Projets de recherche

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.

Code: 07C11AK

In nature, different forms of carbon atoms exist called isotopes. The most common carbon atom has a weight of 12 but a smaller amount naturally exists as a heavier weight of 13. In testosterone, the proportion of carbon13 to carbon-12 present, referred to as the carbon-13/carbon-12 ratio (13C/12C), can be determined by an instrument called a gas chromatograph combustion-isotope ratio mass spectrometer (GC-C-IRMS) The 13C/12C content of testosterone produced in our bodies is ultimately made from carbon broken down from our dietary intake. However, pharmaceutical testosterone is synthesized from soya-plant material that has low 13C/12C ratio. A similarly low 13C/12C ratio of testosterone in urine therefore indicates that the testosterone is of pharmaceutical origin, i.e. a doping offence has occurred. The amount of testosterone in urine is very small, even following its administration, and the separation required to purify and concentrate it for IRMS analysis is time consuming and laborious. A simple and rapid preparatory technique is highly desirable. We will manufacture polymers, which are imprinted with the molecular shape of testosterone, so that testosterone from urine can quickly fit into these imprints, like pieces from a jigsaw. The testosterone can then be easily removed for analysis by GC-C-IRMS. Further development will allow the polymer to be coated onto a small glass bar for stirring in the sample itself prior to the bar being directly transferred into a heated inlet (called a thermal desorption unit) on the GCC-IRMS, where all the testosterone is vaporized for analysis. This ‘on-line process’ is very rapid and all the isolated testosterone is analyzed thus increasing sensitivity. Finally, to prove that an abnormal 13C/12C ratio of the testosterone targeted is not because of impurities present, we intend to simultaneously analyze a portion of the sample with a standard type of spectrometer (quadrupole) attached to the GC-C-IRMS.

Main Findings

Comparison of WADA statistics for adverse findings with those of ten years earlier shows that there has been little change in the ranking of anabolic steroids, with testosterone continuing to account for the most common finding. It is analytically more challenging to prove the administration of testosterone, as it is also naturally produced within the body. An analytical approach that can distinguish administered testosterone from that naturally-produced is by determining the carbon fingerprint of testosterone present in urine. Testosterone has 19 carbon atoms, but collectively the carbon atoms in pharmaceutical testosterone are less heavy than testosterone produced in our body. The difference in heaviness is due to whether or not a carbon atom is present with an extra neutron in its nucleus, that is whether it is the heavier ‘carbon-13’ rather than the ‘carbon-12’. The relative amount of carbon-13 to carbon-12 present, referred to as the carbon-13/carbon-12 ratio (13C/12C), can be determined by an instrument called a gas chromatograph-combustion-isotope ratio mass spectrometer (GC-C-IRMS). For doping control purposes, this approach is used to prove whether an athlete has broken the rules by administering testosterone. The concentration of testosterone in urine is very small, even following its administration (doping), and extensive sample work-up is required to purify and concentrate the testosterone from urine prior to carbon isotope analysis. To reduce the labor and simplify the process, the WADA-accredited at King’s College London (UK) in collaboration with the University of Leeds, manufactured a polymer (a macromolecule) into which the molecular shape of testosterone was imprinted, so that testosterone molecules from urine can quickly fit into these imprints, like jigsaw pieces. The testosterone can then be easily removed for analysis by GC-C-IRMS. The work was challenging, but ultimately successful. Even so, to make the process effective, more R & D work is required, so that an optimized polymer can then be applied by loading into small cartridges through which urine can flow with the testosterone being easily extracted for analysis. In addition to the polymer investigation, the researchers also reconfigured the design of the instrument so that enhanced steroid purification can also be performed within the GC-C-IRMS, a process called multi-dimensional gas chromatography, and they added a different type of mass spectrometer (quadrupole analyzer) to assist with detection. These modifications to the instrument, which are cost-effective and simple to adopt by other laboratories, adds to the certainty that the testosterone measured by isotope analysis is pure, in keeping with the gold standard approach by WADA-accredited laboratories.

Code: 07E04KR

Sildenafil (phosphodiesterase-5 inhibitor) is recognized as treatment for pulmonary hypertension and erectile dysfunction. Ingestion at therapeutic dose shows profound improvement in pulmonary artery pressure, cardiac output, VO2peak, and exercise capacity at hypoxic conditions; at sea level breathing 10% O2 and at Mount Everest base camp (>5,000 m) in subjects free of lung disease. Olympic Nordic venues are often at mild altitude; the Salt Lake Olympic Nordic venue is approximately 1,700 m and the Turin venue is at 1,540 m altitude. Although the Vancouver Nordic venue is at 860 m altitude, significant reductions in VO2peak (-5.9%) and maximal 5-min cycle ergometry performance (total kJ, -3.6%) has been documented at simulated 580 m altitude. Therefore, further studies are needed to characterize potential ergogenic effects of sildenafil at mild altitude. Likewise, short-term inhalation of concentrated ambient air particles (PM) promotes vasoconstriction of small pulmonary arteries and produces pathologic features consistent with pulmonary hypertension. We recently found that breathing high levels of combustion-derived PM during exercise caused a significant (~5%) decrease in 6 min cycle ergometry work output. Since the pulmonary vasculature is a target for effects of ambient PM from fossil fuel combustion, oral sildenafil may enhance performance at ice rink venues (resurfaced with fossil-fueled machines) and at the upcoming Beijing and London Olympics venues which are likely to have high levels of PM. The aims of this project are to determine if a therapeutic dose of sildenafil enhances exercise performance and aerobic capacity 1) in high PM pollution and 2) at mild simulated altitude consistent with Olympic venue altitudes, 3) elucidate a potential mechanism, and 4) confirm measurement of sildenafil and metabolites in plasma and urine. These studies will provide evidence detailing performance enhancement from prior-to-competition ingestion of oral sildenafil at 1) high air pollution venues and at 2) mild altitude.

Main Findings

Exercise performance in this study was not significantly decreased in high particulate matter (PM) air for placebo or treatment groups. Although mean performance was 10 kJ lower for high pollution exercise, individual variability did not allow statistical significance. However, when data was analyzed after separating individuals into responders and non-responders as previously done in other studies, significant improvement in performance was noted for those who had a positive response to sildenafil. We further identified increased in mean pulmonary artery pressure (PAP) 1 h after high pollution exercise, but not after exercise in clean air that was associated with decreased performance. Ingestion of 50 mg sildenafil blunted the observed increase in PAP after high PM exposure exercise. The pulmonary artery pressures we recorded were normal for healthy young males, even after the increases recorded from high PM exposure exercise. However, the 18% change in PAP observed from high pollution exercise was significant (p=0.05) and likely affected exercise performance. In conclusion, oral ingestion of sildenafil citrate blunted the increase in pulmonary artery pressure resulting from exercise in high emissions particulate and was likely responsible for a noted improved exercise performance in the majority of subject in this study. Given the likelihood of international competitions being held in high emission environments, sildenafil citrate could provide an unfair advantage for some.

Code: 07C02FD

Diuretics are an indispensable group of therapeutics used to regulate the excretion of water and salts. By definition diuretics are drugs which increase the urinary flow. In sports diuretics are used for two main reasons: to flush previously taken prohibited substances with forced diuresis and in sports where weight classes are involved to achieve acute weight loss. Diuretics are banned in sport by WADA. Diuretics cover a wide range of chemical products and one important group of diuretics are the thiazides. Because thiazides cover a whole class of structure related compounds, only a few thiazides are mentioned by name in the prohibited list: i.e. bendroflumethiazide, chlorothiazide and hydrochlorothiazide. Thiazides have an amidophenamide (AP) structure in common. AP is described as an aqueous degradation product for thiazides. Because of the potential hydrolysis of thiazides in urine this latter compound should be included in the screening methods for diuretics. Because the information regarding the degradation is limited a study concerning the degradation parameters can provide new and useful insights for doping analysis. In this project the effect of pH and temperature will be investigated. Three thiazides will be included in this study: altizide, hydrochlorothiazide and chlorothiazide. Altizide and hydrochlorothiazide are commercially available on the Belgian market. Chlorothiazide was also included in this study because it can be detected as metabolite for hydrochlorothiazide and bemetizide. Metabolites or degradation products can often be detected for a longer time in urine than the parent compounds. Therefore, in a second part of this project the detection times of metabolites and degradation products for two commercial available thiazide preparations, namely Docspirochlor (containing hydrochlorothiazide) and Aldactazine (containing Altizide), will be investigated.

Main Findings

The goal of this project was to investigate the stability of the thiazide diuretics altizide, hydrochlorothiazide and chlorothiazide both in vitro and in vivo. Not only the degradation of the parent drug was investigated also the formation of the degradation compound 4-amino-6-chloro-1,3-benzenedisulponamide was monitored. The results of the in vitro studies show that the thiazides are degradated faster at higher pH and higher temperature. In particular the lower pH improves the stability. When altizide and hydrochlorothiazide were exposed to UV-light, they photodegradate to chlorothiazide. When the degradation rate between the different compounds was compared for a given temperature and pH, altizide is the most unstable compound. Concentrations ranged between 41-239 ng/mL and 60-287 ng/mL after altizide and hydrochlorothiazide administration, respectively.

Publications

Deventer K, Baele G, Van Eenoo P, Pozo OJ, Delbeke FT. Stability of selected chlorinated thiazide diuretics. J Pharm Biomed Anal. (2009); 49(2):519-24.

Deventer K, Pozo OJ, Van Eenoo P, Delbeke FT. Detection of urinary markers for thiazide diuretics after oral administration of hydrochlorothiazide and altizide-relevance to doping control analysis. J Chromatogr A. (2009); 1216(12):2466-73.

Code: 07C03FD

Recently, a few methods for urinalysis of threshold substances via LC-MS have been developed and published. These methods allow for the direct analysis of urine samples without the need for extraction. The minimal sample preparation of these methods offers several advantages besides cost effectiveness and speed. Indeed, lower sample preparation leads to a reduction in the factors contributing to the measurement uncertainty of a result. Moreover, the required volume of urine for the quantification is limited and the methodology allows for the direct quantification of Phase 2 metabolites. Direct quantification of conjugates is preferred above hydrolysis followed by a quantification of the deconjugated substances since incomplete hydrolysis and or degradation effects (e.g. endogenous steroids) can lead to quantification errors. These errors are an important factor in the bias of current quantitative methods and of the uncertainty estimates. The current project would develop direct LC-MS quantification methods for all WADA threshold substances with a minimal sample preparation procedure. The developed methods would then be implemented and validated in all participating laboratories and a common measurement uncertainty estimate would be made to harmonize methodologies and decision limits (concentration above which a sample can be regarded as exceeding the threshold taking into consideration MU). As such the project would not only harmonize methodologies but also decision criteria leading to a more uniform interpretation of results. Moreover, taking into account the simplified sample preparation and the extensive harmonization of these methods among the participating laboratories it can be expected that the inter-laboratory variation will be lower than currently. Such a reduction in inter-lab variability is a primary objective for an adequate use of individual athlete passports with biometrical data. This hypothesis will be tested in the last phase of the project during which PTsamples will be distributed for every threshold substance to all laboratories. These samples will be quantified using the procedures currently applied in these laboratories as well as with the new unified methodologies. This approach will allow for a direct comparison and evaluation of the effectiveness of the harmonization of methods.

Main Findings

One of the goals of this project was to develop simple and robust quantification methods to quantify all WADA threshold substances using asimplea dilute-and-shoot approach. These methods were developed and validated and MU was estimated for each of them. All were compliant with WADA’s TD’s. Every laboratory implemented the developed methods. A proficiency test was set-up and the values obtained via these methods and the laboratories own methods revealed that there were no clear differences in the quality of the data. No real benefit was obtained and no clear cut reasons could be identified when smaller differences were noticed. Perhaps, this is caused by the high quality standard WADA already demands from accredited methods/laboratories.

Publications/Presentations

Deventer K et al. :Direct quantification of morphine-glucuronides and free morphine in urine using LC-MS/MS. Cologne, 13-02-2011, 29st Cologne Workshop on Dope Analysis.

Deventer, K., Pozo Mendoza, O. J., Verstraete, A., Van Eenoo, P. (2014). Dilute-and-shoot-liquid chromatography-mass spectrometry for urine analysis in doping control and analytical toxicology. TRAC-TRENDS IN ANALYTICAL CHEMISTRY, 55, 1–13.

Deventer, K., Pozo, O., Delbeke, F., Van Eenoo, P. (2012). Direct quantification of morphine glucuronides and free morphine in urine by liquid chromatography-tandem mass spectrometry. FORENSIC TOXICOLOGY, 30(2), 106–113.

Sardela, V., Deventer, K., Pereira, H., Neto, F., Van Eenoo, P. (2012). Development and validation of a ultra high performance liquid chromatography-tandem mass spectrometric method for the direct detection of formoterol in human urine. JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS, 70, 471–475.

Chebbah C1, Pozo OJ, Deventer K, Van Eenoo P, Delbeke FT. Direct quantification of 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid in urine by liquid chromatography/tandem mass spectrometry in relation to doping control analysis. Rapid Commun Mass Spectrom. 2010 Apr 30;24(8):1133-41

Code: 07C01FD

Beta-agonists are substances frequently used for the treatment of asthma. These drugs are most frequently administered by inhalation of aerosol, powder or nebulised solution. Besides the main pharmacological effect, at higher doses, side effects of the use of these products result in anabolic action. Hence, b2-agonists might be misused for their stimulating effect on respiration and growth promoting action when administered in higher doses. As a result, WADA has prohibited the use of these drugs in sports. Exceptions are formoterol, salbutamol, fenoterol, salmeterol and terbutaline which can be used by athletes if a proper medical justification (TUE) is issued. Although this group of medication is frequently declared by athletes on the doping control forms these substances are not always detected. In addition, no minimum required performance levels (MRPL) for laboratories have been presently issued by WADA. Information about the excretion of b2-agonists in urine after the use by different administration routes could result in the establishment of MRPL levels based upon scientific evidence. Therefore this project will focus on the administration of different b2-agonists by different administration routes. In this project 5 b2-agonists will be administered (i.e. salbutamol, formoterol, fenoterol, salmeterol and terbutaline) of which 2 of them will be administered both by inhalation and orally. All these studies will be conducted following a strict research protocol as approved by an ethical committee. As the supposed outcome of this research is a MRPL level, the first step of this project is to develop and validate quantitative analytical methods for all administered b2-agonists (except salbutamol for which these methods already exist).

Main Findings

The goal of this project was to investigate which concentrations can be detected in urine after therapeutical inhalation of β2-agonists. For this purpose a quantitative GC-MS method was developed for the detection of salbutamol. For salmeterol, formoterol and terbutaline LC-MS/MS methods were developed and validated. Since no isotopic labeled internal standard was available for fenoterol, only a semi quantitative LC-MS/MS method could be developed. After the validation, the developed methods were used to investigate the urinary excretion of the β2-agonist after oral administration of 4 mg of salbutamol (Ventolin®) and by inhalation of: Ventolin® (800 μg salbutamol), Serevent® (100 μg salmeterol), Oxis® (18 μg formoterol), Bricanyl® (500 μg terbutaline) and DuoventHFA® (100 μg fenoterol). After oral administration of salbutamol urinary concentrations reached 2626 ng/mL. After inhalation of a high therapeutic dose of salbutamol, the threshold of 1000 ng/mL was almost exceeded for one volunteer (994 ng/mL). The results show that the concentration of salmeterol and formoterol reach a maximum after 1 and 3 hours after ingestion. The highest salmeterol concentration detected was 1.27 ng/mL. For formoterol highest concentration was 11.4 ng/mL. After inhalation of terbutaline and fenoterol highest concentration detected were 197 ng/mL and 58.3 ng/mL, respectively. These concentrations were then compared with routine samples in which an above mentioned β2-agonist was found. In this way samples containing salmeterol (n=45). Formoterol (n=82), terbutaline (n=8) and fenoterol (n=3) were collected and quantified using the developed methods. Concentrations in these samples could not be related to the use of abnormal high doses of β2-agonists. Publications/Presentations

Presentations:

• Presenting “Excretion Studies with β2-Agonists” at the VI Latin American Workshop in Doping Analysis, November 8 to 11, Asunción- Paraguay, 2009.

• Presenting “Excretion Studies with β2-Agonists” at the 28th Workshop on dope analysis, 2010, Cologne, Germany.

Publications:

• Excretion Studies with β2-Agonists, K. Deventer, W. Van Thuyne, O.J. Pozo, P. Van Eenoo, F.T. Delbeke, Proceedings of the 28th Workshop on dope analysis, 2011, Cologne, Germany.

• Quantitative detection of inhaled salmeterol in human urine and relevance to doping control analysis, Submitted to Drug Testing and Analysis.

• Quantitative detection of inhaled formoterol in human urine and relevance to doping control analysis, in preparation.

• Quantitative detection of inhaled terbutaline in human urine and relevance to doping control analysis, in preparation.