Projets de recherche

Code: 13D21DS 

Among the misuse of erythropoietic stimulating agents, the application of substances that induce EPO gene expression by stabilizing HIF 1 as cobalt (II) ions or carbon monoxide inhalation seems to be widely distributed. For athletes, the advantages of the misuse of both substances are low costs, high erythropoietic effectiveness, and no risk of detection, because they are not yet included into the prohibited list of WADA.
The aim of this project is to demonstrate significant effects of cobalt and CO-inhalation on the erythropoietic system which can be detected by the Athlete Biological Bloodpass and direct methods. Special aims are: 1. to monitor the physiological effects of different doses of cobalt and carbon monoxide on plasma-EPO, Hb-mass, and aerobic performance, 2. to prove if the effects of cobalt and CO can be detected by the recently developed Bayesian probabilistic inference techniques, which are the basis for the athletes biological passport, 3. to develop and evaluate direct detection methods for cobalt-misuse, and 4. to evaluate whether the endogenously produced EPO after stimulation by cobalt and CO differs from normal native EPO. 
In a first study the optimal dosage of cobalt and carbon monoxide on endogenous EPO production will be determined. In a second step, application of either cobalt or CO for 3 weeks will provide data on (1.) performance effects of both drugs, (2.) stimulation of endogenous EPO, (3.) changes of hemoglobin mass, and (4) changes of hematological parameters which will be analyzed by the statistics of the athlete biological passport. Cobalt concentration and EPO isoforms will be analyzed in urine and/or blood samples. We expect to develop methods which can be used for the detection of endogenous stimulation of erythropoietin by cobalt and carbon monoxide.

Main Findings: 

Introduction: From the mid 40ies until the late 70ies of the last century cobaltous ions (Co++) were used as an effective drug to treat anemia of different origins. Due to severe side effects this kind of treatment was replaced by other erythropoietic stimulating agents like recombinant human erythropoietin. Co++ acts similarly as a hypoxic stimulus; it stabilizes HIF-1α and HIF-2α and thereby increases the endogenous erythropoietin production. Because of its possible performance enhancing effect Co++ has been added to WADA’s list of prohibited substances and methods. Co++ can be easily purchased and it is recommended from suppliers of nutritional supplements to athletes to boost their performance. The aim of this project was to investigate the effects of low dose Co++ ingestion and to determine the minimum oral dosage which is necessary to increase erythropoietic processes.
Methods: Three sub-studies were carried out: I. Single Co++ dosages between 1mg and 10mg; II. Co++ administration for 5 days with the lowest dosage found to be effective in study I; III. 3-week Co++ administration with the lowest effective dosage (5 mg/day). We determined plasma [EPO], all parameters used for the athlete’s biological passport (ABP) as well as in study III hemoglobin mass and performance. In total, 63 male recreational athletes participated at the studies which were conducted in a double blind design.
Results and discussion: Study I: Single dosages until 2mg Co++ had no erythropoietic effect. Plasma [EPO] increased 5h after the 5mg administration by 18 ±14% (p<0.05) and until 7h following the 10mg Co++ administration by 41 ±15% (p<0.001). Study II: Following the 5-day Co++ application 5mg showed no significant effects on any parameter while 10mg increased [EPO] by 28 ±26% (p<0.05) and the immature reticulocyte fraction by 50 ±21% (p<0.001). [Ferritin] decreased in the 10mg group by 22 ±17ng/ml, p<0.01). Study III: During the 3-week application period plasma [EPO] increased by 30 ±39% (p<0.001) and total hemoglobin mass by 17.1 ±16.8g (p<0.001) while Ferritin decreased by 17 ±18 ng/ml (p<0.01). Although VO2max was not affected, submaximal performance (+11 ±xy Watts at 2 mmol/l lactate, p<0.05) and time until exhaustion (35 ±xy sec., p<0.05) was slightly improved after the 3-week administration period.
Discussion: Co++ administration using dosages 2-5-times above the recommendations of suppliers of nutritional supplements and ~ 2-times above the amount which is considered to be safe and without any side effects in case of life-long daily administration show measurable erythropoietic and performance-enhancing effects. We therefore recommend to install threshold limits for Co++ concentrations in urine and in blood to deter and detect blood manipulation by Co++ administration.

Code: 13D22RV 

Glucocorticosteroids are widely used in sports medicine for the treatment of conditions such as asthma and acute injuries. The use of glucocorticosteroids by oral, intravenous, intramuscular and rectal routes is forbidden, and their use by other routes is allowed. Since some glucocorticosteroids are marketed in different administration forms, the distinction between different routes of administration through the analysis of urine samples is needed. A reporting level of 30 ng/mL for glucocorticosteroids and their metabolites has been established by WADA to detect corticosteroid misuse. Unfortunately, this reporting level is not suitable to distinguish therapeutic use from forbidden administration for all corticosteroids and the investigation of more effective criteria of discrimination is needed. 
The aim of the project is to elaborate analytical strategies to be used in the effective differentiation between legal and forbidden administration routes for synthetic glucocorticosteroids. A comprehensive study of the metabolic pathways of corticosteroids will be performed using liquid chromatography coupled to tandem mass spectrometry. Both phase I (including saturated A-ring and acidic metabolites) and phase II (including conjugates with sulphate and cysteine) metabolism will be studied in several excretion studies already available in our lab for triamcinolone acetonide, betamethasone, prednisolone, methylprednisolone and budesonide. 
Differences between responses of every metabolite detected in the different administration routes will be evaluated. Potential markers will be selected based on these results. Selectivity and sensitivity of these potential markers will be compared with current reporting level. 

Main Findings: 

The objective of the project was to evaluate the current WADA criterion to detect glucocorticoid (GC) misuse.  The urinary profiles of budesonide (BUD), triamcinolone acetonide (TA) and betamethasone (BET) were studied in clinical studies were they were administered by allowed and forbidden administration routes in order to look for the best discrimination markers for each GC. 

For BUD, the metabolite 6β-hydroxy-BUD was confirmed to be the best the marker to detect BUD misuse. However, high 6β-hydroxy-BUD concentrations were found after high-dose inhaled treatments and, for this reason, a reporting level of 40 ng/mL is proposed to discriminate allowed from forbidden administrations. 
For TA, a reporting level of 30 ng/mL of TA did not allow the detection of intramuscular administration. A reporting level of 5 ng/mL for TA was proposed to detect intramuscular use, and to discriminate topical and intranasal from intramuscular administrations. Concentrations obtained after intraarticular and periarticular administrations, which are currently regarded as allowed administration routes, need be taken into account before proposing a definitive reporting level to discriminate allowed from forbidden administrations of TA. 

For BET, a reporting level of 40 ng/mL for BET is proposed to discriminate topical and intranasal from oral and intramuscular administrations. However, concentration values after intraarticular and periarticular administrations are similar to those obtained after intramuscular administrations. 

The results of the present project demonstrate the need of studying the metabolism of each GC after allowed and prohibited administration routes in order to establish the best discrimination criterion for each GC.

Code: 13D14MM 

In the doping control field, the concept of urinary steroid profiling was first introduced in the '80s by Donike et al. to reveal the misuse of testosterone and its precursors. The kinetics of excretion in urine of the target compounds that are part of the steroid profile may be affected by various factors, primarily among them sex, age, ethnicity, physical activity, diet, alcohol consumption, as well as other physiological or pathological conditions. We are focusing our attention on another possible cause of alteration of the urinary steroid profile, that is on the consequences of drug-drug interactions. Examples of agents affecting the urinary steroid profile that are included in the WADA list of banned substances and methods are mainly represented by the masking agent probenecid and by the whole class of the aromatase inhibitors. In addition to those drugs, several classes of compounds not included in the WADA list have been reported to play a significant role on testosterone synthesis and metabolism. An example is represented by the antifungal ketoconazole that acts as inhibitor of the cytochrome P-450-dependent enzymes involved in the hydroxylation during steroid hormone synthesis and in the oxidative/reductive reactions during steroid hormone phase I metabolism. 
Even if the effects of ketoconazole on the steroids hormone synthesis are well known, very little information is available about its impact on the effectiveness of the screening and confirmation strategies routinely followed by the WADA-accredited antidoping laboratories to detect the abuse of androgenic anabolic steroids that are normally present in the human body. This project aims to evaluate the alterations caused by the intake of azole antifungal drugs and to verify whether these effects could in some way affect the reliability and the accuracy of the analytical strategies currently followed to detect doping by testosterone and precursors.

Main Findings: 

The objective of this project was to investigate the effects of azole antifungals administration on the strategies currently adopted by the anti-doping laboratories to report an adverse analytical finding for testosterone related steroids. More specifically, we have considered the effects of the administration of miconazole and fluconazole by different routes and doses on the physiological circadian fluctuation of both the parameters reported in the TD2016EAAS and the endogenous reference compounds reported in the TD2016IRMS. In the first part of this project, the circadian fluctuations of the parameters of the steroid profile selected were evaluated in four male Caucasian subjects for at least five days, and basal ranges of each parameter in each subject were established. These basal ranges were then utilized to evaluate the potential effects of azole antifungals administration on the determination of the parameters selected to detect doping by testosterone related compounds. Finally analytical methods to screen and confirm azole antifungals in human urine were set up and validated. 
The results obtained show that both miconazole and fluconazole are able to alter significantly the key parameters of the steroid profile. More in details, the imidazole antifungal miconazole causes after oral and buccal administration (i) a significant increase of the 5α-androstan-3α,17β-diol/5β-androstan-3α,17β-diol ratio, (ii) a significant decrease of androsterone, etiocholanolone, androsterone/etiocholanolone ratio, androsterone/testosterone ratio, and 5α-androstan-3α,17β-diol/epitestosterone and (iv) a moderate decrease of the 5β-androstan-3α,17β-diol and 5α-androstan-3α,17β-diol. Limited effects were instead registered after dermal intake. These finding can be primarily explained by the ability of miconazole and its metabolites in altering the kinetic/efficacy of de-glucuronidation of the endogenous steroids by β-glucuronidase. The oral intake of the triazole antifungal fluconazole, instead, increase the urinary levels of androsterone, etiocholanolone, androsterone/etiocholanolone ratio, androsterone/testosterone ratio, 5α-androstan-3α,17β-diol, 5β-androstan-3α,17β-diol, 5α-androstan-3α,17β-diol/5β-androstan-3α,17β-diol ratio, and 5α-androstan-3α,17β-diol/epitestosterone ratio. This finding is not linked to the efficacy of the enzymatic hydrolysis by β-glucuronidase, but might be explaining considering the putative anti-aromatase activity of the triazole antifungal agents. 
These evidences underline the importance to screen for the azole antifungals in the athlete urines collected in occasion of doping control test, to reduce the risk of reporting uncorrected results.

Code: 13D34PV 

In the recent years, the use of performance-enhancing peptides has become a growing issue in human as well as in equine sports drug testing. A product called TB-500, claimed to increase muscle growth and tissue repair in horses and other mammals, is available on the Internet and officially distributed.  Anecdotal use not only in horse racing, but also in human sport has been documented, reaching also worldwide attention from the media. The active content has been identified as the N-terminal acetylated 17-23 fragment of thymosin beta4 (Ac-LKKTETQ) by our group.  
Detection of the intact TB-500 and several metabolites has been achieved in horse urine and plasma, but no data are available for detectability in human. It has already been demonstrated that in vitro/ex vivo studies can be used to obtain reliable indication on the metabolism of small peptides.   
The aims of the project are therefore to 1) characterize human metabolism of TB-500 by using human liver microsomes and human S9 fraction (for in vitro studies) and human plasma/serum (ex vivo studies); 2) synthesize appropriate amounts of certified reference standards of TB-500 and its most representative metabolites by solid-phase synthesis; 3) determination of the detection limits of the relevant metabolites and implementation of the metabolites into peptide-screening methods. The synthesized reference standards will be distributed to other human doping control laboratories.

Main Findings: 

A product called TB-500, claimed to increase muscle growth and tissue repair in horses and other mammals, is available on the internet and officially distributed. It is presented as “the synthetic peptide of the active region of thymosin-beta 4 (Tβ4)”, without any further qualitative description such as amino acid sequence or molecular weight.  
In this project the first goal was to characterize human metabolism of TB-500 by using human liver microsomes and human S9 fraction (for in vitro studies) and human plasma/serum (ex vivo studies). Results of this study show that TB-500 showed serial cleavage at the C-terminus, whereas acetylation of the leucine seemed to provide efficient protection of the N-terminus. Results were similar to those described by Ho et al. in the horse using horse liver homogenate as in vitro model. 
 In a second phase of the project, appropriate amounts of 3 metabolites TB-500 M(1-2), TB-500 M(1-3) and TB-500 M(1-5) observed in the first part of the project were synthesized using typical Fluorenylmethyloxycarbonyl (FMoC)-synthesis strategy. As proposed, the synthesized reference standards were distributed to other human doping control laboratories. Besides the synthesis of the metabolites, a heavy version of the TB-500 (TB-500-d3) was synthesized for use as internal standard. In a final step of the project, the LODs of the synthesized metabolites were determined and implemented into an in-house screening method for the detection of peptides in urine. LODs were 500 pg/mL for TB-500 M(1-2), 100 pg/mL for TB-500 M(1-3) and 50 pg/mL for TB-500 M(1-5).

Code: 13D08AR 

Our research program encompasses projects designed to investigate how the use of drugs (emergency pills and androgens) and genetic variation interfere with the Athletes Biological Passport (ABP). ABP is essential to detect doping with anabolic agents, especially with the decreasing doses evidently used by some athletes. However, there are many factors that may contribute to the inter-individual variability in steroid profiles, i.e. drug use, diseases, genetic variation etc. Preliminary results indicate that the use of an emergency pill influences the steroid profile and confounds the test interpretation. We will therefore investigate how the use of emergency pills affects the excretion of steroids in females. We and others have shown that UGT2B17 genetic polymorphism has a large impact on testosterone doping test. We will continue to evaluate the use of genetic testing of UGT2B17 as well as other polymorphisms in sport samples. Finally we will study how androgens affects the haematological module. 
It is of great importance that the athletes ABP will be able to compensate for all possible variability in longitudinal steroid profiles. More knowledge is therefore needed about how drug use and genetic variation may affect the ABP and hence outcome of doping tests.

Main Findings: 

Study 1: We show that DNA extracted from urine samples is not good enough for individual genetic testing in anti-doping work. We showed that UGT2B17 ins/ins men exert higher T/E than ins/ins females and that some del/del athletes (particularly men in power sports) had T/E ˃0.4. The CYP17A1, and UGT2B7 SNPs investigated could not explain the large inter-individual variation in urinary concentration of epitestosterone. 
Study 2: We have shown that the ABP ratios various randomly throughout the menstrual cycle, epitestosterone being the only metabolite that are significantly altered. The administration of an EC decreased the urinary concentrations of epitestosterone 24 hours after the intake, and it is possible that a use of EC could result in atypical finding in the ABP. 
Study 3: We conclude that testosterone but not nandrolone mediates significant effects on the hematocrit profile and gene expression of the EPO gene in blood. Moreover, testosterone increases the serum levels of the future ABP biomarker P-III-NP. It may be of importance to know how the different modules interact for the interpretation of the ABP results. We will continue to study these biomarkers in relation to each other, both at baseline and after the administration of different doping agents.  

Code: 13B08RG 

Growth hormone secretagogues (GHS) are molecules that stimulate the secretion of human growth hormone from the pituitary. They have proven to be potent agonists of hGH secretion reaching circulating hGH values that are even not easily achievable with rhGH administrations. As such, they are the prime candidate pharmaceuticals to replace rhGH abuse. GHS display large structural diversity and many molecules are under development by the pharmaceutical industry. In order to address the entire family of known and upcoming molecules it can be targeted what all GHS have in common: the interaction with the GHSR1a receptor. Based on this premise we developed and validated a competition assay using a radiotracer, 125I-ghrelin. Given the difficulties for testing radioactivity at some antidoping laboratories, and the recent implementation of chemiluminescent tube-based immunoassays for hGH we have studied the potential replacement of the radiotracer by a chemiluminescent one. Accordingly, the development of a method using biotinylated ghrelin and streptavidin labeled with a chemiluminescent tag could be effective to achieve this purpose. 
The main objectives of this project were: - Obtention of appropriate chemiluminescent tags bound to strep traviding and synthesis, purification, and charactesation of streptavidin conjugated to acridinium, not commercially available.
- Set-up of a competition assay protocol employing different chemiluminescent tags.
- Comparison of the radiotracer and the chemiluminescent tracers.
- Assessment of the chemiluminescent competition assay in urine samples.

Main findings:

The results submitted in this project showed the development of an efficient chemiluminescent method performed in three different chemiluminescent tags: acridinium (AC), alkaline phosphatase (AP) and horseradish peroxidase (HRP). This method, based in the interaction between biotinylated ghrelin and streptavidin labeled with a chemiluminescent tag, was evaluated following different steps. After evaluating biotinylated ghrelin in a competition binding assay, the Ki obtained was compared with the Ki values from competition assays performed with ghrelin without any tag, and no interference in the interaction with the receptor was showed. Biotinylated ghrelin appeared then as a suitable ligand. 
Subsequently, the optimum time of addition of streptavidin was tested to avoid any effect on the recognition of biotinylated ghrelin by the receptor,and it was determined its addition 40 min after of the starting of the competition assay. After synthesis and purification of streptavidin labeled with AC, the protocol was optimized for this and the other two chemiluminescent tags (streptavidin labelled with alkaline phosphatase AP and horseradish peroxidase HRP, respectively) through the evaluation of several conditions as: number of washes, membranes obtained from different amount of cells, alternative binding buffers, amount of ST per sample, presence or absence of detergent and process changes of adding of the chemiluminescent substrates, among others. The optimal protocols for each label were tested in a competition assay with different amounts of GHRP-2 in binding buffer and urine and the results showed the best sensitivity with HRP tag. In fact, the presence of GHRP-2 in urine samples obtained 1.5 hours after intravenous treatment with 100 μg of GHRP-2 from in a clinical trial with sedentary health volunteers treated was detected. 
In conclusion, the results shown in this project demonstrate the development of a chemiluminescent method based on interaction between biotinylated ghrelin and the GHSR1a receptor, which is more sensitive than other non-radioactive methods described so far but has less sensitivity than the radioactive method described previously by our own group.  

Code: 13D25MT 

The misuse of recombinant human insulin in sport has frequently been mentioned by confessing athletes but is not detectable with currently available methodologies. This is mainly due to the fact that there are no measurable differences to the endogenously produced hormone. Several attempts to establish diagnostic marker ratios to other endogenously produced hormones (C-peptide) failed due to unstable or highly variable conditions in the living organism. With the present study we aim to establish potential marker metabolites, which will uncover a surreptitious insulin application. Pilot studies demonstrated that there are substantial differences in the degradation/metabolism of insulin between the endogenously secreted and the recombinant and injected insulin. It is assumed, that these differences are due to the exposition of insulin to specific enzymes after subcutaneous or intramuscular administration. 
Several urinary metabolites were identified in earlier studies by this research group and an extension of this approach with most modern analytical instruments is planned, potentially revealing new indicative and diagnostic metabolites. Therefore, purification of target compounds from urine followed by the determination of insulin metabolite profiles by LC-MS/MS is planned, which might serve as diagnostic tool to uncover the misuse of recombinant human insulin. 

Main Findings: 

This study was conducted to explore the metabolic fate of subcutaneously administered recombinant human insulin. Due to the exposure to endogenous proteases in the subcutaneous tissue, a minor amount of the bioactive peptide hormone is cleaved to its truncated metabolite DesB30 human insulin, which still owns the full biological activity. In addition to that, DesB30 is a known by-product of recombinant insulin preparations (at ~ 1%) and, thus, it can enter the circulation from the injection site.  Post administration of recombinant human insulin, circulating DesB30 was identified in plasma samples of diabetic patients (type II). No specimen obtained from healthy volunteers with endogenous insulin analyzed in the context of this study contained DesB30, not even when the production of insulin was stimulated by ingestion of a concentrated glucose solution (OGTT).  The analysis of athletes’ routine doping control samples yielded analytical findings of trace amounts (=LOD) of DesB30 HI in 3 out of 64 samples. The additional monitoring of the respective C-peptide levels yielded inconspicuous results for all athlete samples but, conversely, suppressed C-peptide levels in diabetic individuals after HI administration.  Thus, the detection of DesB30 in athlete blood samples in combination with C-peptide levels represents a potential combination of markers for the misuse of recombinant human insulin in sports. Due to degradation processes, urine specimens were not found suitable for this approach. Furthermore, special care regarding the storage and sampling conditions are crucial for the collected plasma samples and degradation processes must be avoided by applying appropriate conditions during the whole post-sampling period until analysis. This is important for both inhibiting the degradation of HI to DesB30 HI on the one hand and stabilizing the C-peptide of the other hand. 

Code: 13D23SS

Several elite athletes were tested positive for clenbuterol and claimed that these findings were caused by the consumption of meat containing clenbuterol. From several studies and EU monitoring programs it is shown that clenbuterol may be present in meat and after consumption of meat concentrations of clenbuterol in urine can be found. To discriminate between clenbuterol administrated via a pharmaceutical preparation or by ingestion of contaminated meat products research was started in 2012 (WADA 11A18SS). The focus of this study was to determine if there was a difference between the ratio of the two enantiomers (left- and right-- hand form of clenbuterol) in meat, and if after consumption of meat there was a difference between the ratio of the two enantiomers in urine when compared to the ratio in urine after illegal oral administration of preparations to humans. 
In 11A18SS the proof is given that the hypothesis is feasible and the analytical methods are in place and capable to detect the differences.  In the proposed project the focus will be on establishing a relation between the consumption of contaminated meat and a change in ratio of clenbuterol enantiomers in urine via a controlled experiment.  The project will result in a decision model that can be used to assess the source of an adverse analytical finding for clenbuterol.  A new technique focusing on untargeted analyses will also be tested on the acquired samples. This will take into account if there are any other changes in metabolic profiles after both ways of ingestion and will try to discriminate on this basis. This technique is known as metabolomics. 

Main Findings:

The aim of the project was to confirm the proof of principle from a former project with controlled human trials and to build a decision model to be used in sports doping analysis to distinguish AAF for contaminated meat from pharmaceutical preparations. Meat and liver were collected from an animal experiment.  The meat and liver were used for a controlled human trial in which these tissues were consumed by volunteers. Other volunteers ingested a pharmaceutical preparation containing clenbuterol (racemic mixture) or enantio pure clenbuterol. Urine samples of the volunteers were collected for over a week after consumption. All urine samples were analysed using the methods developed in 11A18SS project. The urine samples were also used for untargeted profiling experiments.  
In meat from the animal experiment had S/R clenbuterol ratio of around 1 and 0.9 depending on the animal. This shows that the enrichment of R-clenbuterol in meat of treated animals is not a process that is stable, reproducible and comparable for individual animals and maybe also depends on the concentration administered to the animal. For bovine liver no information on the ratio was available before. Data from bovine liver showed that in liver the S-enantiomer is either enriched or R-clenbuterol very depleted. This is the opposite of the ratio in veal meat. This means that consumption of liver with incurred residues can possibly lead to an opposite ratio in human urine compared to the consumption of veal meat. It was also shown that preparation (cooking and baking) of the incurred meat and liver had little to no effect on the ratio determined prior to preparation.  
In the administration studies a distinction based on the proportion of S-clenbuterol in the human urine samples was possible between those receiving liver (padmin=0.635±0.004) from the other two groups, Spiropent® tablet (padmin=0.499±0.001) respectively meat (padmin=0.509±0.006). A distinction between the volunteers receiving meat and Spiropent® tablets cannot be made based on the enantiomeric composition, due to the reason that the ingested proportions of S-clenbuterol are too close to each other.  
The analysis focusing on potential metabolites of clenbuterol in a targeted non-targeted design predicted a high theoretical number of metabolites/biomarkers. Unfortunately, not many of the predicted metabolites were found in the urine samples of the volunteers. Based on the outcome of the analysis and the statistical processing there is no grouping of the compounds possible at present. There is no underlying mechanism found what could be used to separate the different treatment groups. So it was concluded that it is not possible to use clenbuterol metabolites to discriminate between intentional and unintentional intake of clenbuterol using non-enantiomeric separation.  
Overall it seemed that inter individual differences between elimination kinetics in bovine animals are present. This made setting an absolute threshold or guideline for discrimination not possible. The method developed in the project can be used when there are adverse analytical findings for clenbuterol to obtain additional information. When the S proportion clenbuterol in human urine resembles 0.5 (the S proportion in a pharmaceutical preparation) this is not definite proof of illegal use. However, if the S-proportion is higher than 0.59 this means that in 95% of the cases this is not due to administration of clenbuterol in (racemic) tablet forms.

Code: 13D31PV

Although recent advances of steroid profiling have shown to substantially increase the detection sensitivity, still, the prevalent of misuse is larger than what doping control laboratories can detect. In particular the introduction of slow-release preparations such as T-gels and steroid patches poses anti-doping analysts for greater challenges as the metabolic footprint of misuse with these preparations is very small. 
In 2009, Bayer introduced a long-acting T undecanoate depot Nebido® for therapeutic treatment of hypogonadism in males. Nebido® should be administered only once in two or three months, which is a much longer release period compared to other long-term T-formulations (e.g. Sustanon®). A dose of 1000mg T undecanoate should be injected in the muscle. It is claimed that the insufficient T-levels are restored and do not exceed normal serum levels and likewise cause less unwanted side-effects. 
Also, it has been shown that such high T undecanoate doses can lead to elevated hematocrit levels. Moreover, sustained elevated T-levels can exert a beneficial anabolic effect on micro-damaged muscle tissue after long exercise to enhance recovery.  Interesting features for endurance athletes, for whom the biological passport is a well-established tool to screen and record their doping test results. In this light, it is assumed that described effects can be noticed in both the steroidal and blood module of the biological passport and help to find Nebido® misuse in this category of athletes.  This study aims to investigate the direct detectability of T-undecanoate as administered with Nebido® in blood as well as the influence on various blood and steroid parameters as recorded in the biological passport. 

Main Findings:

Today the steroid passport is a very performant tool to detect abnormal variations in steroid profile markers. Although recent advances of current methods for endogenous steroids have shown to substantially increase the detection sensitivity, the prevalent of misuse is far larger than what doping control laboratories can detect. Particularly, slow-release preparations pose anti-doping analysts for great challenges as the metabolic footprint of misuse with these preparations can be very small. Nebido (Bayer) is one of such long-acting slow release T preparations, containing T undecanoate, that is used for therapeutic treatment of hypogonadism in males. In sports, slow releasing T formulation are applied to rapid repair of micro damage to muscles after exercise for faster recovery. In addition, it has been shown that such high T doses can lead to elevated haematocrit levels. Moreover, sustained elevated T-levels can exert a beneficial anabolic effect on micro-damaged muscle tissue after long exercise to enhance recovery. Both features are interesting for endurance athletes, whereas T used to be related to cheating power athletes for its anabolic effects.  
The T/E ratio was clearly altered by the use of Nebido as slow release T formulation and remain detectable using longitudinal following during 3 months. Androstanediol  ratio and androsterone over etiocholanolone remain mostly unaltered, despite elevated concentrations. Minor steroid metabolites did not contribute to longer or better detection as likely the major pathways were not saturated. As a result the steroidomic model showed similar sensitivities as those observed for longitudinal evaluation of the T/E ratio.  
A very sensitive method for the detection of T in saliva was developed and applied to the oral fluid samples collected after Nebido. Although, salivary T concentrations increased to 7 fold and detectable using population based reference ranges, the basal values were restored after 14 days. Oral fluid analysis of T did not provide any added value for doping control. Hence, the pharmacokinetics of salivary T are much different compared with the observations for T-gel, also a slow-release formulation. 
Blood passports were created after the Nebido T supplementation and results demonstrated that the blood markers were significantly altered but did not results in atypical passport findings. The reticulocyte production was stimulated in the month after Nebido injection, after which a suppressive effect was observed in the second month. By this time haemoglobin was formed to give rise to an elevated plateau in the blood passport that remained below the upper individual limit. Also the off-score showed the alterations that was typical for bone marrow stimulation. 

Code: 13A08MT

The necessities generated by the high standards and quality requirements of modern sports drug testing entails numerous challenges for doping control authorities as well as accredited laboratories. Among these challenges, several prominent aspects can be approached and solved by collecting and, when indicated, testing an additional/ a complementary specimen referred to as dried blood spot (DBS). 1. An important element of efficient doping controls is the frequent and unpredictable sampling of athletes, especially out-of-competition and concerning e.g. anabolic agents, erythropoiesis-stimulating agents, etc. Here, costs associated with sample collection and analysis are particularly limiting factors.
2. With regard to in-competition controls, the differentiation whether a substance such as a stimulant or cannabinoid (i.e. not prohibited at all times) was present in the athlete's blood at pharmacologically relevant concentrations or not is required. From urine analyses, conclusions as to the blood concentration of a drug is a complex issue and has been subject of numerous studies and discussions in the past.2-5
3. The issue of instable compounds can be solved as e.g. in case of Synacthen, a substance that was confiscated in the Spanish Fuentes scandal and admittedly misused by several athletes. Due to its limited stability in blood and urine, its analysis was hampered despite the availability of analytical methods. DBS collection and analysis is considered as a valuable means to improve currently conducted doping control strategies as it addresses all of the above mentioned aspects. DBS are substantially cheaper than conventional doping control samples and thus larger collectives can be processed; drug concentrations at the time of competition can be determined; and instable analytes can be conserved. As a result, both relevant aspects of doping controls, i.e. deterrence from doping and protecion of honest athletes will be considerably strengthened. 

Main Findings: 

A drop of whole blood dried on filter paper (Dried Blood Spots, DBS) represents an aspiring technique for minimal-invasive sample collection in a multitude of analytical disciplines, e.g., therapeutic drug monitoring, preclinical drug development and diagnostic analysis of metabolic disorders in newborns. DBS sampling is characterized by cost-effectiveness, straightforwardness, robustness and facilitated storage and shipment conditions. 
The present investigation was conducted to highlight the opportunities arising from the implementation of DBS as complementary matrix in doping control programs. Being frequently abused, three model compounds were chosen to represent the classes of anabolic agents (stanozolol and dehydrochloromethyltestosterone) and stimulants (pseudoephedrine). A quantitative method was developed and validated for the detection of the target analytes from DBS using liquid chromatography coupled to high resolution/ high accuracy tandem mass spectrometry. The imprecision of the assay amounted to < 8% for intraday and < 18% for day-to-day measurements. Highly purified DBS sample extracts exhibited no ion suppression effects due to interfering matrix components and provided limits of detection of 20 pg/mL for stanozolol and 0.8 ng/mL for DHCMT and pseudoephedrine, respectively, notwithstanding an overall recovery of 26%. Deuterium-labeled internal standards were used to yield reliable quantitative results (accuracy 84-125%). Stability of the analytes was shown for at least 28 days at room temperature.  
Proof-of-principle for the method presented was substantiated by means of the analysis of authentic specimens obtained from administration studies with stanozolol, DHCMT and pseudoephedrine. The results provided, to our best knowledge, unprecedented detection windows for the tested anabolic agents accomplished by DBS sampling to support out-of-competition control efforts for the tested anabolic agents. Furthermore, the unambiguous proof of pharmacologically relevant blood concentrations at given urinary analyte levels are noteworthy for the improvement of in-competition controls, e.g., with regard to stimulant analysis.