Christiane Ayotte

Testing for natural and synthetic anabolic agents in human urine

This paper describes a comprehensive method for the detection of natural and synthetic anabolic agents, including some veterinary preparations such as trenbolone, zeranol (a non-steroidal agent) and clenbuterol (a beta 2-agonist). For the natural steroids such as testosterone, the precise determination of urinary androgens during routine procedures allowed the description of statistical distribution of relevant parameters of the endogenous steroid profile amongst male athletes. The validity of the results is discussed, taking into account some factors that may cause the degradation of the specimen.

Studies on anabolic steroids : I. Integrated methodological approach to the gas chromatographic-mass spectrometric analysis of anabolic steroid metabolites in urine

The analytical and methodological imperatives for large-scale and routine gas chromatographic-mass spectrometric screening of anabolic steroid urinary metabolites are described. Several aspects of their isolation, enzymatic hydrolysis, derivatization and metabolism in humans are discussed. Gas chromatographic-mass spectrometric data illustrating artifacts arising from enzymatic hydrolysis of 3 beta-ol-5-en steroids, and describing new metabolites of boldenone, methanedienone and stanozolol, as well as the conversion of norethisterone into 19-nortestosterone metabolites through de-ethylation at C-17, are presented. The analytical approach developed for gas chromatographic-mass spectrometric screening of anabolic steroids is based on the sequential selection-ion monitoring of specific and discrete ion groups characteristic to the steroids of interest under high-resolution chromatographic conditions. The major analytical and methodological requirements necessary to provide irrefutable evidence, in the case where the presence of a synthetic anabolic steroid or a testosterone to epitestosterone ratio higher than 6:1 is suspected in a given urine specimen, are also discussed.

Nutritional supplements and doping.

ContextThe problems of doping in sport and the increasing use of nutritional supplements by athletes are issues that intersect to the degree that a large number of supplements may contain substances that are banned in sport. Many supplements contain substances that are associated with significant health hazards. Athletes consuming such supplement products may jeopardize their sporting status, and their health. ObjectivesTo clarify and summarize the current status of dietary supplements in general, and to describe specific problems that can be associated with supplement use so that sport physicians might be better prepared to address these issues with their athlete-patients. Data SourceAn analysis of recent and relevant literature accessed through MEDLINE, and interactions with clinicians, laboratory scientists, colleagues, and athletes. ConclusionsThe dietary supplement industry is completely unregulated in the United States; as a consequence, an abundance of supplement products of dubious value, content, and quality are now available around the world. It is known that many supplement products contain substances that are prohibited in sport—typically stimulants or anabolic steroid precursors. Many supplements contain substances (e.g., ephedrine) that have been associated with significant morbidity and mortality. Sport practitioners have particular responsibilities in addressing this issue. Athletes need to be aware of the problems that can follow supplement use, and sport authorities need to ensure that nutritional education and guidance for athletes is of the highest standard. The need for the appropriate regulation of dietary supplements is emphasized.

Determination of the origin of urinary norandrosterone traces by gas chromatography combustion isotope ratio mass spectrometry

On the one hand, 19-norandrosterone (NA) is the most abundant metabolite of the synthetic anabolic steroid 19-nortestosterone and related prohormones. On the other hand, small amounts are biosynthesized by pregnant women and further evidence exists for physiological origin of this compound. The World Anti-Doping Agency (WADA) formerly introduced threshold concentrations of 2 or 5 ng of NA per ml of urine to discriminate 19-nortestosterone abuse from biosynthetic origin. Recent findings showed however, that formation of NA resulting in concentrations in the range of the threshold levels might be due to demethylation of androsterone in urine, and the WADA 2006 Prohibited List has defined NA as endogenous steroid. To elucidate the endogenous or exogenous origin of NA, (13)C/(12)C-analysis is the method of choice since synthetic 19-nortestosterone is derived from C(3)-plants by partial synthesis and shows delta(13)C(VPDB)-values of around -28 per thousand. Endogenous steroids are less depleted in (13)C due to a dietary mixture of C(3)- and C(4)-plants. An extensive cleanup based on two high performance liquid chromatography cleanup steps was applied to quality control and doping control samples, which contained NA in concentrations down to 2 ng per ml of urine. (13)C/(12)C-ratios of NA, androsterone and etiocholanolone were measured by gas chromatography/combustion/isotope ratio mass spectrometry. By comparing delta(13)C(VPDB)-values of androsterone as endogenous reference compound with NA, the origin of NA in doping control samples was determined as either endogenous or exogenous.

Detection of clenbuterol residues in hair

Sixty rats were grown in the presence of 10 (n = 30) and 100 (n = 30) micrograms kg-1 body mass of clenbuterol for a period of 10 d. An immunoextraction step coupled with a competitive enzyme immunoassay allowed the quantification of clenbuterol in hair upon 20 (10 micrograms kg-1) and 30 d (10 micrograms kg-1) after the last dose. This accumulation in hair contrasts with the rapid clearance in tissues. The nature of the immunoreactive material was confirmed by mass spectrometry.

Low LC‐MS/MS detection of glycopeptides released from pmol levels of recombinant erythropoietin using nanoflow HPLC‐chip electrospray ionization

The test used by anti-doping laboratories to detect the misuse of recombinant erythropoietin (rhEPO) is based on its different migration pattern on isoelectric focusing (IEF) gel compared with the endogenous human erythropoietin (hEPO) that can possibly be explained by structural differences. While there is definitely a need to identify those differences by LC-MS/MS, the extensive characterization that was achieved for the rhEPO was never performed on human endogenous EPO because its standard is not available in sufficient amount. The goal of this study was to develop an analytical method to detect pmol amounts of N-linked and O-linked glycopeptides of the recombinant hormone as a model. Using a nanoflow HPLC-Chip electrospray ionization/ion trap mass spectrometer, the diagnostic ion at m/z 366 of oligosaccharides was monitored in the product ion spectra to identify the four theoretical glycosylation sites, Asn24, Asn38, Asn83 and Ser126, respectively, on glycopeptides 22-37, 38-55, 73-96 and 118-136. With 3 pmol of starting material applied on Chip, only the desialylated N-glycopeptides 22-37 and 38-55/38-43 could be observed, and of all the glycan isoforms, those with the smaller structures were predominantly detected. While the preservation of the sialic acid moieties decreased the detection of all the N-glycopeptides, it allowed a more extensive characterization of the O-linked glycopeptide 118-136. The technique described herein provides a mean to detect glycopeptides from commercially available pharmaceutical preparations of rhEPO with the sensitivity required to analyze pmol amounts of hEPO, which could ultimately lead to the identification of structural differences between the recombinant and the human forms of the hormone.

Detecting the Administration of Endogenous Anabolic Androgenic Steroids

The detection of the administration of an androgen such as testosterone that could be present normally in human bodily fluids is based upon the methodical evaluation of key parameters of the urinary profile of steroids, precisely measured by GC/MS. Over the years, the markers of utilization were identified, the reference ranges of diagnostic metabolites and ratios were established in volunteers and in populations of athletes, and their stability in individual subjects was studied. The direct confirmation comes from the measurement of delta (13)C values reflecting their synthetic origin, ruling out a potential physiological anomaly. Several factors may alter the individual GC/MS steroid profile besides the administration of a testosterone-related steroid, the nonexhaustive list ranging from the microbial degradation of the specimen, the utilization of inhibitors of 5alpha-reductase or other anabolic steroids, masking agents such as probenecid, to inebriating alcohol drinking. The limitation of the testing strategy comes from the potentially elevated rate of false negatives, since only the values exceeding those of the reference populations are picked up by the GC/MS screening analyses performed by the laboratories on blind samples, excluding individual particularities and subtle doping. Since the ranges of normal values are often described from samples collected in Western countries, extrapolating data to all athletes appears inefficient. Furthermore, with short half-life and topical formulations, the alterations of the steroid profile are less pronounced and disappear rapidly. GC/C/IRMS analyses are too delicate and fastidious to be considered for screening routine samples. An approach based upon the individual athletes steroid profiling is necessary to pick up variations that would trigger further IRMS analysis and investigations.

Profiling of 19-norandrosterone sulfate and glucuronide in human urine: Implications in athlete's drug testing

19-Norandrosterone (19-NA) as its glucuronide derivative is the target metabolite in anti-doping testing to reveal an abuse of nandrolone or nandrolone prohormone. To provide further evidence of a doping with these steroids, the sulfoconjugate form of 19-norandrosterone in human urine might be monitored as well. In the present study, the profiling of sulfate and glucuronide derivatives of 19-norandrosterone together with 19-noretiocholanolone (19-NE) were assessed in the spot urines of 8 male subjects, collected after administration of 19-nor-4-androstenedione (100mg). An LC/MS/MS assay was employed for the direct quantification of sulfoconjugates, whereas a standard GC/MS method was applied for the assessment of glucuroconjugates in urine specimens. Although the 19-NA glucuronide derivative was always the most prominent at the excretion peak, inter-individual variability of the excretion patterns was observed for both conjugate forms of 19-NA and 19-NE. The ratio between the glucuro- and sulfoconjugate derivatives of 19-NA and 19-NE could not discriminate the endogenous versus the exogenous origin of the parent compound. However, after ingestion of 100mg 19-nor-4-androstenedione, it was observed in the urine specimens that the sulfate conjugates of 19-NA was detectable over a longer period of time with respect to the other metabolites. These findings indicate that more interest shall be given to this type of conjugation to deter a potential doping with norsteroids.

Detection of nandrolone metabolites in urine after a football game in professional and amateur players: a Bayesian comparison

Nandrolone (19-nortestosterone) is a widely used anabolic steroid in sports where strength plays an essential role. Once nandrolone has been metabolised, two major metabolites are excreted in urine, 19-norandrosterone (NA) and 19-noretiocholanolone (NE). In 1997, in France, quite a few sportsmen had concentrations of 19-norandrosterone very close to the IOC cut off limit (2ng/ml). At that time, a debate took place about the capability of the human male body to produce by itself these metabolites without any intake of nandrolone or related compounds. The International Football Federation (FIFA) was very concerned with this problematic, especially because the World Cup was about to start in France. In this respect, a statistical study was held with all football players from the first and second divisions of the Swiss Football National League. All players gave a urine sample after effort and around 6% of them showed traces of 19-norandrosterone. These results were compared with amateur football players (control group) and around 6% of them had very small amounts of 19-norandrosterone and/or 19-noretiocholanolone in urine after effort, whereas none of them had detectable traces of one or the other metabolite before effort. The origin of these compounds in urine after a strenuous physical activity is still unknown, but three hypotheses can be put forward. First, an endogenous production of nandrolone metabolites takes place. Second, nandrolone metabolites are released from the fatty tissues after an intake of nandrolone, some related compounds or some contaminated nutritive supplements. Finally, the sportsmen may have taken something during or just before the football game.

Studies on anabolic steroids: V. Sequential reduction of methandienone and structurally related steroid A-ring substituents in humans: gas chromatographic—mass spectrometric study of the corresponding urinary metabolites

The biotransformation of methandienone (17 beta-hydroxy-17 alpha-methylandrosta-1,4-dien-3-one) in human adults, more particularly the sequential reduction of its A-ring substituents, was investigated by gas chromatography-mass spectrometry. Two pairs of 17-epimeric tetrahydro diols resulting from the stereoselective reduction of the delta 4- and 3-oxo groups and of the delta 1-function were characterized. The major diols were 17 alpha-methyl-5 alpha-androstane-3 alpha,17 beta-diol and 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol, which were both excreted in the conjugate fraction in a 1:3.8 ratio. The immediate metabolic precursors of the 5 beta-diol, namely 17 beta-hydroxy-17 alpha-methyl-5 beta-androsta-1-en-3-one and 17 alpha-methyl-5 beta-androsta-1-en-3 alpha,17 beta diol and their corresponding 17-epimers, were also identified in post-administration urine samples. These data indicated that reduction of methandienone A-ring substituents proceeds according to the sequence. delta 4-, 3-oxo- and delta 1-. The A-ring reduction products of the structurally related steroids mestanolone, 17 alpha-methyltestosterone and oxymethone were also characterized and provided further analytical and metabolic evidence supporting the proposed route of methandienone A-ring reduction. It was also demonstrated using synthetic 17 beta-sulfate conjugates of methandienone and 17 alpha-methyltestosterone that their corresponding 17-epimers are formed by nucleophilic substitution by water of the labile sulfate moiety. The steroidal metabolites were identified on the basis of their characteristic mass spectral features and by comparison with authentic reference standards. Metabolic pathways accounting for the occurrence of the metabolites of interest in post-administration urine samples are proposed.