Previous methods focused on CIR profiling of only testosterone, whereas the described method allows δ13C profiling for a variety of steroid esters. However, the limiting analytical issue of the low amount of ester that could be hydrolysed in any single experiment only allowed measuring of the δ13C values for nandrolone and boldenone and could not be scaled up for analysis for δ2H. The higher levels of analyte required for δ2H analysis for nandrolone and boldenone also means that there are serious issues with measuring these steroids in urinary samples for δ2H in the low ranges needed (2 ng/mL to 10 ng/mL for nandrolone and 5 ng/mL to 30 ng/mL for boldenone Stable isotope ratio analysis of Nandrolone and Boldenone preparations.  
This research was the first comprehensive δ13C profiling study of synthetic nandrolone and boldenone preparations. The results demonstrate that CIR analysis enables the detection of administered nandrolone and boldenone since available data for current preparations shows δ13C values differ significantly from values that can be considered endogenous. Most importantly for doping control, proof of concept was accomplished. The finding that there were no nandrolone or boldenone preparations in this study were found to possess δ13C values within the endogenous zone strengthens current protocols for the confirmation of anti-doping violations. The results from this study provide intelligence for the field of sports drug testing and will be made available to WADA accredited laboratories.

Code: 11D10DT 

Hair samples are approved as alternative biological matrices in doping control and provide potential access to retrospective long-term information on substance abuse. Nevertheless its application in doping control is restricted to few applications, mainly due to considerably (esp. compared to urine) low target concentrations of xenobiotics. Basic lipophilic compounds are generally preferentially integrated into hair and clenbuterol in particular represents an outstanding candidate for hair incorporation due to its intense melanin-binding. In spite of its low therapeutic dosages, single or occasional therapeutic administrations resulted in positive identification of clenbuterol in hairs. As a consequence of a recent accumulation of low-level findings of clenbuterol in doping controls, hair analyses were suggested to narrow down potential origins. This is apparently a reasonable approach because of the excellent incorporation rate and detection limit. However, the quantitative interpretation of hair analyses is mainly emerged from the field of drugs of abuse and focused on high dosages and long term abuse. 
There is little information on quantitative evaluations of single and/or sub-therapeutic levels.  It’s therefore the aim of this project to collect data on the occurrence of clenbuterol in hair and its statistical significance after low-level incorporation of clenbuterol. From a practical point of view, it is intended to propose threshold values for the quantitative differentiation of therapeutic or abusive applications of clenbuterol and evaluate influencing parameters (hair pigmentation). 

Main Findings: 

Clenbuterol is a well-known 2-agonist, which is banned for use in sports and strictly regulated for the use in livestock industry. Parallel to improvements of clenbuterol detection limits, an increasing number of positive results were found in doping control samples and in samples from residents or travelers from high-risk countries and were often suspected to be caused by the illegal use of clenbuterol, e.g. for cattle fattening. 
In this project asensitive LC-MS/MS method was developed to detect low clenbuterol residues in hair with adetection limit of 0.02 pg/mg. The present study consists of two parts: first, a sub-therapeutic application study and second, a study with volunteers, who were subject of a high contamination risk. 
In both parts hair and urine samples were analyzed. In case of the application study serum was additionally analyzed. For the first part, a total dosage of 30 μg clenbuterol was applied to 20 healthy volunteers on five subsequent days. One month post administration clenbuterol was detected in the proximal hair segment (0-1 cm) in concentrations between 0.43 and 4.76 pg/mg. No statistical differences were detected between gender or hair pigmentation (brown vs. dark brown color). Serum and urine samples were taken 90 min after each applicationand a correlation between dosage, measured clenbuterol concentration and their inter-individualvariance was observed. For the second part, samples of 66 Mexican soccer players were analyzed. In 89 percent of these volunteers, clenbuterol was detectable in their hair at concentrations between 0.02 and 1.90 pg/mg. A comparison of both parts showed no statistical difference between sub-therapeutic application and contamination. In contrast, discrimination from typical abuse of clenbuterol is apparently possible.

Code: 11C8MT 

The enzyme Sirtuin 1 (SIRT1) modulates many metabolic processes in response to low nutrient availability in the organism. It is assumed to play a major role in regulation of many cell-processes such as insulin secretion and cell death. SIRT1 activators such as SRT1720 mimic, by affecting the enzyme SIRT1, a calorie restriction and produce beneficial effects on insulin sensitivity, fatty acid oxidation in skeletal muscles, liver and brown adipose tissue, liponeogenesis in white adipose tissue and mitochondrial biogenesis.   
Therefore medical research focuses on SIRT1 activators for treating metabolic diseases like Diabetes type 2, adiposity and inflammation. At present several SIRT1 activators are testing in phase-I and phase-II clinical studies.  
Concerning a potential misuse in sport, some studies indicate that SIRT1 activators affect the switch of muscle fibers that enhance whole-body energy expenditure resulting in increased endurance capacity and motor skills. Therefore SIRT1 activators might pose a threat to the integrity of sport and represent potential doping substances covered by the newly established category S0 (non-approved substances) among the WADA Prohibited List. Consequently, detection methods for blood and urine concerning the intact drugs and respective metabolic products will be developed, requiring the synthesis of model sirtuin activators, their in vitro metabolism (enabling the characterization of urinary metabolites), and finally the implementation of new target analytes in routine doping controls. 

Main Findings: 

Sirtuin-1 activators represent a class of emerging therapeutics aiming at the treatment of different diseases including type-2 diabetes, obesity, and cancer. Main effects of chosen drug candidates are attributed to mitochondrial biogenesis and transformation of muscle fibers from type-II to type-I, resulting in increased energy expenditure derived from lipids.  
Several substances are in advanced clinical trials with an archetypical representative referred to as SRT-1720. Since the detailed structures of the lead drug candidates are not yet disclosed by pharmaceutical companies, four model compounds derived from SRT-1720 were chemically synthesized and characterized with physic-chemical methods including nuclear magnetic resonance spectroscopy and mass spectrometry. The reference substances were subsequently used to establish detection assays from human plasma using standard conditions and instrumentations available in doping control laboratories. Further, the model compounds were subjected to in vitro metabolism to generate potential target analytes to be analysed from human urine. Due to the common and conserved pharmacophore of all studied substances, comparable metabolic pathways were observed and modifications such as hydroxylation, oxidation, reduction, and eventually conjugation were identified. An assay for the analysis of sirtuin-1 activators from human urine was also established and validated, demonstrating its fitness-for-purpose in sports drug testing programs. The obtained data will facilitate and accelerate the inclusion of new drugs and drug candidates with similar nuclei into routine doping controls and the currently available compounds (e.g. SRT-1720) are already now traceable in plasma and urine using the herein developed methodologies

Code: 11A9RV 

Anabolic androgenic steroids and, among them, testosterone are the most important group of forbidden substances detected in antidoping control analyses. The existing marker to detect testosterone misuse (ratio between testosterone and epitestosterone excreted as glucuronides) present some limitations and it is necessary to develop analytical approaches to improve the detection of testosterone misuse. In the last years, LC-MS systems have demonstrated wide possibilities for the elucidation of new phase I and phase II metabolites and the use of this technology has resulted in the detection of previously unreported metabolites. 
The objective of the project will be the evaluation of testosterone metabolites conjugated with glucuronic acid resistant to hydrolysis with enzymes with -glucuronidase activity, that may include mono, bis and diglucuronides. Open screening methods for the detection of glucuronoconjugated metabolites by LC-MS/MS will be developed. The methods will be applied to samples obtained before and after testosterone administration for the identification of the new metabolites. The structure of the metabolites will be characterized using mass spectrometric techniques. The excretion profiles of the identified metabolites in samples obtained after administration of testosterone by different routes (oral, intramuscular, topic) will be compared with those of other markers of testosterone misuse targeted in conventional screening procedures, in order to evaluate their interest as long-term markers of testosterone misuse.  
Finally, a methodology addressed to the reliable detection of identified metabolites in routine antidoping analysis will be developed. 

Main Findings: 

The objective of the project was to evaluate testosterone metabolites conjugated with glucuronic acid resistant to hydrolysis with β‐glucuronidase enzymes. Two testosterone glucuronide metabolites which remain conjugated in urine after hydrolysis with β‐glucuronidase enzymes were detected (G1 and G2) by liquid chromatography tandem mass spectrometry (LC‐MS/MS) using a neutral loss scan method based on losses of 176, 194, 211 and 229 Da, characteristic of steroids conjugated with glucuronic acid. The metabolites were characterized using mass spectrometric techniques. The structure of the phase I metabolites released after chemical hydrolysis of G1 and G2 was confirmed using reference standards as 3α,6β‐dihydroxy‐5α‐androstan‐17‐one (6β‐hydroxy‐androsterone) and 3α,6β‐dihydroxy‐5β‐androstan‐17‐one (6β‐hydroxy‐etiocholanolone), respectively. 
The hydrolysis in urine of G1 and G2 using different amounts of β‐glucuronidase showed that almost no cleavage of these compounds was achieved in conventional analysis conditions. Therefore, due to their resistance to hydrolysis with β‐glucuronidases, these metabolites have been underestimated in previous works using conventional analysis conditions. In contrast to previously published results, an important increase in G1 and G2 concentrations (between 50‐300 folds) was observed after testosterone undecanoate administration when using direct detection of G1 and G2 by LC‐MS/MS.  The concentrations remained elevated for longer times compared with the current marker (T/E ratio). 
The results show the need to quantify G1 and G2 in samples obtained after administration of testosterone and other endogenous steroids, and in samples from athletes to gather additional information to evaluate the usefulness of G1 and G2 to detect the administration of endogenous steroids and their inclusion in the steroidal module of the athlete´s biological passport for longitudinal monitoring.

Code: 11D8GW 

In 2002 The International Olympic Committee – Medical Committee (IOC-MC) established the requirement for athletes using asthma medication (short acting 2-agonists) to present evidence of current asthma, exercise induced asthma (EIA), exercise-induced bronchoconstriction (EIB) or airway hyperresponsiveness (AHR) through the therapeutic use exemptions (TUE) process. The World Anti-Doping Agency (WADA) introduced the IOC-MC policy on inhaled short acting 2- agonists in January, 2009. In January, 2010 WADA withdrew the requirement for an objective airway challenge for inhaled Salbutamol and Salmeterol. In contrast, other commonly used short acting 2-agonists including Terbutaline remained on the restricted list. 
The inclusion of inhaled 2-agonists on the WADA list is based upon health concerns rather than anti-doping concerns with limited evidence available to support an ergogenic effect. To date, the vast majority of available data has been undertaken with inhaled Salbutamol in Caucasian, male athletes with limited knowledge of the ergogenic effect or pharmacokinetics of other short-acting 2-agonists (i.e. Terbutaline), across gender or ethnicity. 
Accordingly, the proposed study aims to quantify Terbutaline limits in the urine after oral and inhaled routes of administration (Study 1) and investigate the ergogenic effect of inhaled Terbutaline on 5 km running performance (Study 2). Both studies will include male and female participants from Caucasian, Asian and Afro-Caribbean communities. 
The proposed studies will assist in the implementation of regulations on the use of inhaled Terbutaline and assist in the resolution of contested doping violations. Ultimately the project will provide more clarity for the use of inhaled Terbutaline by athletes with various forms of asthma and will result in improved quality of care for the athlete. 

Main Findings: 

Elite athletes have a higher prevalence of Exercise-induced bronchoconstriction (EIB) than the general population. Treatment for EIB and asthma includes inhalation of short-acting β2-agonists which act to reverse the bronchoconstriction of the airways. The majority of athletes treat symptoms of EIB through the use of salbutamol however, other β2-agonists, such as terbutaline, are available which are longer acting and have fewer side-effects. In contrast to salbutamol, salmeterol and formoterol, therapeutic doses of the inhaled short-acting β2-agonist, terbutaline still requires a TUE. Limited research exists examining the ergogenic effect of terbutaline. Accordingly, aim of the present study was to examine the effects of 2 mg and 4 mg inhaled terbutaline on exercise performance during a 3km running time-trial.  
Participants (8 males; age: 24.3 ± 2.4 years; weight: 77.6 ± 8 kg; height: 179.5 ± 4.3 cm, and 8 females; age: 22.4 ± 3 years; weight: 58.6 ± 6 kg; height: 163 ± 9.2 cm) completed three 3km time-trial in a randomised, single blind, repeated measures design with a minimum of 7 days between trials. Prior to the test participants were assigned to one of three groups:  (1) 8 inhalations of non-active inhalant (placebo);
(2) 4 inhalations of non-active inhalant plus 4 inhalations of terbutaline (2mg); or
(3) 8 inhalations of terbutaline (4mg). In addition to performance variables, urine concentrations of terbutaline were measured following each trial. There was no significant difference in completion time between trials in either males or females following 2 mg or 4 mg of inhaled terbutaline. 
There was no difference in urine concentration following either 2 mg inhalation or 4 mg inhalation in males or females. There was a high individual variation in urine concentration of terbutaline with a maximum value of 1250 ng/ml-1 following the inhalation of 4 mg of terbutaline. In conclusion, terbutaline, when taken in therapeutic doses (2 mg or 4 mg), does not improve 3 km running time-trial performance in males or females. Elite athletes have a higher prevalence of Exercise-induced bronchoconstriction (EIB) than the general population. Treatment for EIB and asthma includes inhalation of short-acting β2-agonists which act to reverse the bronchoconstriction of the airways. In contrast to salbutamol, salmeterol and formoterol, therapeutic doses of the inhaled short-acting β2-agonist, terbutaline still requires a TUE. Accordingly, the purpose of the present study was to measure the urine concentrations of terbutaline following single and repeated doses of oral and inhaled terbutaline in Caucasian males, Caucasian females, Afro-Caribbean males and Asian males to allow for comparisons between gender and race. 
Twenty-two male and eight female subjects (8 male Caucasian, 8 female Caucasian, 6 male afro-Caribbean, 6 male Asian) provided written informed consent and were recruited for the study. All participants were free from asthma, EIB and AHR confirmed by no history of disease and a negative eucapnic voluntary hyperpnoea (EVH) challenge. Participants were assigned to one of four groups in a cross-over design: 1. Single Oral Administration (SOA): single dose of 5 mg oral terbutaline (Bricanyl, AstraZeneca, UK),
2. Single Inhaled Administration (SIA): 4 inhalations of 0.5 mg terbutaline (Bricanyl Turbohaler, AstraZeneca, UK) totalling 2mg inhaled;
3. Repeated Inhaled Administration (RIA): repeated doses of 1 mg (2 x 0.5 mg inhalations) of terbutaline at 08:00h, 12:00h, 16:00h and 20:00h for 2 days;
4. Repeated Oral Administration (ROA): repeated doses of 5 mg oral terbutaline at 10:00h and 18:00h for 2 days. Following the final dose of terbutaline during each trial, participants were required to provide urine samples at 1h, 3h, 6h and 12h time-points. Urine samples were frozen at -80OC for later analysis of terbutaline. 
The present study demonstrates that both inhaled and oral terbutaline administration results in the presence of terbutaline in urine. Of note, significant differences in urine concentration of terbutaline following inhaled and oral administration were observed. The study identified upper thresholds following oral and inhaled administration which could be used to identify the use of supra-therapeutic doses of terbutaline. The highest urine concentration of terbutaline 
following inhalation was 1,500 ng.ml-1 and for oral administration was 2,000 ng.ml-1
. Gender differences existed between male and female Caucasians following 
multiple oral administration however; further research including both female Asians and female Afro-Caribbean’s is required to fully elucidate any gender differences. Ethnic differences were identified between male Caucasians and male Asians following single inhaled administration. Future research should also examine urine concentrations following a minimum therapeutic dose of inhaled terbutaline versus oral terbutaline and also establish differences in enantiomers in the urine associated with mode of administration.

Code: 11C25FB 

Blood doping is aimed to illicitly increase the oxygen delivery to tissues to improve sport performance, especially in endurance sports/disciplines. At present, WADA-accredited antidoping laboratories can count on several analytical strategies to detect most if not all the different forms of blood doping: these detection strategies may be either “direct” (aimed to detect the “non endogenous” substances administered to obtain the blood-doping effect), and “indirect (where the longitudinal stability of a panel of hematological and/or biochemical parameters is regularly monitored to spot any significant deviation from the individual physiological ranges). 
To the best of our knowledge, the only “masking strategy” for blood doping that has been considered so far is the practice of “blood dilution”. This strategy is performed before a doping control test, and it is obtained by intravenous infusions of physiologic solutions (with or without the addition of modified polysaccharides, used also as plasma volume expanders). This practice is aimed both to dilute specific target analytes, making therefore more problematic their detection by the WADA laboratories, as well as to reduce the value of some diagnostic haematological parameters that are considered in the framework of longitudinal monitoring of the athletes. 
We postulate that an additional, and in principle very effective, masking strategy, would be the administration of supramolecular structures, and primarily among them phospholipidic liposomes, with the objective to reduce the free concentration of circulating peptides, polypeptides and proteins that are analytical targets of both “direct” and “indirect” methods of detection. 
The present project is aimed to verify the effectiveness of this potential masking strategy, and to develop specific, selective, and suitable analytical methods to ensure its detection by the WADA-accredited laboratories. 

Main Findings: 

The detection, and ideally quantification, of doping agents that still remain “invisible” to the anti-doping laboratories are one of the most urgent challenges for the anti-doping community. To be “invisible”, a doping agent needs to satisfy one or more of the following conditions: (i) to be still unknown; (ii) to be identical to an endogenous substance, (iii) to be present in the biological fluids in a concentration smaller than the limit of detection of the available analytical methods, and/or (iv) to be masked by masking agents that are themselves unknown. This research project has specifically addressed this last point. 
In the last few years, novel and potentially more effective masking agents have been considered in sport doping. Among them, drug delivery systems (DDS) seem to be particularly attractive to cheaters. DDS may indeed be used to alter the absorption, release, distribution and excretion profile of prohibited drugs, making their detection by the WADA-accredited laboratories more problematic. 
In this study we have considered a particular class of prohibited drugs vehiculated by drug delivery systems, and, specifically liposomial encapsulated hemoglobins (LEHs). Basically, LEHs, tha are also defined “hemosomes”, are a new form of hemoglobin-based oxygen carriers (HBOCs) that mimic the oxygen diffusivity of erythrocytes, so that they may be considered “artificial red blood cells” rather than “artificial hemoglobins”. Besides their multiple, potential clinical applications, and their extremely promising therapeutic utility, LEHs could be misused as a sport doping practice, since they can increase the oxygen carrying capacity of blood, with the consequent improvement of sport performance, especially in endurance disciplines. Although LEH are not commercially available yet, their preparation is relatively straightforward, and this may increase the interest of those seeking an “invisible” way to take advantage of “blood doping”. 
The main results of the present research project can be summarized as follows: I. Liposome-encapsulated haemoglobins (LEHs) are stable enough to be illicitly used as performance-enhancing drugs.
II. LEHs are “challenging” to detect with current methods for the initial testing analysis are applied.
III. Direct detection of LEHs in blood can be achieved by targeting the intact liposome-hemoglobin complex by flow cytofluorimetry with double coloring. The performance of the methods is further improved by using coated microbeads.
IV. Indirect detection of the intake of LEHs and/or of other liposome vehiculated drugs can be performed by HPLC-MS/MS using a aqueous normal phase, by monitoring the entire phospholipid and sphingomyelin profile in urine. The above results confirm what already reported in a previous research project also funded by the WADA (project code 09D9FB), i.e. that liposomes could represent an effective form of “doping delivery systems”, with potential masking effects. Efficient detection of LEHs requires upgrade of routine detection methods to identify their intake either in blood (by flow cytofluorimetry based techniques) and/or in urine (by HPLC-MS/MS based techniques).

Code: 11A22OP 

Testosterone misuse is the most detected doping offence in screening analysis by means of the ratio between testosterone and epitestosterone (T/E).

Recently, four testosterone metabolites have been reported in our laboratory after alkaline treatment of the sample. In a previous WADA project (reference 10A16OP), we have shown that the detection of these metabolites and the ratio between them is useful for the detection of testosterone misuse after oral and topic administration. Therefore, the addition of these metabolites into screening methods seems to be a promising complement to T/E.

Besides the cases already tested, the measurement of T/E has also additional limitations like (i) its use in samples with low basal T/E or (ii) it can be affected by external factors like alcohol intake. Most of these external factors are related with the glucuronidation process. Since the basic released metabolites are not glucuronidated, they can be less affected by these factors providing a complementary tool for testosterone misuse detection. The knowledge about the phase II metabolic pathway which is associated with the presence of these metabolites can provide useful information for the antidoping control field. Among other adbantages, knowing the phase II metabolite would theoretically allow for including these metabolites in already existing screening procedures.

Therefore, the goal of this follow-up project is to evaluate of the usefulness of the quantitative detection of the metabolites released after basic treatment for the detection of testosterone misuse. In order to achieve this main goal, the project will be divided in three specific objectives: (i) detection of testosterone misuse in population with low basal T/E values, (ii) study of the effect of several factors which can vary the steroidal profile in the values of these metabolites and  (iii) elucidation of the phase II metabolic pathway related with the occurrence of these metabolites.

Main Findings: 

Recently, four new testosterone metabolites (Δ1-AED, Δ6-AED, Δ6-T and Δ15-AD) have been reported in our laboratory after alkaline treatment of the urine. These metabolites and the ratio between them were found to be useful for the detection of oral testosterone misuse. Therefore, the addition of these metabolites into screening methods seemed to be a promising complement to the steroid profile.

The goal of this follow-up project was to evaluate the usefulness of the detection of these metabolites for doping control. For this purpose, the project was divided in three specific objectives: (i) elucidation of the metabolic pathway responsible of the occurrence of these metabolites, (ii) study of the effect of several factors which can vary the steroidal  profile in the values of these metabolites and (iii) detection of testosterone misuse by these metabolites.

We demonstrated that the studied metabolites are produced from the degradation of cysteine conjugates. The formation of these metabolites implies an unreported metabolic biotransformation: 6,7-dehydrogenation as phase I metabolism followed by conjugation with glutathione and subsequent transformation to cysteine conjugates. The postulated pathway was supported by studies with human hepatocyte cells systems. Analogously to testosterone, this pathway might also be present in other steroids, opening the possibility of targeting additional biomarkers. This fact was confirmed by the detection of 24 boldione metabolites (11 conjugates with cysteine and 13 conjugated with N-acetylcysteine).

The influence of several factors in the excretion of the studied metabolites was evaluated. Degradation, freeze/thaw cycles and infradian variability studies showed moderate variations (below 40%) for these metabolites. UGT2B17 polymorphism does not influence the excretion of cysteinyl compounds whereas the intake of exogenous substances (alcohol or 5α-reductase inhibitors) dramatically affects their excretion. During pregnancy only the excretion profile of Δ1-AED increased. Overall, the presented data describes the stability of the urinary cysteinyl steroids under the influence of many factors, proving their potential as suitable parameters to be included in the steroid profile.

The usefulness of cysteinyl markers for the detection of T im and T gel misuse was studied. Among cysteinyl metabolites Δ1-AED/Δ15-AD was shown to be the best marker after T im allowing the detection in all studied cases. The use of this marker also allowed the detection of T gel misuse in almost all volunteers with T/E around 1. Worse results were observed for the detection of T gel misuse in volunteers with T/E below 0.2. Anyway, the use of cysteinyl markers did not increase the detection capabilities of the current steroid profile questioning their usefulness for doping control analysis.