Search

β 2 -adrenergic agonists having the potential to be misused to enhance performance for their thermogenic and anabolic properties are prohibited in sports. Clenbuterol, ractopamine and zilpaterol are utilised legally or illegally as growth promoters of animals raised for their meat. No withdrawal times are imposed for ractopamine prior to slaughter; residues are detected in meat of treated animals, which constitutes a risk of inadvertent consumption. Insufficient information is available on the fate of ractopamine in humans to implement efficient detection in athletes' urine samples. We have developed a confirmation procedure for total ractopamine in urine following the enzymatic hydrolysis of glucuronides and sulphates and the conversion to tri-TMS derivative (limit of identification at 0.15 ng/ml). The sulphates were found to form between 85% to 97% of ractopamine excreted in athletes' urine samples analysed routinely or in volunteers following the administration of a micro-dose of 2.5 μg. Peak levels were reached at 2 to 6 h and decreased rapidly below 1 ng/ml 10 h after dosing. With one exception, the highest level estimated in athletes' samples was 1.2 ng/ml. Zilpaterol was confirmed in a few urine samples collected in the USA and Mexico (highest level 2 ng/ml), while hundreds of athletes' samples were reported to contain clenbuterol by our laboratory over the past 7 years. Most of these cases originated from Mexico (n = 102) and Guatemala (n = 119), often clustered in events during which multiple samples were collected, and for the vast majority, in levels lower than 0.2 ng/ml., (© 2022 John Wiley & Sons Ltd.)

Testosterone doping in sports is detected through the measurement of the carbon isotopic signature (δ 13 C) of testosterone and its metabolites in urine. A critical step in achieving accurate and precise δ 13 C values during compound-specific stable carbon isotope analysis (CSIA) is the removal of interfering matrix components. To this end, the World Anti-Doping Agency (WADA) recommends the use of high-performance liquid chromatography (HPLC) as a method of sample pretreatment. We provide a description of an automated two-dimensional HPLC (2D-HPLC) purification method for urine extracts that has made possible the CSIA of underivatized steroids, requiring only 36 min per sample. Eight urinary steroids including testosterone (T) and dehydroepiandrosterone (DHEA) and four of their metabolites as well as two endogenous reference compounds were collected during HPLC purification. Comparative GC chromatograms are used to contrast the efficiency of two-dimensional (2D) purification to a previously established 1D-HPLC method. The 2D purification leads to improved sample purity while simultaneously decreasing the analysis time, allowing for unprecedented sample throughput. Precision of δ 13 C for all analyzed compounds in negative and positive controls was 0.5‰ or better, which is comparable with the precision of pure reference materials at similar intensities., (© 2020 John Wiley & Sons, Ltd.)

Introduction: The percentage of circulating reticulocytes (RET%) is a useful marker of blood doping in the context of the Athlete Biological Passport (ABP). The viability of the ABP depends on the comparability of sample data obtained across multiple laboratories for a given athlete. With the recent introduction of a different technology for the measurement of reticulocytes, the goal of this study was to compare currently employed Sysmex XT/XE analyzers to the recently introduced Sysmex XN analyzer., Methods: RET% differences were searched in two independent data sets, the first consisting of 95 369 RET% values coming from 29 laboratories located in five continents as part of routine testing for the ABP, the second from a targeted study involving 510 samples analyzed on both a Sysmex XT and XN analyzers by two different laboratories., Results: A relatively small but significant bias of 0.27 ([0.22-0.35] 95% CI) for the first data set and 0.19% ([0.16-0.22] 95% CI) for the second data set was observed with Sysmex XN analyzers returning higher values than Sysmex XT/XE analyzers. This bias appears constant over most of the range of RET% measured in elite athletes., Conclusion: When RET% values are obtained for the same athlete with different technologies (XT/XE vs XN), an adjustment of RET% emanating from the XT/XE instruments through a decrease of 0.22% within the ABP calculated ranges appears to be sufficient to integrate the results from the two technologies., (© 2019 John Wiley & Sons Ltd.)

The presence of erythropoiesis stimulating agents (ESAs) in the urine samples collected from athletes is detected using traditional Western blotting following either size-based separation (SDS/SAR-PAGE) or isoelectric focusing (IEF). Although there is an important testing effort, there is little doubt that ESAs are still abused in sports and that reducing the costs of the tests might increase the number of tests and improve deterrence. The capillary electrophoresis system developed by Protein Simple may be useful to this end. This platform is fully automated and could be easily implemented in anti-doping laboratories, which would contribute to the improvement of the overall assay performance and standardization of the method. Such an automated system could be of interest during major sports events, such as the Olympic Games, where a high number of samples needs to be analyzed in a short period of time. From the experiments conducted so far, we conclude that the technique is promising, with the sensitivity and reproducibility needed to screen ESAs in human urine samples., (© 2018 John Wiley & Sons, Ltd.)

According to the World Anti-Doping Agency (WADA) technical document for erythropoiesis stimulating agents (ESA) analysis (TD2014EPO), double-blotting of serum/plasma samples is mandatory for all analysis by isoelectric focusing (IEF) and for the confirmation procedures (CP) performed by SDS-PAGE or SAR-PAGE. The goal is to prevent potential cross-reactions of the secondary antibody with remaining proteins in the purified samples. To this end, we have developed an immunopurification method of ESA in serum/plasma samples using a combination of streptavidin-coated immunomagnetic beads and biotinylated anti-EPO polyclonal antibodies. Here we report that this immunomagnetic bead-based purification allows the analysis of serum/plasma samples by single-blotting. Serum and plasma samples, either intact or spiked with different ESAs, were immunopurified and analyzed by single-blotting, after SAR-PAGE or IEF using a cross-reaction minimized secondary antibody coupled to HRP. The results show that when samples are immunopurified according to this strategy, there is no non-specific binding when single-blotting is performed after SAR-PAGE. With IEF, we observe a faint smearing, however, in the pH gradient outside the ESA detection region. These interferences did not alter ESA profiles of spiked urinary samples or of samples received for routine testing. This approach was compared to the MAIIA monoliths purification or to the isolation of ESAs with other combinations of immunomagnetic reagents (ie, anti-Mouse IgG-coated magnetic beads and anti-EPO mAb). The recovery of ESAs was shown to be significant for serum/plasma samples. Our results suggest that single-blotting could be performed on serum/plasma samples without non-specific interferences. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

The challenges facing modern anti-doping analytical science are increasingly complex given the expansion of target drug substances, as the pharmaceutical industry introduces more novel therapeutic compounds and the internet offers designer drugs to improve performance. The technical challenges are manifold, including, for example, the need for advanced instrumentation for greater speed of analyses and increased sensitivity, specific techniques capable of distinguishing between endogenous and exogenous metabolites, or biological assays for the detection of peptide hormones or their markers, all of which require an important investment from the laboratories and recruitment of highly specialized scientific personnel. The consequences of introducing sophisticated and complex analytical procedures may result in the future in a change in the strategy applied by the Word Anti-Doping Agency in relation to the introduction and performance of new techniques by the network of accredited anti-doping laboratories., (© 2017 S. Karger AG, Basel.)

Two steroids were identified in a supplement named D-2 following the detection of unknown compounds during the routine testing of an athlete's sample. The main glucuroconjugated metabolites were isolated from this urine by high performance liquid chromatography (HPLC) following enzymatic hydrolysis and identified by gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) analyses as being 2α-hydroxy-5α-androst-3-en-17-one (M1) and 2β,3α-dihydroxy-5α-androstan-17-one (M2). A third metabolite, 3α,4β-dihydroxy-5α-androstan-17-one (M3) was also detected, however in lower amounts. The precursor steroids, 5α-androst-2-en-17-one (1) and 5α-androst-3-en-17-one (2) were present in the first D-2 products offered on the Internet. Later, the corresponding 17-hydroxyl compounds were offered as such or as esters (acetate, cypionate) in different relative ratios. Both M2 and M3 were synthesized from the trans-diaxial hydrolysis of the corresponding 2α,3α- and 3α,4α-epoxides (3). These were excreted in the hours following the controlled administration of the commercial product called D-2 R to a male volunteer and were also produced from the incubation of 1 and 2 with S9 liver fractions. Some preparations contain predominantly the alkene in C-2 and, therefore, an efficient detection method must include both primary metabolites M1 and M2. The latter was found equally in the fractions extracted following the enzymatic hydrolysis with β-glucuronidase and the chemical solvolysis, which may ease its identification. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Recombinant erythropoietin (rhEPO) has been misused for over two decades by athletes, mainly but not only in endurance sports. A direct rhEPO detection method in urine by isoelectric focusing (IEF) was introduced in 2000, but the emergence of third-generation erythropoiesis-stimulating agents and so-called biosimilar rhEPOs, together with the sensitivity of human endogenous EPO (huEPO) pattern to enzymatic activities and its modification following short strenuous exercise, prompted the development of a complementary test based on SDS-PAGE analysis. While Mircera and NESP are easily detected with the existing IEF and SDS-PAGE methods, some samples containing both epoetin-α/β and huEPO present profiles that are still difficult to interpret. As doping practices have moved to micro-dosing, these mixed patterns are more frequently observed. We investigated the impact of enzymatic desialylation on the urinary and serum EPO profiles obtained by SDS-PAGE with the aim of improving the separation of the bands in these mixed EPO populations. We observed that the removal with neuraminidase of the sialic acid moieties from the different EPOs studied reduced their apparent molecular weight (MW) and increased the migration distance between huEPO and rhEPO centroids, therefore eliminating the size overlaps between them and improving the detection of rhEPO., (Copyright © 2013 John Wiley & Sons, Ltd.)

Rationale: The analysis of urinary metabolites of testosterone-related steroids through the measurement of their carbon isotopic signature (δ(13) C) by gas chromatography/combustion/mass spectrometry (GC/C/IRMS) is a confirmation method employed in doping control analyses. Stringent analytical conditions are essential to an accurate and precise analysis as well as the proper selection of the metabolites, which forms the basis of the refined method presented in this paper., Methods: In a simplified approach, following enzymatic hydrolysis and extraction from a relatively low volume of urine sample, a one-step high-performance liquid chromatography (HPLC) purification was developed for seven diagnostic urinary metabolites (TS) including testosterone itself, dehydroepiandrosterone, 5α- and 5β-androstanediol, epitestosterone, androsterone, etiocholanolone and two endogenous reference compounds (ERC), 5β-pregnanediol and 5α-androst-16-en-3β-ol. These steroids were pooled in three fractions and analyzed as such. With regards to the GC/C/IRMS analysis, a multi-level isotopic calibration using the 'identical treatment' principle was created., Results: The proposed isotopic calibration yielded results for purified reference steroids with a precision ≤0.15 and accuracy of ≤0.30 ‰ (between-assay, n = 26). Compared to other common endogenous reference compounds, those selected in this study had δ(13) C values close to the target metabolites which, along with the proposed isotopic calibration, produced narrow reference intervals within ± 3‰ for most diagnostic TS-ERC pairs, in compliance with the requirements of the World Anti-Doping Agency., Conclusions: These carefully controlled analytical conditions are compatible with routine operations, affording accurate and precise results for the more diagnostically relevant metabolites such as testosterone itself and the 5α- and 5β-androstanediols. The values of the TS-ERC pairs measured in reference populations are described and the results from the routine testing of several hundreds of athletes' samples are discussed. Robust, this technique permitted the detection of adverse findings that would have been missed had these low level metabolites not been analyzed., (Copyright © 2013 John Wiley & Sons, Ltd.)

Desoxymethyltestosterone (DMT; 17β-hydroxy-17α-methyl-5α-androst-2-ene) is a designer steroid present in hormonal supplements distributed illegally as such or in combination with other steroids, for self-administration. It figures on the list of substances prohibited in sports and its detection in athlete's urine samples is based upon the presence of the parent compound or the main urinary metabolite, which has not been characterized yet. Following its isolation from cultures of human fresh hepatocytes and S9 fractions of liver homogenates, we were able to identify this metabolite as being 17α-methyl-2β,3α,17β-trihydroxy-5α-androstane. Other minor metabolites were also characterized. The production, isolation, NMR, mass spectral analyses and chemical synthesis are presented., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Due to its stimulatory effects on the central nervous system, and its structural similarity to banned stimulants such as ephedrine and methamphetamine, pseudoephedrine (PSE) at high doses is considered as an ergogenic aid for boosting athletic performance. However, the status of PSE in the International Standard of the Prohibited List as established under the World Anti-Doping Code has changed over the years, being prohibited until 2003 at a urinary cut-off value of 25 µg/ml, and then subsequently removed from the Prohibited List during the period 2004-2009. The re-consideration of this position by the World Anti-Doping Agency (WADA) List Expert Group has led to the reintroduction of PSE in the Prohibited List in 2010. In this manuscript, we present the results of two WADA-sponsored clinical studies on the urinary excretion of PSE and its metabolite cathine (CATH) following the oral administration of different PSE formulations to healthy individuals at therapeutic regimes. On this basis, the current analytical urinary threshold for the detection of PSE as a doping agent in sport has been conservatively established at 150 µg/ml, (Copyright © 2011 John Wiley & Sons, Ltd.)

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 athlete's steroid profiling is necessary to pick up variations that would trigger further IRMS analysis and investigations.

The urinary phase II metabolites of norsteroids, 19-norandrosterone, 19-noretiocholanolone and 19-norepiandrosterone glucuronide and sulphate, were analyzed in samples collected during the pregnancy, following the administration of norsteroids or the consumption of edible parts of non-castrated pig and in athletes' samples in which they were found during routine controls. The level of the sulfo- and glucuroconjugated metabolites was precisely determined by GC/HRMS, after selective hydrolysis. The goal was to evaluate whether the fine analysis of the norsteroid conjugates produced and excreted in different conditions would show a pattern that could be linked to their origin. The delta (13)C values of the metabolites formed following the ingestion of edible parts of non-castrated pig were measured by isotope ratio mass spectrometry. Our results indicated that it is not possible to determine the origin of the urinary metabolites based upon the sole evaluation of the different metabolites and conjugates. The GC/C/IRMS is the only method permitting to distinguish between the exogenous and endogenous origin of the metabolites.

Methyldrostanolone (2alpha,17alpha-dimethyl-17beta-hydroxy-5alpha-androstan-3-one) was synthesized from drostanolone (17beta-hydroxy-2alpha-methyl-5alpha-androstan-3-one) and identified in commercial products. Cultures of cryopreserved human hepatocytes were used to study the biotransformation of drostanolone and its 17-methylated derivative. For both steroids, the common 3alpha- (major) and 3beta-reduced metabolites were identified by GC-MS analysis of the extracted culture medium and the stereochemistry confirmed by incubation with 3alpha-hydroxysteroid dehydrogenase. Structures corresponding to hydroxylated metabolites in C-12 (minor) and C-16 were proposed for other metabolites based upon the evaluation of the mass spectra of the pertrimethylsilyl (TMS-d(0) and TMS-d(9)) derivatives. Finally, on the basis of the GC-MS and (1)H NMR data and through chemical synthesis of the 17-methylated model compounds, structures could be proposed for metabolites hydroxylated in C-2. All the metabolites extracted from hepatocyte culture medium were present although in different relative amounts in urines collected following the administration to a human volunteer, therefore confirming the suitability of the cryopreserved hepatocytes to generate characteristic metabolites and study biotransformation of new steroids.

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.

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., (Copyright 2008 John Wiley & Sons, Ltd.)

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.

Context: The 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., Objectives: To 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 Source: An analysis of recent and relevant literature accessed through MEDLINE, and interactions with clinicians, laboratory scientists, colleagues, and athletes., Conclusions: The 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.

Nutritional supplements are part of the diet of many athletes. With the exception of caffeine and ephedrine alkaloids, most of these products do not contain substances that are prohibited to competing sportsmen. In recent years, androgens, pro-hormones such as DHEA, androstenedione, androstenediol and 19-norsteroids became available for oral self-administration in many countries and on the Internet. Their claimed actions, efficiency or potency, and the possible adverse effects have not been thoroughly investigated by controlled clinical studies. Some products were shown to contain prohibited substances such as ephedrine, caffeine, or steroids, that were not listed on the label. Urine samples collected after the administration of these supplements can test positive. The administration of natural steroids such as testosterone and its precursors cannot be proven by the sole identification of the substances in the urine. The approach to detection is based upon the deviation of selected parameters of the metabolic profiles from the range of values normally found in humans. The individual's norm is also studied to exclude the few cases of systematic and natural excretion of extreme values. The combination of the GC/MS and the GC/C/IRMS offers a powerful tool to discriminate between the natural and synthetic origin of the urinary steroids.

Interval samplings uncover blood diurnal oscillations for several hormones, highlighting the importance of short time intervals in the disclosure of subtle pulsatile patterns of some peptide hormones, namely LH. In a study designed to develop new probes against steroid misuse, venous blood was sampled at 5-min intervals for 4 hours from 12 eugonadal adult male athletes, 6 receiving transcutaneous administrations of testosterone propionate and 6 placebo subjects. Brief supraphysiologic serum testosterone peaks were disclosed, the amplitude and frequency of these peaks being larger for the treated group. No solid explanation could be given to explain these bursts. Neither the binding/dissociation kinetics of SHBG molecules with and without increased circulating level of dihydrotestosterone, nor brief testosterone-inducing LH bursts, nor increased Leydig cell release could be invoked to explain these peaks. Their occurrence, although relatively rare, could represent a threat and lead to improper treatment.

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.

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.

1. There is some evidence that androgens affect dopaminergic function in animals and man. We investigated the effect of methyltestosterone (MT) (30 mg po) on the growth hormone (GH) response to the dopamine (DA) receptor agonist, apomorphine (Apo) HC1 (0.5 mg sc), in 9 normal men. MT was given 2 hr before Apo. 2. The peak plasma MT concentration was present 1 hr after administration (19.9 +/- 19.5 ng/ml; X +/- SD); the concentration at 4 hr was 7.2 +/- 4.9 ng/ml. At the time of Apo administration, plasma MT varied from 6.0-24.1 ng/ml. 3. There was no significant effect of MT on Apo-GH secretion (interaction F(7,56) = 1.08; p = NS). The mean individual peak GH concentration after Apo alone was 20.2 +/- 11.9 (X +/- SD) vs 22.2 +/- 9.9 ng/ml when MT preceded Apo (p = NS). 4. These results suggest that exogenous androgens do not affect DA receptor function in males with normal androgenic function. Lack of effect due to an insufficient dose or duration of administration of MT cannot be excluded.

The metabolism of stanozolol (17 beta-hydroxy-17 alpha-methyl-5 alpha-androstano[3,2-c]pyrazole), an androgenic-anabolic steroid widely used in sport for the purpose of enhancing performance, was investigated in humans. The analysis method was based on the use of solid-phase extraction on the Sep-Pak C18 cartridge, enzymic hydrolysis of steroid conjugates and high-resolution gas chromatograph-mass spectrometric (GC-MS) analysis of trimethylsilylated steroid extracts. After administration of a single 20-mg oral dose, twelve metabolites including unchanged stanozolol were recovered predominantly from the conjugated steroid fraction and characterized by GC-MS. In the unconjugated fraction, 16 alpha-hydroxystanozolol, 17-epistanozolol, stanozolol and 3'-hydroxy-17-epistanozolol were the most abundant metabolites. In the aglycone fraction, 16 alpha- and 16 beta-hydroxystanozolol, stanozolol and 3'-hydroxystanozolol were the most abundant metabolites. Other metabolites resulted from regioselective hydroxylation of stanozolol at C-4, whereas other were 17-epimers of 3'- and 16 alpha-hydroxystanozolol. Further hydroxylation leading to the formation of four isomeric dihydroxylated metabolites was also observed. They were tentatively assigned the structures of 3',16 alpha-, 4 beta,16 alpha-, 3',16 beta- and 4 beta,16 beta-dihydroxystanozolol. The mass spectral features of their bis-N,O-trimethylsilyl derivatives obtained under electron-impact ionization are presented. The effect of pH on the relative recovery of some of these metabolites is also presented. The usefulness of this analytical methodology for the detection and identification of stanozolol urinary metabolites in doping-control situations is demonstrated. The metabolism of stanozolol is also discussed, and metabolic pathways accounting for the formation of its biotransformation products are proposed.

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.

The metabolism of 17 alpha-methyl-17 beta-hydroxy-2-oxa-5 alpha-androstan-3-one (oxandrolone) in man has been investigated by gas chromatography/mass spectrometry. After oral administration of a 10 mg dose to man, five metabolites were detected in the free fraction of the urinary samples. Oxandrolone, the major compound excreted in urine, was detected within 72 h after administration. During this period 35.8 and 8.4% of the administered dose was excreted as unchanged oxandrolone and 17-epioxandrolone, respectively. In addition, minute amounts of 16 alpha- and 16 beta-hydroxyoxandrolone and a delta-hydroxy acid resulting from the hydrolysis of the lactone group of oxandrolone were detected in the urine samples 8-60 h after administration. Furthermore, the susceptibility of oxandrolone to hydrolysis was investigated under several pH conditions. Extraction and fractionation of steroidal metabolites was achieved by using C18 and silica Sep Pak chromatography. The mass spectra of the metabolites are presented and major fragmentation pathways discussed.