Projets de recherche

Code: 11C12AB 

The anti-doping community is focusing efforts on developing methods to detect gene doping, a new threat to the world of sport. 
Methodology with potential to detect ‘doping’ genes in blood samples from athletes has recently been developed by us and other researchers. It involves PCR assays targeting intronless cDNA sequences that are present in doping genes but absent in intron-containing endogenous genes.  Acceptance of this methodology for routine gene doping testing requires reference materials as controls to ensure the method performs as intended. A positive control containing the cDNA sequence for the candidate doping gene is commonly used in method development. Inadvertent cross-contamination of a tested sample with this type of control would lead to a false positive test result. Hence, it is not suitable for routine testing because of legal implications. 
We propose to develop a DNA reference material for a positive control in gene doping testing that will overcome this problem. Using erythropoietin as a model doping gene, we will produce a reference material that will be detectable by the PCR assays with similar specificity and sensitivity as the doping gene. However, the reference material and gene doping products will differ, allowing easy discrimination between true positive and false positive test results. We will characterise this reference material for purity and quantity using latest technologies including digital PCR and next generation sequencing. In addition, using this reference material we will in vitro validate the complete erythropoietin gene doping detection method from sample processing to PCR detection. 
This model system will serve as a prototype for preparing reference materials for detecting doping with other candidate genes. 
The proposed research is crucial in the development of a routine method for direct detection of gene doping and for ensuring the method is reliable, reproducible and robust and will withstand legal scrutiny. 

Main Findings: 

The anti-doping community is focusing efforts on developing methods to detect gene doping, a new threat to the world of sport. Recently, methodology with potential to detect ‘doping’ genes in blood samples from athletes has been developed by us and other researchers. 
Acceptance and implementation of this methodology for routine gene doping testing requires Reference Material (RM) as a control to ensure the method performs as intended. Existing ‘in-house’ controls based on complementary DNA (cDNA) for targeted genes will not withstand legal scrutiny since any cross-contamination will generate ‘false positive’ test results. 
In this project, we generated and characterised a DNA RM that will overcome this problem. Using EPO as a model doping gene, we produced three forms of the RM. Each form is detectable by the PCR assays that target a doping EPO gene. However, the products generated in these assays from the RM and a doping gene differ, allowing easy discrimination between true positive and ‘false positive’ test results. We characterised these three forms of the RM for purity, quantity and stability, and studied their performance in gene doping detection assays. Finally, the three forms were in vitro validated in the complete EPO gene doping detection method from sample processing to PCR detection. 
This model system will serve as a prototype for preparing RM for detecting doping with other candidate genes. The produced RM is crucial in the development and acceptance of a routine test for direct detection of gene doping and for ensuring the method is reliable, reproducible and robust and will withstand legal scrutiny.

Code: 11A17PV

Anabolic steroids are the most frequently detected prohibited substances in sports. Analysis for these substances is routinely performed using gas chromatography-mass spectrometry (GCMS) and liquid chromatography mass spectrometry (LC-MS). Although the detection capabilities of LC-MS are limited for the detection of steroids with androstanediol as a back bone to their structure, LC-MS is often more sensitive for many other steroids. Moreover, recently several publications have shown that LC-MS can detect steroids (glucuronides) directly in urine without any sample preparation.

The advantages for such a direct urinalysis are multi-fold. It reduces costs, required volume of sample and analysis time, but also it allows for the direct detection of intact conjugated steroids rather than the liberated steroid molecules after hydrolysis.

In the past several small scale attempts have been made to detect these intact steroid conjugates in urine. These attempts were quite successful, but did not meet WADA’s minimum required performance limits. Recently however several scientific improvements have been made, including more sensitive instruments, optimized mobile phase compositions (e.g. methanol
instead of acetonitrile), which now allow for the detection of several steroids well below WADA’s MRPL. These preliminary results (e.g. 19-norandrosterone glucuronide) show that the direct urinalysis of steroid conjugates at concentrations at or below the MRPL is achievable.

Main Findings: 

The aim of this project was to study intact conjugated anabolic steroids in urine in comparison with the traditional methods where the aglycones are detected after enzymatic hydrolysis. This approach permitted the detection of anabolic steroids misuse by liquid chromatography mass spectrometer since the glucuronide and sulfate groups are easy to ionize by ESI. Consequently, steroids with a sulphated or glucuronidated androstane framework could be detected. Research resulted in the development of a multi-target analytical method for the screening of different exogenous anabolic steroids and also for the quantification of endogenous anabolic steroids.

The use of a dilute-and-shoot strategy allowed to minimize sample preparation yielding a short turnaround time. Additionally a small volume of urine can be used. This is a great advantage compared with traditional methodologies where liquid-liquid extraction or solid phase extraction after enzymatic hydrolysis and detection by LC-MS or GC-MS (after derivatisation step) is applied.

The use of excretion studies or synthesis in vitro are of great interest since the number of conjugated anabolic steroids commercially available is still very low. With this methodology new conjugated anabolic steroids were detected and included in the multitarget method.

Code: 11D7MD 

This project, which constitutes a follow-up from two previous works funded by WADA aims at widening our understanding of the effects of glucocorticoids (GC) on muscle function, exercise performance, postural control and health risks. Indeed, we demonstrated in our previous works that short-term but not acute GC administration improves performance during endurance exercise lasting at least 40 minutes, without any gender effect. However, no study, to our knowledge, has focused on the ergogenic effects of GC during repeated bouts of high-intensity exercises. In the same way, whether the numerous deleterious effects of chronic (>1 month) GC intake are well known, there is no consensus on health risks after short-term (1 week) systemic GC intake. 
We therefore propose to study whether short-term GC administration would improve performance during repeated bouts of high-intensity exercise. In parallel, we will investigate with complementary methods the repercussions of GC administration on biomechanical (especially the magnitude of the braking of the fall during the swing phase), and physiological parameters (cardio-respiratory, muscular and inflammatory) in order to assess both GC ergogenic effects and health risks induced by short-term high dosage treatment.

Main Findings: 

In the project “Effects of glucocorticoid during repeated bouts of high-intensity exercise” we tested the ergogenic effects and health risks of short-term GC administration. Physiological and biomechanical parameters were analyzed. Several results can be highlighted: 
- No ergogenic effect of short-term systemic GC (i.e. 60 mg prednisone/day/7 days) was found during repeated bouts of brief intense exercise. Muscle oxygenation parameters measured with the near infrared spectroscopy technique were not altered by short-term GC administration at rest and during repeated bouts of high-intensity exercise. These suggest that GC treatment had no effect on the blood oxygen transport capacity and on the muscle oxygen extraction during exercise. The unaltered oxygen availability at the muscle level seems to be in accordance with the lack of improvement in performance during the last sequence of hopping with the GC treatment.  
- Regarding hormone responses during exercise, while no change was found in saliva testosterone, GC intake induced changes in saliva cortisol and DHEA concentrations. In parallel, blood IL-6 and IL-10 were altered at rest and during the exercise performed. It is interesting to note that the anti-inflammatory effects of prednisone were maximal and stable from the beginning of treatment, in the resting and exercise conditions. However, hormonal concentrations continued to decline during short-term intake. Lastly, our study shows that short-term prednisone treatment affects the circadian pattern of saliva DHEA but not testosterone in our recreationally trained subjects.  
- Regarding baroreflex sensitivity (BRS), heart rate (HR) and systolic blood pressure (SBP) variability (HRV, SBPV) short-term GC intake did not change SBP. However, a low frequency (LF) SBPV increase associated with a LF-BRS decrease and a HR increase was observed, indicating a sympathetic cardiovascular stimulus.  
- Regarding EMG activities in the ankle extensors EMG during single leg hopping, and balance control during single stance of the first step in gait initiation, no GC effect was noted compared to placebo.

Code: 11A3JP 

Recent advances in liquid chromatography–mass spectrometry (LC-MS) have opened the possibility of converting many of the doping control analysis methods to LC-MS platforms. This would increase flexibility and selectivity for many analyses, while obviating the need for derivatization in most cases. A limitation to this switch is the established sample preparation strategies required for the numerous doping control methods. Developed for GC-MS analysis, they are time consuming and not always appropriate for LC-MS analysis. While the LC-MS technology holds the promise of providing a universal screening tool, the varied requirements for sample preparation for the numerous tests limit this potential. Professor Pawliszyn's group has long recognized the bottle-neck that sample preparation presents for the analytical laboratory, and have made significant strides in developing solid phase microextraction (SPME) technology to simplify sample preparation for both GC-MS and LC-MS, while maintaining sensitivity and enhancing options for automation.  
In this project we will evaluate automated SPME technology as a universal sample preparation strategy, coupled with LC-MS as a universal tool for identification and quantification of prohibited substances. We will develop validated methods for urine analysis for a range of prohibited substances from the S1, S3, S4, S5, S6, S7, S8, S9 and P2 categories. We will apply enzymatic deconjugation of phase II drug metabolites as required and investigate guidelines for confirmatory analysis. SPME technology is also readily applicable to plasma and whole blood analysis and has significant advantages relative to SPE. We will evaluate the performance of the system in these matrices as well. At the conclusion of the project we will have sufficient data to evaluate the suitability of automated SPME coupled with LC-MS as a universal tool in prohibited substance screening. If judged suitable, it will be immediately applicable for introduction to the screening program in WADA accredited facilities. 

Main Findings: 

• The potential of automated solid phase microextraction (SPME) in thin film geometry as simple and convenient sample preparation approach for urine, plasma and blood analysis was demonstrated.
• SPME conditions for each of the studied matrices were carefully optimized. The effect of experimental parameters such as pH, ionic strength, centrifugation and temperature was investigated.
• Protocols for the multiclass analysis of 110 and 25 prohibited substances in urine and plasma, respectively, were developed and validated.
• In the case of urine, 100 out of 110 compounds showed R2 above 0.991, intra and inter day precision was below 20 % in most cases, and in terms of accuracy only 6 compounds exhibited more than 20 % deviation from their nominal concentration value. Regarding LOQ, only 15 out of 110 compounds did not meet the MRPL values stipulated by WADA; however, as presented in the final report, by using a more sensitive mass spectrometry analyzer WADA requirements can be fulfilled.
• In the method developed for plasma analysis, satisfactory results in terms of linearity (R2> 0.99), inter and intra-day accuracy (85 – 130 %), precision (<20 %) and limits of quantitation (0.25 – 10 ng/mL for most compounds) were found.
• The suitability of the automated system to incorporate a hydrolysis step before SPME extraction was also verified.
• The most important advantages of SPME in doping controls can be listed as follows:
1. Minimum sample handling before SPME extraction. No need of centrifugation or protein precipitation (no clogging issues as in SPE).
2. High-throughput analysis. The automated 96 Concept system allows simultaneous preparation of 96 samples.
3. Efficient sample clean-up. Elimination or reduction of possible ionization suppression or enhancement effects.
4. No breakthrough volume issues. The open bed geometry of SPME allows simultaneous quantitation of a wide range of substances without concerning about extraction phase saturation.

Code: 11A25AR 

Anabolic androgenic steroids (AAS) behave differently in the human body. The human organism deals with these compounds differently in respect of uptake, distribution into different organs, metabolism and excretion. We recently demonstrated that ¾ of Oriental people have a severely compromised capacity to excrete testosterone in the urine compared to only 10 % in people from the west. This is a confounder in the doping test program and therefore it is urgent to find new biomarker and approaches. 
In the way towards personalised test programmes, Bayesian inference techniques are known to suit particularly well. Another approach is to verify the origin of testosterone by using IRMS. 
To further improve the new individualised steroid profile passport, we will conduct humanstudies with nandrolone and different doses of testosterone in order to assess the sensitivity and specificity of IRMS and to learn more how administrated AAS are metabolized.

Main Findings:

The urinary testosterone glucuronide to epitestosterone glucuronide (T/E) ratio is a biomarker included in Steroid Module of the Athlete Biological Passport (ABP) which has improved doping tests for steroids. However, the ratio is greatly affected by a genetic deletion polymorphism in the UGT2B17 enzyme which is the major catalyst of testosterone metabolism. Suspect urine doping tests are further analyzed with gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) to determine the origin of the androgen.  
We investigated the sensitivity of the steroidal module and the IRMS analysis in subjects administered with three doses of testosterone enanthate (500, 250, and 125 mg), in relation to the UGT2B17 polymorphism. All subjects carrying the UGT2B17 enzyme reached the traditionally used threshold, a T/E ratio of 4, after all three administered doses, whereas none of the subjects devoid of this enzyme reached a T/E of 4. However, using the ABP and IRMS analysis, all three doses generated a positive result with a high degree of sensitivity. Our results demonstrate that administration of one single dose, as small as 125 mg testosterone enanthate, could be detected with the ABP in combination with IRMS. Since IRMS is sensitive to testosterone doping independent of UGT2B17 genotype, also very small changes in the steroidal passport should be investigated with IRMS.  In our study of the excretion profile of nandrolone and the 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) metabolites after one single i.m. dose of 150 mg nandrolone decanoate (ND) to healthy volunteers we were able to demonstrate that 19-NA is detectable for 9 months in about half of the individuals. This is the first study where GC-C-IRMS analysis has been performed after an i.m administration of ND in a controlled study group. The absolute δ19-NA (‰) was in the same range as observed previously, with values between -29.1 -and -34.7 in 19-NA positive samples confirming the presence of 19-NA of exogenous origin. The use of the well characterized androsterone (A) as an endogenous reference compound or any other compound not modified by exogenous administration is also possible. Here we show that 11-oxoandrostenedione and pregnanediol may also serve as endogenous reference compounds to detect exogenous origin of 19-NA 3-9 months after one single dose of 150 mg ND. Interesting to note, exogenous 19-NA was traced in GC/C/IRMS in a sample where 19-NA was below the decision limit.   
In summary, GC/C/IRMS analysis confirmed the presence of exogenous derived steroids in both our studies, regardless of UGT2B17 genotype, dose, and time, further supporting the strength of using this methodology. 

Code: 11B8CG

Luteinizing hormone (LH) is a naturally occurring hormone which is secreted by the pituitary gland. In males LH stimulates testosterone production by the testes. Recent research has proven that the monitoring of LH is a valuable tool for both the detection of anabolic steroid abuse; where suppression of LH is observed, and gonadotrophin releasing hormone abuse, where elevation of LH levels is observed. The ability to use LH as a marker for the detection of the abuse of other doping substances is limited by the currently available testing techniques. There are numerous commercially available immunoassays but these are only marketed for the testing of serum samples. The use of the available immunoassays for monitoring of LH in urine needs to be carefully cross validated for comparison to ensure suitably of the results for anti doping.

Furthermore to better understand why there are such differences in the concentrations detected by different immunoassays it is proposed to purify and sequence from urine both naturally present LH as well as from recombinant LH excretion studies. The proposed research will harmonize the measurement of LH in urine for anti doping as well as gain a better understanding of the measurand which will lead to better detection methodologies.

Main Findings: 

The Immulite and Delfia assay are able to detect LH in urine even after extended periods of storage at 40°C, 21°C, 4°C and -20°C. The results for the Delfia stability study reflect what was observed for the Immulite stability study, that LH is unstable at room temperature but although it is also unstable when frozen, quantifiable amounts are still able to be detected with the Delfia assay and the Immulite assay. Statistical analysis of 1000 athlete samples gave correlation factors for both the Immulite and the Delfia, enabling a more accurate comparison of results between the two assays. This will be particularly useful to the anti-doping community as different laboratories use only one or the other of the assays for detecting LH in urine. For anti-doping purposes urine is stored frozen which is where urinary endogenous LH is degrading. Sequencing of the beta subunit of LH revealed the C-terminal and N-terminal of the protein are still largely intact meaning that changes to the protein chain must be occurring within the protein backbone. The tertiary structure of LH may be allowing certain areas to more readily degrade due to turns and disulphide bond placements in the structure as well as the influence of post translational modifications such as the single N-linked glycan on the beta subunit.

Code: 11D5VB

Elite athletes have a high prevalence of asthma and asthma-like symptoms as well as a high use of anti-asthmatic therapy. Salbutamol and salmeterol, has been removed from the prohibited list and these drugs can now be used more freely, also by athletes with asthma-like symptoms only. Performance enhancing effects of beta2-agonists are, though, to some degree contradictory, and the scientific problems concerning beta2-agonists are as follows:

1. A recent systemic review by Pluim et al (2011), they points out that the current literature concerning possible performance enhancing effects of systemic beta2-agonists is weak and calls for future studies with the use of more reliable, valid and sensitive performance protocols.

2. Furthermore, Pluim et al (2011) showed that the training level of the participants in the salbutamol studies have been low to moderate, which is not representative for elite athletes.

3. In another recent review, Collomp et al (2010), reports that no studies, has thoroughly investigated the ergogenic effects of terbutaline after oral administration, neither on exercise performance nor on metabolic parameters and body composition. Hypothesis:

Beta2-agonists have acute and chronic intracellular actions on skeletal muscle in animal studies, which could take place in human skeletal muscles as well, and by that induce 1) muscle strength and performance, and 2) increase volume of the muscles.

The therapeutic doses used in most former studies might have been too low to induce performance-enhancing action, whereas multi-pharmacy with the use of maximal allowed doses of each of the beta2-agonists might have additive effect. Acute use of mixed Beta2-agonists may enhance exercise performance in elite athletes.

Aim:

The aim is therefore to investigate acute, as well as, chronic effects of daily beta2-agonists use on skeletal muscle hypertrophy, muscle fiber characteristics, intracellular adaptations and exercise performance in healthy trained and elite trained athletes.

Main Findings

The primary purpose of the project was to investigate acute and chronic effects of beta2-agonists in skeletal muscle and on performance in healthy trained men. We observed that acute combined inhalation of salbutamol, salmeterol and formoterol, within the current therapeutic dosing limits, enhanced arm ergometer performance and muscle strength, but not swim endurance performance, in elite swimmers. Furthermore, we observed that acute systemic beta2-agonist treatment with terbutaline augmented ion handling of skeletal muscle, which was associated with an enhancement in knee-extensor exercise performance. We also observed that acute systemic beta2-agonist treatment with salbutamol modified expression of genes involved in growth of skeletal muscle in recovery from resistance exercise. When administered chronically, treatment with terbutaline and salbutamol augmented the hypertrophic response to 4 and 11 weeks of resistance training, respectively. However, the larger gains in muscle mass induced by these beta2-agonists did not translate into any superior improvements in muscle strength compared to resistance training with placebo. In fact, chronic beta2-agonist treatment repressed maximal oxygen consumption (V̇O2max) relative to lean body mass. Muscle fiber-type distribution shifted towards a fast-twitch phenotype with salbutamol treatment, while no significant change was observed for terbutaline. In conclusion, our observations support the restriction towards systemic administration of beta2-agonists on the prohibited list. Our data also suggest that a therapeutic dosing limit should be introduced for terbutaline. Combined inhalation of several beta2-agonists may also be of concern, at least during competitions of short and intense duration.

Code: 11B15MU 

Alms of our research are a) methodology development for comprehensive differentiation of endogenous and exogenous hCG, LH and ESA by glycane profiling, b) construction of lectin-carbohydrate Interaction database that allows rapid identification of WADA prohibited glycoprotein hormones, and c) development of sample preparation procedure to allow detection of recombinant glycoprotein hormones by normal immune-assays currently equipped in WADA accredited laboratories. Result verification by second lmmuno assay is necessary when commercial immuno assay kit is used for doping tests, but the verification is not always easy because hCG is extensively metabolized or degraded in vivo. Misuse of biosimilar EPO having different isoform compositions from reference EPO became known since 2007 and caused sometimes in identification difficulties. A sole human cell derived EPO (Dynepo) was withdrawn fromthe market in 2007, and currently, commercial glycoprotein hormones are largely manufactured from CHO cell line. Many of other glycoprotein hormones such as hCG and LH from human has been replaced by the recombinant products. It is reported that CHO cells do not normally express siatyl-alpha2-6 transferase. We have analyzed sialyl- alpha2-6-linked hexose of glycoprotein hormones by the reactivity with 45 array of lectins, and found that human origin EPO and hCG has Sialyl- afpha2- 6-linked galactose/galactosamine residues but CHO-derived glycoprotein hormones are lacking this moiety. Thus, origins of glycoprotein hormones were successfully identified. In this project, we plan to construct stereo-specific sample preparation procedure using lectin beads/columns, gathering lectin­carbohydrate spectra by analyzing LH, hCG and ESA from human and CHO-cell line, and development of antibody assisted lectin micro array test system to identify recombinant glycoprotein hormones.

Main Findings: 

Our results of lectin-glycan interaction monitoring confirmed that origin dependent difference of human glycoprotein hormones, and genetically manufactured corresponding glycoprotein preparation arises not from gene expression but from the posttranslational modification process. Synthesis of core peptide is genetically controlled but the addition of 0-and-N-Linked glucan to the core peptide depends on the available sialyl transferase (ST) and the substrate carbohydrates in the culture medium. Use of SSA, SNA or TJA-1 lectin for isolation of endogenous and exogenous glycoprotein by capturing Sialya2-6Gal/GalNac as the tag carbohydrate allows origin dpendent separation of ESAs and Gonadotropins. Hormones in isolated recombinant or human fraction are to be asssayed by means of normal immunoassays just for detection. By this configuration, both peptide and carbohydrate moieties of the target compounds are to berecognized, thus, highly specific lectin-antibody identification can be achieved. Because overall results of lectin fractionation coupled with immunoassay shows the origin dependent difference of glycans, the positive finding of glycoproteins in the fraction 1 and 2 can be considered as adverse analytical finding regardless of the hormone concentration. The results represented a possibility to establish high throughput origin specific screening of the glycoprotein hormones by lectin column fractionation followed by normal sandwich immuno assay kits or immuno assay instruments

Code: 11C14NL 

MicroRNAs (miRNAs) are small (19 to 25 nucleotides) noncoding transcripts involved in many cellular and physiological mechanisms such as erythropoiesis. Recently, a new class of miRNAs was found in cell-free body fluids such as serum and plasma. These new class of miRNAs are called "circulating miRNAs". Circulating miRNAs have been found as very stable, specific and sensitive biomarkers. Therefore, they could be altered in a specific manner in doping interventions such as erythropoiesis-stimulating agents (ESA). 
First promising results have been obtained from our laboratory related to the utilization of circulating miRNAs to detect ESA abuse. 
After microarray profiling and RT-qPCR analysis, an increase of miR-144 was observed up to 27 days after ESA injection. Interestingly, miR-144 has been reported to be essential in erythropoiesis in human and other organisms. 
In this project, we plan to investigate more in details the pre-analytical and analytical characterization of the use of miR-144 as long-term biomarker for the detection of ESA. Indeed, we plan to test different biological matrix, extraction methods and the stability in order to facilitate the future utilization of miR-144 as indirect biomarker. Moreover, negative population will be tested to study, more in details, background noise and inter-individual variability of miR-144 concentration in plasma to define threshold limit.

Main Findings: 

In this project, different pre-analytical tests have been performed to characterize two selected miRNA. These two miRNAs were potential biomarkers for the detection of ESAs abuse and autologous blood transfusion, respectively. 
Extraction protocol was demonstrated to be the most important step in miRNAs analysis. Standard extraction protocol is based on phenol/chloroform extraction followed by silica columns purification. Although very efficient, the use of these toxic solvents should be handled carefully. To prevent the use of phenol/chloroform and to decrease the cost of analysis, different extraction procedures were tested. Since circulating miRNAs are very stable, extraction protocol based on boiling was possible. Comparison with standard method demonstrated that heat-and-shoot protocol was efficient only for minute amount of plasma. In contrast, with a volume of 100 µl standard method is close to 10 fold times better regarding qPCR signal. Plasma matrix is known to contain reverse transcription and PCR enzyme reaction. Since more inhibitors were present in 100 µl of plasma than 12 µl could explain this observation. Thus, boiling is sufficient to counteract inhibitors of enzyme reaction only in small volume.  
Minimally invasive test such as fingertip prick test possess some advantages. We observed that heat-and-shoot and standard method could be used to extract circulating miRNAs from digital blood. Digital blood collections are beneficial in clinics but this utility in anti-doping is questioned as some athletes preferred standard blood collection to fingertip prick test because of the pressure applied on fingers when they compete or train. In addition, it is difficult to analyse several variable in small amount of plasma and thus, combination of biomarkers are not possible. 
Circulating miRNAs have been demonstrated to be very stable. From our experiment, one of the miRNAs was unstable after heating sample at 60°C with the 3 pool of plasma tested. In contrast, the other one showed a good stability even in extreme conditions. This observation supported the use of the circulating miRNA as an efficient biomarker to detect autologous blood transfusion.

Code: 11A23JM 

The objective of this project is to further strengthen the ability of anti-doping laboratories to unambiguously identify mis-use of the anabolic steroid nandrolone for athletic performance enhancement. This will be achieved through preparation of a reference material that has an accurately known value for the ratio of stable isotopes of carbon in the chemical residue found 
in urine after nandrolone is metabolised by the body. The presence in a urine sample of 19- norandrosterone glucuronide, the main urinary metabolite of nandrolone, is indicative of the use of this prohibited drug. Under certain circumstances 19-NA may be present at low concentrations in samples of human urine for reasons unconnected with doping. To confirm Adverse Analytical Findings (AAFs) for samples containing 19-NA at concentrations between 2 
ng/mL and 10 ng/mL, WADA Technical document TD2010NA requires that carbon isotope ratio analysis be performed to demonstrate that the metabolite has not been produced naturally in the urine. Provided that other criteria are fulfilled, samples with 19-NA concentrations in this range are only 
to be reported as an AAF if the carbon isotope ratio of endogenous androsterone in the sample is greater than 3 per mille (‰) different to that 
of the 19-NA detected. 
In this project a stable solution of 19-NA glucuronide with an accurately assigned value for the carbon isotope ratio of the steroid will be prepared. This can then be added by laboratories to a urine CRM free of 19-NA but containing the endogenous reference compound for this analysis 
(androsterone). The freeze-dried urine CRM MX005, prepared to validate detection of testosterone abuse, has already been assigned a carbon isotope ratio value for androsterone in a previous WADA-funded project and could be used for this purpose. 

Main Findings: 

A certified reference material (CRM) has been prepared in the form of a solution with a metrologically-traceable reference value for the carbon isotope delta value 
(δ13CVPDB) of 19-norandrosterone (19–NA) in its glucuronide conjugate (19–NAG). The CRM has been packaged in 800 amber glass ampoules containing 1 mL of the solution and is now available to WADA-accredited laboratories. It consists of 19-NAG in water containing 20% methanol at a concentration equivalent to 201  ng/mL 19-NA (as the unconjugated steroid).  The certification of the isotope ratio of the 19-NA component of 19-NAG was complicated by the requirement to hydrolyse and remove the glucuronide moiety prior to measuring the isotope ratio of the steroid. The hydrolysis can introduce impurities that must be fully separated from the 19-NA prior to isotope ratio measurement. This was achieved by solvent extraction prior to gas chromatography coupled with combustion isotope ratio mass spectrometry  (GC/C/IRMS). Calibration of the GC/C/IRMS was performed using the isotope reference material CU/USADA 34-1, permitting metrological traceability to the international carbon isotope ratio reference standard VPDB. Potential sources of bias in the reference method including isotopic fractionation during extraction, internal standard selection and reproducibility of measurement were investigated. The homogeneity and stability of the CRM was verified by analysis of randomly selected vials after storage at -80 °C, -20 °C and +40 °C for periods up to 10 months. 
The certified property value (with uncertainty at the 95% level of confidence) for 19-NA in the CRM is provided in the following table: 

The CRM is intended for fortification by end users into urine material known to be free of 19-NA in which the δ13CVPDB value for an endogenous reference compound such as androsterone has been well characterised. It is designed to assist laboratories in validation and quality control of methods for determination of the carbon isotope ratio of 19–NA in urine at concentrations in the range 2 – 15 ng/mL as required by WADA technical documents TD2010DL and TD2014NA. The use of the glucuronide conjugate for fortification into urine will permit full validation of the sample workup in analytical methods for confirmation of the presence of exogenous 19-NA in its most abundant metabolised form, 19-NAG