Projets de recherche

Code: 08A17PT

The detection and quantification of small molecule doping agents has become a relatively simple process with mass spectrometry regarded as the definitive, analytical methodology. The threat from biological agents such as proteins, peptides or genetic manipulation, provides a much greater analytical challenge for most doping control laboratories. As a result of this much research has been expended into the use of endogenous proteins as so called biomarkers of abuse.Until recently the only effective tool for quantitative analysis of large molecule targets has been enzyme linked immunosorbent assays (ELISA) or similar immunoassay based techniques. These assays are very effective for the analysis of large molecules, particularly where sandwich assays, involving the use of two complimentary antibodies, are employed. While it is likely that immunoassays will remain the standard for routine quantitation of single proteins, great strides are being taken in the development of quantitative methods based upon mass spectrometry, a highly sensitive and selective technique widely applied in the fields of pharmaceutical and sports testing. More recently, developments in sample handling techniques, chromatographic stationary phases and triple quadrupole instruments have made the quantification of proteins by mass spectrometry a reality which could be transferred into the majority of doping control laboratories. A major advantage of using LC-MS is that it provides an independent means of detecting the target protein compared to immunoassay and is a potential gold standard for validating these methods. This is reinforced by the ability to monitor several peptide fragments from the same protein to increase the confidence in the analytical result and it is also possible to analyse multiple proteins in a single analytical run. The aim of this project is to develop a generic LC-MS approach to the quantification of a range of growth hormone (GH) biomarkers currently used or proposed for use in sports drug testing. The method will be designed for use in a typically equipped doping control laboratory and will make as little use as possible of specialised equipment. 

Main Findings: 

The aim of this project was to develop a single methodology based upon LC-MS capable of detecting multiple biomarkers of the administration of growth hormone (GH). The preferred aim was to do so without recourse to antibodies or similar bio-reagents for which continuation of supply could be an issue. Potential applications of the developed method would be as a reference method for the detection of GH biomarkers or possibly for use as a confirmatory procedure for the same.  The approach to the project was broken down into: • the development LC-MS methods to detect the proteins of interest via proteotypical peptides,
• to obtain stable isotope labelled versions of the targeted peptides,
• to develop a generic extraction protocol capable of isolate all the analytes in a single fraction
• to validate the resultant methodology                                                                                                                                   The markers chosen were based upon those previously identified as providing the most definitive proof of GH abuse PIIINP, IGF-I and its binding proteins including IGFBP-3. In addition a number of additional potential biomarkers (IGFBP-2, IGF-II LRH) were included in the assay development phase. In the case of PIIINP, direct determination of potential prototypic peptides using traditional approaches proved impossible given the low levels of standard material available. Significant effort was expended identifying possible approaches to hydrolysis of the protein. By means of computer simulation (in-silico digestion) the most likely enzyme for cleaving PIIINP to produce sufficiently selective proteolytic peptides for use in an assay was determined. The chosen protease, Glu-C, was utilised but detection of the expected peptide fragments remained elusive. Finally, a custom synthesis of the expected peptide was commissioned and using this standard it proved possible to detect a target peptide for the analysis of PIIINP using LC-MS. While a target peptide is now available, this was accomplished a stage in the experimental work to prevent full development of an assay to be undertaken. Having developed LC-MS methodologies for the detection of proteotypic peptides of IGF-I, IGFBP-3 and related peptides attention was switched to optimisation of extraction methodologies to isolate the proteins from human plasma.  In practice, it was found that isolation of IGF-I was related to the concentration of the IGFBP’s present in the sample unless steps were taken to completely disrupt the binding of these proteins. Optimised conditions for extraction of IGF-I could be obtained by acidification of the sample and extraction on a polymeric phase SPE cartridge. This approach gave only low and variable extraction efficiency for the IGFBP’s and a second methodology optimised for these analytes was developed.  Analysis of samples for IGF-I in both plasma and serum using this approach proved successful. Reasonable results could also be obtained for the binding proteins with acceptable data obtained for calibration lines and QC’s. All indications are that these methods, if applied in the correct manner, would provide the basis for successful analysis of IGF-I and IGFBP-3 in plasma / serum samples.  The developed methodologies for IGF-I, the IGF binding proteins and related biomarkers could be used as the basis of confirmatory methods for these markers in human plasma / serum or as reference methods with which immunoassay based methods can be calibrated or controlled. In order to do so reference matrix with known levels of each marker would prove highly beneficial and aid the introduction of LC-MS methodologies into WADA sports testing laboratories. While it proved impossible to develop a methodology for PIIINP in the time frame available, an appropriate proteotypic peptide has been identified which could form the basis for the development of an LC-MS based assay. Publications are in preparation detailing the methodologies developed and initial investigation of PIIINP and this target peptide.

Code: 08A15MT 

This project comprises a combination of the analysis of different prohibited peptide hormones (for example, but nor limited to: sunthetic insulin analogues,synacthen, gonadorelin, synthetic IGF analogues) in plasma and/or urine samples. Former studies provided information about the expected concentratons thatt were estimates in low fol/mL range, their chromatographic separation and their mass spectrometric characterisation. Thus, an efficient and highly specific sample preparation procedure with nanotechnology based immuniaffinity purification employing coated magnetic beads and appropriate primary antibodies will be developed. The subsequent determination and identification of peptides will be performed by means of liquid chromatography - tandem mass spectrometry. The major goal of the planned project is the simultaneous purification and determination of a variety of structurally and physiologically different bioactive peptide hormones from biological fluids, and te installation of a comprehensive screening tool dedicated to the detection of prohibited compounds with peptide-based structure using mass spectrometric approaches is aimed. The assay shall utilize commercially available reagents and equipment only in order to allow a rapid and facile transfer to other anti-doping laboratories, and detailed instructions and protpcpls shall enable a fast implementation of a complementary and highly specific screening tool in sports drug testing laboratories. 

Main Findings: 

Bioactive peptides such as insulins, the synthetic adrenocorticotrophic hormone analogue Synacthen, Gonadorelin (LHRH), Growth Hormone Releasing Hormones (GHRHs) and insulin-like growth factors (IGF-1 and derivatives) provide a reasonable potential for the misuse as performance enhancing agents and are prohibited in elite sports according to the list of banned substances established by the World Anti-Doping Agency. Currently, the determination of these analytes is possible by dedicated assays only or methods are even not at hand so far. In the present project, a procedure to determine several prohibited peptides, which are excreted into urine (e.g. Gonadorelin, Human insulin, Humalog (Insulin Lispro), Apidra (Insulin Glulisine), Novolog (Insulin Aspart), Lantus (Insulin Glargine), Porcine Insulin, Synacthen, IGF-1, longR3-IGF-1, Geref and CJC-1295), was developed. The method enables the effective, highly sensitive and specific screening for several different target analytes that are simultaneously purified and analysed by means of immunoaffinity purification, subsequent liquid-chromatographic separation and high resolution / high accuracy mass spectrometric determination at low pg/mL concentrations. Central aspect of the approach is the combination of immunoaffinity purification with mass spectrometry. Employing different specific antibodies coupled to paramagnetic beads, the simultaneous isolation of all targets in one sample preparation procedure was accomplished. In general, the approach is extendable to any banned peptide if adequate antibodies are available. At the present status of the project the above mentioned analytes (11 prohibited peptides, 5 internal standards) are covered  and the method is fully validated under consideration of qualitative result interpretation. 

Code: 08B03RK

Luteinizing hormone (LH) is one of the prohibited gonadotrophins named in the WADA 2008 Prohibited List. LH is a naturally occurring hormone which is secreted by the pituitary gland. In males LH stimulates testosterone production by the testes. Whilst LH has been prohibited in sport for some years, it is only relatively recently that human LH has become available as a commercial pharmaceutical product due to recombinant biotechnology. Thus there is now a pressing need to establish valid reference range(s) for endogenous LH levels in urine to assist in the detection of doping with recombinant LH as well as to improve the standardisation of urine LH measurement as an adjunct to detection of suppressed LH levels that accompany doping by use of exogenous androgens and hCG. Whilst there are several commercially available immunoassays optimised for serum but which can be used to measure LH in urine, the numerical values obtained from the different kits are not in good agreement. There is also the question of whether kits calibrated using natural pituitary-purified LH give accurate results when measuring degradation metabolites of recombinant LH excreted in urine. Thus there is a need to establish comparability of LH measurement amongst the over 30 WADA laboratories who use a variety of techniques for the measurement of LH. This project will measure LH in a range of urines using a variety of commercial assays in order to establish comparability of measurement and establish upper and lower reference ranges for normal subjects. An attempt will also be made to determine if any assays or combination of assays can be used to validly detect recombinant LH as well as distinguishing reliably from endogenous LH. 

Main Findings: 

The primary objective of this project was to determine which method or methods that can be successfully used to measure luteinizing hormone (LH) in urine. The measurement of LH is important for two reasons. The first is that the administration of testosterone and other androgenic hormones leads to a suppression of the secretion of endogenous LH and the second is the recent availability of recombinant LH which may elevate LH values. There are several different methods used by WADA laboratories to measure LH in urine all of which are based on antibody reactions. However it was clear that the different methods can give very different numerical values. Controlled studies using a variety of samples and a number of methods have shown that the Siemens Immulite is the preferred method for measuring LH in urine particularly when the suppression of LH is being used as an aid to detect doping with testosterone. Most other methods are not useful for this purpose because the LH or at least that portion of the LH molecule to which the methods respond is not stable in urine and hence low and apparently suppressed values can and do result from degradation on storage. The values of LH in urine as measured by the Immulite are relatively stable on storage for at least two weeks at room temperature and for months at -200C. 
Studies involving the administration of recombinant LH have shown that the injection of the recommended dose of LH does not lead to any elevation of measured LH in urine. This was not unexpected given the dose and the rate of natural LH production. A short term elevation of urinary LH was observed when thirty two times the recommended dose was injected. A method using SDSPAGE with Western blotting has been developed which can distinguish between pituitary and recombinant LH in both standards and in samples extracted from urine. It is clear from this work that the LH excreted in urine has a different apparent molecular weight to the parent LH.  
As a result of this project it is now feasible to use LH suppression as an adjunct procedure with T/E measurement to detect testosterone doping and to detect and confirm the use of large doses of recombinant LH. 

Code: T08A02MS

The aim of the project is to evaluate the robustness of the method with athlete’s samples regarding some collection scenarios and to compare the results obtained in different transport and storage conditions on serum samples. The final aim of the global project is to obtain information on sampling practices also called collection scenarios based on supporting data.

Main Findings: 

• For samples separated immediately after clotting, rhGH detection kit appears to have a slightly better sensitivity when samples are stored frozen compared to cooled.
• For samples arriving after 4 days (96h) and centrifuged upon reception, no differences exist for ratio if samples are analyzed immediately or after 7 days if frozen storage is applied.
• For samples separated immediately after clotting, storage must be made frozen. Under cool conditions, ratios is lower after ten days compared to one day. If frozen, ratios do not present any significant difference when analyzed after one day or ten days.
• If delay between end of collection and centrifugation is below 24 hours, no significant differences of ratio is observed between delays.
• Longer delays (up to 3 days; 72h) leads to lower ratio, but difference is close to analytical variation predicted by rhGH kits supplier’s experts.
• Later, ratios get significantly lower and tends to continuously decrease with time.

Code: R08B02GG

In 2003 the development of the software platform GASepo was submitted and succeeded for a WADA research grant. This included the participation of various experts of 9 WADA accredited labs for the development of this software. After two years GASepo now provides a reliable and easy-to-use software platform for quantitative interpretation of gel images deriving from IEF analysis as well as from SDS-Page analysis for the detection of recombinant EPO. As indicated in the initial grant application, further development of the software is anticipated due to the increasing demands of the various procedures and data from rEPO detection. This proposal targets to a novel and additional way for background subtraction of gel images and sample lanes with a special focus on low abundance gel images. The current version of the GASepo software offers two different ways of automated background subtraction:

1 – Subtraction of the background surface deriving from the lane boundaries

2 – Subtraction of the background surface deriving from an artificially created lane near the lane of interest. The proposed background subtraction module shall allow for subtraction of the background for each band separately. In addition some minor adjustments regarding the flexibility of report creation and the new demands of SDS-PAGE shall be implemented.

Main Findings

The project aimed at functionality enhancement of the existing GASepo software platform for quantitative interpretation of gel images from isoelectric focusing analysis. The GASepo software Version 1 has been previously developed and, in the meantime, has become an internationally accepted tool among the vast majority of the WADA-accredited anti-doping laboratories. Due to the recent development in the EPO doping analytics as well as due to accumulated experience with GASepo gained over time, it was necessary to update selected functionality features and adapt the software to the new generation of operating systems called Windows 7, both in its 32 bit as well as its 64 bit version.

Major and minor software modifications improved the usability and adapted the software to the recent demands of the WADA Technical Document TD2010EPO on EPO detection. Final product of this project is a renewed software version 2.1 of the GASepo platform. The software was validated against a phantom image with known pixel numbers and intensities. A validation report as well as the phantom image for revalidation is included in the software package.

Code: R08B01RH

Growth hormone (GH) is a naturally occurring hormone produced by the pituitary gland. Although banned under the International Olympic Committee (IOC) and World Anti- Doping Agency (WADA) list of prohibited substances, the detection of GH abuse poses a formidable challenge. The GH-2000 and GH-2004 projects have worked to develop a test to detect GH that is based on the measurement of GH-sensitive markers, which rise in response to the administration of GH. The magnitude and duration of the elevation is dependent on the dose of GH given, gender and the individual marker. Men were more sensitive to the effects of GH than women. IGF-I and P-III-P were the best of these markers and were selected to construct formulae that gave improved discrimination between those taking GH and those taking placebo compared with a single marker.

The results of the GH-2000 and GH-2004 projects have been reviewed by a panel of international experts at IOC, WADA and USADA workshops in Rome (April 1999), Dallas (March 2004), Nashville (May 2005), Austin (June 2006), Chicago (June 2007) and London (April 2008). These meetings have confirmed the method’s scientific validity and independent confirmation of this approach has been provided by the Australian-Japanese consortium and Kreischa Institute. The major impasse in implementing this methodology is the need for two immunoassays to be used for each analyte. Furthermore, the original IGF-I assay used in the GH-2000 study is no longer available. It is therefore necessary to validate two new assays for IGF-I and a further P-III-P assay to overcome this hurdle. The aim of the current study is to do this. This will be achieved by simultaneously measuring IGF-I and P-III-P by two commercial immunoassays for each protein on samples obtained from 500 elite athletes.

Main Findings

This report presents new threshold levels for the GH-2000 detection algorithm estimated for each assay combination using reference dataset of 496 samples from elite athletes and a false positive rate of 1 in 10,000 (99.99% specificity). A total of 4 different assay methodologies were used for this study; two for IGF-I and two for P-III-NP concentrations. In addition to the proposed threshold levels estimates, a sample size uncertainty limit has been added. Although this study was not designed to test the sensitivity of the test, it is noteworthy that we correctly identified Terry Newton as having been doping with GH and also identified another rugby league player with a suspicious finding.

Code: 08A07LM 

Testosterone is the most commonly reported of all banned substances with 1,124 cases in the WADA statistics for 2006.  As testosterone is a natural hormone, detection requires analysis with highly selective techniques to demonstrate the presence of it and its metabolites outside of normal ranges.  The outcomes of this project will further increase the ability of WADA to identify cheating by the use of testosterone and related compounds. 
A key component in identifying the banned use of the natural hormone testosterone for performance enhancement is the ability of WADA-accredited laboratories to accurately determine the changes that occur in carbon isotope ratios after doping. When an athlete takes synthetic testosterone it has a different 13C/12C ratio to that of the testosterone produced naturally in an athlete’s body. The measurement of these 13C/12C ratios is a complex technical task as the changes in ratio are very small and only specialised mass spectrometers can measure this change.   
A thorough review of the factors affecting accuracy in 13C/12C ratio results will be performed in this project to provide insight into the aspects of the methodology that contribute most to any measurement uncertainty. This will allow the methods to be further optimised to provide even greater accuracy and increase the probability of detection of doping.  

Main Findings: 

The first part of the project investigated opportunities to minimise the uncertainty associated with measurements of stable carbon isotope ratios of steroids related to testosterone in urine. A rigorous investigation of the inputs to a measurement uncertainty budget compliant with the JCGM Guide to Uncertainty in Measurement (GUM) 1 was performed. Optimised analytical method conditions were identified experimentally and a new instrument calibration method was developed that minimises measurement bias and facilitates uncertainty estimation. Using method validation data generated in this project, the combined uncertainty associated with results from the optimised analytical method employing this calibration approach was compared with that obtained when using the calibration method commonly employed in instrument software.
A new approach has been developed for the calculation of carbon isotope ratios when using GC-C-IRMS. The optimised approach uses a single point calibration rather than a linear calibration, and the measurement equation has been re-evaluated to avoid introducing a bias when the sample and internal standard 45R′ ratios do not match. While unmatched sample and internal standard 45R’ ratios do not introduce a bias when using this new approach, matching these ratios does improve the measurement uncertainty.
A number of factors in the sample preparation were identified that influence measurement uncertainty. These sample preparation steps were investigated and optimised to ensure the lowest possible measurement uncertainty: • SPE of conjugated, unconjugated and derivatised steroids
• Hydrolysis of conjugated steroids
• LLE of unconjugated steroids
• HPLC of unconjugated steroids
• Derivatisation
Validation of the optimised method including investigations of ruggedness, reproducibility and accuracy to allow an estimate of measurement uncertainty associated with a typical result. In developing this estimate, the full measurement equation for calculation of the isotope ratio values was expanded to include factors representing any bias in each of the main terms and validation results were used to provide estimates of the potential size of such bias even when it was not detected.
Complete uncertainty budgets were prepared for the carbon isotope ratios of key analytes and their delta differences in line with the GUM1. Detailed calculation of the sensitivity coefficients for each of the terms of the both measurement equations was required. This was relatively simple for the new measurement equation but required the use of mathematical software for the equation used by the IRMS instrument software. Uncertainty budgets were prepared for results from each calibration method for the δ13C values of androsterone, etiocholanolone, 11-oxoetiocholanolone, 11β-hydroxyandrosterone, 5β-androstane-3α,17β-diol, 5α-androstane-3α,17β-diol, 5β-pregnanediol, testosterone and dehydroepiandrosterone. They were also prepared for the differences between δ13C values (Δ13C) for androsterone versus 11-oxoetiocholanolone; etiocholanolone versus 11-oxoetiocholanolone; androsterone versus 11β-hydroxyandrosterone; etiocholanolone versus 11β-hydroxyandrosterone; 5β-androstane-3α,17β-diol versus 5β-pregnanediol; and 5α -androstane-3α,17β-diol versus 5β-pregnanediol.
This allowed a comparison of the measurement uncertainty associated with results from the two calibration approaches. The uncertainties are very similar for the two calculation approaches, although the uncertainty when using the new equation is consistently lower

Code: 08A01ZL

The current strategies used for the detection of EPO in sports are labour-intensive, time-consuming and cost-inefficient. The aim of this project is to develop an innovative strategy towards a fast and cost-efficient EPO assay in sports. This strategy is based on two key steps: 1) nanoparticles-based immunomagnetic extraction (NPIME), and 2) capillary zone electrophoresis (CZE) separation coupled with on-line sample concentration (OLSC) and native fluorescence (NF) detection.  First, a NPIME approach will be developed. Using anti-EPO antibody immobilized magnetic-cored nanoparticles, recombinant and natural EPO in samples is specifically extracted onto the nanoparticles and then desorbed into a much smaller volume. This process effectively concentrates EPO and meanwhile eliminates interference from the sample matrix. Second, a CZE-OLSC-NF method will be established. While the CZE separation provides fast profiling of EPO glycoforms, distinguishing recombinant and natural EPO. The OLSC allows for concentrating EPO from a larger injected sample volume into a much narrower zone before separation, thus significantly reduces the detectable concentration level. The NF detection selectively detects EPO with high sensitivity. Finally, the developed NPIME approach and the CZE-OLSC-NF method will be combined in off-line mode and applied to EPO spiked urine or urine samples of EPO injected patients. The proposed strategy can provide two significant advantages: speed and overall cost-efficiency. Our goal is to reach a total analysis time of less than 4 hours and an overall limit of detection (LOD) of 10-11 M at the end of the project proposed. The overall performance can be further effectively improved by in-depth optimization of the individual techniques. Also, the sensitivity can be further significantly improved by using a deep UV laser as the excitation light source. Thus the strategy proposed can be eventually developed into a reliable and robust analytical method for anti-doping analysis of EPO in the near future. 

Main Findings: 

The current EPO assay is associated with apparent drawbacks: labour-intensive, time-consuming and cost-inefficiency. This assay involves four key component methods: 1) isoelectric focusing (IEF), 2) ultrafiltration, 3) double blotting, and 4) on-gel chemiluminescence detection. The four key component methods are essential for specific and sensitive detection of EPO; however, they are all labour-intensive and time-consuming.  The aim of the project is to explore straightforward and effective substitutes for these sub-methods and then integrate them into a fast and cost-efficient approach. Nanoparticles (or microbeads)-based immunomagnetic extraction (NPIME or MBIME) is used as a replacement of ultrafiltration and double blotting. Capillary zone electrophoresis (CZE) is used as a substitute of IEF and the combination of native fluorescence (NF) and on-line sample concentration (OLSC) is used as an alternative for the on-gel chemiluminescence detection. We have successfully synthesized immunoaffinity magnetic nanoparticles (MNPs) using anti-alpha fetoprotein IgG as a model antibody. However, when the established method was transferred to anti-EPO antibody, the obtained immune-MNPs failed to recognize EPO. The reason for this seems that the antibody used was too susceptible to the reaction conditions for the immobilization. To overcome this issue, we turned to commercial protein A-coated magnetic beads and developed MBIME approach for specific extraction of EPO, which could enrich EPO by one order of magnitude. Besides, we successfully established an in-lab built deep UV laser-induced fluorescence (deep UV-LIF) detection system for detection of the native fluorescence of EPO. It enhanced the detection sensitivity by one order of magnitude as compared with UV absorbance detection. CZE was an effective replacement of IEF, which permitted complete resolution of EPO glycoforms within 25 min. OLSC could enhance the detection sensitivity by 50-100 fold, however, when used for the sample prepared by the MBIME approach, it resulted in notable loss in separation resolution. So it was excluded as a sub-method for the integration. Based on the component approaches established, the finally integrated approach, MBIME-CZE-deep UV LIF, allowed for the extraction, separation and detection of EPO glycoforms within 6.5 hours. However, the detection sensitivity was enhanced only by two orders of magnitude, which is far from the requirement for the analysis of real samples. To further develop the proposed strategy into practical approach for real sample assay, solutions to overcome the experienced issues were found. As human urine exhibited very significant matrix effect, antibody without cross reactivity to other proteins is a critical requirement for specific extraction. Besides,high binding affinity is a critical requirement for more efficient extraction. Furthermore, inertness under immobilization conditions is essential for the synthesis of active immune-affinity MNPs. An anti-EPO antibody that can meet these requirements is the key for the practical use of this approach.

Code: T08A04MS

The aim for the second step is to obtain statistically significant comparison between different delays of transport. The most important point to use for this comparison will be the obtained ratios calculated for both GH detection kits. The aim of the third step is to compare ratios obtained from samples analysed as A-sample, and samples analysed as B-sample after a frozen or cold storage of 10 days.

Main Findings

• For samples separated immediately after clotting, rhGH detection kit appears to have a slightly better sensitivity when samples are stored frozen compared to cooled.

• For samples arriving after 4 days (96h) and centrifuged upon reception, no differences exist for ratio if samples are analyzed immediately or after 7 days if frozen storage is applied.

• For samples separated immediately after clotting, storage must be made frozen. Under cool conditions, ratios is lower after ten days compared to one day. If frozen, ratios do not present any significant difference when analyzed after one day or ten days.

• If delay between end of collection and centrifugation is below 24 hours, no significant differences of ratio is observed between delays.

• Longer delays (up to 3 days; 72h) leads to lower ratio, but difference is close to analytical variation predicted by rhGH kits supplier’s experts.

• Later, ratios get significantly lower and tends to continuously decrease with time.

Code: 08A16CG 

The overall aim of the proposed project is to apply NMR spectroscopy and pattern recognition (PR) methods as innovative method for anti-doping screening and the elucidation of the overall biochemical effects of different doping methods.
This project will test the hypothesis that there are quantifiable variations in the urinary excretion of low molecular weight metabolites, induced by physiological response to doping, and that these changes could be related to different doping methods. By this means doping could be monitored without searching a specific compound but evidenced as a whole, in a fingerprinting approach, through NMR spectra of biofluids and subsequent multivariate analysis. As a first approach, this project will focus the detection of the use of designer steroids, by examining athlete’s positive samples for synthetic steroids like stanozolol.
The project includes the use of the large sample databank of Doping Control Laboratory of Athens, the generation of NMR spectral database and the consecutive analysis as well as the discovery of novel relevant biomarkers. The specific objectives are to apply biofluid NMR spectroscopy and Pattern Recognition in order to build multivariate biochemical models based on the use of prohibited formulations by athletes and to create databases of NMR spectra of characterised biofluids in selected human states. The derived models will be further applied to classify unknown samples according to primary and secondary metabolite variations induced by doping. Data will be analysed using a variety of standard methods to investigate multivariate analysis mapping of endogenous biochemical profiles. The new NMR-based metabonomic approaches will be compared with conventional methods, based on clinical chemistry and mass spectrometry and their relative usefulness in the field of anti-doping control will be evaluated.
To our knowledge the multiparametric approach proposed in this project has not been done before in humans. Such studies could lead to new strategies to complement antidoping control based on the Metabonomic approach maximizing the reliability of screening practices in use.

Main Findings: 

Metabonomics is well established as a powerful method for the evaluation of the metabolic profile of an organism, proffering a holistic view of an organism’s response to different exogenous factors. The aim of the project was to examine whether the NMR based metabonomics approach could be used as complementary tool to the existing methods on the basis of a non-targeted profiling analysis that is able to capture the metabolic signature of athletes who have utilized exogenous steroids. The applicability of the method was tested in a cohort of 263 human urine samples of both men and women athletes targeted to doping controls. Among them, 59 had been reported as positive to the official doping controls for the application of exogenous anabolic steroids.
The study included the NMR measurements of all samples and the chemometric analysis of the resulted spectroscopic data set. The latter consisted of complex 1H NMR spectra containing hundreds of signals from both endogenous and exogenous metabolites and was analyzed by multivariate statistical tools in two ways:
i) using data reduction of the original matrix to a lower dimension, followed by Principal Components Analysis (PCA), Partial Least Square–Discriminant Analysis (PLS-DA) and Orthogonal PLS-DA (OPLS-DA) and ii) using a pre-processed full spectroscopic matrix with peak alignment followed by PCA and interval based Extended Canonical Variate Analysis (iECVA). The method i) resulted in models with partial grouping betweenn the originally classified groups, while method ii) provided a clearer classification that highlighted significant differences of metabolites, such as creatine, creatinine, hippurate, and
acetate among the groups of athletes. It is worth noticing that the samples derived from positive athletes displayed a significant higher variance comparing to the “negative” ones in all developed statistical models. This depicts one limitation of the present study, which arises from the low homogeneity of the sample pool and the lack of sufficient metadata that could be added to the statistical models development.
It is concluded that the NMR based metabonomics approach could be used as an ultra fast and cost effective predictive tool in anti-doping control that could highlight those samples originated from doped athletes among a larger cohort of samples on the basis of their metabolic fingerprint. In order to be fully applicable, the samples collection should be accompanied with extended metadata information that could be utilized in the multivariate statistical analysis.