Presence of ß2 -agonist growth promoters in human urine samples: GC-MS/MS evaluation of the excretion profiles of ractopamine administered in microdoses.

ß2 -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.

Two-dimensional high performance liquid chromatography purification of underivatized urinary testosterone and metabolites for compound-specific stable carbon isotope analysis.

Testosterone doping in sports is detected through the measurement of the carbon isotopic signature (δ13 C) of testosterone and its metabolites in urine. A critical step in achieving accurate and precise δ13 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 δ13 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.

Standardization of reticulocyte counts in the athlete biological passport.

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.

Detection of erythropoiesis stimulating agents in urine samples using a capillary Western system.

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.

Immunomagnetic beads-based isolation of erythropoietins from urine and blood for sports anti-doping control.

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.

Challenges in Modern Anti-Doping Analytical Science.

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.

Detection of 5α-androst-2-en-17-one and variants: Identification of main urinary metabolites in human urine samples by GC-MS and NMR.

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 2ß,3α-dihydroxy-5α-androstan-17-one (M2). A third metabolite, 3α,4ß-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 ß-glucuronidase and the chemical solvolysis, which may ease its identification. Copyright © 2016 John Wiley & Sons, Ltd.

A simplified and accurate method for the analysis of urinary metabolites of testosterone-related steroids using gas chromatography/combustion/isotope ratio mass spectrometry.

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 5ß-androstanediol, epitestosterone, androsterone, etiocholanolone and two endogenous reference compounds (ERC), 5ß-pregnanediol and 5α-androst-16-en-3ß-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 5ß-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.

Desialylation improves the detection of recombinant erythropoietins in urine samples analyzed by SDS-PAGE.

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/ß 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.

Characterization of desoxymethyltestosterone main urinary metabolite produced from cultures of human fresh hepatocytes.

Desoxymethyltestosterone (DMT; 17ß-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-2ß,3α,17ß-trihydroxy-5α-androstane. Other minor metabolites were also characterized. The production, isolation, NMR, mass spectral analyses and chemical synthesis are presented.

Determination of urinary concentrations of pseudoephedrine and cathine after therapeutic administration of pseudoephedrine-containing medications to healthy subjects: implications for doping control analysis of these stimulants banned in sport.

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

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

Identification of drostanolone and 17-methyldrostanolone metabolites produced by cryopreserved human hepatocytes.

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.

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.

Excretion of norsteroids' phase II metabolites of different origin in human.

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.

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.

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.

Nutritional supplements and doping.

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.