Projets de recherche

Code: T08C01FD 

Anabolic steroids are amongst the most misused substances in doping control and are intensively metabolized in humans. Adequate screening for misuse of these substances therefore relies on the detection of metabolites in urine samples collected from athletes.
Most of the studies investigating the metabolism of pharmaceutically available steroids were performed in the 1980’s via gas chromatography mass spectrometry (GC-MS). This research resulted in the selection of appropriate metabolites for the detection of steroid misuse. Over the years the selection of metabolites was further elaborated to include several metabolites that can result in prolonged detection times. Over the last decade, liquid chromatography tandem mass spectrometry (LC-MSn) was introduced as a screening technology in doping control laboratories world-wide. As a result several steroids that are difficult to detect via GC-MS became readily detectable. However, it should also be noted that several easily detectable metabolites by GC-MS are virtually undetectable via LC-MS. Indeed, both GC-MS and LC-MS are compatible techniques and the simultaneous application of both technologies is needed to cover the detection of all categories of structures. Recently, it has been shown that precursor ion scanning via LC-MS can be used to detect new steroid metabolites and the use of this technology has resulted in the detection of previously unreported metabolites. These results have also illustrated the limitations of GC-MS for the detection of steroid metabolites. Indeed, some highly abundant metabolites in the LC-MS analysis could hardly be detected by GCMS. This has highlighted the need for a re-investigation of steroid metabolism to allow for their adequate detection by LC-MS. Preliminary results for several steroids indicate that detection times could be prolonged considerably if more appropriate LC-MS metabolites are selected.

Main Findings: 

The use of LC-MS/MS in neutral loss and precursor ion scan modes allows for the detection of steroid metabolites. Several strategies based on this methodology have been applied for metabolic studies of some anabolic steroids. Previously unreported metabolites have been detected. In the case of methyltestosterone one of these metabolites increased the detectability of the steroid misuse. For stanozolol, the screening in negative ionization mode for one of the detected metabolites also improved the detection to stanozolol misuse. Several real samples have been analysed for these metabolites showing their applicability. For other steroids, additional metabolites have been found although more experiments are needed in order to prove their usefulness for doping control analysis.

Code: 08A13UF 

The analysis of 13C/12C is a well proven and powerful method to detect abuse of endogenous steroids. However, the method is rather complicated as compared to more classical analytical techniques. Successful application requires skilled and
experienced technicians and scientists. However some possible problems appear to be exaggerated. This has led partly to the impression that only measurements obtained under absolutely perfect conditions can be trusted. Self evidently this has been exploited to dishonestly charge the method. The project aims to find out to which degree 13C/12C analyses are impaired when the conditions are not ideal.

Main Findings: 

• Chromatographic resolution in GC-C-IRMS is significantly impaired by ineffective combustion.
• Lack of oxygen and the state of the furnace themselves are more significant than e. g. combustion temperatures in this respect.
• By contrast, the effects of dead volumes, cold spots etc. seem to be less important.
• Observed peak overlap which is induced by cold spots (and possibly also by dead volumes) does not necessarily result in invalid data.
• This even applies when the (true) δ13C-values of incompletely resolved signals differ largely.
• Nonetheless, poor peak shapes and insufficient resolutions require careful investigation of the corresponding causes.
• In order to still benefit from analyses obtained from poorly resolved chromatograms, incomplete combustion must definitely be excluded.
• The peak shapes of highly oxidized compounds are a sensitive indicator for the effectiveness of combustion, increasing peak tailing corresponding inefficient combustion. This however might confound with the effects of higher retention times.
• The δ13C-values of lowly oxidized compounds are more sensitive to incomplete combustion than those of highly oxidized compounds.  However, these conclusions must be confined to steroids currently.

Code: 07E03HG

In some sports about 100% of athletes take nutritional supplements (NS) without any reflection of the risk/benefit relation. One of the risks connected with the use of NS is the risk of inadvertent doping originating either from contaminated or faked NS. An international IOC study performed in 2002 has shown that about 15% of nonhormonal nutritional supplements contained anabolic androgenic steroids (mainly prohormones) not declared on the label. Since that time athletes have been warned by their federations, information systems have been established, legislation towards anabolic-androgenic steroids as nutritional supplements has been changed e.g. by the anabolic steroid act 2004 in the USA and some companies have improves their quality control systems. The question is: has the situation on nutritional supplement market improved or got worse? To answer this question on an international level an extended follow-up of the 2002 IOC study should be performed. NS from different countries and from internet sources should be purchased and analysed for prohormones, classic anabolic steroids, new designer steroids, β2-agonists and stimulants. The results should be used to educate athletes to reduce the non-reflected use of NS, to force the industry for the improvement of quality control systems for NS and to motivate governmental institutions to regulate and restrict the market for NS.

Main findings

Previous studies have shown that supplements may be contaminated with anabolic androgenic steroids so that these supplements present a considerable risk of inadvertent doping for athletes involved in a doping-control system. The purpose of the present study was to investigate the international supplement market for an update on the contamination rate of nutritional supplements. A total of 597 supplement samples were obtained from 17 countries and from the Internet. Samples were analyzed for presence of 43 different anabolic-androgenic steroids by gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. For most substances, the limit of detection was in the range of 10-50 ng/g or lower. In four samples, the presence of a prohibited substance was confirmed (Androsta-1,4-diene-3,17dione (twice), DHEA, and Androsta-1,4,6-triene-3,17-dione). A total of 561 samples were found to be negative of the selected anabolic agents and in 32 samples, no analytically acceptable result was obtained. The present results indicate that the prevalence of supplement contaminations has decreased in recent years. Nevertheless, supplement contaminations and adulterations still present a risk of inadvertent doping to athletes who are involved in a doping-control system. Further studies are necessary to identify the risk of contamination for further prohibited substances.

Code: 07A08JS

Recently, autologous blood transfusion is one of biggest challenges for abuse detection. For reasons of convenience and safety, autologous transfusions, in which the cells are the athlete's own, are reportedly far more common than homologous transfusions, in which the cells belong to someone else with compatible blood. So far there is no adequate and unequivocal method to confirm illicit boosting of an athlete's red blood-cell via transfusion of one’s own blood. Presently our institution is running the WADA sponsored project “Overall approach for blood transfusion detection (autologous/homologous)”. As a complementary approach the present project aims to add another alternative to the possible ways to demonstrate autologous blood transfusion developing tests, based on comparison of red blood cell membrane proteomes of fresh red blood cells and stored red blood cells for different times, at standard storage conditions. Several studies employing different strategies describe in details normal human erythrocyte proteome, while some studies have postulated changes in erythrocyte proteome after red blood cell storage (Anniss et al.; Manojkumar et al.) This project is presented with an idea to identify detectable changes in erythrocyte membrane proteome before and after standard storage procedures in order they can be used as a methods to identify autologous blood transfusion. For such purpose we will apply proteomics methods such as 2D electrophoresis, 2D-DIGE, mass spectrometry of identified proteins. The project is presented as a preliminary step of one year in order to test the hypothesis. If, as expected, differential proteome appears as an actual possibility next step would be to develop adequate tests which would employ flow cytometry and proper markers in order to demonstrate existence of different populations of erythrocytes in the blood of the same subject, which would confirm anterior autologous transfusion.

Main Findings

In the present project we employed two complementary proteomic strategies in order to get an insight to the changes occurring at the membranes of the RBC over prolonged storage time. 2D gel electrophoresis as a strategy of protein separation has its drawbacks such a difficulties to separate efficiently extremely basic, acidic, or hydrophobic proteins. So, it was necessary to complement it with isotope tagging for relative and absolute quantitation (iTRAQ) methodology, which is peptide oriented and helps to achieve satisfactory resolution of the changes occurred. Not a single method is sufficient to follow all the changes happening. From the application of each one of the methodologies, several important proteins appear as serious candidates for further targeting. However, within the same 2D spot it is possible to identify several proteins with different degree of certainty, even though that experimentally some of them may be discarded taking into account molecular mass, sequence coverage, % or score number and other indicators. When it was possible we compared also graphs of corresponding spots (increase or decrease of spots volume ratio) and ratios of iTRAQ results when corresponding identified proteins appeared in both of them. According to the results from our studies and also available results of other authors we propose to group observed findings such as:

• Transmembrane proteins

• Cytoskeletal proteins

• Other proteins

• Other investigator’s findings related to our studies

Code: T07B01JS 

The aim of this proposal is the characterisation of distinct antibodies that specifically recognise growth hormone isoforms. Some of them are to be employed in an ELISA-based test to detect growth hormone abuse by athletes. In addition, other antibodies with different specificities and different origin will be studied. For the characterisation of the antibodies the technique of surface plasmon resonance will be employed. In a straight-forward strategy the antibodies will be immobilised onto the carboxymethylated surface through standard amide-bond ligation chemistry. Different chips (each one with four channels, including controls) will be required to respond to different aspects of the characterisation. Available GH isoforms structurally related to the pituitary GH-N form will be flown across for the determination of specificity, cross-reactivity and to determine kinetic parameters. Also the effect of other growth-hormone related substances, both structurally and functionally, may be included in the study.

Main Findings

A rigorous study on the binding characteristics of thirteen different monoclonal antibodies, that address growth hormone isoforms, has been successfully conducted. In this study, antibodies were compared using the Surface Plasmon Resonance method and this comparison helped in the selection of hGH antibodies for the purpose of anti-doping testing.

Code: 07C12JS

Glucocorticoids are potent anti-inflammatory and immunosuppressive agents used to treat a broad variety of medical conditions. Due to their ability to alleviate pain and enhance the athlete’s concentration capacity during strength and endurance competitions, these drugs have became doping substances. Consequently, the systemic administration of these steroids is forbidden by WADA, and their use requires a therapeutic use exception approval. However, topical preparations when used for dermatological, auricular, nasal, ophthalmic, buccal, gingival and perianal disorders are not prohibited and do not require any form of therapeutic use exemption. Since some glucocorticoids are marketed in both systemic and topical forms, the distinction between different routes of administration through the analysis of urine samples is necessary. Currently, no methodology is available to address this discrimination. Recently, it has been discovered that the misuse of a testosterone gel can be distinguished from an oral or intramuscular testosterone administration through the changes in the steroid profile. In particular, a characteristic increase in the excretion of the 5α-metabolites of testosterone versus their 5β counterparts has been detected. There are two genes encoding two distinct isoenzymes of 5α-reductase that are differentially expressed in human tissues. The type 1 isoenzyme is transiently expressed in newborn skin and scalp, and permanently expressed in skin from the time of puberty. Type 2 is the predominant isoenzyme detectable in male accessory sex glands and in the prostate. Both enzymes are expressed in the liver. No difference between the ability to reduce cortisol or testosterone, to 5α-tetrahydrocortisol, or androsterone respectively, has been proved for 5α-reductase enzymes. In that sense, patients affected by a 5α-reductase deficiency (type 2 isoform deficient) can be easily diagnosed by finding either low 5α-tetrahydrocortisol/5β-tetrahydrocortisol or low androsterone/etiocholanolone urinary ratios. Thus, it is to be expected the topic use of a corticosteroid will produce an increase in the excretion of the 5α/5β ratios similarly at what it has been observed for testosterone. As a consequence, a distinction between topical and systemic use of corticosteroids could be accomplished.

Main Findings

The application of corticosteroids from a potential doping point of view in athletes is considered different based on the different routes of application. Systemic use is considered prohibited but topical use is permitted. This dichotomic situation , easy from regulatory side, is very difficult to face by antidoping laboratories, as no analytical distinction so far has been approved to distinguish both routes of application. The purpose of the present project was to afford insight into the possibility to discriminate between prohibited and permitted forms of use of corticosteroids based on metabolic findings in urine. Initial hypothesis was based on the differential appearance of reduced metabolites. Primary focus was directed to 5α and 5β reduced metabolites based on findings for other steroids. This study needed the synthesis of authentic standards for proper chromatographic identification, which was carried out by reaction with sodium borohydride. However, when actual excretion urines after administration of corticosteroids were studied, none of these metabolites were detected. Focus was then moved to other forms of metabolic reduction, especially on C20. Also the possibility to study metabolites originated by oxidative metabolism had to be considered. In order to detect as much metabolites of corticosteroids as possible, a series of innovative methodologies based on LC/MS were developed, based on precursor ion scan and neutral fragments losses. An exhaustive methodology was proposed. When it was applied to different corticosteroids administered orally, they were able to detect the presence of some known but many unknown new metabolites. In fact, as much as 28 metabolites were detected for prednisone, 20 for triamcinolone and 28 for methylprednisolone. The structure of many of those metabolites, however, was not fully identified with the data afforded by the LC/MS approach alone. Taking methylprednisolone as the target compound, an experimental approach combining data from LC/MS and new data generated by GC/MS (methyl-oxime trimethyl-silyl derivatives), it was possible to ascertain the structure of up to 15 metabolites (many unknown so far). Main routes of metabolism identified after oral application were those based on reduction of C20, on oxidation of C6, on C11, on C16, on methyl linked to C6, and the formation of double bond between C6 and C7. When methylprednisolone was administered topically in a relatively high dose (5g), none of the metabolites were present in urine. Only in one patient receiving repetitive massive doses of topical methylprednisolone, some metabolites were present but in very low amounts. There is no reason for an active athlete to receive those massive administrations. Thus, in conclusion, to differentiate a systemic from a topical administration of corticosteroids in sport, the establishment of a threshold concentration in urine appears as the decision of choice. It is worth to summarize that the present project, in addition to contribute to the analytical distinction between different routes of administration of corticosteroids in sport, has afforded fundamental information previously unknown regarding the human metabolism of the family of synthetic corticosteroids.

Publications:

• Pozo O, Ventura R, Monfort N, Segura J, Delbeke FT. Evaluation of different scan methods for the urinary detection of corticosteroid metabolites by liquid chromatography tandem mass spectrometry. J Mass Spectrometry 2009; 44(6): 929-944.

• Pozo O, Marcos J, Ventura R, Segura J. Using complementary mass spectrometric approaches for the determination of methylprednisolone metabolites in human urine. Rapid Communications in Mass Spectrometry 2011, to be submitted

Code: 07C09MT

Insulin, a potentially performance enhancing agent, is prohibited for nondiabetic athletes according to the WADA list of banned substances since 1999. Despite the already existing methods for the mass spectrometric determination of the chemically modified synthetic insulin analogues in regular doping control samples, an approach to uncover the misuse of recombinant human insulin is missing. Insulin is a peptide consisting of an Achain (21 AA) and a B-chain (30 AA), which are connected by two disulfide bonds. It is endogenously produced in the pancreas from the single chain precursor proinsulin after cleavage into insulin and C-peptide (31 AA). In healthy individuals insulin and C-Peptide is secreted in equimolar amounts from the vesicles of the Langerhans` islets cells into the bloodstream. Thus, the ratio of these peptides in human plasma is supposed to be constant and a significant shift towards higher insulin amounts should provide a reliable hint for a surreptitious insulin application. Existing ELISA diagnosis kits for determination of insulin and C-peptide will be utilized to assess the physiological ranges of the ratio in regular plasma specimens and doping control samples. Moreover, specimens obtained from patients being treated with recombinant human insulin will be measured and serve as “positive control” samples. The planned methodology, which has commonly been used for clinical or forensic purposes, could also serve as a screening procedure for plasma doping control specimens to indicate the misuse of any kind of exogenous insulin supplement that cross-reacts with the employed ELISA.

Main Findings

The determination of insulin/ C-peptide ratio in doping control specimens is not a promising approach to uncover the misuse of insulin(s) in sport due to insignificant changes upon insulin administration and a great influence of concurrently provided carbohydrates. Future studies that may provide a more helpful tool are planned and include the examination of the autoimmune status of the volunteers and the determination of potentially formed anti-insulin-antibodies occurring in plasma from patients that were treated with exogenous human insulin. It is known that these antibodies are produced endogenously and may enable the detection of insulin applications in an 'indirect' fashion.

Code: 07C25CG

Detection of anabolic steroids and their long term metabolites that are usually excreted in very low concentrations, remains one of the major challenges facing WADA Accredited laboratories. Though LCMS is nowadays one of the most powerful analytical techniques, its applicability on the detection of anabolic steroids is limited by the ionisation problem that anabolic steroids present with soft ionization sources like ESI or APCI. A number of papers have been published dealing with the ionization problem of anabolic steroids. Different ionization techniques, mobile faces additives and specific transitions have been adopted by several authors in order to meet sensitivity criteria for these compounds. Despite the progress that has been done in the field, the difficulties in ionization of anabolic steroids have lead to LCMS methods that deal with limited number of analytes. On the other hand, several authors have used derivatization, prior to LCMS analysis, in order to enhance sensitivity for difficult ionized molecules. The incorporation of an easily ionized group, like a secondary amino group, that is practically 100% ionized in acidic mobile faces, enhances by orders of magnitude the sensitivity of their detection. This project will investigate the enhancement of sensitivity in the detection of anabolic steroids by LCMS, after derivatization, in ESI and APCI mode for doping control purposes. Several derivatization reagents and conditions will be tested in order to find the most suitable(s) for the detection of anabolic steroids by LCMS well below their MRPL. A screening method for anabolic steroids in LCMS will be developed. The sensitivity of the method will be compared with that of GCHRMS. The developed method will be validated. The final method will be tested for its applicability in the detection of corticosteroids in order to investigate the possibility of creation of a combined LCMS screening method incorporating the whole range of both anabolic steroids and corticosteroids.

Main Findings

The current research program examined the possibility to enhance ESI LC/MS detection of anabolic steroids, a class of prohibited substances with limited soft ionisation capacity, compared to other classes of prohibited substances. The way to achieve the enhancement of sensitivity is through the enhancement of the ionisation capacity by the appropriate derivatisation. After an extensive method development stage comprising 4 phases, 3 equivalent methods of 2-steps derivatisation are proposed aiming the hydroxy and keto groups of steroidal structure. The results presented are showing significant improvement in the detection of anabolic steroids steroids in ESI+ LC/MS. Only 3-OH stanozolol fails to comply the WADA MRPL in urine samples, but this fact should be considered with the fact that the analysis was performed on a LC ion trap MS in full scan mode without any MSMS sensitivity enhancement.

Code: 07C24LM

A key component in identifying the illegal use of the natural hormone testosterone for performance enhancement is the ability of WADA-accredited laboratories to accurately determine the very small changes that occur in carbon isotope ratios after doping. This project aims to enhance a previously prepared Certified Reference Material (CRM) of testosterone in urine by providing a very well characterised reference value for the carbon isotope ratios of steroids related to testosterone abuse in the CRM. The availability of a CRM with well-defined carbon isotope ratios traceable to international standards with low uncertainty will permit laboratories to unequivocally demonstrate the reliability of their methods and ensure comparability of results from different laboratories. This material will be the first certified reference material of this type available in a urine matrix.

Main Findings

The δ 13C reference values assigned in this project to key urinary steroids in the urine matrix CRM, NMIA, MX005 will assure accredited laboratories meet the requirements of WADA Technical Document TD2004EAAS in determining whether results of isotope ratio measurements are consistent with administration of a steroid. The CRM can be used as a quality control and calibration material for isotope ratio measurements at key delta values of interest. The carbon isotope ratios of androsterone and etiocholanolone in the CRM are excellent positive and negative controls, respectively, for the prescribed threshold of 3% difference with respect to endogenous reference steroids. Despite the expanded uncertainty being relatively large, these two steroids are above and below the threshold at the 95% level of confidence. In addition, the certified delta value for androsterone of -27.9% is very close to that at which the results of an analysis must be reported as "inconclusive" when the low concentration of ERCs prevents determination of a delta difference. The use of the CRM in anti-doping programs will lead to improved comparability of results between laboratories for longitudinal studies.

Code: 07C10JA

One of the aims of our ongoing (LIVE) study financed by WADA has been to find microbial contaminants of urine samples. We developed an in vitro simulation system for urine to mimic the storage and transportation conditions of urine prior to testing. Our hypothesis was that yeasts and bacteria are the most probable contaminants responsible for the adverse reactions in urine samples. Our preliminary results suggest that a complex microbial community such as that found in human saliva and feaces has a potential to modify the steroid profile of urine. The abovementioned findings have motivated us to accelerate the development in the area of microbial contamination and focus efforts on the real doping control samples. Here we are proposing an one year research project with following objectives;

(i) Identify contaminating microbes from real doping control samples (sent to Helsinki doping control site)

(ii) Analyze the source of contamination for those samples

(iii) Find the most important contamination parameters that affect the reliability of doping control analyzes

(iv) Design routine laboratory test to find seriously contaminated doping control samples

(v) Design procedures to eliminate microbial contamination or reduce its risk to the doping control analyzes

Main Findings

The microbial characterization of urine samples indicates that urogenital and gastrointestinal tract act as most probable contamination sources. All major microbial groups detected can be explained by non-intentional contamination sources. Intentional contamination is an existing possibility, but based on the results of this project we would put the research effort on the bacteria representing natural, unintentional contaminants. Contaminated samples dominated by Lactobacillaceae and Enterococcaceae did not harbor as high microbial numbers as those dominated by Enterobacteriaceae and Pseudomonadaceae. Overall, the detected bacteria are known capable of altering the steroid profiles, emphasizing the importance of high hygiene level at sampling for a reliable doping control. However, in optimal conditions a low bacterial amount can increase exponentially to high levels in a short period of time. Elevated pH is one of those indicators which are used in doping control laboratories to recognize microbial contamination. This screening parameter may be used together with a number of other criteria, e.g. the presence of free steroids in a urine sample, but has very limited selectivity alone. Abnormal smell or turbidity does not correlate with microbial growth. Several studies have been carried out for the stabilization of human urine samples. None of the investigated physiological methods, including sterilization by filtration, ultraviolet radiation, or ultrasonication, have succeeded in preventing microbial growth. Chemical methods have been shown to be more efficient, but the introduction of any chemical substance into athletes’ samples after collection may be difficult to approve legally. Consequently, rapid freezing has proved to be the only feasible method for stabilizing samples and preventing microbial activity.

Publications: Ojanperä S, Leinonen A, Apajalahti J, Lauraeus M, Alaja S, Moisander T, Kettunen A. Characterization of microbial contaminants in urine. Drug Test. Anal. In press.

Presentations: Alaja S, Apajalahti J, Leinonen A, Kettunen A, Ojanperä S, Kuuranne T, Lauraeus M. Characterization of microbial contaminants in urine. Poster in the Manfred Donike Workshop – 28th Cologne Workshop on Dope Analysis, 2010, Köln, Germany.