Projets de recherche

Code: 10D4GG

Analytical attempts to monitor the application of testosterone doping -constituting as much as 66% of the all analytical findings for anabolic agents - focus mainly on the profiling of testosterone, its precursors and metabolites in urine samples, especially but not limited to the ratio of testosterone and epitestosterone. The establishment of reference ranges including a large variety of endogenous steroids shall contribute to a better detection of abnormal concentrations or ratios.

Urine as a sample matrix for investigating doping practices collects substances which have already passed the body, being like a waste bin their final destination and a remote collection matrix relating to the application of doping substances. Especially in the case of endogenous substances like steroids the influence of metabolism and excretion is levelling possible markers.

Leveling effects are expected to be smaller in serum samples. Especially in the case of parenteral application, normally having a lower amount of substance to enter the body due to the fact, that the liver passage represents no barrier and first pass metabolism, serum is expected to yield a better detection potential. Testosterone esters, the pharmaceutical form of testosterone applied within the frame of this project, besides unmodified testosterone, can be detected in serum samples, whereas they are undetectable in urine.

Due to the implementation of blood profiling as well as the detection of substances with minimal renal passage the access to blood and serum samples grows to get common practice and increasing importance in doping control. Steroid profiling is part of the concept of the athlete’s passport.

The aim of the proposed project is to target unique markers for parenteral application of testosterone preparations. To reach this goal three different parenteral routes of testosterone application are monitored. In addition a comparison to an oral application of a testosterone ester is done.

Main Findings

The aim of the project was to target endogenous serum markers for application of testosterone preparations. In a clinical study, endogenous hormones in serum were monitored after the application of four different testosterone formulations; gel for transdermal delivery, transdermal patch, intramuscular injection, and oral tablets.

The analytes testosterone (T), epitestosterone (EpiT), androsterone (A), etiocholanolone (Etio), dihydrotestosterone (DHT), androstenedione (Adione), 5α-androstane-3α,17β-diol 5β-androstane-3α,17β-diol(5β-diol), dehydroepiandrosterone 17-hydroxyprogesterone(17-OH-Prog),progesterone (Prog), luteinizing hormone (LH), follicle stimulating hormone (FSH) and sex-hormone-binding globulin (SHBG) were included in the project. The samples were assayed for LH, FSH, SHBG, and Prog using immunological methods. For all other analytes, a sensitive method was developed employing liquid-liquid extraction (LLE), derivatization, and liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS).

The study was open and randomized with no blinding procedure. It was divided into two periods during which each participant, randomly assigned, received an oral formulation in one of the periods, and a parenteral application (patch, gel or intramuscular injection) in the other period. All together 12 volunteers (men) participated in the study, and serum samples were collected for 4 weeks after drug application.

Changes in hormone serum concentrations and ratios were assessed using Wilcoxon matched pair test and Friedman ANOVA.

The serum concentrations did not significantly change for any of the hormones measured during the study. Furthermore, no significant changes were observed for the ratios T/LH, T/Prog, T/SHBG and DHT/T. A significant increase was, however, observed for the ratio T/17-OH-Prog in subjects treated with testosterone gel, compared to baseline values. In subjects treated with tablets, patch and intramuscular injections, a trend towards increased T/17-OH-Prog ratios was observed, but the increase was not statistically significant. The findings on elevated T/17-OH-Prog ratios are in accordance with previously published studies [1, 2].

Code: 10A17NB

Since the beginning of the fight against doping, urine is considered to be the preferred biological fluid in which to examine the most commonly used performance enhancing compounds. This was an obvious choice because urine acts as a waste bin where xenobiotics are deposited by the organism. The first analytical techniques used in the fight against doping were thus tailored to detect drugs or drug metabolites at concentrations that can be found in urine. The use of this biological fluid, which could be collected non-invasively during anti-doping tests, became prevalent. Samples could be easily prepared from the urine matrix, and the detection range of performance enhancing drugs was such that it was possible to demonstrate their in-competition use to enhance performance.

Nowadays, analytical techniques have significantly improved in terms of sensitivity and specificity. These improvements allow the scientists to detect forbidden drugs at very low concentration and for a longer time period in multiple different matrices. Even if the blood collection is an invasive procedure, the simplicity of the collection protocol is an important aspect to consider for athletes and doping control officers. Furthermore blood is becoming a biological matrix more and more considered within the antidoping and sport organizations and thus the detection in blood of substances included in the WADA Prohibited list is becoming evident.

The aims of this pilot study are to evaluate the sensitivity and specificity detection, in urine and blood, of 15 different compounds that are the most reported as adverse analytical findings by the WADA laboratories. Samples will be collected after a single dose administration to 3 volunteers for each substance. Finally, we will compare two analytical techniques (LC-MSMS and GC-MSMS) to appraise their use in the fight against doping in urine and blood matrices.

Main Findings

During this project, analytical developments for the detection in blood of compounds like the major endogenous and exogenous steroids as well as opioids and clenbuterol were done. The quantification of endogenous steroids linked to testosterone metabolism is well documented and robust in urine matrix. The blood concentrations of these compounds are important also to give another tool to resolve the difficult topics of testosterone and related substances abuse in sports. The future advent of the steroid module of the athlete passport as well as the establishment of reference values for the athlete population was the main motivation of this part of the project. Sample preparation was the main challenge to solve as the extraction of the investigated compound from the blood matrix is totally different than the one used for urine preparation. Different approaches were tested and a simple extraction using a solid support for a liquid extraction (SLE) was selected. Then limit of detection, linearity and first validation data are promising for a near future accomplishment of this part of the project. Collaboration with the London Anti-Doping laboratory is in progress to compare the two different approaches for the quantification of testosterone and epitestosterone in blood. Results are supposed to be available in the following months. The main exogenous steroids were detected in blood by LC-MS/MS in pg/mL ranges. For all the screened substances a LOD of 100 pg/mL was reached and for few specific compounds a 10 pg/mL LOD was obtained. Comparison of the sensitivity and the time for sample preparation bet ween LC-MS/MS and GC-MS/MS revealed that the first one is more suitable and allowed to reach a lower LOD for the exogenous steroids. Opioids quantification in blood was developed and totally validated. Comparison with immunological tests and the GC/MS quantification showed a good correlation indicating that the use of blood for the opioids detection is fit for purpose. Clenbuterol quantification has been developed to evaluate the complementary information that blood could represent in the context of food contamination or doping abuse in case of adverse analytical finding with clenbuterol. Again, first data obtained regarding LOD, linearity and excretion study samples analyses showed that the developed sample preparation and LC-MS/MS analytical tool were adapted to this purpose. This work gave the opportunity to the laboratory to handle with blood matrix for the detection and quantification of forbidden substances. Perspectives are to continue on this way in order to finish the ongoing (LIVE) validation process. Future implementation of blood analyses for the detection of drug of abuse in sports will be of high interest.

Code: 10A21JY

The aim of our project is to prepare metabolites of beta-blockers timolol, bisoprolol, labetalol and carteolol as reference substances to be used in various doping analytical applications. The results of this project will be directly and immediately applicable to the fight against doping in sports. All the above mentioned drugs are included in the World Anti-Doping Agency’s list of prohibited substances.

According to the data available (WADA), there have been many positive doping findings where athletes have been caught using these drugs during recent years. In doping analysis, the analytical data obtained from the sample are compared to the data obtained from a negative and positive reference. According to the WADA’s International Standard for Laboratories and ISO 17025 standard, well-characterized reference materials are recommended to be used as references in the analysis. Availability of well-characterized reference material increases reliability and legal defensibility of confirmation analyses. Furthermore, reference materials could also be used in quality assurance and in the development of new analytical methods.

The work will be carried out in close collaboration with two research organizations: Faculty of Pharmacy of the University of Helsinki and United Medix Laboratories, Helsinki, Finland. The Doping Control Laboratory of the United Medix Laboratories has been IOC accredited laboratory since 1983 and is now accredited by WADA.

Main Findings

Beta blockers are a class of antihypertensive drugs that were originally developed for treatment of angina pectoris. They have multitude of other indications such as coronary artery disease, congestive heart failure, ischemic heart disease and management of cardiac arrhythmias. By using beta blockers athletes can control effects of performance anxiety such as nervousness, hand tremor and high heart rate.

In doping analysis, the analytical data obtained from the sample are compared to the data from a negative and positive reference. According to the WADA’s international laboratory standard and ISO 17025 standard, well-characterized reference materials are recommended to be used as references in the analysis.

The aim of this study was to synthesize phase I metabolites of bisoprolol and timolol, however the synthesis of a timolol metabolite could not be completed. The synthesized bisoprolol metabolite was characterized by NMR and mass spectrometry and compared to the metabolites extracted from urine samples to confirm the structure. Stability studies of the metabolite were also performed. Samples of O-desisopropylbisoprolol were provided to the accredited doping laboratories at the Cologne Workshop for Dope Analytics 2013.

Code: 10C24ZN

Detection of autologous blood transfusion is probably currently the greatest challenge in the doping control. For the reasons of convenience, for its considerable security and the difficulty of detecting it, autologous transfusion has become the method chosen by many athletes to illegally increase their oxygen delivery capacity. This project aims is to study the proteome changes of erythrocytes during storage, prior to (re) infusion of the blood to the same athlete.

For the project were selected proteins associated with the erythrocyte membrane, because they are easily available and have been well characterized in previous studies with methodologies such as 2D electrophoresis, DIGE, liquid chromatography coupled with mass spectrometry, and other combinations of the above.

The studies made so far can be classified broadly as protein-centered or peptide-centered. In the first case, the proteomics approach is based on the separation of membrane proteins from the sample by 2D electrophoresis; in the second case, the most powerful among those described is the isobaric tagging (iTRAQ), which allows quantitative change detection in multiple samples.

The results of our preliminary studies, as well as those available from other authors, identified proteins which can be grouped as follows:

• cytoskeletal proteins,

• transmembrane proteins

• other proteins related to our research findings by other researchers. To continue our study, we consider efforts in two directions:

First, to validate the results obtained, confirm the same with the largest number of subjects,

Second, to verify by independent technical changes detected in the membrane by proteomic methods, we use Western blot and/or flow cytometry. Ultimately, the goal is to develop a reliable test for the analysis of doping by autologous transfusion, using appropriate markers from membrane proteins or the cytoskeleton of erythrocytes, whose levels vary due to storage of blood in standard transfusion conditions.

Main Findings

Autologous blood transfusion doping (ABT) is defined as the transfusion of stored red blood cells (RBCs) from the same individual. The World Anti-Doping Agency (WADA) includes ABT in the List of Prohibited Substances but no official method exists yet to directly detect it.

In this project the focus was directed towards quantification of erythrocyte membrane protein changes after transfusion. Previously described data showed that some proteins translocate from cytosol to the membrane during erythrocyte storage under standard blood banking conditions. Based on this data, we have selected a panel of proteins to be further characterized. The list included peroxiredoxin-2, glyceraldehyde-3-phosphate dehydrogenase, catalase, tropomodulin-1, sorcin and annexin-7. Using immunoblotting we have observed that in general these proteins were already present in fresh blood samples and their levels increased on the membrane of the erythrocyte during in storage. Additionally, when stored RBC concentrates were mixed in vitro with fresh blood simulating a transfusion procedure, an increase of the marker proteins in the mixtures was observed. However, when blood from recently transfused patients was analyzed, there was no a clear trend in the increment or decrement of the marker proteins. On the other hand, we have observed that the training for endurance disciplines as compared to non-endurance disciplines (both in aquatic sports) seem to influence the basal concentrations of some of the marker proteins, especially those linked with the oxidation protection of RBC.

Based on all the above considerations, it is doubtful whether the changes in marker RBC membrane proteins by translocation from cytosol to membrane may afford useful methodology for detecting blood transfusion in antidoping control by analyzing blood collected from a transfused athlete. However, the analysis of a transmembrane protein, glycophorin A, unraveled modifications that modify protein molecular weight during blood bag preparation or storage conditions, suggesting that characteristic changes on erythrocyte surface are occurring. Therefore, the characterization of the time sequence line of these modifications produced on integral membrane proteins could represent a useful approach to detect autologous blood transfusion.

Code: 10D7GW

In 2002 The International Olympic Committee (IOC) established the requirement for athletes 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 policy on inhaled short acting 2-agonists in January, 2009. However, they have since abandoned the requirement of an objective airway challenge for inhaled salbutamol and salmeterol since January, 2010. The inclusion of inhaled 2-agonists is based upon health concerns rather than anti-doping concerns however; evidence is only available for the performance enhancing effect of inhaled short acting 2-agonists in endurance sports at low to moderate doses and based on athletes taking a one off dose of inhaled 2-agonists. The vast majority of this work has been undertaken in Caucasian, male athletes with limited knowledge of the ergogenic effect or pharmacokinetics in females or other races. The current research team currently has a WADA grant investigating one-off high doses of short-acting 2-agonists on performance in endurance running and football specific trials across race and gender. At present there are no investigations examining the chronic use of short or long acting inhaled 2-agonists on performance.

Main Findings

Between 2002 and 2010 The World Anti-Doping Authority (WADA) and The International Olympic Committee (IOC) established the requirement for athletes to present evidence of current asthma, exercise induced bronchoconstriction (EIB) or airway hyper responsiveness (AHR) through the Therapeutic Use Exemption (TUE) process. These regulations, guided by the IOC Medical Commission (IOC-MC), were based on health not doping (performance enhancing) concerns for athletes in light of a marked increase in the notification by athletes for the use of inhaled short acting β2-agonist from 3.7% at the Atlanta Olympic Games, 1996, to 5.7% at the Sydney Olympic Games. Our group along with others have demonstrated that the requirement of demonstrable evidence through the TUE process improves the quality of care for athletes and has improved the diagnostic sensitivity and specificity of asthma, EIB or AHR. Whilst there appears to be no ergogenic effect from acute, single-dose inhaled salbutamol, no study has investigated the impact of chronic inhalation of the WADA daily upper limit of 1600 µg (~16 inhalations of a standard salbutamol inhaler) in association with endurance and strength and power performance. Sixteen trained male athletes provided written consent and agreed to take part in the study (mean + SD: age 20.1 ± 1.6 years; height 179.9 ± 8.2 cm; weight 74.6 ± 9.1 kg). Participants entered into a 6-week, 4 times per week training study having been assigned to one of two groups in a double blind design. Group 1 (n=8) inhaled 4 x100 μg of placebo, via pocket chamber, 4 times per day for 6 weeks (PLA). Group 2 (n=8) inhaled 4 x100 μg of Salbutamol, via pocket chamber, 4 times per day for 6 weeks (SAL). Pre- and post-training endurance, power and strength and body composition was assessed. Results demonstrated an improvement in O2peak; 3 km running time-trial performance; 1 RM bench press and leg press; and peak extension and flexion torque. Body composition remained unchanged across the study period. Of note, no difference in any endurance; strength and power; or body composition measure was noted between Salbutamol and Placebo groups pre-, during, or post-intervention. In conclusion, there was no improvement in endurance, or strength and power performance following the inhalation of 1600 µg of salbutamol per day for six weeks in non-asthmatic males. This would suggest that the current WADA recommendations, which allow athletes to inhale up to 1600 µg per day is sufficient to avoid an ergogenic impact on endurance and, strength and power performance. Future studies in long term use of inhaled salbutamol focusing on strength and power performance with a greater training volume may be required. Data from this study will assist WADA in the implementation of regulations on the use of inhaled short acting β2-agonist and assist in the resolution of contested doping violations.

Code: 10D9MM

Cytochrome P450 (CYP450) enzymes are essential for the metabolism of many drugs. Although this class has more than 50 enzymes, six of them metabolize 90% of known drugs, with the two most significant enzymes being CYP3A4 and CYP2D6. Genetic variability in these enzymes may influence the individual response to commonly prescribed drug classes. Moreover, CYP450 enzymes can be inhibited by several drugs, resulting in clinically significant drug-drug interactions that can cause alteration in metabolic routes of absorption and elimination with consequent pharmacological effect or toxic reactions. The extent to which a CYP450 inhibitor affects the metabolism of a drug depends on different factors such as the dose and the ability of the inhibitor to bind to the enzyme.

In anti-doping testing, the knowledge of the pharmacokinetics of a drug/class of drugs is a key component of the analytical strategies for the detection of banned drug administration. Athletes may intentionally take advantage of CYP450 inhibition by co-administrating different drugs to obtain an alteration of the metabolism of the banned drug(s), making more complicated the detection by the anti-doping laboratories. This project is designed to provide information on the role that inhibitors of the most abundant CYP450 enzymes may have in doping scenarios. Specifically, the metabolic profile of selected banned compounds, with special emphasis on threshold compounds and to compounds that are excreted in urine mainly as CYP450 metabolites will be assessed individually and in the presence of selected CYP inhibition agents in vitro, in order to establish if the co-administration of CYP inhibitors with doping agents could be used by athletes as masking strategy.

Main Findings

This project was designed to investigate the role that inhibitors of the most abundant CYP450 enzymes may play in doping scenarios. The in vitro metabolic profile of some representative banned compounds was investigated, with special emphasis to compounds that are excreted in urine mainly as CYP450 metabolites, specifically evaluating the effect of selected, non-banned drugs that are also CYP450 inhibitors, with the aim of verifying whether the co-administration of CYP inhibitors with doping agents could be used by athletes as a masking strategy.

In the first part of this project the in vitro metabolism protocol using either pooled human liver microsomes or recombinant human CYP450 isoenzymes (CYP3A4, CYP2D6, CYP2C9 and CYP2C19) was optimized and validated with five representative banned compounds (the selective oestrogen receptor modulator toremifene, the anabolic agents stanozolol, methandienone and the glucocorticoids ciclesonide and deflazacort) in order to obtain a good correlation with the metabolism reported in humans. The optimized in vitro model was subsequently utilized in presence of non banned medicaments commonly used by athletes (primarily among them non antifungals, antiacid and antidepressant agents) to investigate their effects on the in vitro metabolic profile and on the activity of the CYP450 isoforms involved in the phase I metabolism of the selected banned agents.

The in vitro model set up in this study showed good correlation with the previously described metabolism in humans. The CYP450 isoforms involved in the phase I metabolism of the selected banned compounds are the CYP2C9, the CYP2C19, the CYP2D6 and the CYP3A4 isoforms.

On the basis of our results i) the co-administration of banned compounds with antifungals or antidepressants could lead to an incorrect interpretation of the analytical results, producing a masking effect based on the alteration of the phase I metabolic pathways; ii) the number of the markers of drug abuse currently selected during routine analyses should be expanded as much as possible to include also the parent compounds and the possible additional metabolites produced by alternative routes; iii) being the inclusion in our normal routine screening method by LC-MS/MS of the CYPs inhibitors considered in this study very straightforward, if so allowed by the WADA rules, a monitoring study on the real occurrence of CYP inhibitors in the urine samples analyzed by the WADA laboratories would markedly enhance the statistical and epidemiological relevance of our in vitro observations.

Code: R09B2JD

In addition to the increasing number of different recombinant erythropoietins (rhEPO) that are present on the market at the present time, peptide-mimetics of these hormones producing identical effects but presenting unrelated molecular structures will be soon commercialized. Due to this difference in structure, such mimetics cannot be detected by the test used for anti-doping control of rhEPO. Specific methods have to be developed.

One of these peptide mimetics, peginesatide (formerly known as Hematide™), produced by the biopharmaceutical company Affymax is now investigated in Phase 3 trials for the potential treatment of anemia associated with chronic kidney disease (CKD). This drug figures on the WADA list of prohibited substances in sport.

Affymax Company is collaborating with WADA in order that anti-doping laboratories have a test to detect Hematide at their disposal.

Affymax has developed different antibodies directed against Hematide and has tested these antibodies in two different methods, ELISA and immunoblot following SDS electrophoresis.

This project was a collaborative study including Affymax and the anti-doping laboratories of Lausanne (Switzerland) and Châtenay-Malabry (France) and was intended to validate the two different methods proposed by Affymax in two different laboratories.

The aim of the project was to investigate, eventually improve and provide a full validation of the two methods in order that they can be used for detection of the drug in blood samples as screening(ELISA) and confirmation (SDS electrophoresis) tests.

Investigations of ELISA and SDS electrophoresis for eventual improvements were especially performed by the Swiss and French laboratories respectively. Validation of both methods was performed by both laboratories.

Main findings

After some modifications of the SDS electrophoresis method, our laboratory has fully validated both this method and the ELISA method as transmitted by the laboratory of Lausanne.

Our investigations have shown that both methods are specific and robust and can thus be applied for anti-doping control of Hematide using either plasma or serum samples.

Their limit of detection are similar (0.1 ng/mL for ELISA and <0.25 ng/mL for SDS electrophoresis). They have been used in a blind test performed on blood samples from a clinical trial and provided by Affymax to our laboratory. Neither false negative nor false positive results have been observed.

The window of detection was 21 to 28 days following an injection of 0.05 mg/kg (IV).

Code: 09A26WS 

Stimulants generally belong to the substances prohibited in Sports. Among these there are several compounds that are easily available as over-the-counter drugs, that may be present in certain foodstuffs, or that may be produced in the human body from precursors in the diet. This gives rise to the possibility of accidental doping offenses on the one hand. But on the other hand, it also enables deceptive athletes to fraudulently relate a positive test result to dietary habits. 
Hence, in case of a positive test for stimulants, it will be most advantageous to be able assign the relevant compounds to defined sources. Also the possibility to exclude certain sources will be most useful. 
Problems like this can be addressed by stable isotope analysis. The elements hydrogen, carbon, nitrogen, and oxygen make up stimulants and related substances. These elements feature more than one isotope, atoms with slightly differing masses.  The ratios of these isotopes depend on the primary source of the molecule. But they also may be changed during biosynthesis or degradation. 
The project aims to exploit the information present in what is often called "isotope fingerprint". By stable isotope analysis we hope to be able to detect accidental application of prohibited substances as well as pretended pleas. 

Main Findings: 

• Methods have been developed for the 13C=12C and 15N=14N analysis of pure and urinary ephedra alkaloids.
• Comparably large amounts of the compounds are required especially for 15N/14N analysis. Valid 15N/14N analysis roughly requires 3 µg of any single ephedra alkaloid compound to be injected into the GC-C-IRMS system.  In practice, this will be rarely achievable, in particular for the most interesting compounds NE and NPE when formed during metabolism.
• The methodology has been developed also to analyze synephrine and octopamine.  Purity and in particular recovery are however far too poor for stable isotope analysis. This has therefore not been tested for synephrine and related compounds.• 15N/14N analysis of ephedra alkaloids is fundamentally not suited for the purposes intended here. First of all, metabolism induces excessive nitrogen isotope fractionation. The fractionation is time dependent.   After already short periods the 15N=14N ratios of the excreted drug will not correspond to that of the administered compound at all. In addition, there seems to be significant variation in the 15N=14N signatures of various drugs and pharmaceuticals.  Eventually, no sufficiently definite 15N=14N isotopic link between drug and metabolite can be established. This would have to be evaluated much differently if valid 15N=14N analysis of ephedrine metabolites was possible.
• 15N/14N isotopic fractionation during metabolism of ephedra alkaloids appears to be inverse. This means that the parent compound becomes 15N depleted. By contrast, “normal” isotope effects result in heavy isotope enrichment of the reagent because the isotopically lighter species tend to react faster. Inverse isotope effects have been rarely observed. This is therefore highly interesting in basic research but beyond the scope of the study.
• If at all, source assignment of urinary ephedra alkaloids can be based on 13C/12C analysis. Fractionation of carbon isotopes appears comparably small. In addition 13C/12C analysis in practice is much less demanding. In respect to ephedra alkaloids and similar compounds the amount required for valid analysis is ca. 20 less as compared to 15N/14N analysis.
• Combined 15N/14N and 13C/12C isotope fingerprinting appears promising for source assignment of unmetabolized ephedrine pharmaceuticals and preparations. This, however, is beyond the scope of the study.

Code: T09E3MT 

The Influence of orally avallable drugs on the productivity of selected genes has been demonstrated manifold In the past. and an endogenoue substance termed AICAR has bean found to be of considerable Interest ln the treatment of the metabolic syndrome associated with type-II diabetes, obesity, etc. The stimulation of fat utillzatlon and the Increased production of mitochondria generated conslderabla concerns whether this substance might be misused in sports also since laboratory rodents demonstrated significantly enhanced endurance performance arter a 4-weaks treabnant with AICAR, and the Worid Anti-Doping Agency has banned Its use since January 2009. Dua to the natural occurrence of AICAR as a (by-)product in purine biosynthesis, a quantitative determination of natural urinary AICAR values In healthy Individuals Is necessary to provide a means to uncover the llllclt administration of this drug, which should increase the renal allmlnatlon and, thus, the urine concentration of AICAR, slgnlflcantly. 
Prellmlnary data have demonstrated that urinary AICAR levels vary consldarably depending on the studied population (athletes, healthy lndlvlduals, vitamin B12 or follc acid daflclent persons, etc.), and AICAR concentrations higher than 7,500 ng/mL ware detected In athletes' doping control samples. Since It can not be excluded that some athletes misuse AICAR already today, urinary AICAR levels of healthy Individuals shall be determined to obtain reliable reference values. Within the planned project. a cohort of approxlmately 500 sport students shall be analyzed for urinary AICAR levels including pre- and post-exercise samples, males and females, as well aa different sport dlsclpllnaa (endurance, strength, and game sporta) to allow a statlstlcal evaluation of the obtained results and enable the consideration of threshold value(s) that are Indicative for AICAR misuse ln sporta. Future projects might further elucldate the option to differentiate endogenous AICAR from the synthetically derived analog by Isotope-ratio mass spectrometry. 

Main Findings: 

The adenosine monophosphate activated protein kinase (AMPK) activator 5-amino-4-imidazolecarboxyamide ribonucleoside (AICAR) was found to significantly enhance the endurance of rodents even under sedentary conditions. Thus, usage of this substance was classified as gene doping and AICAR was added to the list of prohibited substances of the World Anti-Doping Agency (WADA). Due to the endogenous production of AICAR in healthy humans, considerable amounts are present in the circulation and, thus, are excreted into urine. Considering these facts, the present study was initiated to establish reference values of renally cleared AICAR in elite athletes. Therefore a quantitative analytical method by means of isotope-dilution liquid chromatography coupled to tandem mass spectrometry, following a sample preparation consisting of a gentle dilution of native urine, was developed. Doping control samples of 500 healthy volunteers were analysed and AICAR concentrations in urine were determined. The statistical evaluation showed a significantly better distribution (normality for log-transformed values) for creatinine corrected data compared to density correction. Data evaluation of the analysis of 500 urine samples yielded a mean concentration of 863 ng/mL with sd=462 ng/mL (corrected via density) or 552 ng/mg with sd=290 ng/mg (corrected via creatinine). Computing the 99.99% reference intervals values for the creatinine corrected amounts of 3361 ng/mg were obtained and these calculations for the present dataset suggest that amounts of urinary AICAR higher than 3500 ng/mg are not consistent with an endogenous production in healthy humans.

Code: 09A23CH

To facilitate the continued anti-doping efforts it is becoming important to make the best use of the limited analysis resources available for doping detection. The primary tools for doping analysis consist of equipment that requires highly skilled operators and the analyses are often expensive and/or time consuming. As such the number of samples that can be tested for any one event is limited; affording those doping a possibility of not being tested. To overcome this deficiency we propose a method to both increase the amount of testing and focus the high quality testing on those samples most likely to be actual doping cases. 
This will be accomplished through the use of highly rapid capillary electrophoretic separations; capable of identifying key indicators of blood transfusion and other methods of enhancing oxygen delivery in the blood. The benefits of capillary electrophoretic separations are numerous; the high speed of the separations, the capability of having portable instruments and the low cost of operation. Capillary electrophoresis based separations can act as frontline screening systems to identify those blood samples that present signs of doping; flagging them for further analysis, while eliminating the bulk of the clean samples from further required testing. The advantage in the size of the capillary electrophoretic systems is the ability to perform multiple analysis with as little as a single drop of blood from the athlete; a much less invasive and rapid sampling method. 

Main Findings:

The objective of this project is to investigate how effectively capillary electrophoresis (CE) can be applied to the analysis of athlete blood samples for the identification of blood dopants. This work has specifically targeted three blood dopants: autologous blood transfusions, hemoglobin based oxygen carriers (HBOCs), and perfluorocarbon emulsions (PFC). The benefits of working with CE include the ability to perform very rapid separations, and the versatility to handle both molecular samples (i.e. HBOCs) and cellular samples (i.e. red blood cells). Furthermore, the benefits of CE separations can be translated from bench-top instruments to lab-on-a-chip devices, for what is often termed “point of care” analyses. This could be highly advantageous in anti-doping analyses, where the samples could be rapidly tested with instrumentation brought to the site of the athletic competition, likely reducing the delay in detecting those using performance enhancing agents. 
For our work, we have focused on the use of traditional, bench-top, CE instruments, as they provide the greatest flexibility in method development. In investigating the three above mentioned blood dopants we have been able to successfully apply CE to two of the three dopant methods. The one method that has been found to be incompatible with CE analysis were the PFC; we found that their inability to remain suspended in a blood sample lead to difficulties in obtaining reproducible injections into the CE. Furthermore, their significantly greater density than red blood cells (RBCs) allows for their rapid, initial identification simply through centrifugation, rendering CE analysis superfluous. The remaining dopants, HBOCs and autologous blood transfusions, have shown much better success in detection through CE. In this past year our work on the detection of HBOCs, mixed into fresh blood samples in vitro, was published in Electrophoresis (DOI:10.1002/elps.201100506). In that work we were able to detect the presence of HBOCs at concentrations down to 5.5 g/L of whole blood, an amount below a 5% increase in total hemoglobin concentration. The CE method that we have developed for the detection of autologous blood transfusions continues to show great promise. This analysis is based on the different electrophoretic mobilities experienced by RBCs of different sizes. As circulating RBCs in athletes tend to be newer, larger cells, whereas RBCs taken from storage tend to be substantially smaller, we are able to exploit the differences in size and mobility of the RBCs to identify the presence of RBCs from storage.