Projets de recherche

Code: 14A30JM

The project has two main goals. Firstly, the project aims to evaluate the potential of 3α-glucuronide-6β-hydroxyandrosterone (6OH-A-3G) and 3α-glucuronide-6β-hydroxyetiocholanolone (6OH-Etio-3G) for the screening of endogenous anabolic androgenic steroid misuse. For this first purpose, a quantitative method validated during the project 12A13OP will be applied to several samples from excretion studies already available in our laboratory. Secondly, the project also aims to characterize, elucidate and synthesize the marker M170T298. For this purpose, a large range of analytical strategies will be applied e.g. different derivatization steps, an in-depth study of accurate mass and tandem mass spectra. Once elucidated, the marker will be synthesized and characterized by NMR. After the synthesis of the marker, it would be useful to develop a quantitative method and to check its potential for other administration routes of testosterone. It is intended to reach these goals in a follow-up project once the objectives of the present project are achieved.

Main Findings

[3:22 PM] Meirotti, Luciana

In previous WADA funded projects (11A9RV and 12A13OP), our research group revealed that two testosterone metabolites resistant to enzymatic hydrolysis: 3α-glucuronide-6β-hydroxyandrosterone (6OH-A-3G) and 3α-glucuronide-6β-hydroxyetiocholanolone (6OH-Etio-3G) were present in urine. We characterized them, synthesized them and validated a method for their quantitation. However, their potential usefulness for doping control analysis was not fully evaluated.
On the other hand, common investigations for the screening of testosterone misuse are focused on metabolites with a predicted structure. Thus, unpredicted markers remain not evaluated. In order to cover this gap, we performed a preliminary metabolomic study which showed the presence of a marker (code M170T298) which is a potential urinary marker for testosterone administration. In order to confirm this fact, the characterization, elucidation and synthesis of this marker are required.
Thus, the project has two main goals. Firstly, the project aims to evaluate the potential of 6OH-A-3G and 6OH-Etio-3G for the screening of EAAS misuse. Secondly, the project also aims to characterize, elucidate and synthesize the marker M170T298.
Regarding the first goal, the usefulness of 6OH-A-3G and 6OH-Etio-3G was tested in three scenarios: (i) oral administration, (ii) intramuscular administration and (iii) gel administration. After oral administration of testosterone undecanoate we found that four markers containing either 6OH-A-3G or 6OH-Etio-3G presented detection windows (DWs) larger for all volunteers than those obtained by T/E and comparable to those reported by using cysteinyl metabolites. After intramuscular administration, markers containing either 6OH-A-3G or 6OH-Etio-3G provided detection windows that were similar or longer than those obtained by markers currently included in the steroid profile and clearly higher than those obtained by cysteinyl markers. Finally, the administration of testosterone gel could be screened by markers containing either 6OH-A-3G or 6OH-Etio-3G reaching similar to those obtained by markers currently included in the Athlete Biological Passport.
Regarding the second goal, during this project, we have characterized, elucidated and synthesized the marker M170T298. Our research shows that this marker is 1-cyclopentenoyl glycine (1-CPG) which is probably coming from the metabolism of the cypionic acid released after the hydrolysis of testosterone cypionate. Therefore, it can be valuable to detect the misuse of testosterone cypionate. This fact has been confirmed by analysis of samples collected after testosterone cypionate administration. The determination of 1-CPG provided the longest DWs for these samples

Code: 14D11AR 

Our research program encompasses projects designed to investigate how low doses of topical testosterone interfere with the urinary steroid profile and Athletes Biological Passport (ABP). ABP is essential to detect doping with anabolic androgenic steroids, especially with the use of low-dose-testosterone being increasingly used by some athletes. 
In addition to genetic variation, there are many other factors that may contribute to the inter- and intra-individual variability in the steroid profile, i.e. concomitant drug use, diseases, menstrual cycle, and pregnancy. The latter two conditions have a marked influence on the metabolism and endocrinology of estrogens, progestagens, and peptide hormones (LH,FSH, hCG). However, very little is known about the natural androgen disposition in these situations. In view of the increasing use of LDD it is important to know the natural profile of androgens in the early post-conceptional phase when women still may not know, or know of the pregnancy. There is a dearth of information how pregnancy influences the androgen profile, but it is likely to confound the test interpretation. We will therefore study the androgen profile in females (no dose administration!) in the post-conceptional phase and first trimester.
It is of great importance that the athletes ABP will be able to compensate for all possible variabliity in longitudinal steroid profiles.  More knowledge is therefore needed about how drug use and pregnancy influence the ABP results and hence the outcome of doping tests. 

Main Findings: 

The low-dose androgen doping strategy practiced by certain individuals may be difficult to detect, particularly in women during the menstrual cycle and even more so in the early post-conceptional phase because of extensive hormonal changes. Even though doping puts the fetus at danger, highly motivated sports women may disregard this risk. Alternatively, they may be unaware of conception early in pregnancy. It is probable that exogenous androgens will interfere with the hormonal balance in pregnant women. For obvious reasons there is only sparse information on that in the literature.  
We have investigated the natural androgen excretion profile in women in different phases of pregnancy, and the potential effect on the testosterone/epitestosterone (T/E) ratio used in doping tests. First, pregnancy altered the excretion of glucuronide (G) and sulfate (S) conjugated androgen metabolites. Using an LC-MS/MS method we found that both epitestosterone-S (-S = sulfate conjugate) and epitestosterone-G (-G = glucuronic acid conjugate) were significantly increased throughout the trimesters, being normalized post-partum. Importantly, some of the urinary profiles of steroid ratios in the ABP were altered in pregnant women when compared to the same women after delivery, or with non-pregnant women. Two of the ratios were altered during pregnancy; T/E was lower, whereas A/Etio was higher in pregnant women. Prior information about pregnancy would make the interpretation of the test results safer since pregnancy has such an impact on the ABP steroid profile.  
The steroid profile was also studied in relation to ethnicity and genetic variation. The results show that pregnant women of Asian origin exert higher urinary concentrations of epitestosterone-G than Caucasians. The genotype – phenotype relation between UGT2B17 deletion polymorphism and T/E ratio was disrupted during pregnancy. 
19-Norandrosterone increases in pregnancy and, if above the Decision Limit (DL), analysis of hCG is performed. If the concentration is above 15ng/mL, an IRMS analysis needs to be done in order to confirm if 19-NA is of exogenous origin. In the first trimester 19-NA was below the DL in 55 % of the women. None of the pregnant women reached 15 ng/mL in the first trimester, but 45 % of them were above the DL. The median 19-NA concentrations were increased in the second and third trimester, and in the second and third trimester 56 and 71 %, respectively, of the pregnant women reached the individual DL.
Early pregnancy is a condition that perturbs the hormonal balance and affects the interpretation of common doping tests. Our results are informative about the critical points to be considered in relation to doping tests in fertile female athletes.

Code: T14M02AB 

Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles and is prohibited in males according to the World Anti-Doping Agency list of prohibited substances. Immunoassays are currently used by anti-doping laboratories to measure urinary hCG but results can vary widely among laboratories due to differences in cross-reactivity against different molecular forms of hCG (isoforms). We recently developed a sequential immunoextraction method with liquid chromatography tandem mass spectrometry (LC-MS/MS) detection for quantification of intact hCG, hCG free β-subunit and β-subunit core fragment in urine. Data from hCG excretion studies demonstrated that intact hCG should be monitored for detection of doping with hCG. In order to apply the LC-MS/MS method for confirmation of immunoassay screen positive hCG results a threshold concentration needs to be established. In preliminary studies we found that the current threshold concentration of 5 mIU/mL applied to immunoassays is not appropriate when measured by LC-MS/MS and needs to be significantly lower. 
In the present study we plan to determine the concentration of intact hCG in 600 non-doping male urine samples using the recently developed immunoextraction method with LC-MS/MS detection. Half of the urine samples will be from normal male volunteers and the other half from non-doping male athletes. The data from this study will be immediately used to determine the appropriate threshold concentration for confirming doping with hCG. 

Main Findings:

The concentrations of intact hCG in the male urine samples are provided in the attached file and are graphically displayed in Figure 1. Of the 570 male urine samples, 243 had undetectable intact hCG concentrations (<0.02 IU/L) not displayed on the graph. The remaining 327 urine samples had intact hCG concentrations ranging from 0.02 to 0.50 IU/L.  Of the 590 total samples, 542 (95.1%) had intact hCG concentrations <0.01 IU/L. Of the remaining 28 samples, 27 had intact hCG concentrations >0.01 and <0.26 IU/L. Only sample had an intact hCG concentration of 0.50 IU/L (27 year old, not shown on graph). Given these data, we recommend an extremely conservative intact hCG cutoff of 2 IU/L to be used during confirmation testing for detection of doping with hCG. At a concentration of 2.5 IU/L the imprecision of the method was 11.4%, when 3 separate urine samples were analyzed on 5 different days.

Code: 14A11CA 

The project that we propose implies the development of a novel two-dimensional HPLC purification method for the carbon isotopic analysis of two groups of molecules, corticoids and low concentrated steroids. Prednisolone is a direct metabolite of the synthetic corticoid prednisone but it can also be derived from the dehydrogenase bacterial enzymatic activity of cortisol. In the same manner cortisone, the parent molecule of cortisol, could produce considerable quantities of prednisone as well, yielding false positive cases. Additionally, numerous methods for the purification and C analysis of boldenone and nandrolone metabolites have been developed in the past years; however, to our knowledge, none yet has proven to produce measurements as precise and accurate as the testosterone metabolites, being unquestionably more concentrated. Furthermore two HPLC purifications are commonly required for their purification, from which testosterone metabolites are left aside due to the complexity of the separation even if the latter come necessary in the detection of multi-positives cases. To ascertain the exogenous origin of the corticoids, difficult to chromatographically purify due to their higher polarity, and to efficiently purify boldenone or nandrolone metabolites including testosterone metabolites, the development of a novel two dimensional HPLC method through the heart-cutting technique is sought for a rapid purification of those compounds from the urine matrix without any derivatization. The rationale behind this method development is (i) to save instrumental time for the purification of the compounds, (ii) obtain the same purification power as with two subsequent HPLC cleansing runs and (iii) to gain intensity, precision and accuracy owed to fewer manipulations and chemical reactions. Finally, we wish to validate the methods by confirming the absence of isotopic fractionation through the analysis of all the tested metabolites using the developed HPLC technique and to publish the methods used for routine δ13C measurements. 

Main Findings: 

The use of compound specific isotope analysis (CSIA) to investigate stable isotopic abundances of is still considered a niche discipline for well-trained specialists (Brand 2012) and indeed, the implementation of reliable and robust IRMS methods has proven to be quite a challenge for anti-doping labs. 
Possibly the most critical aspect of CSIA is achieving adequate purification of compounds prior to IRMS analysis since unlike most other mass spectrometric methods, the specificity of CSIA analysis hinges on achieving baseline separation of target GC peaks from all other carbon-containing molecules in the urinary matrix. Insufficient purification results in peak contamination and inconsistent or irreproducible δ13C values. The world anti-doping agency (WADA) recommends the use of high performance liquid chromatography (HPLC) for sample cleanup, however many WADA-accredited labs suggest more than just one purification step (Piper et al. 2008) or their methods require long HPLC columns and extended purification time (75 minutes, Ouellet, LeBerre and Ayotte, 2012). 
This WADA project provides a description of an automated two dimensional HPLC purification (2D-HPLC) method for urine extracts that has made possible the highest throughput CSIA purification of urine extracts described thus far by WADA-accredited labs, requiring only 35 minutes per sample or approximately 20-25 samples/day. In contrast to previously established CSIA methods, no sample manipulation is required between purification steps (e.g. Piper 2008). Six urinary steroids including testosterone/DHEA and 4 metabolites (5α-androstanediol, 5β-androstanediol, androsterone and etiocholanolone) as well as two endogenous reference compounds (pregnanediol and 3α-hydroxy-5α-androst-16-ene) were eluted and collected during HPLC purification. 
Comparative GC chromatograms are used to contrast the efficiency of 2D purification to a previously established 1D HPLC method (Ouellet et al 2014). While each sample requires less than half the time of 1D purification, sample purity is actually improved. Precision of δ13C for all analyzed compounds in negative and positive controls was 0.3‰ or better, which is comparable to the precision of pure compounds at similar concentrations. The appeal of 2D-HPLC is indeed that a properly configured method can easily surpass the efficiency of the highest performing 1D system – producing clean peaks in much shorter run times. No extra expenditure is incurred by the use of 2D-HPLC, aside from the initial instrument cost. The potential for a dramatic increase in the IRMS sample load offered by 2D-HPLC would be especially useful during international sporting events, during which a large number of tests are required over very short time delays. Increased sample throughput makes possible a larger number of IRMS tests, necessarily improves the likelihood of detection of the abuse of testosterone or testosterone-related steroids.

Code: 14C13AB 

Gene doping is believed to become a new threat to sport and the anti-doping community has been focusing efforts on developing a test for its detection. Methodology to detect doping genes in athletes’ blood already exists and work is underway to validate an erythropoietin gene doping test for implementation in testing laboratories. 
Recently, we expanded our detection capability to include four additional candidate genes with potential to increase muscle size and strength. Using commercial PCR assays, we developed a multiplex ‘anabolic gene doping detection panel’ which targets genes for insulin-like growth factor-1, growth hormone, growth hormone releasing hormone and follistatin. The panel allows simultaneous detection of several ‘sport-specific’ genes in one sample, reducing the test’s cost and turn-around-time. 
Acceptance of this method for routine testing requires suitable reference materials (RM) as controls to ensure the method performs as intended. During method development, cDNA-based controls for each gene are commonly used, but these are unsuitable for routine testing, because inadvertent sample contamination with such control would lead to a false-positive test result. 
We propose to develop a single synthetic DNA-RM for testing doping with four ‘anabolic’ genes that will overcome this problem. The RM will incorporate four modified sequences detectable by the ‘anabolic gene doping detection panel’ with similar specificity and sensitivity as each doping gene. However, the products from the RM and the doping genes will differ, allowing discrimination between true-positive and false-positive test results. We will characterise the RM for purity, quantity, homogeneity and stability using digital PCR and other molecular techniques. Furthermore, using this RM we will validate the multiplex ‘gene doping detection panel’ using a model in vitro system. 
This research is crucial in the development of a reliable routine method for detection of gene doping with several genes that could be potentially used in ‘power’ sports. 

Main Findings: 

Gene doping is believed to be a new threat to sport and our laboratory has been focusing efforts on developing tests for its detection. Through these efforts, the first test for erythropoietin gene doping was developed and is currently being implemented in testing laboratories. To expand our detection capability to other genes, we recently developed an ‘anabolic’ gene doping detection panel which targets doping genes that have potential to increase muscle size and strength. The panel consists of commercial PCR assays that were optimised and validated to work equally well when each assay is used on its own or in combination with one or two other assays to allow doping genes to be analysed in a sample simultaneously.

To use the panel of PCR assays in routine testing, suitable reference material(s) (RM) are required for use in method’s quality controls. In this project, we generated a single synthetic DNA RM that can be used in testing doping with any of the four ‘anabolic’ genes using their specific PCR assays performed separately. A characteristic property of this RM is that, due to its unique design, it does not generate a false-positive test result in a routine laboratory setting if an athlete’s sample is inadvertently contaminated with the RM. 

The current study expands the arsenal of gene doping detection targets and has made significant contribution to progress the developed method towards a reliable, reproducible and robust routine test for detecting doping with any of four ‘anabolic’ genes. This test could be included in the fight against doping in sport in the near future.

Code: T14M04WS

The noble gas xenon has been used as a narcotic agent for more than 50 years and both effects and side-effects have been carefully studies.  Besides its anesthetic properties, xenon proved to be beneficial for patients during a surgical intervention and postoperative by stimulating different relevant metabolic pathways.  As the stimulations include the so called hypoxia-inducible factor 1α and as a direct consequence EPO, xenon might be of interest for high level athletes, too.

During the Winter Olympic Games first rumors spread regarding the misuse of xenon by elite athletes.  As neither xenon nor any gas has ever been in the scope of doping control analysis, the challenge for WADA accredited laboratories will be to develop and validate methods suitable for the detection of xenon from different biological matrices.  For blood and serum it has recently been demonstrated that xenon can be analyzed by gas chromatography/high resolution-high accuracy mass spectrometry (GC/TOF-MS) and also, much more complicated, by offline isotope ratio mass spectrometry.  The sensitivity of the GC/TOF-MS approach was sufficient to detect xenon in a blood sample drawn 24 h after  termination of narcosis in a patient.

Now our goals will be to investigate the suitability of urine samples to detect xenon misuse, to further increase the sensitivity by improving sample preparation and mass spectrometric conditions, to fully validate our approach to make it suitable for doping control analysis and to search for and implement a possible internal standard to enhance reproducibility of the results.  This will also encompass studies on the stability of the highly volatile xenon in urine specimens in order to elucidate if and how the analysis of this special analyte can be implemented into existing routine doping control procedures.

Main Findings:

On September 1st 2014, a modified Prohibited List as established by the World Anti-Doping Agency (WADA) has become effective featuring xenon as a banned substance categorized as hypoxia-inducible factor (HIF) activator. Consequently, the analysis of xenon from commonly provided doping control specimens such as blood and urine is desirable, and first data on the determination of xenon from urine in the context of human sports drug testing are presented.    

In accordance to earlier studies utilizing plasma as doping control matrix, urine was enriched to saturation with xenon, sequentially diluted, and the target analyte was detected as supported by the internal standard d6-cyclohexanone by means of gas chromatography/triple quadrupole mass spectrometry (GC/MS/MS) using headspace injection. Three major xenon isotopes at m/z 128.9, 130.9 and 131.9 were targeted in (pseudo) selected reaction monitoring mode enabling the unambiguous identification of the prohibited substance. Assay characteristics including limit of detection (LOD), intraday / interday precision, and specificity as well as analyte recovery under different storage conditions were determined. Proof-of-concept data were generated by applying the established method to urine samples collected from 5 patients before, during and after (up to 48 h) xenon-based general anesthesia.   

Xenon was traceable in enriched human urine samples down to the detection limit of approximately 0.5 nmol/mL. The method’s intraday and interday imprecision values were found below 25%, and specificity was demonstrated by analyzing 20 different blank urine samples that corroborated the fitness-for-purpose of the analytical approach to unequivocally detect xenon at non-physiological concentrations in human urine. The patients’ urine specimens returned ‘xenon-positive’ test results up to 40 h post-anesthesia, indicating the limits of the expected doping control detection window. This time window was comparable to the one obtained in blood specimens.

Since xenon has been considered a prohibited substance according to WADA regulations in September 2014, its analysis from common specimens of routine sports drug testing is desirable. In previous studies, its traceability in whole blood and plasma was shown, and herein a complementary approach utilizing doping control urine samples for the GC/MS/MS analysis of xenon is reported.

Code: 14B12RG

The aim of all kinds of blood manipulation is to increase the total hemoglobin mass (tHb-mass), which is directly correlated to maximum aerobic power and hence performance. 
To minimize these illegal practices we recommend monitoring tHb-mass of endurances athletes over time Serial measurements of tHb-mass can also be used to demonstrate objectively that an athlete has or has not used blood doping practices. 
The practicability of a 15NO-rebreathing method in analogy to the established optimized CO-rebreathing method was evaluated in a scientific project by Prof. Schmidt, Prof. Bloch and Dr. Gäbler and funded by WADA (2010-2012). 15NO has several advantages compared to CO, which could lead to a broad acceptance of the method in federations and athletes. 
The amount of tracer gas which has to be inhaled can be 4000-fold reduced, avoiding a toxic load for the athlete. Furthermore, NO has a 200-fold higher affinity to hemoglobin reducing the influence of possible confounding factors. We expect the NO-rebreathing technique using 15NO as innovative tracer gas as an optimal method to determine tHb-mass. 
The practicability of the new 15NO-method is still limited by the sensitivity of the detector. Physiological loss mechanisms and handling of the blood samples as test routine method require a higher sensitivity than commercially available.  With the further improvement of the 15NO-sensor, setup in the first project, we expect to meet the demanding detection limit for a tHb-mass routine test. As a consequence tHb-mass can be introduced as a key parameter into the athlete’s biological blood pass. 

Main Findings:

We are developing a new detection method for the determination of total hemoglobin mass (tHb-mass) using isotopically labelled nitric oxide (15NO) replacing the tracer gas carbon monoxide in the established CO rebreathing method. Using isotopes allows the reduction of the inhaled amounts of toxic tracer gases below international maximum allowable concentrations. In a first project we did not achieve the required sensitivity to determine the basal Hb15NO concentration. Therefore we improved in the actual follow-up project the sensitivity of our laser spectrometer integrating an optical resonator into our spectrometer setup. 
We were able to improve the sensitivity of our detector by a factor of 67.8 compared to the previous sensitivity. The achieved sensitivity limit corresponds to the application of 1 ml 15-nitrite solution with a concentration of 1.5 nM.  
With the obtained sensitivity we were able to detect the basal Hb15NO concentrations of n=6 volunteers. The mean value of the basal Hb15NO concentration was 1.63 nM (± 0.16 nM). We tried to determine the tHb-mass of the volunteers applying 15 ppmV of 15NO in a 3 l anesthetic bag following the CO-rebreathing protocol. Additionally we applied 10 ppmV of 15NO continuously over a period of 60 minutes.  
Following the inhalation process we observed a transient Hb15NO peak with a fast rise during the rebreathing process. Stopping the rebreathing protocol after two minutes leads to a decrease of the measured Hb15NO concentration. Ten minutes after the start of the inhalation process the Hb15NO values have reached again basal levels.  
During the continuous 15NO inhalation we also observed a fast rise of the Hb15NO level followed by a slow decrease over the complete inhalation time.  In both approaches the calculated tHb-masses were too high by several orders of magnitude. In literature several interactions of nitric oxide with different hemoglobin states or forms are described which would explain the overestimated tHb-masses. In order to achieve correct tHb-masses all the relevant pathways have to be considered and the different losses of the administered tracer gas (conversion to 15-nitrate and 15-nitrite etc.) have to be quantified.

Code: 14A24JM

The aim of this project is to utilise high-accuracy Reference Measurement Procedures (RMPs) developed at the National Measurement Institute of  Australia (NMIA) to assign reference values with low measurement uncertainties for eight target analytes in World Anti-Doping Agency (WADA) External Quality Assessment Scheme (EQAS) samples. The target analytes are androsterone (A), etiocholanolone (E), testosterone (T), epitestosterone (epi-T), 5a-androstane-3a,17b-diol (5a-Adiol) and 5b-androstane-3a,17b-diol (5b-Adiol), 19-norandrosterone (19-NA) and the T/Epi-T ratio. Target setting using reference values in the WADA EQAS will enhance the value of the program permitting an objective evaluation of the performance of WADA-accredited laboratories. Accuracy-based grading provides a more robust and transparent indication of competency than consensus-group grading.

Main Findings:

Reference values for the mass fractions and mass concentrations of testosterone (T), epitestosterone (EpiT), 5α-androstane-3α,17β-diol (5α-Adiol), 5β-androstane-3α,17β-diol (5β- Adiol), androsterone, etiocholanolone, and 19-norandrosterone (19-NA) and the T/EpiT ratio in human urine samples were determined for Cycle 3 of the 2016 World Anti-Doping Agency External Quality Assurance Scheme (WADA EQAS) for Longitudinal Steroid Profiling. Reference values were determined using a Reference Measurement Procedure (RMP) based on the technique of isotope dilution with gas chromatography tandem mass spectrometry (GC-MS/MS) analysis. The reference values are metrologically traceable to the SI units for mass (kg) and volume (m3) within their stated uncertainties. The measurement uncertainties were determined at a level of confidence of 95%.

Code: 14D06RV

The misuse of testosterone and other endogenous anabolic androgenic steroids is detected through alterations in the urinary steroid profile. The steroid profile, composed of concentrations and ratios of endogenous steroid hormones, has been implemented by WADA in the athlete´s biological passport. 
Metabolites included in the steroid profile have both gonadal and adrenal origin. Administration of glucocorticosteroids inhibits the hypothalamic-pituitary-adrenal axis by negative feedback, and reduces the adrenal steroid production. Due to significant adrenal origin of some of the metabolites included in the steroid profile (androsterone, etiocholanolone, 5α-androstane-3α,17β-diol, and 5β-androstane-3α,17β-diol and epitestosterone), it might be expected that reduction in the production of androgens by adrenal glands will have an effect on the urinary steroid profile, mainly in women where the relative importance of androgens generated in the adrenal cortex is greater. Due to the wide use of glucocorticosteroids in sports, its effect on the steroid profile deserves to be studied. 
The aim of the project is to investigate the impact of the administration of glucocorticosteroids by systemic routes on the parameters of the steroid profile in healthy volunteers. The effect of single systemic doses (intramuscular or oral doses) of synthetic glucocorticosteroids on the different parameters of the steroid profile will be evaluated in men and women. 

Main Findings: 

The steroid profile is a powerful tool to detect the misuse of endogenous anabolic androgenic steroids (EAAS) in sports. Glucocorticoids (GCs), which are only prohibited in competition using systemic administration routes, inhibit the hypothalamic-pituitary-adrenal axis. Due to the partial adrenal origin of the compounds included in the steroid profile, the administration of GCs might affect their excretion in urine and, therefore, modify the steroid profile. The aim of the present work was to investigate if GCs administered by either systemic or local routes could affect the steroid profile. 
Three of the most frequently detected GCs in sports (prednisolone, betamethasone and triamcinolone acetonide) were administered to healthy male and female volunteers (n=40) using different administration routes (topical, oral and intramuscular administrations at different doses). In total, 66 administration studies were performed. Urine samples were collected before and after GCs administration. The steroid profile (testosterone T, epitestosterone E, androsterone A, etiocholanolone Etio, 5α-androstane-3α,17β-diol 5αAdiol and 5β-androstane-3α,17β-diol 5βAdiol) was measured by gas chromatography-mass spectrometry.
The excretion rates of the steroid profile metabolites decreased after systemic GC administration (oral and intramuscular administrations). This excretion decrease was found to be associated with the dose and the administration route. However, the ratios evaluated on the steroid profile module of the athlete's biological passport model were not altered.
The results obtained show that GC administration does not distort the establishment of normal ranges of T/E, 5αAdiol/5βAdiol, A/T, A/Etio or 5αAdiol/E ratios. Therefore, GCs administration does not need to be considered a confounding factor in the steroid profile evaluation

Code: 14D04MT 

Growth homone releasing hormones (GHRH) induce growth hormone (hGH) secretion and are prohibited as performance enhancing agents as they act as classical releasing factors of hGH. The availability and the effects are extensively discussed in several internet blogs and black-market platforms. Three synthetic analogues to GHRH (Geref, Tesamorelin and CJC-1295) have been shown to be available to athletes aiming at undermining the doping control system, whereby Geref and Tesamorelin are approved as therapeutics and CJC-1295 has been in clinical trials. Generally, only limited pharmacokinetic data are available for all three peptide-based drugs and their metabolism, particularly their renal elimination after standard therapeutic administration, is largely unknown. This hinders an effective analysis in blood or urine samples for these compounds due to the lack knowledge about relevant metabolites to screen for.

The main parts of the planned project are thus the characterization of metabolites of all GHRH analogues in a rat model (in-vivo) and human blood specimens (in-vitro). Subsequently, effective target peptides (intact drug or derived metabolites) will be evaluated in different biological fluids allowing to establish an initial test method as well as confirmatory procedure inclusive of all relevant target analytes.

Main Findings: 

The family of growth hormone releasing hormones is prohibited in sport due to performance enhancing properties, attributed to enhanced endogenous growth hormone production and/or secretion. Most prominent candidates include the single-chain peptidic drugs/drug candidates Geref (Sermorelin), Tesamorelin, CJC-1295 and CJC-1293. Effective doping control analysis is featured by sensitive determination of the most reliable target metabolites in the respective biological fluid (urine or blood). Thus, the knowledge about the metabolism of a prohibited substance is crucial. In this study, detailed analytical information about the target peptides and their potential metabolites after in vitro experiments and literature review was obtained. Most promising analyte candidates for doping control purposes were synthesized and characterised by mass spectrometry. Several sample preparation strategies were evaluated using solid phase extraction, protein precipitation, ultrafiltration prior to immunoaffinity purification (with magnetic beads or MSIA) and liquid chromatographic-mass spectrometric detection. All developed methods showed sufficient sensitivity to cover the estimated concentrations after administration, and the implementation into existing procedures for other prohibited peptides (insulins, synacthen, etc.) is enabled. The procedures were validated for qualitative result interpretation and considered “fit for purpose” for doping control analysis.