Projets de recherche

Code: 11A25AR 

Anabolic androgenic steroids (AAS) behave differently in the human body. The human organism deals with these compounds differently in respect of uptake, distribution into different organs, metabolism and excretion. We recently demonstrated that ¾ of Oriental people have a severely compromised capacity to excrete testosterone in the urine compared to only 10 % in people from the west. This is a confounder in the doping test program and therefore it is urgent to find new biomarker and approaches. 
In the way towards personalised test programmes, Bayesian inference techniques are known to suit particularly well. Another approach is to verify the origin of testosterone by using IRMS. 
To further improve the new individualised steroid profile passport, we will conduct humanstudies with nandrolone and different doses of testosterone in order to assess the sensitivity and specificity of IRMS and to learn more how administrated AAS are metabolized.

Main Findings:

The urinary testosterone glucuronide to epitestosterone glucuronide (T/E) ratio is a biomarker included in Steroid Module of the Athlete Biological Passport (ABP) which has improved doping tests for steroids. However, the ratio is greatly affected by a genetic deletion polymorphism in the UGT2B17 enzyme which is the major catalyst of testosterone metabolism. Suspect urine doping tests are further analyzed with gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) to determine the origin of the androgen.  
We investigated the sensitivity of the steroidal module and the IRMS analysis in subjects administered with three doses of testosterone enanthate (500, 250, and 125 mg), in relation to the UGT2B17 polymorphism. All subjects carrying the UGT2B17 enzyme reached the traditionally used threshold, a T/E ratio of 4, after all three administered doses, whereas none of the subjects devoid of this enzyme reached a T/E of 4. However, using the ABP and IRMS analysis, all three doses generated a positive result with a high degree of sensitivity. Our results demonstrate that administration of one single dose, as small as 125 mg testosterone enanthate, could be detected with the ABP in combination with IRMS. Since IRMS is sensitive to testosterone doping independent of UGT2B17 genotype, also very small changes in the steroidal passport should be investigated with IRMS.  In our study of the excretion profile of nandrolone and the 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) metabolites after one single i.m. dose of 150 mg nandrolone decanoate (ND) to healthy volunteers we were able to demonstrate that 19-NA is detectable for 9 months in about half of the individuals. This is the first study where GC-C-IRMS analysis has been performed after an i.m administration of ND in a controlled study group. The absolute δ19-NA (‰) was in the same range as observed previously, with values between -29.1 -and -34.7 in 19-NA positive samples confirming the presence of 19-NA of exogenous origin. The use of the well characterized androsterone (A) as an endogenous reference compound or any other compound not modified by exogenous administration is also possible. Here we show that 11-oxoandrostenedione and pregnanediol may also serve as endogenous reference compounds to detect exogenous origin of 19-NA 3-9 months after one single dose of 150 mg ND. Interesting to note, exogenous 19-NA was traced in GC/C/IRMS in a sample where 19-NA was below the decision limit.   
In summary, GC/C/IRMS analysis confirmed the presence of exogenous derived steroids in both our studies, regardless of UGT2B17 genotype, dose, and time, further supporting the strength of using this methodology. 

Code: 11B8CG

Luteinizing hormone (LH) is a naturally occurring hormone which is secreted by the pituitary gland. In males LH stimulates testosterone production by the testes. Recent research has proven that the monitoring of LH is a valuable tool for both the detection of anabolic steroid abuse; where suppression of LH is observed, and gonadotrophin releasing hormone abuse, where elevation of LH levels is observed. The ability to use LH as a marker for the detection of the abuse of other doping substances is limited by the currently available testing techniques. There are numerous commercially available immunoassays but these are only marketed for the testing of serum samples. The use of the available immunoassays for monitoring of LH in urine needs to be carefully cross validated for comparison to ensure suitably of the results for anti doping.

Furthermore to better understand why there are such differences in the concentrations detected by different immunoassays it is proposed to purify and sequence from urine both naturally present LH as well as from recombinant LH excretion studies. The proposed research will harmonize the measurement of LH in urine for anti doping as well as gain a better understanding of the measurand which will lead to better detection methodologies.

Main Findings: 

The Immulite and Delfia assay are able to detect LH in urine even after extended periods of storage at 40°C, 21°C, 4°C and -20°C. The results for the Delfia stability study reflect what was observed for the Immulite stability study, that LH is unstable at room temperature but although it is also unstable when frozen, quantifiable amounts are still able to be detected with the Delfia assay and the Immulite assay. Statistical analysis of 1000 athlete samples gave correlation factors for both the Immulite and the Delfia, enabling a more accurate comparison of results between the two assays. This will be particularly useful to the anti-doping community as different laboratories use only one or the other of the assays for detecting LH in urine. For anti-doping purposes urine is stored frozen which is where urinary endogenous LH is degrading. Sequencing of the beta subunit of LH revealed the C-terminal and N-terminal of the protein are still largely intact meaning that changes to the protein chain must be occurring within the protein backbone. The tertiary structure of LH may be allowing certain areas to more readily degrade due to turns and disulphide bond placements in the structure as well as the influence of post translational modifications such as the single N-linked glycan on the beta subunit.

Code: 11D5VB

Elite athletes have a high prevalence of asthma and asthma-like symptoms as well as a high use of anti-asthmatic therapy. Salbutamol and salmeterol, has been removed from the prohibited list and these drugs can now be used more freely, also by athletes with asthma-like symptoms only. Performance enhancing effects of beta2-agonists are, though, to some degree contradictory, and the scientific problems concerning beta2-agonists are as follows:

1. A recent systemic review by Pluim et al (2011), they points out that the current literature concerning possible performance enhancing effects of systemic beta2-agonists is weak and calls for future studies with the use of more reliable, valid and sensitive performance protocols.

2. Furthermore, Pluim et al (2011) showed that the training level of the participants in the salbutamol studies have been low to moderate, which is not representative for elite athletes.

3. In another recent review, Collomp et al (2010), reports that no studies, has thoroughly investigated the ergogenic effects of terbutaline after oral administration, neither on exercise performance nor on metabolic parameters and body composition. Hypothesis:

Beta2-agonists have acute and chronic intracellular actions on skeletal muscle in animal studies, which could take place in human skeletal muscles as well, and by that induce 1) muscle strength and performance, and 2) increase volume of the muscles.

The therapeutic doses used in most former studies might have been too low to induce performance-enhancing action, whereas multi-pharmacy with the use of maximal allowed doses of each of the beta2-agonists might have additive effect. Acute use of mixed Beta2-agonists may enhance exercise performance in elite athletes.

Aim:

The aim is therefore to investigate acute, as well as, chronic effects of daily beta2-agonists use on skeletal muscle hypertrophy, muscle fiber characteristics, intracellular adaptations and exercise performance in healthy trained and elite trained athletes.

Main Findings

The primary purpose of the project was to investigate acute and chronic effects of beta2-agonists in skeletal muscle and on performance in healthy trained men. We observed that acute combined inhalation of salbutamol, salmeterol and formoterol, within the current therapeutic dosing limits, enhanced arm ergometer performance and muscle strength, but not swim endurance performance, in elite swimmers. Furthermore, we observed that acute systemic beta2-agonist treatment with terbutaline augmented ion handling of skeletal muscle, which was associated with an enhancement in knee-extensor exercise performance. We also observed that acute systemic beta2-agonist treatment with salbutamol modified expression of genes involved in growth of skeletal muscle in recovery from resistance exercise. When administered chronically, treatment with terbutaline and salbutamol augmented the hypertrophic response to 4 and 11 weeks of resistance training, respectively. However, the larger gains in muscle mass induced by these beta2-agonists did not translate into any superior improvements in muscle strength compared to resistance training with placebo. In fact, chronic beta2-agonist treatment repressed maximal oxygen consumption (V̇O2max) relative to lean body mass. Muscle fiber-type distribution shifted towards a fast-twitch phenotype with salbutamol treatment, while no significant change was observed for terbutaline. In conclusion, our observations support the restriction towards systemic administration of beta2-agonists on the prohibited list. Our data also suggest that a therapeutic dosing limit should be introduced for terbutaline. Combined inhalation of several beta2-agonists may also be of concern, at least during competitions of short and intense duration.

Code: 11B15MU 

Alms of our research are a) methodology development for comprehensive differentiation of endogenous and exogenous hCG, LH and ESA by glycane profiling, b) construction of lectin-carbohydrate Interaction database that allows rapid identification of WADA prohibited glycoprotein hormones, and c) development of sample preparation procedure to allow detection of recombinant glycoprotein hormones by normal immune-assays currently equipped in WADA accredited laboratories. Result verification by second lmmuno assay is necessary when commercial immuno assay kit is used for doping tests, but the verification is not always easy because hCG is extensively metabolized or degraded in vivo. Misuse of biosimilar EPO having different isoform compositions from reference EPO became known since 2007 and caused sometimes in identification difficulties. A sole human cell derived EPO (Dynepo) was withdrawn fromthe market in 2007, and currently, commercial glycoprotein hormones are largely manufactured from CHO cell line. Many of other glycoprotein hormones such as hCG and LH from human has been replaced by the recombinant products. It is reported that CHO cells do not normally express siatyl-alpha2-6 transferase. We have analyzed sialyl- alpha2-6-linked hexose of glycoprotein hormones by the reactivity with 45 array of lectins, and found that human origin EPO and hCG has Sialyl- afpha2- 6-linked galactose/galactosamine residues but CHO-derived glycoprotein hormones are lacking this moiety. Thus, origins of glycoprotein hormones were successfully identified. In this project, we plan to construct stereo-specific sample preparation procedure using lectin beads/columns, gathering lectin­carbohydrate spectra by analyzing LH, hCG and ESA from human and CHO-cell line, and development of antibody assisted lectin micro array test system to identify recombinant glycoprotein hormones.

Main Findings: 

Our results of lectin-glycan interaction monitoring confirmed that origin dependent difference of human glycoprotein hormones, and genetically manufactured corresponding glycoprotein preparation arises not from gene expression but from the posttranslational modification process. Synthesis of core peptide is genetically controlled but the addition of 0-and-N-Linked glucan to the core peptide depends on the available sialyl transferase (ST) and the substrate carbohydrates in the culture medium. Use of SSA, SNA or TJA-1 lectin for isolation of endogenous and exogenous glycoprotein by capturing Sialya2-6Gal/GalNac as the tag carbohydrate allows origin dpendent separation of ESAs and Gonadotropins. Hormones in isolated recombinant or human fraction are to be asssayed by means of normal immunoassays just for detection. By this configuration, both peptide and carbohydrate moieties of the target compounds are to berecognized, thus, highly specific lectin-antibody identification can be achieved. Because overall results of lectin fractionation coupled with immunoassay shows the origin dependent difference of glycans, the positive finding of glycoproteins in the fraction 1 and 2 can be considered as adverse analytical finding regardless of the hormone concentration. The results represented a possibility to establish high throughput origin specific screening of the glycoprotein hormones by lectin column fractionation followed by normal sandwich immuno assay kits or immuno assay instruments

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