Projets de recherche

Code: 14A08OM

For sports drug testing, the errorless discrimination of rEPOs from endogenous human EPO is important. The current gel electrophoretic methods are also labour-intensive for anti-doping laboratories. 
Mass spectrometry-based procedures provide strong identification power, robustness and reproducibility in sports drug testing. Recently, we have developed a high-sensitivity and high-throughput qualitative detection method for human urinary NESP using LC-MS/MS. This was the first report of successful practical application of the mass spectrometric identification of NESP in human urine. 
Following this outcome, the aims of this study are to optimize the NESP detecting method for a robust implementation in WADA accredited doping laboratories, and further to establish a novel detection method for other rEPOs, such as CERA in human matrices by LC-MS/MS for doping control purposes. 
CERA is a long-acting erythropoietin derivative with methoxy polyethylene glycol butanoic acid linking to the N-terminal amino group or the ε-amino group of any lysine, predominantly Lys45 and Lys52, in the protein molecule. The structure of CERA is different from that of human EPO and epoetins. After enzymatic digestion, the target Pegylated peptides will be searched by the LTQ Orbitrap high resolution hybrid mass spectrometer for discriminating between CERA and endogenous EPO. Furthermore mass spectrometric glycoform profiling of rEPOs (e.g. epoetins) will be performed by LC-ESI-MS/MS. After enzymatic digestion of core protein and glycan of rEPOs, the target digested glycosylated peptides will be identified by the LTQ Orbitrap high resolution hybrid mass spectrometer (Thermo) and SynaptG2 HDMS TOFMS (Waters) for discriminating between epoetins and endogenous EPO. We focus the differences of syalylation, N-glycosilation, O- acetylation of sialic acids and O-glycosilation between epoetins and human EPO, and we have an optimized CID technique for discriminating N-glycopeptides by LC-MS. 

Main Findings: 

The current analytical methods for recombinant human erythropoietin (rEPO) are mainly gell electrophoretic methods. Mass spectrometry is necessary for  the reliable identification of rEPOs in doping control. The aim of this research project is to develop the mass spectrometric detection method for human sports drug testing.  
The research comprises three topics, i.e., 'Mass spectrometric characterization of rEPOs and their biosimilars', 'Improvement of the LC-MS/MS method for detecting darbepoetin alfa in human urine', and 'Mass spectrometric approach for detecting CERA'. 
It has been recognized that EPO biosimilars typically have the same amino acid sequence as the innovator product, and that they have slight differences in overall chemical structure.  As expected, the biosimilars of darbepoietin alfa have different glycan distributions compared with the originator. Interestingly, the biosimilars of darbepoietin alfa and a biosimilar of epoetin alfa contain not only des-arginine product comprising 165 amino acids, but also a C-terminal arginine product comprising 166 amino acids, in contrast to the results in which the originator recombinant EPO products. darbepoetin alfa and CERA contain only des-arginine EPO comprising 165 amino acids. The presence of C-terminal arginine EPO in human matrices might be evidence of administration of EPO biosimilars.  
We established a high-quality and more robust UPLC-MS/MS method for detecting darbepoetin alfa using a deutrated internal standard. The lower detection limit was 1 pg/ml. The detection window in urine from patients treated with NESP was estimated to be up to 16 days after administration. The present method could be a useful tool for detecting the biosimilars of darbepoetin alfa for doping control testing because the method does not depend on the glycan profiles.  CERA and epoetin beta representing human EPO could be successfully discriminated using  LC-MS/MS with in-source CID. However, this mass spectrometry-based technology needs to be improved in terms of sensitivity for detecting CERA in human biological fluids. 

Code: T14M01GP 

Human chorionic gonadotropin (hCG) may be abused by male athletes in sports and is included in the banned substance list of the World Anti-Doping Agency. An ti - soping laboratories mainly use immunoassays to quantify hCG but cross-reactivity with the different forms of hCG can constitute a poblem for quantitative hCG determination especially in urine samples. Choriogonadotropin protein is a heterodimer composed of an alpha chain, whjich is also common to thyrotropin, lutropin, follitropin, and a beta chain, which confers its specific biological activity. The alpha and beta subunits are non-covalently linked and carry numerous disulfide bonds. the 2 chains also harbour carbohydrate moieties: 2 N-glycosylations ans 4O- glycosylations on the beta subunit and 2 N-glycosylations on the alpha subunit. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers analytical specificity superior to that of immunoassays and can be alternative method for wuantification of hCG in athletes biological samples. for optimal assay accuracy and reliability, a stable isotope labeled internal standard should be used. In a precious project, we developed a protpcol to produce an isotopically-labeled version of hCG (PSAQ standard). The choriogonadotrophin heretodimer was successfully expressed in mammalian cells and purified. Stable isotope incorporation was determined to be greater than 98%. The goal of this project is to produce 200 to 500μg of hCG PSAQ standard according to the protocol previously developed to deliver different anti-doping laboratories. 

Main Findings: 

Recombinant hCG protein was produced and labelled in HEK293 cells. The choriogonadotrophin heterodimer was successfully expressed and purified using Ni-IMAC resin. To obtain higher purity level, the sample was submitted to a second purification step using Ion Exchange Chromatography (IEX). The purity was estimated to be greater than 95%.

In this program, to increase the sequence coverage, a sample of hCG was treated with a  combination of five enzymes to remove N-linked glycans and many common O-linked glycans.  Following deglycosylation, hCG alpha and beta subunits sequences were verified by LC-MS/MS analysis after reduction/alkylation and in-gel trypsin digestion of the purified protein.  The sequence coverage of CGB is greatly improved after treatment with the Deglycosylation Mix compared to the previous feasibility study where the protein was only digested with PNGase F.  Stable isotope incorporation was estimated using MS data generated by LC-MS/MS analysis.  
hCG PSAQ™ standard will be delivered in aliquots of 20 μg.  We recommend to store at -80°C upon receipt.

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%.