Projets de recherche

Code: T09E1MB

Research in recent years demonstrated that the analysis of changes in the hGH isoform pattern occurring after administration of recombinant hGH can be used to detect hGH doping. Two pairs of immunoassays based on monoclonal antibodies have been developed, which can be used as two independent tests to measure the relative abundance of the monomeric 22 kD isoform over all other isoforms in a serum sample. Practically, a ratio is calculated between the results from the “rec” assay (employing antibodies preferentially recognizing the 22kDa isoform) and the “pit” assay (employing antibodies recognizing a wide spectrum of pituitary isoforms of GH). Details of the method have been published (Wu et al., 1999; Bidlingmaier et al. 2000, 2001, 2003, 2007, 2009). The method has been implemented in most WADA accredited laboratories and could theoretically be used for detecting cheating athletes.

An important aspect to make test results court proof is the reliability and quality of the normative data underlying the cut-off criteria developed to define an adverse analytical finding. Several studies have been performed to investigate the isoform composition and the respective rec/pit ratios in samples taken at rest and after exercise in healthy subjects, recreational and elite athletes from several sports disciplines and from different ethnic background. The differences found in the rec/pit ratios between the groups were comparably small, and it could be clearly shown that injection of rhGH leads to a strong increase in the ratio exceeding the normal variability.

To further strengthen the scientific evidence for the cut-off, the project proposed here aims to investigate the rec/pit ratio under clinical conditions which might be used as an argument by an athlete to explain an adverse analytical finding (=increased ratio) in front of a court. Investigations of molecular isoforms of hGH under different pathological conditions are scarce. Some papers exist about the 20 kD isoform in acromegaly, when endogenous GH secretion is increased (Murakami 2000, 2004; Tsushima 1999; Boguszewski 1997). The findings were not consistent, pointing to a similar variability in the isoforms in acromegalic patients as in normal subjects. However, the assays used in these papers are completely independent from the isoform assays used in WADA accredited laboratories. So, it remains to be clarified if specific pituitary diseases, but also other conditions where hGH secretion is affected, have an influence on the rec/pit ratios.

In the proposed project, samples from different clinical studies will be investigated. The conditions of interest are:

- early pregnancy (40 samples).

- acromegaly (30 samples)

- other pituitary diseases (non-functioning adenomas, Cushing adenomas etc., 20 samples total)

- diabetes (both, type I and type II, 20 samples total)

The samples will be analyzed by both rec/pit assay combinations (termed “kit1” and “kit2”). The two kits involve different antibodies and are used for screening and confirmation test procedures in WADA accredited laboratories.

Main Findings

From the data obtained during the project it is reasonable to conclude that diabetes and cortisol producing pituitary adenomas (Cushings disease) have no detectable impact upon the GH isoform pattern in serum samples as measured by the rec- and pit-assays used for the differential immunoassay approach.

During pregnancy, where pituitary GH secretion is gradually reduced and placental GH secretion increased with increasing gestational age, the situation is different for kit 1 and 2: Whereas the antibodies used in the rec2- and pit2-assay as well as the pit1-assay apparently are not influenced by the changes in the molecular forms of circulating GH, the presence of GHV seems to reduce the signal obtained from the rec1-assay. Overall, this leads to a remarkable reduction in the rec1/pit1-ratio especially after gestational week 10/11, probably because GHV concentrations reach higher levels. However, it is important that there is no risk of a false positive doping test result arising from this situation: Although for ethical reasons it is impossible to formally test recombinant GH applications in pregnant females, the only possible impact of the phenomenon on a doping test would be false negative test results because of falsely low ratios.

In active acromegaly, there was an intriguing difference between the findings in females and males: Whereas the ratios in female patients were completely normal, the mean ratios in males where somewhat higher than expected. However the samples were collected over several years, and not in a controlled doping test setting. Therefore, results have to be interpreted with caution. Nevertheless - although the vast majority of the ratio results were in the expected range, and even the highest ratios were far below the cut offs used to define an AAF - the observation in males deserves further investigation. It is noteworthy that the highest ratio was observed in a patient with a particular aggressive and big tumor, which was continuously growing and required repeated surgery plus cranial irradiation therapy in addition to medical treatment. Because of the paucity of scientific studies investigating the GH isoform composition in acromegaly it cannot be ruled out a priori that some subtypes of pituitary tumors preferentially synthesize the 22 kD GH isoform. Alternatively, the continuous presence of high circulating GH levels might affect the formation of dimers and multimers of GH in selected patients, thereby potentially leading to altered rec/pit ratios.

However, it is clear that the results presented here come from severely ill patients suffering from an extremely rare, but easily detectable disease because of the dramatic consequences of the pituitary tumors. Even if this is a population of extreme cases, no rec/pit ratio came into the range seen after recombinant GH administration. Therefore, the observation seems to be of some scientific interest but very likely does not represent a real practical problem in doping controls.

Code: 09B7CR

The detection of doping with recombinant peptide and protein hormones (e.g. erythropoietin – Epo, human growth hormone - hGH) is one of the most challenging analytical problems in doping control. The WADA-accredited method for the detection of doping with recombinant human erythropoietins (rhEpo) is based on isoelectric focusing (IEF) in carrier ampholytes. 
Due to the risk of suffering a stroke or heart attack some athletes already admitted (e.g. the triathlet Lisa Huetthaler; published interview) having used anticoagulants (e.g. aspirin) in combination with Epo-doping. Heparin is one of the oldest and cheapest anticoagulants. 
Our preliminary results showed that heparin has an at least threefold destructive effect on the Epo IEF-method, i.e. either (1) the Epo-profile gets completely destroyed or smeared, (2) the NESP-profile gets shifted to the endogenous area, or (3) a negative profile gets shifted to the basic area and thus leads to a false positive result.
The project investigates the effect of different heparin pharmaceuticals (unfractionated heparins,  fractionated “low molecular mass” heparins) on both the Epo IEF-PAGE and SDS/Sarcosyl-PAGE methods and after administration of heparin to humans. Both biological matrices will be studied, urine and blood serum/plasma. Additionally, the capability of Epo immunoaffinity purification on the removal of heparin will be studied. What we already know is that ultrafiltration is not able to remove higher molecular mass heparins from urinary Epo. 
Hence, the effect of a targeted enzymatic degradation of heparins on various Epo IEF and SDS-PAGE profiles (endogenous, recombinant) will be studied. This latter strategy might be a solution to the destructive effect of heparins on Epo analysis. 

Main Findings: 

The detection of doping with recombinant peptide and protein hormones (e.g. erythropoietin - Epo, human growth hormone - hGH) is one of the most challenging analytical problems in doping control. The WADA-accredited method for the detection of doping with recombinant human erythropoietins (rhEpo) is based on isoelectric focusing (IEF) in carrier ampholytes.
Due to the risk of suffering a stroke or heart attack some athletes already admitted (e.g. the triathlete Lisa Hütthaler; published interview) having used anticoagulants (e.g. aspirin) in combination with Epo-doping. Heparin is one of the oldest and cheapest anticoagulants. Our results showed that heparin has an at least threefold destructive effect on the Epo IEF-method, i.e. either (1) the Epo profile gets completely destroyed or smeared, (2) the NESP profile gets shifted towards the endogenous area, or (3) a negative profile gets shifted to the basic area and thus leads to a false positive result. The project investigated the effect of different heparin pharmaceuticals (unfractionated heparins, fractionated “low molecular mass” heparins) on the analysis of endogenous and recombinant epoetins by IEF-PAGE and SDS/SAR-PAGE with spiked samples and after administration of heparin to humans. Both, urine and blood serum were used as sample matrices. It  was demonstrated that Epo immunoaffinity purification is able to remove heparin and prevents its harmful effect on the Epo IEF profile. Alternatively, targeted enzymatic degradation of heparins prior to IEF- or SDS/SAR-PAGE can be used to reverse the destructive effect of heparin. No destructive effects of heparin were observed for SDS- and SAR-PAGE of EPO standards and urine/serum samples (independently of that result, urine and serum samples have to be immunoaffinity purified in order to avoid gel overloading with high abundant proteins)

Code: 09A11GR 

Many new compounds—chemically different drugs—have recently been included in the list of prohibited substances published by the World Anti-Doping Agency. Current applications of HPLC–MS in today’s anti-doping laboratories are based predominantly on the utilization of high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) systems. With these systems, known drugs or metabolic products are determined by measuring precursor/product ion pairs, providing the utmost sensitivity but also restricting the technique to a limited number of compounds.
To overcome this problem, Georgakopoulos and colleagues have recently proposed the use of orthogonal-axis high-resolution time-of-flight mass spectrometry (OTOF), which allows a broader range of doping agents. Significantly improving this methodology, we have evaluated HPLC/in-source CID atmospheric pressure chemical ionization (APCI) orbitrap mass spectrometry with accurate mass measurements for its screening potential for agents with antiestrogenic activity, beta2-agonists, exogenous anabolic steroids, and other anabolic agents to take advantage of its high resolution, sensitivity, and full scan acquisition. Furthermore, we plan to refine significantly this methodology in our proposed study.
The aims of the proposed research study are the following:
Development and optimization of HPLC/ESI–, APCI–, and APPI HRMS (MS/MS) methods with respect to eluent composition, ion source parameters, and fragmentation. The detection limits and specificity of the methods will be compared. A full description and validation as a general screening method based on SPE and HPLC/orbitrap mass spectrometry with different ionization techniques for doping agents, including anabolic steroids, beta2-adrenergic agonists, SARMs, agents with antiestrogenic activity, diuretics, stimulants, beta-blockers, and cannabinoids. Validation of the method will consist of investigation of specificity, analytical
recovery, limit of detection, and repeatability.
Finally, a comparison of the HPLC/orbitrap mass spectrometry and HPLC/MS/MS methods for the qualitative screening of prohibited substances in urine will be performed.

Main Findings: 

1) The Multipole RF Amplitude, Multipole 00, Lens 0, Multipole 1 and Front lens voltages have dramatic effect on the Orbitrap sensitivity.
2) The research results shown that at least 113 compounds(including metabolites) out of 131 can be detected in urine at concentrations corresponding the MRPL by HPLC-APCI/Orbitrap mass spectrometry.
3) It was observed that most diuretics containing double bonded sulfur atoms could not be detected by HPLC–APCI/Orbitrap mass spectrometry because they did not ionize under APCI conditions used.
4) The proposed HPLC–ESI/Orbitrap mass spectrometry with high-pH mobile phase was demonstrated to be effective for comprehensive screening of banned substances and their metabolites.
5) Ammonium hydroxide (pH=10.3) was found to be necessary for the sensitive detection of anabolic agents and other doping substances in terms of the S/N ratio.
6) Contrary to common expectations, high-pH mobile phase do not affect the responses of β-blockers and corticosteroids in positive ESI.
7) The results demonstrated that at least 121 compounds (including metabolites) out of 131 can be detected in urine at concentrations corresponding the MRPL by HPLC–ESI/Orbitrap mass spectrometry with high-pH mobile phase.
8) It was observed that steroids without any protonable function (e.g 17α-methyl5α-androstane-3α,17β-diol) gave good photoionization yields in solvent mixtures of 2-propanol/acetonitrile/water and propanol/ethanol/water.
9) 2-propanol/acetonitrile/water (37.5:12.5:50) was found to be necessary for the sensitive detection of doping substances in terms of the S/N ratio.
10) For maximum sensitivity, the APCI probe nebulizer (shared by APPI) is titled toward the cone face as close as possible but far enough to minimize the nebulized mobile phase spraying directly on the cone face.
11) The research results shown that doping substances (including metabolites) with diverse structures and polarities can be analyzed in urine with 100% detection rate at concentrations corresponding the MRPL by HPLC-APPI/Orbitrap mass spectrometry.
12) The results demonstrated that 225 compounds (including metabolites) out of 225 can be detected in urine at concentrations corresponding the MRPL by HPLC– APPI/Orbitrap mass spectrometry with Hypercarb column.
13) Our results confirm the previous findings about the lower susceptibility to matrix effect of APPI.
14) The specificity of the method HPLC–APPI/Orbitrap mass spectrometry with hypercarb column was extremely good and did not represent a limiting factor for selectivity.
15)Our results shows that APPI is a valuable tool for day-to-day usage in doping control because it is able to successfully to ionize more compounds, with greater structural diversity, than the other two ionization techniques

Code: 09D5CG

Luteinizing hormone (LH) is a peptide hormone which is abused by athletes to either induce the production of endogenous hormones such as testosterone to hide the use of anabolic agent use which would normally suppress the production of LH. In the case of the low molecular weight LH receptor agonists they would induce the production of endogenous hormones such as testosterone in a similar way as LH. These compounds are banned in the WADA list (S2. Hormones and related substances) as they have a similar biological effect as the use of LH. 
A method of detection for these compounds needs to be direct as their effect, the excess production of endogenous hormones, cannot be proven by analysis techniques which test for the endogenous hormones. In this proposal several compounds which have been identified as having high LH receptor agonist activity will be synthesized. An instrumental analysis technique based on liquid chromatography with tandem mass spectrometry will be developed for the analysis of urine samples. 

Main Findings: 

Luteinizing hormone (LH) is a peptide hormone which is abused by athletes to either induce the production of endogenous hormones such as testosterone or it can also be used to hide the use of anabolic agents which would normally suppress the production of LH. LH is a naturally occurring hormone which is secreted by the anterior pituitary gland. In males LH acts on the Leydig cells of the testis, stimulating testosterone production, while in females, LH plays an integral part in the ovulation cycle. In the case of the low molecular weight (LMW) LH receptor agonists they would induce production of endogenous hormones such as testosterone in a similar way as LH. These compounds are banned in the WADA list (S2. Hormones and related substances) as they have a similar biological effect as the use of LH.  
The method of detection for these compounds needed to be direct as their effect, the excess production of endogenous hormones, cannot be proven easily by analysis techniques which test for the endogenous hormones. In this project several compounds which had been identified as having high LH receptor agonist activity were synthesised. These compounds fall into two groups, pyrazoles and the Org series. For this project Pyrazole 10, Pyrazole 25, Org 41841 and Org 43553 were synthesised. An instrumental analysis technique was developed using High Resolution Mass Spectrometry coupled with Liquid Chromatography (LC/HRMS) for the analysis of urine samples spiked with the LMW LH receptor agonists. In vitro enzyme analysis was undertaken to identify possible metabolites for the LMW LH receptor agonists. The proposed analysis methodology was validated using the parameters of specificity, linearity, limit of detection (LOD), precision and recovery. The LOD for each compound was determined at 2 ng/mL for Pyrazole 10, Pyrazole 25 and Org 43553 and 5 ng/mL for Org 41841. The analysis technique will be able to be immediately implemented into laboratory screening tests for small molecules using liquid chromatography with mass spectrometry detection. A sample of Org 43553 has been distributed to all WADA laboratories.

Code: 09A20RV 

There is a need for doping control laboratories to improve the detection capabilities of the administration of anabolic androgenic steroids (AAS). Most AAS are extensively metabolized. Studies on steroid metabolism have been traditionally performed using GC-MS methods. Recently, LC-MS systems have demonstrated wide possibilities for the elucidation of new phase I metabolites and the use of this technology has resulted in the detection of previously unreported metabolites. Phase II metabolic reactions of AAS have been normally studied by using specific hydrolysis of the conjugated metabolites present in urine extracts. In most cases, enzymes with –glucuronidase activity were used, thus mainly conjugates with glucuronic acid have been studied up to now using both GC-MS and LC-MS/MS approaches.
Sulfate metabolites are known to be important for some endogenous steroids and they have also been described for exogenous AAS. However, the study of sulfates has been limited by the difficulties of their efficient hydrolysis to the phase I metabolites detected by GC-MS or LC-MS/MS. In spite of this, long-term sulfated metabolites have been reported for some AAS.
The objective of the project will be the evaluation of the phase II metabolism of AAS to look for sulfate conjugates of steroid metabolites that could be used as long-term markers of AAS misuse. A methodology based on the direct analysis of sulfate conjugates by LC-MS/MS will be developed. The different scan methods will be developed to identify unknown sulfate metabolites in administration study samples of different AAS. The excretion profiles of the identified sulfate metabolites will be compared with those of other steroid metabolites targeted in conventional screening procedures, in order to evaluate their interest as long-term markers of steroid misuse. Finally, a methodology addressed to the reliable detection of identified sulfate metabolites in routine antidoping analysis will be developed.

Main Findings: 

The objective of the project was to study the phase II metabolism of anabolic androgenic steroids (AAS) to look for sulphate conjugated metabolites that could be used to improve detection capabilities of AAS misuse. A methodology based on the direct analysis of sulphate conjugates by LC-MS/MS was developed. A liquid-liquid extraction with ethyl acetate was used to extract steroid sulphates from urine samples. Based on the common mass spectrometric behavior of steroid sulphates, precursor ion and neutral loss scan methods, and selected reaction monitoring methods including theoretical transitions of potential metabolites, were used to detect new sulphate metabolites in post-administration samples. AAS studied were boldenone, boldione, methyltestosterone and metandienone. Boldenone sulphate and epiboldenone sulphate were identified as minor metabolites of boldenone in humans. These metabolites were detected in urine during the same time as the main metabolites (boldenone and 5β-androst-1-en-17β-ol-3-one, BM1, detected in the glucuronide fraction). The analysis for the absence of these sulphates could be used as additional criterion of the endogenous origin of boldenone and BM1 in samples with low concentrations of these metabolites before going to GC/C/IRMS analysis. For boldione, seven metabolites conjugated with sulphate were detected. Three novel sulphate metabolites of methyltestosterone were identified and one of them was detected in urine for long time after administration, increasing the retrospectivity of the detection between two and three times with respect to other metabolites described. For metandienone, seven sulphate metabolites were detected in post-administration samples and one of them, was detected in urine for long time after administration, doubling the detection time compared to the last long-term metabolite described (the same phase I metabolite excreted in the glucuronide fraction). 
The results of the project demonstrate the importance of sulphatation as a phase II metabolic pathway for AAS and the interest to study this metabolic fraction to look for new metabolites of AAS to improve the capabilities (e.g. long-term metabolites) of the detection of these compounds in doping controls.

Code: 09B8FG 

Erythropoietin (EPO) is an hormone regulating the synthesis of red blood cells, thus increasing the ability of blood to carry oxygen. Its analogues could be used by athletes in endurance sport and therefore appear in the WADA list of banned substances. Nowadays there are direct and indirect methods for the detection of erythropoietin. 
The direct method developed by Lasne, is divided into four steps: urine concentration, isoelectric focusing separation, double blotting and detection by chemiluminescence. The development of other forms of recombinant EPO, and the need to increase the window of detectability of EPO administration, led to the need of updating the Lasne method to ensure the highest efficacy and efficiency of the anti-doping analysis. For instance, due to the differences in the apparent molecular mass of recombinant and endogenous erythropoietins, the administration of some recombinant EPOs can also be detected by SDS PAGE, another tool to clarify the profile of the suspicious samples. 
The overall aim of the project is to study the effect of some specific modifications of the Lasne method, in order to (i) reduce the overall time of the analysis, (ii) simplify the sample treatment process, (iii) improve the limit of detection, and (iv) increase the robustness of the results.  Particularly, we plan to evaluate the effect of some modification at the level of (i) the preparation of the gel, (ii) the incubation steps respective to the blotting process, and (iii) the antibody incubations following the second blotting step. The effectiveness of the proposed solutions will be tested on a significant number of reference samples, of spiked urines and of negative and positive samples coming from our routine activity.

Main Findings: 

In the first part of this project we focused on the possibility of reducing the overall analytical times of the method for the detection of erythropoietins (EPOs), possibly completing the procedure in less than one working day from the start of the analytical process. The method is basically the same originally proposed by Lasne et al. and presently implemented by the WADA accredited laboratories, with few significant modifications involving the use of a novel blotting system based on vacuum technologies (namely, the SNAP i.d.® system). The system allows in principle a significant shortening of the time required for blocking, washing and antibody incubations, and we have considered its potential effectiveness if used after the first blotting step of the isoelectrofocusing (IEF) procedure. By applying a vacuum to actively drive reagents through the blotting membrane, the time of incubation for the blocking step and the washing step are indeed drastically shortened. 
Furthermore, since the differences between the apparent molecular masses of endogenous and recombinant erythropoietins and analogues make possible to discriminate the endogenous and some exogenous EPOs by SDS-PAGE, the utility of the SNAP i.d.® system was verified also for this procedure. Finally, due to the recent developments in the field of electrophoretic EPOs detection, the utility for the SDS/Sarcosyl-PAGE procedure was also investigated.  The alternative procedures involving the SNAP i.d.® system in the IEF and SDS/Sarcosyl-PAGE methods were validated in terms of sensitivity, specificity and robustness using blank urine samples spiked with biological reference preparation of exogenous erythropoietin (BRP, rEpo) or darbepoetin α (NESP) or epoetin δ (Dynepo) or CERA and urine samples from antidoping controls found to be negative during the screening analysis. The effectiveness of the newly developed approach has been also evaluated analyzing urine samples from excretion studies. 
The results showed that even if the time of each step is drastically reduced (the use of SNAP i.d.® system reduces the time for the complete analysis five to six hours) the limits of detection, the specificity and the robustness of the newly developed procedure are very similar to those obtained by the method currently used by the WADA accredited laboratory of Rome.  We have validated both the IEF method and the SDS/Sarcosyl-PAGE method with the use of the SNAP i.d.® system.  The shortening of the time required to complete the procedure may reveal particularly valuable especially on the occasion of major international events, where the analytical workload drastically increase and, in parallel, the reporting times are very compressed. 
In the second part of the study we have evaluated the effect of some specific modifications of the procedure, in particular different reagents: a secondary antibody goat anti-mouse IgG (H+L) biotin conjugated different from the one currently in use in our laboratory purchased from Pierce/Thermo, two different streptavidin-peroxidase adducts (one complexed and one conjugated) and a specific monoclonal antibody anti-hEPO from mouse (Clone 23H2) on a gelatinous support inside a column to purify the urine samples were tested.  The results obtained showed no significant advantage with respect to the reference procedure, although the results obtained the monoclonal antibody anti-hEPO used for immunopurification step is not good to purify urine samples but we have preliminarily obtained good results using it as primary antibody. 

Code: 09D3PB

In the last years the WADA has founded studies of our groups aiming to develop in vitro test systems for the structure independent identification of anabolic substances. Our test system is a stable transfected yeast transactivation system for the identification of substances with affinity to the androgen receptor. Using this test system we could identify and characterise several designer steroids. In the last funding period we further characterized SC and started with the construction of a new reporter gene system in Schizosaccharomyces pombe. SC was able to detect anabolic steroids and their metabolites with a high specificity and sensitivity in urine of abusers (Zierau et al. 2008). Even selective androgen receptor modulators (SARMs) could be detected with SC. In excretion studies with Methyltestosterone in close cooperation with the doping control lab cologne SC was able to detect 1-Testosterone abuse up to 307 hours (GCMS detection limit was 118 hours). Treatment of the urine (concentration, purification) further increases the sensitivity of SC. Using new reporter gene plasmids we could reduce the duration time of the test from 2 days down to 18 h. In addition SP was successfully generated and is now ready to be further characterized. Reaching these milestones, our future aim is to use the SC to supplement GCMS techniques in routine doping analytics. Therefore we want to develop a standard routine procedure protocol to use the system in routine analysis. We also want to further enhance the sensitivity of the system by validation the newly generated SP system. In addition our SC system will be used to identify new long-term metabolites of anabolic steroids. So SC will in addition further improve the sensitivity of the GCMS detection systems.

Main findings

The results of this research Project present biologic and pharmacological “in vitro” and “in vivo” evidence that increases of EPO secretion during limited oxygen availability may be affected by extracellular adenosine generation and signaling. Therefore, we suggest that adenosine may be a doping agent “in vivo” since bone marrow is an hypoxic tissue. The A3 receptor does not appear to be involved in these direct effects of adenosine in HeL023 differentiation. On the contrary, the A3 receptor appears to be the mediator for the increase in EPO production induced by Adenosine.

The molecular signaling induced by Adenosine to increase EPO release by A3 receptors promotes HIF-1 expression. In particular, Adenosine can affect hematopoiesis at three levels: 1) increasing EPO concentration; 2) increasing ET-differentiation and 3) impairing MK-commitment. Adenosine, through A1 and A3 receptors, improves ET differentiation, accelerating the early commitment signaling. Adenosine may impair MK differentiation tuning PM fate toward the ET commitment. The mechanism appeared to involve the distal molecular effectors of EPO signaling pathway. Moreover, Adenocard increases EPO blood levels. Even if the increase was of modest entity, it is possible that adenosine, given as a unique bolo, has been rapidly degraded in few hours “in vivo”. We suggest that a chronic Adenocard treatment may generate a greater EPO increase with relevant physiological effects on PM cells

Code: 09E12RM

The fatigue that accompanies prolonged exercise in most often ascribed to events occurring in the periphery (glycogen depletion, dehydration). Recent evidence, however, suggests that the central nervous system plays a critical role, especially during exercise in the heat. This is an important consideration, as many major championships take place during the summer, often in countries with warm climates.

Many stimulants acting on the CNS are included in the WADA Prohibited List; they can enhance performance and may pose a risk to health. Pharmacological manipulation of the CNS function can influence mood, the sensation of effort and/or thermal stress, cognitive function and tolerance to pain and discomfort. Drugs that manipulate brain neurotransmitters, in particular serotonin, dopamine and noradrenaline, have been shown to influence exercise performance, but many of these agents are not currently included on the Prohibited List. Many of these drugs are used in the treatment and management of a wide variety of psychiatric disorders, so the pharmaceutical industry is constantly producing novel, more selective and more powerful agents, with the potential for marked effects on exercise performance. Athletes may have legitimate reasons for their use (under Therapeutic Use Exemption; TUE), but the inappropriate use of these agents by athletes to enhance performance is a distinct possibility.

These drugs, which are readily accessible over the internet, may also pose a risk to health as the normal limits to body temperature may be exceeded. At least one high profile death during the Tour de France has been ascribed to hyperthermia consequent upon amphetamine use.

This project will investigate the performance effects of drugs affecting the CNS, and will also consider the various factors (gender, environment etc) that may influence their efficacy. These findings will help determine the suitability of this class of drugs for future inclusion on the Prohibited List.

Main findings

Stimulant medications enhance the activity of the central and peripheral nervous systems, producing a variety of effects including increased alertness, motivation and arousal, elevated heart rate and blood pressure, altered mood and the perception of a diminished requirement for food and sleep. Since these responses are likely to influence athletic performance, the aim of this project was to examine evidence that pharmacological manipulation of the central nervous system can produce improvements in exercise performance. Anecdotal reports, evidence from screening procedures used by WADA-accredited laboratories, as well as TUE application statistics, provide some evidence that athletes may be increasingly turning drugs of this nature. Of course, athletes may have legitimate reasons for the use of these drugs, but these agents may also be used with the sole aim to enhance performance. Of particular concern would be medications employed in the treatment of attention deficit hyperactivity disorder (ADHD) and narcolepsy.

The present series of studies provide clear evidence to support the inclusion of Bupropion on future Prohibited Lists. The findings of the studies presented here, along with our previously published work, demonstrate that bupropion can produce a significant, measureable and consistent improvement in exercise performance in warm conditions when taken at the maximal therapeutic dose (+7-9% vs a placebo condition). This response is apparent in both male and female participants, and does not appear to be influenced by pre-exercise nutritional intake. Increased exercise performance following administration of bupropion was associated with the attainment of higher core temperatures and heart rates during the later stages of exercise. This occurs without any apparent change in the subjects’ perceived exertion or thermal stress. It is distinctly possible that the use of bupropion and methylphenidate by athletes competing in warm conditions will increase susceptibility to heat illness and potentially fatal heat stroke.

Contrary to the findings of a recently published report, ingestion of tyrosine (the precursor of dopamine) produced no effect on physical performance during an exhaustive cycle test; this response may be due to rate limiting steps in catecholamine synthesis from tyrosine. The administration of the Parkinson’s drug, Sinemet (containing L-Dopa), also failed to significantly influence exercise performance, but performance may have been limited by side effects reported by some participants. S-adenosylmethionine (SAM) has become popular as an alternative therapy for the treatment/management of depression and other related disorders. Despite some effects on the physiological response to exercise, there was no evidence of a benefit to exercise performance following a 7 days of administration.

In conclusion, it appears that bupropion can produce a measureable and consistent improvement in exercise performance in warm conditions when taken at the maximal therapeutic dose. With this in mind, inclusion of bupropion on future Prohibited Lists appears to be warranted. Substances that alter catecholamine production do not appear to consistently influence performance, but further investigation is justified.

Code: 09A05DP 

This project aims at four goals: 
A. Interpretation and evaluation of precursor ion scanning chromatograms (urine profile recognition) 
In the field of anti-doping control, chromatograms from target steroid-analysis are generally evaluated and interpreted visually by the analyst which is trained for this purpose. 
A similar approach will be adopted for evaluating the precursor scanning chromatograms. To get acquainted with the chromatograms, around 50 blank samples will be analysed.  In a second step precursor ion scan chromatograms obtained from controlled administration studies (e.g. methyltestosterone, methanedienone,…) and from adverse analytical findings from routine target screening will also be investigated to get acquainted with positive samples. The applicability of instrument software for this purpose will be evaluated.  
B. Application to real samples
Urine-samples will be analysed. These samples will include all out of competition samples and both out of competition samples and in competition samples   
C. Study of the influence of different population factors in the detectability of different markers for several anabolic steroids 
A single dose of the previously studied steroids (stanozolol and methyltestosterone) will be administered to six volunteers belonging to different population groups. Urine will be collected for three weeks and a method including all feasible markers will be applied. The best marker(s) will be selected based on the results obtained. This procedure will also be followed if promising new markers are found in the re-evaluated steroids. 
D. Re-evaluation of the metabolism of additional steroids by LC-MS/MS looking for alternative markers for the detection of steroid misuse 
Three or four additional steroids will be re-evaluated via the analysis by LC-MS. These steroids will be selected based on the availability of excretion studies in both laboratories.  
Appropriate precursor scan and/or neutral loss scan methods will be applied for each steroid depending on their structure. Feasible metabolites will be characterized by MS techniques. The metabolic nature of these metabolites will be determined, if necessary, by the analysis of chimeric mouse urine after the drug administration. A full excretion study will be analyzed in order to determine the long term metabolites. 

Main Findings: 

Anabolic steroids with a 3-keto-4-ene structure have in common that they fragment by LC-ESI-MS/MS at high collision energy in tree common ions: methyltropylium (m/z 105), tropylium (m/z 91) and phenyl ion (m/z 77). The use of a precursor ion scan method for these 3 ions would allow to detect new/unknown AAS and metabolites with 3-keto-4-ene structures. 
In this project, the potential of approaches based on precursor ion scan for doping control purposes have been evaluated in two scenarios: (i) routine application and (ii) metabolic studies of AAS.
In the routine application, the potential use of the approach in two laboratories (Gent and Barcelona) was explored. The analysis of a common set of 12 samples allowed for the confirmation of the suitability of the method for the detection of positive samples for known steroids. The analysis of this common set of samples also revealed several limitations in the harmonization between laboratories regarding relative retention times and the precursor ions observed. 
During the whole project, a total of 1911 routine-samples were analyzed using the established precursor scan protocol by the two laboratories. These samples covered both samples from athletes from high-risk sports (strength sports) and urines samples from athletes who tested positive for known steroids. Seven suspicious samples were detected. Despite the application of several analytical strategies such as product ion scan in both positive and negative ionization mode with accurate mass measurements and GC-MS, none of them could be clearly assigned to belong to the AAS family. Regarding metabolic studies, the method allowed for the detection of several metabolites for boldione, 4-chloro-methanedienone and clostebol. Several previously unreported metabolites were detected and characterized.  
Finally, the effect of population factors in the detectability of long-term metabolites was evaluated in two different scenarios: stanozolol and clostebol. Results showed that both 17-epistanozolol-N-Glucuroinide and 4-chloro-5-androst-3β-ol-17-one 3β-sulfate are the long-term metabolites for stanozolol and clostebol irrespective of the ethnicity of the volunteer. 

Code: 09B3JP

The analytical strategy of doping control relies on the availability of reference standards against which compare the results obtained after the application of any procedure. The situation with the endogenous urinary EPO standard (NIBSC 2nd international reference preparation) needs careful attention: - the urinary standard (uEPO) from NIBSC is coming to an end and according to the correspondence held with NIBSC, there are no plans to produce a new batch.
- At this stage, only 2 vials (20 IU) per laboratory and year will be provided.
- The NIBSC uEPO standard was produced by unspecific protein concentration from urine of anaemic patients. Hence it contains very high concentrations of proteins other than uEPO.
- Despite all efforts devoted so far, there has not been an appropriate characterisation of the origin of the charges responsible for its IEF behaviour.
- The comparison between any standard obtained from patients and a standard obtained from healthy volunteers has to be performed.
- An appropriate analytical standard of serum or plasma EPO is needed and has to be developed.               The technology for EPO immunopurification has already been developed and the members of the research team have first-hand experience in the field. However, its up-scaling for large volume processing (e.g. ten litres or urine per day) has to be developed tested properly. 
With this background, the aim of the project is: - developing the methodology for producing a urinary EPO standard by processing urine from patients and healthy volunteers.
- producing immunopurified urinary and serum EPO standards, available for doping control purposes.              As secondary objectives, the daily variation of their urinary EPO profiles will be studied and, using EPO from patients producing elevated concentrations of the structural characterisation of EPO will be approached.

Main Findings: 

The general aim of the project was the development of a large scale inmunopurification procedure and its application for the preparation of a relatively large scale batch of endogenous EPO standard in urine and, if feasible, plasma from healthy volunteers and patients producing large amounts of this glycoprotein.
The immunopurification procedure developed implied a sample pretreatment includingdissolution under alcaline conditions of the sediment/precipitates present in urine, followed by a thorough multi‐step filtration (first, a filtration through a triple glass fibre filter AP25 (Millipore), 2 µm AP20 (Millipore) and 0.5 µm RW06 (Millipore); second a 0.22 µm Millipak filter unit).  
Immunopurification of urinary volumes between 1.5 and 2 L was achieved using 2 mL Sepharose columns containing as much as 12 mg of anti‐EPO monoclonal antibody. Columns needed to be used at 4ºC, overnight at a 2‐3 mL/min flow rate. Recovery, during the development phase showed to be roughly around 50% for solutions of up to 80 IU/L.  
However when real urine samples were treated, it soon became evident that the urinary matrix was able to block the columns, even after substantial filtration. Tamm‐Horsfall protein, the most abundant glycoprotein in urine, could have been part of the problem. Extensive washing of the immunoaffinity columns, changing of filtering procedures as well as heating the urine samples seemed to delay the problem, but did not solve it.
On the other hand, it was impossible to find proper patients or volunteers with elevated EPO excretion. Patients from clinical hematology, pneumology or oncology resulted in depleted EPO concentrations. Finally it was decided to use urine from healthy volunteers with serious implications regarding the amount of EPO that could be reached.  Immunopurified EPO was very sensitive to preparation conditions with significant losses due to irreversible adsorption on glass or plastic vials.
Overall, recovery was below 5% and only a very small, symbolic, batch of 15 vials of 1.6 IU/vial of liophylized material could be obtained in a buffer composition compatible with electrophoretic or mass spectrometric methods.
Obtaining reasonable amounts of this glycoprotein standard, and at a reasonable cost, would imply an international approach with access to a number of high EPO excreting individuals under proper long term conditions.