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Seized designer supplement named “1-Androsterone”: Identification as 3β-hydroxy-5α-androst-1-en-17-one and its urinary elimination
Abstract
New analogues of androgens that had never been available as approved drugs are marketed as "dietary supplement" recently. They are mainly advertised to promote muscle mass and are considered by the governmental authorities in various countries, as well as by the World Anti-doping Agency for sport, as being pharmacologically and/or chemically related to anabolic steroids. In the present study, we report the detection of a steroid in a product seized by the State Bureau of Criminal Investigation Schleswig-Holstein, Germany. The product "1-Androsterone" of the brand name "Advanced Muscle Science" was labeled to contain 100mg of "1-Androstene-3b-ol,17-one" per capsule. The product was analyzed underivatized and as bis-TMS derivative by GC-MS. The steroid was identified by comparison with chemically synthesized 3β-hydroxy-5α-androst-1-en-17-one, prepared by reduction of 5α-androst-1-ene-3,17-dione with LS-Selectride (Lithium tris-isoamylborohydride), and by nuclear magnetic resonance. Semi-quantitation revealed an amount of 3β-hydroxy-5α-androst-1-en-17-one in the capsules as labeled. Following oral administration to a male volunteer, the main urinary metabolites were monitored. 1-Testosterone (17β-hydroxy-5α-androst-1-en-3-one), 1-androstenedione (5α-androst-1-ene-3,17-dione), 3α-hydroxy-5α-androst-1-en-17-one, 5α-androst-1-ene-3α,17β-diol, and 5α-androst-1-ene-3β,17β-diol were detected besides the parent compound and two more metabolites (up to now not finally identified but most likely C-18 and C-19 hydroxylated 5α-androst-1-ene-3,17-diones). Additionally, common steroids of the urinary steroid profile were altered after the administration of "1-Androsterone". Especially the ratios of androsterone/etiocholanolone and 5α-/5β-androstane-3α,17β-diol and the concentration of 5α-dihydrotestosterone were influenced. 3α-Hydroxy-5α-androst-1-en-17-one appears to be suitable for the long-term detection of the steroid (ab-)use, as this characteristic metabolite was detectable in screening up to nine days after a single administration of one capsule.
... In common with the spectra of steroids with enol-TMS groups only at C17, the molecular and [M − 15] + ions are abundant and there is a characteristic ion at m/z 169. Two very prominent ions at m/z 290 and 275 characterize the 3-TMSO-Δ 5function (Goudreault & Massé, 1991;Parr et al., 2011;. The ion at m/z 290 represents loss of C1-C4 with the 3-TMSO-group (CH 2 = CH-CH(OTMS) = CH 2 ) from the A ring (ion of type iy). ...
... These ions are missing from the spectrum of 3β,17dihydroxy-5α(and 5β)-androstan-1-ene (139) (Parr et al., 2011; where, instead, prominent ions (in decreasing order of abundance) at m/z 143,142 (more prominent in the spectrum of the 5β-isomer), 127 and 129 appear (Fig. 44b). Again formation of these ions appears to involve rupture of the A-ring but, in this case, with charge retention on the A ring to give an ion such as CH 2 = CH-(C = OTMS) + -CH 3 for m/z 143 as shown by incorporation of a methyl group at C2 (Goudreault & Massé, 1991). ...
... Positive ion EI spectra of the TMS derivatives of (a) 3β,17dihydroxy-5α-androstan-1,16-diene (138). (Adapted from Parr et al. (2011)), (b) 3β,17β-dihydroxy-5α-androstan-1-ene 139), (Adapted from Parr et al., (2011)) and (c) 3α-hydroxy-2-methyl-5α-androstan-17-one (140). (Adapted from Goudreault and Massé (1991)). ...
This review describes the mass spectral fragmentation of trimethylsilyl (TMS) and related alkylsilyl derivatives used for preparing samples for analysis, mainly by combined gas chromatography and mass spectrometry (GC/MS). The review is divided into three sections. The first section is concerned with the TMS derivatives themselves and describes fragmentation of derivatized alcohols, thiols, amines, ketones, carboxylic acids and bifunctional compounds such as hydroxy- and amino-acids, halo acids and hydroxy ethers. More complex compounds such as glycerides, sphingolipids, carbohydrates, organic phosphates, phosphonates, steroids, vitamin D, cannabinoids, and prostaglandins are discussed next. The second section describes intermolecular reactions of siliconium ions such as the TMS cation and the third section discusses other alkylsilyl derivatives. Among these latter compounds are di- and trialkyl-silyl derivatives, various substituted-alkyldimethylsilyl derivatives such as the tert-butyldimethylsilyl ethers, cyclic silyl derivatives, alkoxysilyl derivatives, and 3-pyridylmethyldimethylsilyl esters used for double bond location in fatty acid spectra. © 2019 Wiley Periodicals, Inc. Mass Spec Rev 0000:1-107, 2019.
... [18] Two steroids were extracted from the Furazadrol supplement. b both 3α and 3β isomers are reported [19] c the 5α isomer is reported [21] Results ...
... This is a similar steroid to that extracted from Supplement L. In this study, we show that Supplement L is 4-fold less intrinsically potent than Supplement F and moreover shows inactivating metabolism, in keeping with previous reports that 1-Androsterone undergoes extensive metabolism. [19] Further investigation of these two steroids is now needed to identify structural differences that lead to different bioactivities. ...
Sport supplements containing steroids never approved for therapeutic use have the potential for abuse by athletes. Most are marketed online and may contain undisclosed steroids yet are readily available despite lacking toxicological or pharmacological evaluation. In this study, 18 supplements purchased online underwent organic solvent extraction to isolate any steroids they contained. From the 18 supplements, 19 steroids were identified and for each, its intrinsic androgenic potency was determined by a yeast cell (Saccharomyces cerevisiae) androgen bioassay and its potential androgenic potency was determined by a liver (HuH7) cell androgen bioassay. The yeast bioassay showed that of the 19 steroids tested, 6 demonstrated strong intrinsic bioactivity, with 4 metabolically activated to even stronger androgens. Moreover, 4 steroids with moderate and 1 with intrinsically weak androgenic bioactivity were activated to more potent androgens. Finally, 8 steroids were metabolically inactivated or deactivated into weaker androgens. Our results show that Internet-sourced sport supplements may contain intrinsically strong androgens, or precursors that can be metabolized to them. These potentially potent pharmacologically active steroids are being used without regulatory control or consumer awareness of their potential adverse effects. Copyright © 2016 John Wiley & Sons, Ltd.
... It is known that the detection of a new agent in both, urine sample(s) from the anti-doping control tests and supplement(s) available on the black market, is quite common [5,11,17]. The researches on substances banned in sport and detected in supplements, are often carried out together with the excretion studies [5,[22][23][24][25][26][27]. The form of an investigated compound in urine and its excretion concentration profile after the administration of the supplement containing the banned compound, is useful knowledge in the anti-doping control. ...
... The form of an investigated compound in urine and its excretion concentration profile after the administration of the supplement containing the banned compound, is useful knowledge in the anti-doping control. The quantitative analytical methods of doping agents in urine (a matrix of samples the most frequently collected in the anti-doping control [28]) were mainly performed with the use of such analytical systems as GC-MS/MS, GC-MS and LC-MS/MS in the case of anabolic agents [16,[25][26][27], and GC-MS, GC-NPD, LC-MS/MS and HPLC in the case of stimulants [5,14,23,24]. ...
The quantitative analysis of a new designer doping agent, 2-ethylamino-1-phenylbutane (EAPB) and its metabolite, 2-amino-1-phenylbutane (APB) in urine samples, and the determination of EAPB in dietary supplement samples, have been presented. The main purpose of the present study was to develop simple and reliable gas chromatography-mass spectrometry method (GC-MS) for excretion study following a single oral administration of dietary supplements containing EAPB. Three analytical methods for the determination of EAPB in urine and supplement samples, and APB in urine samples using the GC-MS system, have been validated. The method of the determination of EAPB in supplement samples was applied to analyze seventeen dietary supplements, CRAZE and DETONATE. Two other methods were used to determine the urinary excretion profile of EAPB and APB in the case of three healthy volunteers and, on further investigation, it was applied to the anti-doping control in sport. Quantification was obtained on the basis of the ions at m/z 86, 58 and 169, monitored for EAPB, APB and diphenylamine (used as an internal standard), respectively. The limits of detection and quantification were 2.4 and 7.3μg/g for EAPB in the case of supplement analysis, 2.9 and 8.8ng/mL for EAPB in the case of urine analysis, and 3.2 and 9.7ng/mL for APB. The other validation parameters as linearity, precision and trueness have been also investigated with the acceptable results. The extraction yield of all presented methods was above 69%. EAPB was detected in fourteen analyzed supplements (not included EAPB in their labels) and its content varied between 1.8 and 16.1mg/g. Following oral administration of three supplements with EAPB to one male and two female volunteers, the parent compound of EAPB and its metabolite were monitored and the excretion parameters as the maximum concentration of the analyte in urine (2.2-4.2μg/mL for EAPB; 1.1-5.1μg/mL for APB) and the time for the maximum height of the excretion peak (2-8h and 22h in one case for EAPB; 20-22h and 4h in one case for APB) have been indicated. EAPB and APB were detected at the level above 50ng/mL (50% of the minimum required performance level for stimulants in the anti-doping control in-competition in sport) in the urine up to 46-106h and 58-120h, respectively. Additionally, the result of the anti-doping control during swimming competition of one athlete, whose urine sample was analyzed for stimulants and narcotics, has been presented. The qualitative and quantitative analyses of new designer agents in urine samples and the excretion studies of these substances are of a great importance in the anti-doping control in sport. Moreover, the presentation of detection examples of these agents in supplements that haven't got included an information about them in the labeling, make athletes (and other supplement customers) more and more aware of the risk of the supplement use and possible health and doping consequences.
Copyright © 2015 Elsevier B.V. All rights reserved.
... Some PS manufacturers responded by hiring chemists to reverse engineer existing PS and AS compounds to make them compatible with federal law (4). Many of these efforts, which required the imposition of slight modifications to the structure of existing anabolic compounds, were directed toward developing precursors of 17hydroxy-5␣-androst-1-en-3-one (33). More commonly known as 1-testosterone, this required the substitution of a 1,2-double bond for the 4,5-double bond that was normally found in the A ring of testosterone. ...
... As such, this PS is prohibited from athletic use. Prior research has shown that a single 115-mg dose of this PS (subjects ingested 330 mg/day in the present study) remains detectable in the urine for a full 7 days following supplementation (33). Therefore, athletes are strongly cautioned against using this or any other PS. ...
Purpose: Prohormone supplements(PS) are recognized not to impart anabolic or ergogenic effects in men, but the research supporting these conclusions is dated. The Anabolic Steroid Control Act was amended in 2004; the viability of PS have not been evaluated since that time. Methods:17 resistance-trained males(23±1yrs; 13.1±1.5% body fat) were randomly assigned to receive either 330mg/d 3b-hydroxy-5a-androst-1-en-17-one(PROHORMONE; n=9) or sugar(PLACEBO; n=8) p.o. and complete a 4 week(16 session) structured resistance-training program. Body composition, muscular strength, circulating lipids, and markers of liver and kidney dysfunction were assessed at study onset and termination. Results:PROHORMONE increased lean body mass by 6.3±1.2%, decreased fat body mass by 24.6±7.1%, and increased their back squat 1-RM and competition total by 14.3±1.5% and 12.8±1.1%; respectively. These improvements exceeded(p<0.05) PLACEBO, who increased lean body mass by 0.5±0.8%, reduced fat body mass by 9.5±3.6%, and increased back squat 1-RM and competition total by 5.7±1.7% and 5.9±1.7%; respectively. PROHORMONE also experienced multiple adverse effects. These included a 38.7±4.0% reduction in HDL (p<0.01), a 32.8±15.05% elevation in LDL (p<0.01), and elevations of 120.0±22.6% and 77.4±12.0% in LDL/HDL and C/HDL; respectively(both p<0.01). PROHORMONE also exhibited elevations in serum creatinine (19.6±4.3%;p<0.01) and AST(113.8±61.1%;p=0.05), as well as reductions in serum albumin (5.1±1.9%;p=0.04), ALP(16.4±4.7%;p=0.04), and GFR(18.0±3.3%;p=0.04). None of these values changed(all p>0.05) in PLACEBO. Conclusion:The oral PS 3b-hydroxy-5a-androst-1-en-17-one improves body composition and muscular strength. However, these changes come at a significant cost. Cardiovascular health and liver function are particularly compromised. Given these findings, we feel the harm associated with this particular PS outweighs any potential benefit.
... Some PS manufacturers responded by hiring chemists to reverse engineer existing PS and AS compounds to make them compatible with federal law (4). Many of these efforts, which required the imposition of slight modifications to the structure of existing anabolic compounds, were directed toward developing precursors of 17hydroxy-5␣-androst-1-en-3-one (33). More commonly known as 1-testosterone, this required the substitution of a 1,2-double bond for the 4,5-double bond that was normally found in the A ring of testosterone. ...
... As such, this PS is prohibited from athletic use. Prior research has shown that a single 115-mg dose of this PS (subjects ingested 330 mg/day in the present study) remains detectable in the urine for a full 7 days following supplementation (33). Therefore, athletes are strongly cautioned against using this or any other PS. ...
Purpose: Prohormone supplements(PS) are recognized not to impart anabolic or ergogenic effects in men, but the research supporting these conclusions is dated. The Anabolic Steroid Control Act was amended in 2004; the viability of PS have not been evaluated since that time. Methods:17 resistance-trained males(23±1yrs; 13.1±1.5% body fat) were randomly assigned to receive either 330mg/d 3b-hydroxy-5a-androst-1-en-17-one(PROHORMONE; n=9) or sugar(PLACEBO; n=8) p.o. and complete a 4 week(16 session) structured resistance-training program. Body composition, muscular strength, circulating lipids, and markers of liver and kidney dysfunction were assessed at study onset and termination. Results:PROHORMONE increased lean body mass by 6.3±1.2%, decreased fat body mass by 24.6±7.1%, and increased their back squat 1-RM and competition total by 14.3±1.5% and 12.8±1.1%; respectively. These improvements exceeded(p<0.05) PLACEBO, who increased lean body mass by 0.5±0.8%, reduced fat body mass by 9.5±3.6%, and increased back squat 1-RM and competition total by 5.7±1.7% and 5.9±1.7%; respectively. PROHORMONE also experienced multiple adverse effects. These included a 38.7±4.0% reduction in HDL (p<0.01), a 32.8±15.05% elevation in LDL (p<0.01), and elevations of 120.0±22.6% and 77.4±12.0% in LDL/HDL and C/HDL; respectively(both p<0.01). PROHORMONE also exhibited elevations in serum creatinine (19.6±4.3%;p<0.01) and AST(113.8±61.1%;p=0.05), as well as reductions in serum albumin (5.1±1.9%;p=0.04), ALP(16.4±4.7%;p=0.04), and GFR(18.0±3.3%;p=0.04). None of these values changed(all p>0.05) in PLACEBO. Conclusion:The oral PS 3b-hydroxy-5a-androst-1-en-17-one improves body composition and muscular strength. However, these changes come at a significant cost. Cardiovascular health and liver function are particularly compromised. Given these findings, we feel the harm associated with this particular PS outweighs any potential benefit.
... Because these emerging drugs are devised specifically to evade detection, they impose a considerable burden on antidoping methods [33]. A recently launched paradigm is ''1-androsterone'', administration of which modifies the urinary steroid profile, and in particular the ratios of androsterone/etiocholanolone and 5a-/5b-androstane-3a,17b-diol and the concentration of 5a-dihydrotestosterone [34]. Meanwhile, 3a-hydroxy-5a-androst-1-en-17-one, a characteristic metabolite, is likely to play an important role by permitting a wider time frame of detection of steroid abuse, since screening performed as much as 9 days after a single administration of one capsule enabled its detection [34]. ...
... A recently launched paradigm is ''1-androsterone'', administration of which modifies the urinary steroid profile, and in particular the ratios of androsterone/etiocholanolone and 5a-/5b-androstane-3a,17b-diol and the concentration of 5a-dihydrotestosterone [34]. Meanwhile, 3a-hydroxy-5a-androst-1-en-17-one, a characteristic metabolite, is likely to play an important role by permitting a wider time frame of detection of steroid abuse, since screening performed as much as 9 days after a single administration of one capsule enabled its detection [34]. ...
Though we may still sing today, as did Pindar in his eighth Olympian Victory Ode, "… of no contest greater than Olympia, Mother of Games, gold-wreathed Olympia…", we must sadly admit that today, besides blatant over-commercialization, there is no more ominous threat to the Olympic games than doping. Drug-use methods are steadily becoming more sophisticated and ever harder to detect, increasingly demanding the use of complex analytical procedures of biotechnology and molecular medicine. Special emphasis is thus given to anabolic androgenic steroids, recombinant growth hormone and erythropoietin as well as to gene doping, the newly developed mode of hormones abuse which, for its detection, necessitates high-tech methodology but also multidisciplinary individual measures incorporating educational and psychological methods. In this Olympic year, the present review offers an update on the current technologically advanced endocrine methods of doping while outlining the latest procedures applied-including both the successes and pitfalls of proteomics and metabolomics-to detect doping while contributing to combating this scourge.
... A larger increase in serum creatinine levels was observed in a small 4-week placebo-controlled trial with resistance-trained men randomized to 330 mg daily of the oral prohormone 3bhydroxy-5a-androst-1-en-17-one (1-androsterone) or placebo (38). Bioactivation of the prohormone into the potent anabolic steroid 17b-hydroxy-5a-androst-1-en-3-one (1-testosterone) results from oxidation at carbon 3 of the A-ring and reduction at carbon 17 of the D-ring of the steroid nucleus (156). In those receiving 1-androsterone, serum creatinine levels increased significantly from 97.3 mmol/L (1.1 mg/dL) to 115.0 mmol/L (1.3 mg/dL). ...
Anabolic-androgenic steroids (AAS) are a class of hormones that are widely abused for their muscle-building and strength-increasing properties in high, nontherapeutic, dosages. This review provides an up-to-date and comprehensive overview on how these hormones work and what side effects they might elicit. We discuss how AAS are absorbed into the circulation after intramuscular injection or oral ingestion and how they are subsequently transported to the tissues, where they will move into the extravascular compartment and diffuse into their target cells. Inside these cells, AAS can biotransform into different metabolites or bind to their cognate receptor: the androgen receptor. AAS and their metabolites can cause side effects such as acne vulgaris, hypertension, hepatotoxicity, dyslipidemia, testosterone deficiency, erectile dysfunction, gynecomastia, and cardiomyopathy. Where applicable, we mention treatment options and self-medication practices of AAS users to counteract these side effects. Clinicians may use this review as a guide for understanding how AAS use can impact health and to assist in patient education and, in some cases, the management of side effects.
... With the current targeted approaches, a known AAS can be detected by comparison with a certified reference material. The discovery of tetrahydrogestrinone (THG) misuse by several elite athletes in 2003 [13][14][15], followed by other previously unknown steroids, like desoxymethyltestosterone (DMT, Madol, Pheraplex) [16] and 1-androsterone [17], first unearthed the issue of the abuse of designer steroids in sports. ...
Predictive models have been developed for the early identification of novel anabolic androgenic steroids and to obtain information on their molecular structure. To this purpose, gas-chromatographic and mass spectrometric characteristic parameters of 133 anabolic androgenic steroids have been specifically considered. Starting from Principal Component Analysis, different chemometric methods were applied, such as classification and clustering techniques, outlining a spectral and structural characterization for each steroid subclass, and considering the contribution of more than 30 variables. Mass spectrometric data on the TMS-derivatives of the target steroids were obtained by gas chromatography coupled to quadrupole-time of flight mass spectrometry using electron ionization. Steroids included in the training set were grouped in 5 subclasses according to their structural similarity, and the experimental data, processed by the chemometric models, allowed the identification of class-specific common fragments and spectral trends.
The results of this study, validated on a test set of 23 steroids, have confirmed that the proposed approach allows tracing novel “designer anabolic steroids”, including those previously unknown new structures that may have been designed and illicitly synthesized to be invisible to the current anti-doping tests.
... Others are registered only for veterinary use in some countries, but not in Switzerland (boldenone, trenbolone, clenbuterol) ( Kicman, 2008). Other substances are new analogues of androgens, structurally modified, and marketed as "nutritional supplements" or "prohormones" (e.g., 1-androsterone, 7-keto-DHEA) ( Delbeke, Van Eenoo, Van Thuyne, & Desmet, 2002;Parr et al., 2011). Drug candidates in this group, lacking clinical approval and therefore not intended for human use, include SARMs, which bind with high affinity to the androgen receptor and thus have comparable effects with AAS, but due to high tissue specificity, the undesired androgenic side effects are significantly reduced ( Geldof et al., 2016;Geyer, Schanzer, & Thevis, 2014;Krug et al., 2014;). ...
Background:
Substances developed for therapeutic use are also known to be misused by athletes as doping agents and, outside of regulated sport, for image-enhancement. This has generated a market for counterfeit doping substances. Counterfeit doping agents may be of poor pharmaceutical quality and therefore constitute health risks to consumers.
Objectives:
This study aims to investigate the pharmaceutical quality of 1,190 doping products seized at the Swiss border.
Methods:
Swiss customs authorities seize incoming shipments potentially containing doping agents. Qualitative and semiquantitative analyses were performed in order to test for prohibited doping substances. The main analytical methods utilized for characterizing confiscated compounds were liquid chromatography-high resolution mass spectrometry, polyacrylamide gel electrophoresis with subsequent in-gel tryptic digestion and identification of peptidic compounds using nanoliquid chromatography-tandem mass spectrometry, and electrochemiluminescence immuno assay.
Results:
For 889 (75%) of the analyzed products, the label suggested the content of anabolic agents, for 146 samples (12%) peptide hormones or growth factors, and for 113 items (9%) antiestrogens, aromatase inhibitors or other metabolic modulators. For the majority of the investigated products, the pharmaceutical quality was an unsatisfactory standard: nonapproved substances were detected and less than 20% of the products contained the claimed substance in the respective amount.
Conclusion:
A comprehensive sample of confiscated doping products was analyzed, allowing for monitoring of developments regarding the use of doping substances in Switzerland and for anticipating future trends and challenges in sports drug testing. An alarming number of tested products was of substandard pharmaceutical quality.
... [38] It has been reported to be a component of dietary supplements such as 1-androsterone (Advanced Muscle Science) and 1-AD (ErgoPharm) which typically contain Δ1-testosterone in addition to one or more of the following compounds: 5α-androst-1-ene-3,17-dione, 5αandrost-1-ene-3,17-diol, or 3β-hydroxy-5α-androst-1-en-17-one, all of which are metabolized to Δ1-testosterone in vivo. [111,112] Following an equine in vivo controlled oral administration (800 mg, 2 Thoroughbred geldings), [113] Δ1-testosterone has been reported to be excreted as a range of unconjugated, sulfateconjugated and glucuronide-conjugated metabolites which were detectable up to 72 h post-administration. After phase I metabolism, the parent compound as well as eight metabolites were identified as follows: four reduced metabolites (5α-androst-1-ene-3α,17β-diol, 5α-androst-1-ene-3β,17α-diol, 5α-androst-1-ene-3β,17β-diol, and epiandrosterone (3β-hydroxy-5α-androstan-17-one)), three doubly reduced metabolites (5α-androstane-3β,17β-diol, 5α-androstane-3α,17α-diol, and 5α-androstane-3β,17α-diol), and one hydroxylated metabolite. ...
In recent years the potential for anabolic steroid abuse in equine sports has increased due to the growing availability of "designer steroids". These compounds are readily accessible online in "dietary" or "nutritional" supplements and contain steroidal compounds which have never been tested or approved as veterinary agents. They typically have unusual structures or substitution and as a result may pass undetected through current anti-doping screening protocols, making them a significant concern for the integrity of the industry. Despite considerable focus in human sports, until recently there has been limited investigation into these compounds in equine systems. In order to effectively respond to the threat of designer steroids, a detailed understanding of their metabolism is needed to identify markers and metabolites arising from their misuse. A summary of the literature detailing the metabolism of these compounds in equine systems is presented with an aim to identify metabolites suitable for incorporation into screening protocols by anti-doping laboratories. The future of equine anti-doping research is likely to be guided by the incorporation of alternate testing matrices into routine screening, the improvement of in vitro technologies that can mimic in vivo equine metabolism, and the improvement of instrumentation or analytical methods that allow for the development of untargeted screening, and metabolomics approaches for use in anti-doping screening protocols.
... Identication of the four free metabolites was obtained through LC-QTOF-MS analysis of target ions and GC-MS analysis of TMS ethers together with TMS enol-TMS ethers, and by comparison of mass spectral data with reference data from the literature. 13,20 M1. M1 exhibited the [M + H] + at m/z 289.2160 (C 19 H 28 O 2 ) in a full scan mode (Fig. S1 †), indicating the same MM as unchanged 1-testosterone (C 19 H 28 O 2 ). ...
In this study 1-testosterone urinary metabolic profiles were investigated by liquid chromatography quadruple time of flight mass spectrometry (LC-QTOF-MS) using full scan and targeted MS/MS techniques with accurate mass measurement. 1-Testosterone was administered to two healthy male volunteers. Liquid-liquid extraction (LLE) and direct-injection were applied to processing urine samples. Chromatographic peaks for potential metabolites were found by using the theoretical [M-H] - as target ion in full scan experiment and their actual deprotonated ions were analyzed in targeted MS/MS mode. Four free reported metabolites (M1, M2, M3 and M4) were found for 1-testosterone together with an unreported sulfate-conjugated metabolite (S1). Due to the absence of useful fragment ion to illustrate the sulfate structure, gas chromatography mass spectrometry (GC-MS) instrument was applied to obtain structural details of the trimethylsilylated phase I metabolite released after solvolysis. Thus its potential structure was proposed particularly by a combined MS approach. All the metabolites were evaluated in terms of how long they could be detected and S1 was considered to be the long term metabolite for 1-testosterone misuse detected up to 15 days by LLE and 10 days by direct-injection analysis after oral administration.
... 17β-Hydroxy-2α,17α-dimethyl-5α-androstan-3-one 2) Methasterone [122,[129][130][131][132][133][134] 17β-Hydroxy-17α-methyl-5α-androst-1-en-3-one 1) [131] 4,17β-Dihydroxyandrost-4-en-3-one 1) 4-Hydroxytestosterone [135,136] 5α-Androstane-3β,17α-diol [137] Androst-4-ene-3β,17α-diol [137] 5β-Androst-1-ene-3β,17β-diol [137] 5β-Androst-1-ene-3α,17β-diol [137] 17β-Hydroxy-5α-androstano-[3,2-c]-pyrazol 2) Prostanozol [130] 6α-Methylandrost-4-ene-3,17-dione 2) [102,130,132] 3β-Hydroxy-5β-androstan-17-one Epietiocholanolone [132] 17β-Hydroxy-17α-methyl-5β-androstan-3-one 5β-Mestanolone [132] 17α-Methyl-5α-androst-2-en-17β-ol Desoxymethyltestosterone, DMT, Madol [132,138,139] 4-Chloro-17α-methylandrost-4-ene-3α,17β-diol [133] Androst-4-ene-3,6,17-trione 6-Oxoandrostenedione, 6-Oxo [140,141] Androsta-1,4,6-triene-3,17-dione 2) Androstatrienedione [142][143][144] 3β-Hydroxyandrost-4-ene-7,17-dione 7-Keto-DHEA [145] 6α/β-Bromoandrost-4-ene-3,17-dione 6-Bromandrostenedione [139,146] 17α-Methyl-5α-androstane-3α,17β-diol [146] Estra-4,9-diene-3,17-dione Trenbolox [123,139,147] 17β-Acetoxy-17α-methylandrost-5-ene-3β,7β-diol MbAEt [139] 3β-Hydroxy-5β-androstan-17-one Epiandrosterone [139] 2α,3α-Epithio-17α-methylandrostan-17β-ol 2) 2a,3a-Epithio-17a-methyletiocholanol [123,139] 3-Tetrahydropyranoloandrost-4-ene-6,17-dione 6-KetoDHEA [139] Androsta-1,4-diene-3,17-dione Boldione [139] 3β,7β-Dihydroxyandrost-5-en-17-one 7β-Hydroxy-DHEA [139] 3α-Acetoxy-5α-androstan-17-one Androsterone acetate [139] 5α-Androst-1-ene-3β,17β-diol 1) [139] 17β-Hydroxy-17α-methyl-5α-androstan-3-oxime 2) Mestanolon-oxim [139] 5α-Androstane-3,17-dione-bis-oxim [139] 17β-Hydroxy-5α-androstano-[3,2-c]-isoxazol 2) [123,139,[147][148][149] 17β-Hydroxy-5α-androstano-[2,3-d]-isoxazol 2) [123,139,[147][148][149] 6ξ-Hydroxyandrost-4-ene-3,17-dione [123,142] 3ξ-Hydroxyandrost-4-ene-6,17-dione 6,17-Keto-etiocholeve-3-ol-tetrahydropyranol [144] 4,17α-Dimethylestra-1,3,5-trien-17β-ol M1,4ADD (Methyl-1,4-androstadiene-3,17-diol) [150] 17β-Hydroxy-17α-methylandrosta-4,6-dien-3-one Jungle Warfare [123,139,151] 3β-Hydroxy-5α-androst-1-en-17-one 1-Androsterone, 1-DHEA [139,152,153] ...
Anabolic androgenic steroids (AAS) are some of the most common performance
enhancing drugs (PED) among society. Despite the broad spectrum of adverse effects and legal
consequences, AAS are illicitly marketed and distributed in many countries. To circumvent existing
laws, the chemical structure of AAS is modified and these designer steroids are sold as nutritional
supplements mainly over the Internet. Several side effects are linked with AAS abuse. Only little is
known about the pharmacological effects and metabolism of unapproved steroids due to the absence
of clinical studies. The large number of designer steroid findings in dietary supplements and the
detection of new compounds combined with legal loopholes for their distribution in many countries
show that stricter regulations and better information policy are needed.
... In our second study we investigated effects of a single treatment of the anabolic steroid 1-AD in healthy male volunteers on the the HPGa. The molecular structure of 1-AD is similar to the endogenous pro-hormone androstenedione and is sold as 'dietary supplement' to promote muscle mass [32]. As shown in Fig. 5 for most of the analysed serum parameters, circadian secretion pattern could be detected. ...
Analysing effects of pharmaceutical substances and training on feedback mechanisms of the hypothalamic-pituitary-gonadal axis may be helpful to quantify the benefit of strategies preventing loss of muscle mass, and in the fight against doping. In this study we analysed combined effects of anabolic steroids and training on the hypothalamic-pituitary-gonadal axis. Therefore intact male Wistar rats were dose-dependently treated with metandienone, estradienedione and the selective androgen receptor modulator (SARM) S-1. In serum cortisol, testosterone, 17β-estradiol (E2), prolactin, inhibin B, follicle-stimulating hormone (FSH), luteinizing hormone (LH), Insulin-like growth factor 1 (IGF-1), and thyroxine (T4) concentrations were determined. Six human volunteers were single treated with 1-androstenedione. In addition abusing and clean body builders were analysed. Serum concentrations of inhibin B, IGF-1, cortisol, prolactin, T4, thyroid-stimulating hormone (TSH), testosterone and LH were determined. In rats, administration of metandienone, estradienedione and S-1 resulted in an increase of muscle fiber diameter. Metandienone and estradienedione but not S-1 administration significantly decreases LH and inhibin B serum concentration. Administration of estradienedione resulted in an increase of E2 and S-1 in an increase of cortisol. Single administration of 1-androstenedione in humans decreased cortisol and inhibin B serum concentrations. LH was not affected. In abusing body builders a significantly decrease of LH, TSH and inhibin B and an increase of prolactin, IGF-1 and T4 was detected. In clean body builders only T4 and TSH were affected.
Copyright © 2015. Published by Elsevier Ltd.
... [2][3][4] In recent years, many new designer agentsmostly steroids but also stimulantshave been detected in nutritional supplements. [5][6][7][8][9][10][11] Many cases of substances identified during routine anti-doping control in that period were related to new stimulants as methylhexaneamine, β-methylphenethylamine, and N,α-diethyl-phenylethylamine. [12][13][14][15][16][17] The routine anti-doping testing process in the WADA-accredited laboratory in Warsaw (Poland) indicated a new designer agent, N,Ndimethyl-2-phenylpropan-1-amine (NN-DMPPA), in the urine of four athletes (two of them failed urine drug tests in 2013, the others in 2014). ...
Reports of new designer agents banned in sport being detected in supplements widely available for athletes are constantly emerging. The task of anti-doping laboratories is to control athletes for the presence of substances listed by the World Anti-Doping Agency (WADA) and those that are structurally/biologically similar to them. Recently, a new designer stimulant, N,N-dimethyl-2-phenylpropan-1-amine (NN-DMPPA), was detected by the WADA accredited anti-doping laboratory in Warsaw during routine anti-doping control. The urine samples from four athletes were analyzed in the screening method for stimulants and narcotics and the presence of NN-DMPPA was detected. The identity of NN-DMPPA was confirmed by gas chromatography-mass spectrometry using a synthesized reference standard. The measured concentrations of NN-DMPPA were between 0.51 and 6.51 µg/mL. The presence of the NN-DMPPA compound has been detected in the ‘nutritional supplement’ NOXPUMP that had been purchased in a store in Poland. NN-DMPPA at 121.7 µg/g was indicated in the investigated supplement together with another banned stimulant β-methylphenethylamine. The presence of this new stimulant was not indicated on the labelling of the supplement, a situation which is not unusual within this market. Thus, it is important to make athletes aware of the risk related to the use of supplements. Moreover, specific legistation dealing with the commercialization of drugs banned for sport should be undertaken. Copyright © 2014 John Wiley & Sons, Ltd.
... Different research groups from the antidoping community have investigated the presence of doping agents in nutritional supplements. The presence of prohibited substances not declared on the labels can be explained by contaminations and adulterations (Cavalcanti, Leal, Garrido, Padilha, & Aquino Neto, 2012;Kim et al., 2011;Kohler et al., 2010;Maughan, Greenhaff, & Hespel, 2011;Maughan & Shirreffs, 2012;Parr et al., 2011). As highlighted by Maughan, King, & Lea, (2004), the innocent ingestion of prohibited substances is not an acceptable excuse, and athletes testing positive are liable to penalties (Maughan et al., 2004). ...
This article presents the prevalence of stimulant doping among Brazilian athletes, the analytical approaches used, as well as a general evolution of the detectability of the stimulants being used. Results from the Brazilian accredited doping control laboratory are compared with the global statistics disclosed by the World Anti-Doping Agency. The high prevalence of stimulant doping in Brazil can be attributed to several reasons, including "self-administration," a "body-shaping" culture, and the use of nutritional supplements.
... methandienone leading to 17α-methyl-5zandrostan-3α,17β-diol) [22] as well as for endogenous like compounds. For this last compounds, recently Parr et al. [25] showed that after the administration of 3β-hydroxy-5α-androst-1-en-17one in addition to some already described metabolites common to boldenone metabolism and other 'synthetic' metabolites, Colour online, B&W in print Figure 2. Time course of the concentrations of 5β-androstane-3α,17βdiol (closed circles), 5α-androstane-3α,17β-diol (opened circles) and the 5β/5α ratio (closed triangles) after the administration of 100 mg of boldione 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 changes on the endogenous steroid profile was observed with the formation of 5α-reduced compounds, namely androsterone (3α-hydroxy-5α-androstan-17-one) and 5α-andostan-3α,17β-diol. ...
Boldione is an anabolic androgenic steroid (AAS) related to boldenone, androstenedione, and testosterone bearing two double bonds in C1 and C4 positions. Boldione is rapidly transformed to the well-known AAS boldenone, being both compounds included in the list of prohibited substances and methods published yearly by the World Anti-Doping Agency (WADA). After the administration of boldione to a male volunteer, the already described urinary metabolites of boldenone produced after reduction in C4, oxydoreduction in C3 and C17, and hydroxylation have been detected. In addition, minor new metabolites have been detected and their structure postulated after mass spectrometric analyses. Finally, the reduction of the double bound in C1 produces metabolites identical to the endogenously produced ones. A method based on gas chromatography coupled to isotope ratio mass spectrometry (GC/C/IRMS) after a urine sample purification by high performance liquid chromatography (HPLC) permitted to confirm the main synthetic like boldione/boldenone metabolite (17β-hydroxy-5β-androst-1-en-3-one) and boldenone at trace levels (< 5 ng/mL) and then to establish its synthetic or endogenous origin, and to determine the exogenous origin of metabolites with the same chemical structure of the endogenous ones. The detection of pseudoendogenous androgens of synthetic origin partially overlapped boldenone and its main metabolite detection, being an additional proof of synthetic steroids misuse. By the use of IRMS, the correct evaluation of the modifications of the steroid profile after the administration of synthetic AAS that could be converted into endogenous like ones is possible. Copyright © 2013 John Wiley & Sons, Ltd.
... A and Etio, the final products of metabolism of testosterone, are considered some of the stable parameters of steroidal profile [27]. Administration of 1androsterone, a designer supplement available under the brand name ''Advanced Muscle Science'' and dehydroepiandrosterone (DHEA) significantly increased the A/Etio ratio to approximately twice their basal levels [29,30]. Dihydrotestosterone (DHT), a biologically active metabolite of testosterone, is often being misused in sports for its anabolic and psychotropic benefits. ...
Steroid profiling is the most versatile and informative technique adapted by doping control laboratories for detection of steroid abuse. The absolute concentrations and ratios of endogenous steroids including testosterone, epitestosterone, androsterone, etiocholanolone, 5α-androstane-3α,17β-diol and 5β-androstane-3α,17β-diol constitute the significant characteristics of a steroid profile. In the present study we report the influence of various oxidizing adulterants on the steroid profile of human urine. Gas chromatography-mass spectrometry analysis was carried out to develop the steroid profile of human male and female urine. Oxidants potassium nitrite, sodium hypochlorite, potassium permanganate, cerium ammonium nitrate, sodium metaperiodate, pyridinium chlorochromate, potassium dichromate and potassium perchlorate were reacted with urine at various concentrations and conditions and the effect of these oxidants on the steroid profile were analyzed. Most of the oxidizing chemicals led to significant changes in endogenous steroid profile parameters which were considered stable under normal conditions. These oxidizing chemicals can cause serious problems regarding the interpretation of steroid profiles and have the potential to act as masking agents that can complicate or prevent the detection of the steroid abuse.
In 2020, the confirmation of the non-endogenous origin of several pseudo-endogenous steroids by means of isotope ratio mass spectrometry (IRMS) was recommended by the World Anti-Doping Agency (WADA), in addition to previously established target analytes for IRMS in sports drug testing. To date, however, IRMS-based methods validated in accordance with current WADA regulations have not been available. Therefore, the aim of this research project was the development and validation of a method to determine the carbon isotope ratios (CIR) of all newly considered pseudo-endogenous steroids, encompassing the anabolic androgenic steroids comprising a 1-ene-core structure (5α-androst-1-ene-3β,17β-diol, 5α-androst-1-ene-3,17-dione (1AD), 17β-hydroxy-5α-androst-1-en-3-one, 3α-hydroxy-5α-androst-1-ene-17-one (1AND), and 3β-hydroxy-5α-androst-1-ene-17-one (1EpiAND)), as well as steroids referred to as hormone and metabolic modulators (androsta-1,4,6-triene-3,17-dione (TRD) and its main metabolite 17β-hydroxy-androsta-1,4,6-triene-3-one) and 6α- and 6β-hydroxy-androst-4-ene-3,17-dione. With peak purity of target analytes being critical for IRMS analyses, a twofold high-performance liquid chromatography (HPLC)-based sample purification was employed, with all analytes being acetylated between the first and second HPLC fractionation. Using established gas chromatography/combustion/IRMS (GC/C/IRMS) instrumentation, limits of quantification were estimated at 10 ng/mL for a 20 mL urine aliquot for all analytes, except for 1AND (20 ng/mL), and combined measurement uncertainties were estimated between 0.4 and 0.9 ‰. For proof-of-concept, samples collected after the single oral administration of a nutritional supplement containing 1AD and 1EpiAND were analyzed as well as existing excretion study urine samples obtained after the administration of 4-androstenedione and TRD. Based on the obtained results, the developed method was considered to be fit-for-purpose.
Androgens, both steroidal and non‐steroidal in nature, are among the most commonly misused substances in competitive sports. Their recognized anabolic and performance enhancing effects through short‐ and long‐term physiological adaptations make them popular. Androgens exist as natural steroids, or are chemically synthesized as anabolic androgenic steroids (AAS) or selective androgen receptor modulators (SARMs). In order to effectively detect misuse of androgens, targeted strategies are used. These targeted strategies rely heavily on mass spectrometry and detection requires prior knowledge of the targeted structure and its metabolites. While exquisitely sensitive, such approaches may fail to detect novel structures that are developed and marketed. A non‐targeted approach to androgen detection involves the use of cell‐based in vitro bioassays. Both yeast‐ and mammalian cell androgen bioassays demonstrate a clear ability to detect AAS and SARMS, and if paired with high resolution mass spectrometry can putatively identify novel structures. In vitro cell bioassays are successfully used to characterize designer molecules, and to detect exogenous androgens in biological samples. It is important to continue to develop new and effective detection approaches to prevent misuse of designer androgens and in vitro bioassays represent a potential solution to non‐targeted detection strategies.
Continuous research on metabolomics is of utmost importance in the field of anti-doping control, including updating the selection of the most appropriate markers of prohibited substances, testing specimens and analytical strategies. Among metabolomics research topics, nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in equine medicine and have large diversity in their chemical structures; hence, their metabolism has been widely elucidated to construct appropriate anti-doping tests. This article provides an overview of the analytical methodologies used in anti-doping analysis and the metabolic research of NSAIDs concerning issues related to the use of alternative testing matrices, sample preparation strategies and possible analytical techniques. In addition, the metabolic pathways of chosen NSAIDs in horses, associated analytical problems and future research directions are discussed.
Both athletes and the general population use nutritional supplements. Athletes often turn to supplements hoping that consuming the supplement will help them be more competitive and healthy while the general population hopes to improve body image or vitality. While many supplements contain ingredients that may have useful properties, there are supplements that are contaminated with compounds that are banned for use in sport or have been deliberately adulterated to fortify a supplement with an ingredient that will produce the advertised effect. In the present study, we have used yeast- and mammalian cell androgen bioassays to characterize the androgenic bioactivity of 112 sports supplements available from the Australian market, either over the counter or via the Internet. All 112 products did not declare an androgen on the label as an included ingredient. Our findings show that 6/112 supplements had strong androgenic bioactivity in the yeast cell bioassay, indicating products spiked or contaminated with androgens. The mammalian cell bioassay confirmed the strong androgenic bioactivity of 5/6 positive supplements. Supplement 6 was metabolized to weaker androgenic bioactivity in the mammalian cells. Further to this, Supplement 6 was positive in a yeast cell progestin bioassay. Together, these findings highlight that nutritional supplements, taken without medical supervision, could expose or predispose users to the adverse consequences of androgen abuse. The findings reinforce the need to increase awareness of the dangers of nutritional supplements and highlight the challenges that clinicians face in the fast-growing market of nutritional supplements.
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 HPLC following enzymatic hydrolysis and identified by GC-MS and 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 synthesised 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.
N,N-dimethyl-2-phenylpropan-1-amine (NN-DMPPA) is a new designer stimulant prohibited in sport in-competition according to the List of Prohibited Substances and Methods published by the World Anti-Doping Agency (WADA). The first published data on the excretion study of NN-DMPPA to support the knowledge of NN-DMPPA in routine anti-doping control have been presented. The reliable gas chromatography-mass spectrometry quantitative method (GC-MS) has been validated and applied to the excretion study of NN-DMPPA. The validation parameters of the GC-MS method for determination of NN-DMPPA in human urine were the linear calibration range of 100 to 7500 ng/mL, the LOD of 13.9 ng/mL and the LOQ of 42.2 ng/mL. According to the obtained repeatability, intermediate precision, and trueness, the applied GC-MS method was precise and accurate. Urine samples from three volunteers in the excretion study were collected for 5 days after single oral administration of the supplement NOXPUMP containing NN-DMPPA. The obtained results showed the maximum concentration of NN-DMPPA (189–303 ng/mL) in urine samples at a time of 2–3 h post-administration. The NN-DMPPA concentration in urine samples was higher than 50 ng/mL until 22–23 h after the dietary supplement ingestion. This means that according to the WADA rules the use of a supplement containing NN-DMPPA may be related to a positive case when athletes took this supplement in-competition. Moreover, excretion results demonstrate also that NN-DMPPA may be detected in urine samples by the applied GC-MS method till 46 h after supplement administration. Additionally, the excretion study of β-methylphenethylamine as the second prohibited substance present in the supplement NOXPUMP has been investigated.
Graphical Abstract
Excretion study of new designer stimulant, N,N-dimethyl-2-phenylpropan-1-amine, and β-methylphenethylamine following single oral NOXPUMP supplement dose
Whenever athletes willfully or accidentally ingest performance-enhancing drugs or other banned substances (such as drugs of abuse), markers of those drugs can be detected in biological samples (e.g., biofluids: urine, saliva, blood); in the case of some drugs, that evidence can be apparent for many weeks following the last exposure to the drug. In addition to the willful use of prohibited drugs, athletes can accidentally ingest banned substances in contaminated dietary supplements or foods and inadvertently fail a drug test that could mean the end of an athletic career and the loss of a good reputation. The proliferation of performance-enhancing drugs and methods has required a corresponding increase in the analytical tools and methods required to identify the presence of banned substances in biofluids. Even though extraordinary steps have been taken by organizations such as the World Anti-Doping Agency to limit the use of prohibited substances and methods by athletes willing to cheat, it is apparent that some athletes continue to avoid detection by using alternative doping regimens or taking advantage of the limitations in testing methodologies. This article reviews the testing standards and analytical techniques underlying the procedures used to identify banned substances in biological samples, setting the stage for future summaries of the testing required to establish the use of steroids, stimulants, diuretics, and other prohibited substances.
The abuse of unknown designer androgenic anabolic steroids (AAS) is considered to be an issue of significant importance, as AAS are the choice of doping preference according to World Anti-doping Agency statistics. In addition, unknown designer AAS are preferred since the World Anti-doping Agency mass spectrometric identification criteria cannot be applied to unknown molecules. Consequently, cheating athletes have a strong motive to use designer AAS in order to both achieve performance enhancement and to escape from testing positive in anti-doping tests. To face the problem, a synergy is required between the anti-doping analytical science and sports anti-doping regulations. This Review examines various aspects of the designer AAS. First, the structural modifications of the already known AAS to create new designer molecules are explained. A list of the designer synthetic and endogenous AAS is then presented. Second, we discuss progress in the detection of designer AAS using: mass spectrometry and bioassays; analytical data processing of the unknown designer AAS; metabolite synthesis; and, long-term storage of urine and blood samples. Finally, the introduction of regulations from sports authorities as preventive measures for long-term storage and reprocessing of samples, initially reported as negatives, is discussed.
Androgenic anabolic steroids (AAS) are prohibited in sports due to their anabolic effects. Doping control laboratories usually face the screening of AAS misuse by target methods based on MS detection. Although these methods allow for the sensitive and specific detection of targeted compounds and metabolites, the rest remain undetectable. This fact opens a door for cheaters, since different AAS can be synthesized in order to evade doping control tests. This situation was evidenced in 2003 with the discovery of the designer steroid tetrahydrogestrinone. One decade after this discovery, the detection of unknown AAS still remains one of the main analytical challenges in the doping control field. In this manuscript, the current situation in the detection of unknown AAS is reviewed. Although important steps have been made in order to minimize this analytical problem and different analytical strategies have been proposed, there are still some drawbacks related to each approach.
In sports drug testing the steroid profile is the most versatile and informative screening tool for the detection of steroid abuse. Despite the introduction of observed urine collection procedures by the World Anti-Doping Agency (WADA), chemical manipulation of urine specimens by athletes to conceal drug use still occurs and poses an ongoing challenge for doping control laboratories worldwide. In vitro urine adulteration using highly oxidative chemicals have been reported several times in the past. In this study we report the effect of two oxidising agents, Fenton's reagent and peroxidase–peroxide system on the human urinary steroid profile. Varying concentrations of these oxidants were reacted with urine and the reactions monitored by gas chromatography-mass spectrometry. A significant decrease in the absolute concentrations of androsterone, etiocholanolone, 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol and epitestosterone was observed with consequent alteration of the steroid profile ratios. Adulteration of urine sample with these oxidants can thus mask the abnormality in a steroidal profile following steroid abuse. Drug testing authorities should take into account the effects of these oxidizing adulterants while interpreting the steroid profile data for doping control purposes.
International anti-doping efforts are harmonized and regulated under the umbrella of the World Anti-Doping Code and the corresponding Prohibited List, issued annually by the World Anti-Doping Agency (WADA). The necessity for a frequent and timely update of the Prohibited List (as the result of a comprehensive consultation process and subsequent consensual agreement by expert panels regarding substances and methods of performance manipulation in sports) is due to the constantly growing market of emerging therapeutics and thus new options for cheating athletes to illicitly enhance performance. In addition, 'tailor-made' substances arguably designed to undermine sports drug testing procedures are considered and the potential of established drugs to represent a doping substance is revisited in light of recently generated information. The purpose of the annual banned substance review is to support doping controls by reporting emerging and advancing methods dedicated to the detection of known and recently outlawed substances. This review surveys new and/or enhanced procedures and techniques of doping analysis together with information relevant to doping controls that has been published in the literature between October 2010 and September 2011.
Historically, dope-testing methods have been developed to target specific and known threats to the integrity of sport. Traditionally, the source of new analytical targets for which testing was required were derived almost exclusively from the pharmaceutical industry. More recently, the emergence of designer drugs, such as tetrahydrogestrinone that are specifically intended to evade detection, or novel chemicals intended to circumvent laws controlling the sale and distribution of recreational drugs, such as anabolic steroids, stimulants and cannabinoids, have become a significant issue. In this review, we shall consider the emergence of designer drugs and the response of dope-testing laboratories to these new threats, in particular developments in analytical methods, instrumentation and research intended to detect their abuse, and we consider the likely future impact of these approaches.
Various products containing rarely characterized anabolic steroids are nowadays marketed as dietary supplements. Herein, the designer steroid methyl-1-testosterone (M1T) (17β-hydroxy-17α-methyl-5α-androst-1-en-3-one) was identified, and its biological activity, potential adverse effects, and metabolism were investigated. The affinity of M1T toward the androgen receptor (AR) was tested in vitro using a yeast AR transactivation assay. Its tissue-specific androgenic and anabolic potency and potential adverse effects were studied in a Hershberger assay (sc or oral), and tissue weights and selected molecular markers were investigated. Determination of M1T and its metabolites was performed by gas chromatography mass spectrometry. In the yeast AR transactivation assay, M1T was characterized as potent androgen. In rats, M1T dose-dependently stimulated prostate and levator ani muscle weight after sc administration. Oral administration had no effect but stimulated proliferation in the prostate and modulated IGF-I and AR expression in the gastrocnemius muscle in a dose-dependent manner. Analysis of tyrosine aminotransferase expression provided evidence for a strong activity of M1T in the liver (much higher after oral administration). In rat urine, 17α-methyl-5α-androstane-3α,17β-diol, M1T, and a hydroxylated metabolite were identified. In humans, M1T was confirmed in urine in addition to its main metabolites 17α-methyl-5α-androst-1-ene-3α,17β-diol and 17α-methyl-5α-androstane-3α,17β-diol. Additionally, the corresponding 17-epimers as well as 17β-hydroxymethyl-17α-methyl-18-nor-5α-androsta-1,13-dien-3-one and its 17-epimer were detected, and their elimination kinetics was monitored. It was demonstrated that M1T is a potent androgenic and anabolic steroid after oral and sc administration. Obviously, this substance shows no selective AR modulator characteristics and might exhibit liver toxicity, especially after oral administration.
Dihydrotestosterone (DHT), a biologically active metabolite of testosterone, may be misused in sports to benefit from its anabolic and psychotropic effects. After DHT application, a significant increase of the glucuronides of DHT and its metabolites can be expected for a certain time period depending upon dose, formulation, route of administration, and in case of percutaneous administration the chainlength of the ester. DHT and its metabolites can be monitored by gas-chromatography/mass spectrometry (GC/MS) after enzymatic hydrolysis and trimethylsilylation. To investigate the extent of the alteration of the urinary steroid profile after DHT application, timely controlled experiments have been performed with: a) oral application of [16,16,17-2H3]-DHT, and b) sublingual application of a 25 mg dose of DHT. In the experiment with [16,16,17-2H3]-DHT within 24 hours about 44% of the applied dose was recovered after hydrolysis with beta-glucuronidase from E. coli as di- or tri-deuterated 5 alpha-androstane glucuronides: androsterone (33.2%), 5 alpha-androsta-ne-3 alpha,17 beta-diol (2.5%), 5 alpha-androstane-3 beta, 17 beta-diol (0.9%), DHT (7.2%). Hydrolysis with beta-glucuronidase/arylsulfatase from Helix Pomatia resulted in a about 10% higher yield except for DHT. In the study with sublingual application of 25 mg of DHT the extent of the recovery of DHT and its metabolites was in the same range as for the deuterated DHT. The urinary glucuronide concentrations of DHT, androsterone (AND), 5 alpha-androstane-3 alpha, 17 beta-diol (5 alpha A3 alpha D) and 5 alpha-androstane-3 beta,17 beta-diol (5 alpha A3 beta D) and their ratios to etiocholanolone (ETIO), 5 beta-androstane-3 alpha, 17 beta-diol (5 beta A3 alpha D) and epitestosterone (EPIT) were increased for up to 48 hours after application. For doping control purposes concentrations of DHT, 5 alpha A3 alpha D, 5 alpha A3 beta D and ratios of 5 alpha-metabolites to non 5 alpha-metabolites such as DHT/ETIO, DHT/EPIT, 5 alpha A3 alpha D/5 beta A3 alpha D, 5 alpha A3 beta D/5 beta A3 alpha D, and AND/ETIO outside the reference ranges are a proof for DHT application. Reference ranges for Asian and Caucasian male and female athletes are calculated from data bases of the Asian Games 1994, the previous Asian Games 1990 and the routine doping control samples of Caucasian athletes measured in Cologne 1994. At the occasion of the 1994 Asian Games in Hiroshima alterations in the concentrations and ratios of the DHT depending parameters for outside there reference ranges have been found and have been sanctioned on this basis by the Medical Commission of the Organisation of Olympic Council of Asia (OCA).
1,5alpha-Androsten-3beta,17beta-diol and/or 1,5alpha-androsten-3,17-dione (1AD) is an over-the-counter pro-hormone nutritional supplement designed to enhance strength and performance in athletes. 1AD purportedly mimics the pharmacological activity of testosterone through activation of the pro-hormones to their active form 1,5alpha-androsten-17beta-ol-3-one or Delta(1)-testosterone. This testosterone analogue ostensibly possesses strong androgenic potency without the adverse effects associated with aromatization of testosterone to estrogens. We have developed a highly sensitive and selective liquid chromatography-tandem mass spectrometry assay for the simultaneous determination of 1AD, its analogues, and several structurally related endogenous hormones in plasma and urine. The limits of quantitation for the analytes ranged from 0.25 to 0.5 ng/mL. The accuracy of the assay was 92-113% with a precision of 2-12% relative standard deviation (RSD) for all analytes at 1.0, 5.0, and 15.0 ng/mL, respectively. The interassay precision was 6-16% RSD, and the accuracy was 90-105%. We have used this assay to determine the unconjugated and total (conjugated and unconjugated) concentrations of 1AD, its analogues, androstenediol, androstenedione, testosterone, dihydrotestosterone, and estradiol, in plasma and urine, as well as to investigate the metabolic fate of the three 1AD analogues (diol, dione, and active forms) when incubated with rat liver microsomes or rat testicular homogenates. Concentrations of both unconjugated and total testosterone in plasma were approximately 1.5 ng/mL and ranged from undetectable to 4.1 ng/mL in urine. 1AD and its analogues were not detected in plasma or urine. In vitro metabolism experiments using rat liver microsomes and testicular homogenates provided evidence for the interconversion of the three 1AD analogues, biosynthesis, and decomposition of several endogenous hormones, as well as evidence for 1AD analogue-induced changes in the typical profiles of testosterone and androstenedione in testicular tissue.
The use of anabolic steroids was banned by the International Olympic Committee for the first time at the Olympic Games in Montreal in 1976. Since that time the misuse of anabolic steroids by athletes has been controlled by analysis of urine extracts by gas chromatography—mass spectrometry (GC—MS). The excreted steroids or their metabolites, or both, are isolated from urine by XAD-2 adsorption, enzymatic hydrolysis of conjugated excreted metabolites with β-glucuronidase from Escherichia coli, liquid-liquid extraction with diethyl ether, and converted into trimethylsilyl (TMS) derivatives. The confirmation of an anabolic steroid misuse is based on comparison of the electron impact ionization (EI) mass spectrum and GC retention time of the isolated steroid and/or its metabolite with the EI mass spectrum and GC retention time of authentic reference substances. For this purpose excretion studies with the most common anabolic steroids were performed and the main excreted metabolites were synthesized for bolasterone, boldenone, 4-chlorodehydromethyltestosterone, clostebol, drostanolone, fluoxymesterone, formebolone, mestanolone, mesterolone, metandienone, methandriol, metenolone, methyltestosterone, nandrolone, norethandrolone, oxandrolone and stanozolol. The metabolism of anabolic steroids, the synthesis of their main metabolites, their GC retention and El mass spectra as TMS derivatives are discussed.
Complete 1H and 13C spectral assignments of 17beta- and 17alpha-hydroxy epimers of three biologically active sterols (boldenone, 3-methoxyestradiol and 3-methoxydihydroequilenin) were achieved making use of one- and two-dimensional NMR techniques (1D-HOHAHA, DEPT, COSY, NOESY, TOCSY, HSQC and COLOC).
Tetrahydrogestrinone (18a-homo-pregna-4,9,11-trien-17beta-ol-3-one or THG) was identified in the residue of a spent syringe that had allegedly contained an anabolic steroid undetectable by sport doping control urine tests. THG was synthesized by hydrogenation of gestrinone and characterized by mass spectrometry and NMR spectroscopy. We developed and evaluated sensitive and specific methods for rapid screening of urine samples by liquid chromatography/tandem mass spectrometry (LC/MS/MS) of underivatized THG (using transitions m/z 313 to 241 and 313 to 159) and gas chromatography/high-resolution mass spectrometry (GC/HRMS) analysis of the combination trimethylsilyl ether-oxime derivative of THG (using fragments m/z 240.14, 254.15, 267.16, and 294.19). A baboon administration study showed that THG is excreted in urine.
Madol (17alpha-methyl-5alpha-androst-2-en-17beta-ol) was identified in an oily product received by our laboratory in the context of our investigations of designer steroids. The product allegedly contained an anabolic steroid not screened for in routine sport doping control urine tests. Madol was synthesized by Grignard methylation of 5alpha-androst-2-en-17-one and characterized by mass spectrometry and NMR spectroscopy. We developed a method for rapid screening of urine samples by gas chromatography/mass spectrometry (GC/MS) of trimethylsilylated madol (monitoring m/z 143, 270, and 345). A baboon administration study showed that madol and a metabolite are excreted in urine. In vitro incubation with human liver microsomes yielded the same metabolite. Madol is only the third steroid never commercially marketed to be found in the context of performance-enhancing drugs in sports.
In human sports doping control analysis most of the steroids are analyzed after enzymatic hydrolysis of the glucuronides as per-trimethylsilyl (TMS) derivatives applying gas chromatography-mass spectrometry (GC-MS). According to the recommendations of the World Anti-Doping Agency the identification of analytes should be based on retention time and on mass spectrometric characterization. This study shows that the bis-TMS derivatives of 16 specific C19 steroids, namely the stereoisomers of 5xi-androst-1-ene-3xi,17xi-diol (8 isomers), androst-4-ene-3xi,17xi-diol (4 isomers), and 17xi-hydroxy-5xi-androstan-3-one (4 isomers), reveal very similar mass spectra. As a rule, when taking the retention times, which are provided as Kovac indices for all these isomers, into account, a restriction to two or three possible isomers is possible. Reliable identification should additionally include a comparison of the retention times of the analytes with the reference compounds measured concomitantly. In some cases standard addition may be appropriate. Due to the limited availability, the above mentioned isomers were synthesized by reduction of the corresponding alpha,beta-unsaturated oxo steroids either with K-Selectride or by catalytic hydrogenation (Pd/C as catalyst). The products of the reactions were identified by means of nuclear magnetic resonance (NMR) characterization and by further reduction to the corresponding 5xi-androstane-3xi,17xi-diols and GC-MS comparison with commercially available reference standards.
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