Projets de recherche

Code: 13D18GF 

Synthetic cannabinoids mimic the psychoactive effects of tetrahydrocannabinol (THC) and are found in herbal smoking mixtures, commonly sold as "spice".  Like THC, these compounds are cannabinoid receptor agonists. Today, there is an immense variety of synthetic cannabinoids and spice blends available on the market and new substances constantly appear. Slight chemical modifications of existing compounds make many new cannabimimetics possible. For doping analysis, a fast elucidation of the metabolic pathways of new synthetic cannabinoids is of high importance in order to be able to implement them in doping control procedures. Due to a lack of a toxicological profile, in vivo excretion studies are however difficult to perform. Hence, the current study will employ in vitro metabolism studies on selected cannabimimetics using human cryopreserved hepatocytes. Human cryopreserved hepatocytes are recognized to be a close model to the human liver, showing excellent metabolizing and transporting activities. After hepatocyte incubation and a clean-up procedure, analysis will be performed with liquid chromatography and gas chromatography, combined with mass spectrometry. The selection of the target substances for the in vitro experiments will be based on the frequency of their detection in forensic samples. This is due to a rapidly changing market. The proposed metabolism is verified through the analysis of samples from traffic controls.

Main Findings: 

For doping analysis, the elucidation of the metabolic pathways of new synthetic cannabinoids is of high importance in order to be able to implement them in doping control procedures. Because of the high variability of synthetic cannabinoids and new herbal mixtures in a rapidly changing marked, a fast characterisation of metabolites is required. In terms of non-approved substances like this, though, human excretion studies are difficult to perform due to ethical considerations. In vitro studies, however, can identify human metabolites, and human primary hepatocytes are recognized to be a very close model to the human liver. Furthermore, the improvement of cryopreservation techniques has facilitated the use of cryopreserved hepatocytes which are now commercial available. 
The aim of the current project was to use human cryopreserved hepatocytes to study the metabolism and characterize metabolites of selected synthetic cannabinoids predominating on the marked today. Two frequently reported cannabinoids in herbal smoking mixtures are JWH-307 and AM-694. Thus, these two compounds were selected for the study. 
The incubation mixtures were analysed for substrate and metabolites using liquid chromatography-mass spectrometry (LC/MS/MS) with electrospray ionisation (ESI). Free, glucuronide and sulphate products were separated during sample preparation. Although varied conjugation rates were observed, the majority of the main metabolites were to a high extent excreted as glucuronide conjugates. For all metabolites, the structures are proposed, not confirmed. 
After incubation of AM-694 with human hepatocytes, 10 metabolites were recovered in the extracted culture medium. The most abundant generated products were a defluoroniated monohydroxylated metabolite and a carboxylated metabolite. Furthermore, the results from the hepatocyte assay were compared to analytical results from a forensic AM-694 urine sample. The in vitro data correlated well with observed metabolites in the urine sample. 
After incubation of JWH-307 with human hepatocytes, 22 metabolites were identified. A monohydroxylated compound, a dihydroxylated compound, and three dihydrodiol metabolites were the prevailing metabolites generated in the incubation mixture. 
In conclusion, incubations with human hepatocytes seems to represent a useful model for identifying and predicting major urinary metabolites. 

Code: 13A17MT

Most substances relevant to doping controls undergo considerable metabolism following administration. The generated metabolites are commonly less toxic and more polar which allows for renal clearance. Consequently, in urine the analysis of metabolites is more promising than attempts to detect the ingested or injected compound, particularly when these metabolites are excreted for a longer period of time than the drug. These so-called long-term metabolites are of great interest especially for those doping agents that are predominantly used during out-of-competition periods as for instance in case of anabolic androgenic steroids. 
In order to identify drug metabolites in biological matrices, stable isotope labeling of compounds has proven to be a helpful means within the last 3 decades. Usually, hydrogen is replaced by its heavier isotope deuterium at 3 to 5 locations within the steroid nucleus and the resulting mass shift was detected by means of conventional mass spectrometers. The sensitivity of this approach has recently been considerably improved by using hydrogen isotope ratio (HIR) mass spectrometry instead of conventional mass spectrometers as these HIR-dedicated systems are built to determine the deuterium/hydrogen ratio at natural abundance, i.e. with approximately 1 out of 10000 hydrogen atoms representing a deuterium atom. For deuterium-labeled compounds commonly up to 10 % of hydrogen is substituted and thus enables a highly sensitive detection of any metabolite of the administered compound as long as it carries the deuterium-label.  This feature will be exploited in a novel approach to investigate the metabolism of clenbuterol and nandrolone. Both substances are in the scope of sports drug testing since several years and the systematic study of their metabolism will be conducted to support their long-term detection and determination of respective sources (i.e. endogenous/natural or artificial/contamination). 

Main Findings: 

Employing stable isotope-labeled drugs and GC/IRMS for metabolism studies significantly facilitates the detection of relevant analytes also at lowest abundance in complex matrices such as human urine. In the present research project, the metabolism of two critical anabolic agents, 19-nortestosterone and clenbuterol, was revisited utilizing the recently established approach. Triply deuterated 19-nortestosterone and nine-fold deuterated clenbuterol was used to characterize the drugs’ metabolic profile and to complement and/or corroborate the picture of long-term metabolites as well as the possibilities to differentiate the origin of analytes. In case of 19-nortestosterone, metabolites supporting the classification of low abundant 19-norandrosterone findings as of natural or xenobiotic nature is desirable; in case of clenbuterol, information facilitating the differentiation of contaminated food ingestion from deliberate drug misuse is needed.
The results obtained for both drugs were manifold and a substantial number of known and several yet unknown metabolites as well as sample preparation-derived artefacts were identified. A new 19-nortestosterone metabolite was observed, which was traceable as long as 19-norandrosterone in human urine in the conducted elimination study. The structure of this compound is not entirely clarified yet, and since it appeared to exhibit significant interindividual variability (as suggested by means of routine doping control samples and confirmed adverse analytical findings), its utility for sports drug testing purposes might unfortunately be rather limited. Also for clenbuterol, metabolites were detected and attributed to oxygenation products of the drug. As these are expected to possess different elimination kinetics than clenbuterol itself, a means to differentiate the recent intake of a minute amount (i.e. via food contamination) from remnants of clenbuterol being eliminated from drug abuse weeks ago could be obtained. Here, urinary clenbuterol / metabolite ratios need to be determined in future studies.

Code: 13B10MU 

Prohibited glycoprotein hormones such as ESA, hCG, FSH etc. are currently tested by immunological methods, and the decision is made by immuno-blotting electrophoresis or by comparing concentration with the applicable threshold. However, heterogeneity of the markers or multiple sources of the target compound, e.g. pregnancy, hormone producing tumor, doping etc. can sometime cause in difficulty of decision making. We confirmed that all recombinant glycoproteins from CHO cell line studied were lacking certain human type glycanes, which are not always identifiable by mass spectrometry. Our first year results represented that recombinant and human glycoprotein hormone can be isolated by stereo specific lectin-glycane interactions, and the recombinant fraction does not show any human type glycane specific lectin interactions. Thus, findings of our 1st year project supported the possibility of origin specific detection of glycoproteins by lectin specific extraction coupled to a normal immunoassay instruments that commonly used by all WADA laboratories.

Main Findings: 

Genetically manufactured glycoprotein has the unique isoform profile and the heterogeneity can sometimes cause in interpretation difficulties. Prohibited glycoprotein hormones are currently tested by immunological methods with the result evaluation using the applicable threshold or by comparing the isoform profile with that of the reference standard as no procedure yet available to identify their origins. Glycoproteins from human show a strong lectin-glycan interaction with certain lectins whereas those expressed in CHO-cell lines do not show any interaction with the selected lectins. Immobilized SSA lectin was suspended with a sample aliquot and non-reactive glycoprotein was collected by an exclusion chromatography.  Rest of captured hormone was then eluted with Lactose solution and each fraction was measured by immunoassays. SSA captures LH and FSH from human, and Lutropin (rLH) and Follitropin (rFSH) are collected in the Filtrate. IEF gel electrophoresis western blotting analysis and well-evaluated immunoassays confirmed that FSH in the Filtrate and the Eluate were identical to those of rFSH and uFSH respectively. Pre-analysis lectin fractionation coupled with immunoassay has enabled an origin identification of LH and FSH. Some other possible markers to indicate origin of glycoprotein were evaluated.

Code: 13D13JD 

This research project will contribute to the understanding of inhaling high therapeutic doses of asthma medication (long acting Beta-2-agonists) over a sustained period of time on athletic performance. The majority of research undertaken with inhaled Beta-2-agonists involves acute doses of short acting Beta-2-agonists in male athletes. Investigating acute doses of Beta-2-agonists does not necessarily replicate real life situations, where athletes prescribed Beta-2-agonists will use them on a daily basis for a prolonged period of the time.

The potential performance enhancement from inhaled Beta-2-agonists focuses on increases in skeletal muscle protein, leading to increased muscle mass.  Increased muscle mass is associated with increased muscle force production.  In addition the long term use of Beta-2-agonists may also decrease body fat.  No study has investigated the potential for these performance gains to occur when athletes inhale long acting Beta-2-agonists over a prolonged period of time.

There is limited ecologically valid data available that demonstrates whether long acting Beta-2-agonists, taken over a prolonged period of time is performance enhancing. Further to this there is no data available that details the movement of Beta-2-agonists through the body following long-term use of high therapeutic doses of inhaled long-acting Beta-2-agonists.

This research project will investigate the impact of inhaling the long acting Beta-2-agonists Salmeterol or Formoterol twice daily over a 12 week period on sprint performance, strength, power, recovery and body composition in male and female athletes. In addition the movement of the inhaled long acting Beta-2-agonists through the body will be analysed at 6 weeks and 12 weeks.  The results of this study will provide data to inform the use of inhaled Beta-2-agonists by elite athletes. In particular they will provide data to assist in the implementation of regulations on the use of inhaled Salmeterol and Formoterol and assist in the resolution of contested doping violations.
 

Main Findings: 

The purpose of this study was to contribute to the understanding of inhaling high therapeutic doses of long acting β2-agonists over a sustained period of time on athletic performance. In particular this study investigated the impact of inhaling Salmeterol or Formoterol twice daily over a ten week period on strength, power, recovery and body composition in male and female athletes.
To carry out the investigation into the potential ergogenic effect of long term use of long acting β2-agonists we recruited 38 participants (23 male and 15 female) who had no history of asthma. All participants underwent a eucapnic voluntary hyperpnoea challenge to confirm absence of asthma. Participants completed baseline assessments which included measuring strength and power performance as well as assessing body composition. Participants were randomly assigned to one of three groups: placebo inhaler, salmeterol inhaler (100 µg twice daily) or formoterol inhaler (12 µg twice daily). The participants undertook a 10 week training protocol with a repeat of the baseline assessments taking place at five and ten weeks. Over the course of the training periods participants took their assigned inhaled dose of β2-agonists twice daily.  During the training weeks participants attended three training sessions a week that focused specifically on developing strength, power and sprint performance. Assessments of recovery and mood were recorded via questionnaires during the training period. 
All 38 participants completed all data collection between weeks 0 and 5, however only the male participants completed all 10 weeks. Between week zero and week five 30 m sprint time improved in both the formoterol (–0.29 ± 0.11 s; P<0.049) and salmeterol (– 0.35 ± 0.05 s; P<0.04) groups when compared to the placebo group (+0.01 ± 0.11 s).  However, there were no changes in other strength and power measurements, recovery, mood or sum of 4 skin folds between groups over the course of the 5 weeks between groups. Between weeks 5 and 10 decreases in sprint time in salmeterol and formoterol groups were similar to placebo group in the male participants. 
Our results suggest that large daily doses of salmeterol and formoterol may lead to improvements in sprint performance over a 5 week period. However the improvements in sprint performance are not sustained over a longer period of time.  Future work investigating the mechanisms will provide an understanding how daily use of long acting β2-agonists may influence sprint performance over a sustained period of time.

Code: 13A04JW 

Traditional Chinese Medicine (TCM) has been used in China for thousands of years. Some drugs of TCM contain endogenous steroids such as musk, penis and testes of ox, and oviductus ranae, which make it possible to abuse TCM as doping. In the 2011 FIFA Women's World Cup, adverse analytical findings about the exogenous source of etiocholanolone and other steroids were reported , and musk was regarded as the source of the steroidal preparation. Isotope ratio mass spectrum is applied in doping test to confirm the endogenous steroids abuse by determining the isotopic ratio of 13C/12C, whereas the carbon isotope ratios of steroids in animal materials used in TCM were seldom reported in literature. In view of the fact that TCM materials would be abused as doping, their influences on doping test should be further studied. 
In this project, musk, penis and testes of ox, and oviductus ranae will be collected for the detection of concentrations and carbon isotope ratios of endogenous steroids by GC-MSD and GC/C/IRMS. Excretion study will also be executed to demonstrate the influences of musk intake on the doping test. 
Based on those studies, the possibility of those TCMs abuse as doping will be estimated.

Main Findings: 

Traditional Chinese Medicine (TCM) has been used for a long time. Some TCMs contain prohibited steroids, which make it possible to abuse TCMs as doping. In this study, steroid contents and δ13C-values were analyzed in musk, penis-testes of ox, and forest frog’s oviduct. Excretion study was executed to demonstrate the influences of musk ingestion on doping control.  
  The analysis on 20 batches of musk illustrated that the contents of androgen were greater than that of estrogen and progestin in musk. Etio, 5α-3β17α-diol, 5β-diol, and An were main androgen in musk. IRMS analysis demonstrated that the androgen in wild deer musk possessed δ13C-values in the range of exogenous steroid values, while the steroid δ13C-values in domestic deer musk samples were within the normal range of human body.  
  In penis-testes of ox, An, Etio, DHEA, DHT, 4-AD, T, EpiA, and Preg were detected; Chol had average δ13C-value of -22.6 ‰. In forest frog’s oviduct, Preg and 5β-diol could be identified; Chol bore the average δ13C-value of -27.8 ‰.  
  In musk excretion study, 200 and 100 mg of wild and domestic deer musk samples were administrated by 29 subjects. The urinary profile showed changes, but the changes were inconsistent among subjects. The fluctuations might be proportional to the musk quality. In the IRMS test, the δ13C-values of Etio and 5β-diol were more sensitive than other markers, and AAFs were obtained. It is the first report about the AAFs in the musk administration study. 

Code: 12D14MP

In accordance with the 2012 Prohibited List all 2-agonists are prohibited in sport, in- and out of-competition, except salbutamol, formoterol and salmeterol when administered by inhalation in accordance with the manufacturers recommended therapeutic regime. Whereas for salbutamol and formoterol thresholds differentiating the therapeutic use from misuse are indicated in the List, such threshold for salmeterol has not yet been established and existing data seem inconclusive.

Cytochrome P450 3A4 (CYP3A4) is an enzyme that plays a central role in the metabolism of a wide variety of drugs. P-glycoprotein 1 (P-gp), a protein encoded by the ABCB1 gene, is responsible for the regulation of the distribution of drugs. The activity of CYP3A4 and the expression of P-gp may be responsible for the inter-individual variability of pharmacokinetics (PK) of drugs using these substrates for their metabolic pathway and distribution, respectively. This can be explained due to the genetic polymorphisms of both CYP3A4 and P-gp among individuals and/or drug-drug interactions in the case of co-administration of drugs using CYP3A4. Salmeterol and fluticasone are using the same enzyme isoform CYP3A4 and corticosteroids appear to induce the activity of this enzyme.

This study aims to establish the PK profiles of inhaled salmeterol administered alone or in combination with fluticasone proprionate to healthy volunteers and type the genetic variations of the genes encoding for CYP3A4 enzyme and P-gp transporter protein in the participants of the study. This will allow determining the threshold level of the maximum therapeutic dose of inhaled salmeterol administered alone or in combination with fluticasone and to investigate the role of inhaled corticosteroids as potential masking agents when co-administered with salmeterol. Finally, the study will provide preliminary data on the possible association between genetic polymorphisms of the CYP3A4 and the ABCB1 genes with the PK and excretion profiles of inhaled salmeterol in healthy volunteers.

Main Findings

Results:

As part of the study, liquid chromatography – mass spectrometry (LC-MS) methods for the determination of salmeterol, its metabolite α-hydroxysalmeterol and fluticasone in human urine and plasma were developed and validated. In urine, the Limit of Detection (LOD) was 0.05 ng/mL for salmeterol and fluticasone, and 0.50 ng/ml for α-hydroxysalmeterol, while the Limit of Quantification (LOQ) was 0.10 ng/mL for salmeterol and fluticasone and 1.00 ng/mL for α-hydroxysalmeterol.

At Phase A, the highest observed individual urine concentration of salmeterol when not normalised for specific gravity was 0.56 ng/mL. When all urine concentrations were normalised, the highest concentration observed was 0.61 ng/mL and when only those samples with a specific gravity higher than 1.020 g/mL were normalised the highest concentration observed was 0.53 ng/mL. At Phase B, the highest observed individual urine concentration of salmeterol was 0.91 ng/mL when not normalised for specific gravity, 1.06 ng/mL when all urine concentrations were normalised for specific gravity and 0.79 ng/mL when only those samples with a specific gravity higher than 1.020 g/mL were normalised. No statistically significant differences were found between the concentration of salmeterol at Phase A and Phase B. The reported urinary concentrations of salmeterol represent the free parent compound, only.

At Phase A, the highest observed individual urine concentration of α-hydroxysalmeterol when not normalised for specific gravity was 5.55 ng/mL. When all urine concentrations were normalised, the highest concentration of α-hydroxysalmeterol observed was 6.94 ng/mL and when only those samples with a specific gravity higher than 1.020 g/mL were normalised to a urine specific gravity of 1.020 g/mL, the highest concentration of α-hydroxysalmeterol observed was 5.55 ng/mL. At phase B, the highest observed individual urine concentration of α-hydroxysalmeterol when not normalised for specific gravity was 3.42 ng/mL. When all urine concentrations were normalised, the highest concentration of α-hydroxysalmeterol observed was 11.4 ng/mL and when only those samples with a specific gravity higher than 1.020 g/mL were normalised the highest observed individual urine concentration of α-hydroxysalmeterol was 3.42 ng/mL. No statistically significant differences were found between the concentration of α-hydroxysalmeterol at Phase A and Phase B.

Conclusions:

We propose establishing a threshold for salmeterol and α-hydroxysalmeterol high enough to prevent any adverse analytical findings from the administration of salmeterol up to the maximum therapeutic dose yet able to detect those athletes who use salmeterol in excess doses. Based on the findings of the present study, the data that are available in the literature from other excretion studies and the analysis of routine doping control samples and a possible accumulation rate of 1.3, a urinary threshold concentration of 2.0 ng/mL for salmeterol can be supported.

Code: 12D20ZC

In CHINADA, 25% of the OOC tests is given to target testing each year. Thus, intelligent target test plan (TTP) becomes a crucial research that lays on the daily TDP work. During recent years, we exert our efforts on improving the TTP. Moreover, another way that attracts our attention is to follow up with the lab data, which could be very useful in discovering those potential drug-users. However, the kind and way of put those data into practice shall be pondered deeply over.

In 2010 Laboratory Statistics Report from WADA, the adverse analytical findings of AAS is 60.8% in the whole number of the AAF. Be different from exogenous AAS, endogenous anabolic androgenic steroids are difficult to detect.

It is recommended that a urine sample in which the parameter is met during the screening procedure, be routinely submitted to the IRMS analysis. Even though, some parameters abovementioned are still under the detection line after taken the endogenous AAS by athletes. It is obvious that finding out the athlete’s markers variant longitudinally means much to the antidoping job.

In the project, we will develop the AAF study, the cross-sectional study, and model construction study as well as the longitudinal study. For the results of this project, we hope we can set up the endogenous AAS model of the athletes and find out the accurate time to carry out testing according to the individual AAS model.

Main Findings

In order to fix the development of Athlete Steroidal Passport (ASP) module, the project has been adjusted. Doping risk of sports was assessed firstly. Besides the data of steroid profile from high-risk sports from 2009 to 2014 were collected, the data of non-athletes were also collected for baseline analysis. Based on these results, statistics of intra-individual CV of T/E ratio with sport factor and period factor was conducted to find the high risk period of different sport.

Three levels of doping risk of different sports were classified for in competition and out of competition separately. The results can be helpful the focus on different sports for IC and OOC tests.

The steroid profiles of non-athlete population have been analyzed. The cut-off value of different genotype was defined using cluster analysis. Also, the range of intra-individual CV of T/E ratio was calculated, which was much higher than it was reported before.

The steroid profiles of athlete population were analyzed based on the data of non-athletes. Through the analysis of the suspicious samples from suspicious athletes selected with intra-individual CV of T/E ratio with sport and competition period, the high risk months of each sport have been found. This regularity of each sport can be the guidance of establishing the test distribution plan, not only for target athletes, but also for all the athletes of the same sport.

Code: 12B8FD 

Blood doping is banned by the World Anti-Doping Agency in all sports due to its effects on sport performance, especially in endurance disciplines. WADA accredited laboratories have developed testing methods for the detection of blood doping by eryhtropoietins, synthetic hemoglobins, RSR13, and homologous blood transfusions, while no direct, internationally recognized method is yet available for the detection of autologous blood transfusions. We propose a flow cytofluorimetric approach based on the recognitions of markers of storage in red blood cells. More specifically, markers of apoptosis (namely, phosphatidylserine) and markers to detect reduction of antigen expression on red blood cells membrane (primarily among them CD55 and CD59 proteins) have already been evaluated by our laboratory as potential diagnostic parameters to detect the infusion of previously stored blood, with very promising results. Preliminary data obtained on several different blood samples, tested at different times after collection and in different storage conditions, showed that the selected parameters are significantly modified by red blood cells storage. We are planning to implement the number of markers considered for this study, possibly broadening the panel of diagnostic markers to be monitored to effectively detect the recourse to autologous blood transfusions. Once the most suitable diagnostic markers will be identified and selected, the effectiveness of the approach will be verified on subjects undergoing preoperative autologous donation in the framework of pre and post- surgery practices. 
We strongly believe that the proposed approach could effectively complement the analysis presently under evaluation for the detection of autologous blood transfusions, representing a significant advancement towards the development of a robust and reliable direct method to detect autologous blood transfusion.

Main Findings:

One  of  the  current  challenges  for  the  Antidoping  Laboratories  worldwide  is  the  detection  of  Autologous Blood Transfusion (ABT). At present, ABT can be detected only by indirect methods, which require the longitudinal evaluation of the stability of selected hematological parameters, as in the hematological module of the athlete biological passport.  
This  project  aimed  to  study  and  to  explore  a  multi---parametric  strategy,  based  on  flow  cytofluorimetry, targeting specific morphological/biochemical changes of red blood cells, as well as  the  alteration  of  other  hematological  parameters  (e.g.  the  increase  of circulating  microparticles), consequent to the storage period of the withdrawn blood prior to the reinfusion in  the  receiver  person.  This  approach  is  in  fact  a  “direct”  detection  strategy,  since  it  recognizes  specific changes on the “exogenous” (this meaning the transfused) red blood cells (RBCs).  
We have conducted a series of experiments on human whole blood samples that were focused on the  identification  of  diagnostic  signs  of  red  blood  cells  aging  also  matching  all  the  following  conditions: (i) to be easily detected by flow cytofluorimetry; (ii) to be stable after addition of the stored sample to fresh blood; and (iii) to show a sufficiently pronounced variation, allowing their identification also in mixed blood samples generated after a transfusion practice.   
From the data we obtained two main conclusions can be drawn: i) Flow  cytofluorimetry  is  able  to  identify  multiple  indicators  of erythrocytes  aging  that  are generated after a storage time in the fridge and blood banks condition.
ii) The  signals  of  RBC  aging  that  have  been  detected  in  our experimental  conditions  (I.e.,  by considering a period of storage up to 40 days) are the following:a. reduction of the expression of surface proteins on RBC;
b. moderate increase in the concentration of glycated HB (HbA1c);
c. reduction of RBC main size, that generates a population of smaller and more dense erythrocytes;
d. the  consequent  formation  of  a  population  of  microparticles,  that is  a  direct consequence of red blood cells microvesiculation process. Unluckily,  it  was  not  possible  to  verify  in  vivo  the  observation  recorded  ex  vivo,  as  well  as  to  evaluate  the  potential  effectiveness  of “markers  of  reinfusion”,  due  to  the  lack  of  samples collected from auto---transfused subjects. Experiments in this direction are still in progress, thanks to newly activated, ongoing cooperation with other research groups and clinical laboratories.  
In  spite  of  the  above  limitation,  the  data  we  collected  at  this  stage  are  very  promising  in  the  development of a universal, direct method for detecting both autologous and homologous blood transfusions: based on our results, a detection strategy based on the counting of the number of microvesicles and the counting of red blood cells of the more dense fraction (smaller in size) seem to present a higher diagnostic value than that based on the variation over time of the expression of specific proteins on the red blood cell membrane.  
Finally, it has to be stressed out that our proposed approach needs a solid standardization: indeed, the  same  counting,  when  performed  on  different  flow  cytofluorimetry  instruments,  that  use  different  detectors settings,  can  lead  to  different  measurements,  inevitably  resulting  in  an increase of the inter---laboratory variability of the results. 

Code: 12A18GG

Testosterone is still regarded as the major contributor to steroid doping world-wide. Among the most common forms of application are injections of different sorts of esters. State-of-the-Art detection of Testosterone doping includes the quantification of the Testosterone /Epitestosterone – Ratio (T/E – ratio) as well as subsequent Isotope ratio mass spectrometry (IRMS). Previous published studies of our research group have demonstrated that a comparably high percentage of testosterone preparations do not significantly differ from endogenous values of testosterone and markers of testosterone doping. Consequently when such preparations with endogenous – like 13CVPDB values are applied, IRMS - technology fails to detect testosterone
doping. Direct detection of the testosterone ester leads to an unequivocal proof of doping with testosterone preparations, because such esters are not built endogenously.  Previous studies indicate that a direct detection of testosterone esters in both hair and plasma is possible.
Aim of the proposed project is the investigation and optimisation of the direct detection of testosterone esters in body fluids like serum, whole blood and stabilized blood with an already developed detection method using modern and sensitive technology. The project will gain information on diagnostic windows for detection of doping using testosterone esters and proper sampling conditions. Additional aim of the proposed project is to evaluate the suitability of already collected blood samples in doping control (e.g. samples collected for blood parameter measurement or growth hormone detection) for a possible reanalysis for testosterone esters.

Main Findings:

Injections of synthetic esters of testosterone are among the most common forms of testosterone application. In doping control, the detection of an intact ester of testosterone in blood gives an unequivocal proof of the administration of exogenous testosterone. The aim of the current project was to investigate the detection window for a number of testosterone esters in blood. Furthermore, the suitability of different types of blood collection devices was evaluated.
A clinical study with six participants was carried out, comprising a single injection of a testosterone ester preparation. The enrolled subjects were randomly assigned to receive either a single intramuscular injection of testosterone undecanoante 1000 mg (Nebido®) or a single intramuscular injection of a mixture of four testosterone esters: testosterone propionate 30 mg, testosterone phenylpropionate 60 mg, testosterone isocaproate 60 mg and testosterone decanoate 100 mg (Sustanon®). Three subjects were assigned to each study group and blood was collected throughout a testing period of 60 days. At each sampling, the blood was collected into three different blood collection tubes: Tube A: Vacuette 9 ml serum tube with clot activator and gel separator (Greiner bio-one Vacuette®, Kremsmünster, Austria). Tube B: Vacuette 6 ml plasma tube with sodium fluoride, potassium oxalate, and no gel separator (Greiner bio-one Vacuette®, Kremsmünster, Austria). Tube C: Vacutest 3.5 ml plasma tube with sodium fluoride, disodium ethylenediaminetetraacetic acid (Na2EDTA), and gel separator (Vacutest Kima, Arzergrande, Italy). Additionally, a study on the in vitro degradation of testosterone esters in blood was performed, using the same collection tubes as described above. The applied analytical method included liquid-liquid extraction and preparation of oxime derivatives, prior to TLX-sample clean-up and LC-MS/MS detection.
In the clinical study, the elimination half-lives and detection times depended on the type of testosterone ester administrated. The depot effect of an intramuscular testosterone ester preparation increases in proportion to the length of the ester side chain. This is because the half-life of the absorption increases with longer chains.
As expected, the shortest chained ester, testosterone propionate, showed the most rapid elimination and shortest half-life. Nevertheless, the ester could still be detected for 4-5 days in serum and plasma of all study participants receiving the drug. Testosterone phenylpropionate and testosterone isocaproate were detected for at least 8 days in serum and plasma, whereas testosterone decanoate showed a detection time of 18 days. Testosterone undecanoate was detectable in all post-administration blood samples collected during the whole study period (60 days), thereby giving the longest detection time of the esters investigated.
The stability studies showed that the shorter chained testosterone esters were hydrolysed more rapidly in blood collection tubes not stabilized with NaF (tube A) compared to stabilized tubes (tube B and C). The rate of hydrolysis seems to be dependent on storage temperature. In the clinical study, though, the testosterone ester detection window was not affected by the applied blood collection tube.

Code: 12B11GK

Recombinant human growth hormone (rhGH) has been on the list of forbidden substances since availability of its recombinant form in the early 1990s; however adequate routine doping tests are lacking. The project aims to develop a fast and highly sensitive drug test for detecting two or more hGH-dependent markers in the serum of elite adolescent athletes. In our approach proteomic markers for hGH action such as insulin-like growth factor 1 (IGF-1) and pro-collagen type III N-terminal peptide (P-III-P) will be identified in just a single immunoassay. Considering the desirable reduction of time, costs and workload using FCS instead of other currently available IGF-1 and P-III-NP assays the presented methodology will be an important contribution for a functional doping test for proper use of rhGH. 

Main Findings: 

Two approaches have been developed to detect recombinant hGH in blood in order to control its misuse with the intention of improving athletic performance. Adequate non-invasive tests for human growth hormone (hGH)-dependent markers such as insulin-like growth factor 1 (IGF-1) and pro-collagen type Ill N-terminal peptide (PIIINP) are still lacking. In this one-year project a fast and sensitive bead-based immunoassay for IGF-1 and PIIINP detection in serum was developed based on Fluorescence Correlation Spectroscopy (FCS). FCS was used as a reliable technology for measuring absolute concentrations in the nano-molar range. Three FCS immunoassays - a sandwich and a competitive immunoassay for IGF-1 as well as a competitive PIIINP assay - were established and validated against commercially available ELISA. We were able to detect molarities between 0.5 and lOnM of IGF-1 and between 0.5 and 2.5nM of PIIINP with high accuracy in serum samples.  
Considering the desirable reduction of time, costs and workload using FCS instead of other currently available IGF-1 and PIIINP assays the presented methodology might be an important contribution for proper use of rhGH in sports.