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Studies on the in vivo metabolism of the SARM YK11: Identification and characterization of metabolites potentially useful for doping controls
Abstract
A steroidal compound was recently detected in a seized black market product and was identified as (17α,20E)‐17,20‐[(1‐methoxyethylidene) bis (oxy)]‐3‐oxo‐19‐norpregna‐4,20‐diene‐21‐carboxylic acid methyl ester (YK11). This compound is described to possess selective androgen receptor modulator‐ and myostatin inhibitor‐like properties. As YK11 is an experimental drug candidate and a non‐approved substance for humans, scientific data on its metabolism is scarce. Due to its steroidal backbone and the arguably labile orthoester‐derived moiety positioned at the D‐ring, substantial metabolic conversion in vivo was anticipated.
In order to unambiguously detect urinary metabolites of YK11, an elimination study with six‐fold deuterated YK11 was conducted. Post administration specimens were analyzed using hydrogen isotope ratio mass spectrometry coupled to single quadrupole mass spectrometry to identify metabolites alongside basic mass spectrometric data. Further characterization of those metabolites relevant to sports drug testing was accomplished using gas chromatography/high resolution‐high accuracy mass spectrometry.
Fourteen deuterated urinary metabolites were detected comprising unconjugated, glucuronidated and sulfoconjugated metabolites. As expected, no intact YK11 was observed in the elimination study urine samples. While the unconjugated metabolites disappeared within 24 h post‐administration, both glucuronidated and sulfated metabolites were traceable for more than 48 h. The chemical structures of the two most promising glucuronidated metabolites (5β‐19‐nor‐pregnane‐3α,17β,20‐triol and 5β‐19‐nor‐pregnane‐3α,17β‐diol‐20‐one) were verified by in‐house synthesis of both metabolites and verified by NMR analysis. In order to elucidate their potential in sports drug testing, both were successfully implemented into the currently applied analytical method for the detection of anabolic agents.
... One of the prominent leading compound categories are selective androgen receptor modulators (SARMs) [21]. These agents were developed as more favorable alternatives to AAS, with comparable androgen receptor (AR) affinity and minimal androgenic impacts [25,26]. Many SARMs exist as non-steroidal compounds (quinolones, tetrahydroquinolones, tricyclics, bridged tricyclics, aryl propionamides, aniline, diaryl aniline, bicylclic hydantoins, benzimidazole, imidazolopyrazole, indole, and pyrazoline derivatives), which in-part mediate their unique effects [4,21]. ...
... Unfortunately, the attractive aspects of SARMs have also garnered attention as a novel recreational performance enhancing compounds [27]. In 2008, WADA banned SARMs in absolute due to their inherent abuse risk and the then-present detection of various SARM metabolites in athlete urine samples in 2010 [26,33,37]. Consequently, SARM misuse has steadily increased over the last decade, whereby nearly 40 cases were reported via WADA doping control sample analysis in 2016 [38]. ...
... Consequently, SARM misuse has steadily increased over the last decade, whereby nearly 40 cases were reported via WADA doping control sample analysis in 2016 [38]. Similar to AAS and other ancillary performance enhancing substances, SARM providers are commonly found on the internet and small laboratories both within and outside the US are able to synthesize these compounds for global distribution [26,27]. ...
Anabolic androgenic steroids (AAS) are testosterone and testosterone-derivative compounds sporadically employed by athletes and increasingly used recreationally to acquire a competitive edge or improve body composition. Nevertheless, users are subject to undesired side effects majorly associated with tissue-specific androgen receptor (AR) binding-mediated actions. More recently, selective AR modulators (SARMs) have gained popularity towards delivering androgen-associated anabolic actions with hopes of minimal androgenic effects. While several SARMs are in preclinical and clinical phases intended for demographics subject to hypo-gonadism, muscle wasting, and osteoporosis, several athletic organizations and drug testing affiliates have realized the increasingly widespread use of SARMs amongst competitors and have subsequently banned their use. Furthermore, recreational users are haphazardly acquiring these compounds from the internet and consuming doses several times greater than empirically reported. Unfortunately, online sources are rife with potential contamination, despite a prevailing public opinion suggesting SARMs are innocuous AAS alternatives. Considering each agent has a broad range of supporting evidence in both human and non-human models, it is important to comprehensively evaluate the current literature on commercially available SARMs to gain better understanding of their efficacy and if they can truly be considered a safer AAS alternative. Therefore, the purpose of this review is to discuss the current evidence regarding AAS and SARM mechanisms of action, demonstrate the efficacy of several prominent SARMs in a variety of scientific trials, and theorize on the wide-ranging contra-indications and potential deleterious effects, as well as potential future directions regarding acute and chronic SARM use across a broad range of demographics.
... Conversely, the SARM ostarin (enobosarm) has been found as an undeclared ingredient of many dietary supplements. There have thus been increasing efforts focusing on isolating SARM metabolites for drug testing [68,69], with multiple SARMs having already been added by the World Anti-Doping Agency (WADA) to their "prohibited list" [70]. ...
Cachexia is a complex wasting syndrome, accompanying a variety of end-stage chronic diseases, such as cancer, heart failure and human immunodeficiency virus (HIV) infection/acquired immunodeficiency syndrome (AIDS). It significantly affects patients’ quality of life and survival. Multiple therapeutic approaches have been studied over time. However, despite promising results, no drug has been approved to date. In this review, we examine and discuss the available data on the therapeutic effects of androgens and selective androgen receptor modulators (SARMs) for cachexia.
... The in vivo metabolism of the steroidal SARM YK-11 and the anabolic steroids methylstenbolone and trenbolone was investigated by Piper et al. by analysing post-administration urine specimens. Several metabolites, relevant to sports drug testing, were characterised using GC-HRMS (as trimethylsilyl derivatives) and/or LC-HRMS instruments [18,161,162]. Moreover, GC-HRMS and GC-MS/MS techniques were recently employed for detecting novel A-Ring reduced metabolites of the anabolic steroids methyltestosterone and metandienone [163]. ...
Performance and image enhancing drugs are misused by top athletes, young people and amateurs like bodybuilders. Their misuses lead to adverse analytical findings in sport but also physical and mental health risks. Anti-doping laboratories must set up analytical strategies and develop precise, robust and sensitive methods to detect all analytes prohibited by the World Anti-Doping Agency (WADA). In recent years, high resolution mass spectrometry (HRMS) systems have emerged and are gradually replacing more traditional systems for screening and quantification. The targeted compounds in this review are the drugs prohibited by the WADA in-and out-of competition and glucocorticoids. Advanced technologies were applied for anti-doping, forensic and clinical purposes. The contribution of HRMS in the analysis of doping products allows a retrospective analysis to detect new metabolites in order to increase the detection windows and to identify new synthetic products. These systems are also used for new applications like study of metabolomics, characterisation of metabolites and applications in alternative matrices (hair, dried blood spots). Moreover, the misuses of these substances lead the consumers to purchase on the black market.
... Therefore, a GC Orbitrap system with an alternative ion source design favoring higher abundances of ions with larger m/z values was used, and the measured TMS derivatives further exhibited a reduced in-source fragmentation compared to acetates (Piper et al., 2018(Piper et al., , 2019. In addition, trimethylsilylation of steroidal analytes is routinely used, thus offering a platform for implementing potential new target analytes into sports drug testing methods. ...
Trenbolone is a synthetic anabolic-androgenic steroid, which has been misused for performance enhancement in sports. The detection of trenbolone doping in routine sports drug testing programs is complex as methods utilizing gas chromatography/mass spectrometry are complicated by unspecific derivatization products and artifacts, and liquid chromatography/mass spectrometry-based assays have shown to allow for comparably high limits-of-detection only. The number of previously reported metabolites in human urine is limited, and most analytical methods rely on targeting epitrenbolone, trenbolone glucuronide, and epitrenbolone glucuronide. In order to probe for the presence of additional trenbolone metabolites and to re-investigate the metabolism, an elimination study was conducted. One single dose of 10 mg of 5-fold deuterated trenbolone was administered to a healthy male volunteer and urine samples were collected for 30 days. For sample processing, published protocols were combined considering unconjugated, glucuronic acid-, sulfo- and alkaline-labile conjugated steroid metabolites. The sample preparation strategy consisted of solid-phase extractions, liquid-liquid extractions, metabolite de-conjugation, HPLC fractionation, and derivatization. Analytical methods included gas chromatography/thermal conversion/hydrogen isotope ratio mass spectrometry combined with single quadrupole mass spectrometry as well as liquid chromatography/high accuracy/high resolution mass spectrometry of the hydrolyzed and non-hydrolyzed samples. Twenty deuterium-labeled metabolites were identified including glucuronic acid-, sulfo- and potential cysteine-conjugates, and characterized by parallel reaction monitoring experiments yielding corresponding product ion mass spectra. Main metabolites were attributed to trenbolone-diol and potential trenbolone-diketone derivatives excreted as glucuronic acid and sulfo-conjugated analytes with detection windows of 5, respectively 6 days. Further characterization was conducted with pseudo MS³ experiments of the intact conjugates and by comparison of resulting product ion mass spectra with reference material.
... ARE-luciferase reporter assay in HEK293 cells suggested that the active constituent of YK11 was 2a. Although YK11 is sold via the internet as an anabolic steroid, it is an experimental drug candidate and a non-approved substance for humans [10][11][12]. It should be noted that YK11 has not been studied in humans or even animals. ...
The palladium catalyzed carbonylation of 1 using the chiral ligand L1 afforded 2 in a highly diastereoselective manner. The stereochemistry of the major diastereomer 2a was determined by X-ray crystallographic analysis. AR luciferase reporter assay studies suggested that 2a was the active constituent of YK11 (2).
... As such, efforts are currently focused on continuing to isolate metabolites from novel SARMs for drug testing (89,90), and multiple SARMs have already been added by the World Anti-Doping Agency (WADA) to the Prohibited List (91). Consequently, several athletes have been suspended for testing positive for SARM metabolites; an NBA athlete was recently suspended for taking LGD-4033 (92), while an NCAA basketball player and four UFC fighters have been penalized for trace amounts of ostarine (Enobosarm) in their systems (88,93). ...
Selective androgen receptor modulators (SARMs) are small molecule drugs that function as either androgen receptor (AR) agonists or antagonists. Variability in AR regulatory proteins in target tissues permits SARMs to selectively elicit anabolic benefits while eschewing the pitfalls of traditional androgen therapy. SARMs have few side effects and excellent oral and transdermal bioavailability and may, therefore, represent viable alternatives to current androgen therapies. SARMs have been studied as possible therapies for many conditions, including osteoporosis, Alzheimer's disease, breast cancer, stress urinary incontinence (SUI), prostate cancer (PCa), benign prostatic hyperplasia (BPH), male contraception, hypogonadism, Duchenne muscular dystrophy (DMD), and sarcopenia/muscle wasting/cancer cachexia. While there are no indications for SARMs currently approved by the Food and Drug Administration (FDA), many potential applications are still being explored, and results are promising. In this review, we examine the literature assessing the use of SARMS for a number of indications.
... Fragkaki et al. reported on the preferred traceability of LGD-4033 in human urine by means of a bishydroxylated metabolite of LGD-4033, which allowed detection windows of 20 days in a pilot elimination study. 2 Piper et al. focused on a new steroidal SARM candidate referred to as YK-11, whose metabolic fate was studied by means of hydrogen isotope ratio mass spectrometry, revealing 5-19nor-pregnane-3α,17β,20-triol and 5-19-nor-pregnane-3α,17β-diol-20-one as a potential target analytes for routine doping control analyses of human urine. 3 The use of surrogate organisms, here zebrafish, was presented by Sardela et al., 4 demonstrating that general metabolic patterns can, to a certain extent, be simulated using this comparably simple model, and the utility of in vitro metabolism in doping controls was outlined in a comprehensive study by Mazzarino et al., who focused on a new class of doping agents collectively considered as REV-ERB agonist. 5 Another substance being studied extensively was bemitil, which has been added to WADA's monitoring program in 2018. ...
Selective androgen receptor modulators (SARMs) are performance‐enhancing drugs (PEDs) that stimulate anabolism, increase muscle mass and strength and promote recovery from exercise. The use of SARMs in sports is considered doping and is strictly prohibited by the World Anti‐Doping Agency (WADA) and the International Federation of Horseracing Authorities (IFHA). To monitor the abuse of SARMs in sports, it is essential to develop advanced, selective and sensitive analytical methods that provide reliable results. This review evaluates the advances in this area, with a focus on the identification of target analytes related to SARMs, such as SARMs, their metabolites or markers. The aim is to identify targets that could extend the detection windows of SARMs, provide scientific support for results management and/or offer an indirect biomarker‐based approach to doping control. This review also aims to evaluate the current liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) methods developed for the monitoring of SARMs in different biological matrices, including traditional matrices such as urine and serum/plasma samples, as well as alternative matrices such as dried blood spots, hair and nail samples.
Selective androgen receptor modulators (SARMs) are prohibited by the World Anti‐Doping Agency (WADA) since 2008. Similarly, to anabolic androgenic steroids (AAS), SARMs are detrimental to health not only in athletes but also in the general population. However, studies of the occurrence of SARMs outside of sport are scarce. Swedish healthcare samples from the Drugs of Abuse Laboratory at Karolinska were analyzed using WADA‐accredited screening methods at the Doping Control Laboratory in Stockholm to estimate the frequency of SARM use outside of the WADA laboratories. Twenty (4%) of the male urine samples ( n = 542) were positive for SARMs, whereas none of the analyzed female samples ( n = 100) contained any SARMs. The top three SARMs found were LGD‐4033 followed by RAD140 and ostarine. Two or more SARMs were found in >50% of the SARM‐positive samples. AASs were identified in 40% of samples containing SARMs. A difference between genders was observed where 34% male and 7% female samples contained AAS. Many samples displayed testosterone/epitestosterone values indicative of testosterone intake, without presence of other AAS, and hence, there is a risk that these samples are being falsely reported as negative. Our results indicate that SARM use might be a concern outside of sport. Subsequently, in addition to AAS, the healthcare system should also be informed about SARM abuse and the associated adverse side effects.
The possible performance‐enhancing effects and medical benefits of ecdysterone (ECDY) have been discussed several times throughout the last decades. In 2020, the World Anti‐Doping Agency include ECDY in their monitoring programme and continued this prevalence study until now. Only little is known about the human metabolism of ECDY besides the first study performed on human subjects in the field of sports drug testing that was already conducted in 2001. Aim of this study was the in‐depth investigation on human ECDY metabolism to improve its detectability and to support the decision‐making processes as to how ECDY can be implemented most effectively into sports drug testing regulations. In a first trial, one male volunteer was administered with threefold deuterated ECDY to enable the detection and potential identification of all urinary metabolites still comprising the deuterium label by employing hydrogen isotope ratio mass spectrometry and high‐resolution/high‐accuracy mass spectrometry. Samples were collected for up to 14 days, and metabolites excreted unconjugated, glucuronidated, and sulphated were investigated. The detected deuterated metabolites were confirmed in a second administration trial encompassing two male and one female volunteers. After the administration of 50 mg of unlabelled ECDY, urine samples were collected for up to 7 days. Besides the already described urinary metabolites of ECDY, more than 20 new metabolites were detected encompassing all expected metabolic conversions including side chain cleavage at C21. A large interindividual variation in the amounts of excreted metabolites was visible, and considerable differences in abundances of early‐ and late‐excretion phase metabolites were observed.
YK-11 is a steroidal selective androgen receptor modulator, a compound class prohibited in both equine racing and human sports because of their potentially performance enhancing properties. YK-11 is easily accessible via internet-based supplement vendors making this compound a possible candidate for doping; however, its phases I and II metabolism has not yet been reported in the horse. The purpose of this study was to investigate the in vivo metabolites of YK-11 in urine and plasma following oral administration with three daily doses of 50 mg to two Thoroughbred horses. In vitro incubations with equine liver microsomes/S9 were also performed for use as metabolite reference materials; however, this resulted in the formation of 79 metabolites with little overlap with the in vivo metabolism. In plasma, parent YK-11 and seven phase I metabolites were detected, with five of them also observed in vitro. They were present nonconjugated in plasma, with one metabolite also indicating some glucuronide conjugation. In urine, 11 phase I metabolites were observed, with four of them also observed in vitro and six of them also detected in plasma. Nine metabolites were excreted non-conjugated in urine, with two of them also indicating some sulfate conjugation. Two minor metabolites were detected solely as sulfate conjugates. The most abundant analytes in urine were a mono-O-demethylated breakdown product and di-O-demethylated YK-11. The most abundant analytes in plasma were two isomers of the breakdown product with an additional hydroxylation reaction, which also provided the longest detection time in both matrices.
For decades, anabolic androgenic agents have represented the substance class most frequently observed in doping control samples. They comprise synthetic and pseudoendogenous anabolic androgenic steroids and other, mostly non-steroidal compounds with (presumed) positive effects on muscle mass and function. While exogenous substances can easily be detected by gas/liquid chromatography and mass spectrometry, significantly more complex methodologies including the longitudinal monitoring of individual urinary steroid concentrations/ratios and isotope ratio mass spectrometry are required to provide evidence for the exogenous administration of endogenous compounds.
This narrative review summarizes the efforts made within the last 5 years to further improve the detection of anabolic agents in doping control samples. Different approaches such as the identification of novel metabolites and biomarkers, the acquisition of complementary mass spectrometric data, and the development of new analytical strategies were employed to increase method sensitivity and retrospectivity while simultaneously reducing method complexity to facilitate a higher and faster sample throughput.
Probing for evidence of the administration of prohibited therapeutics, drugs and/or drug candidates as well as the use of methods of doping in doping control samples is a central assignment of anti-doping laboratories. In order to accomplish the desired analytical sensitivity, retrospectivity, and comprehensiveness, a considerable portion of anti-doping research has been invested into studying metabolic biotransformation and elimination profiles of doping agents. As these doping agents include lower molecular mass drugs such as e.g. stimulants and anabolic androgenic steroids, some of which further necessitate the differentiation of their natural/endogenous or xenobiotic origin, but also higher molecular mass substances such as e.g. insulins, growth hormone, or siRNA/anti-sense oligonucleotides, a variety of different strategies towards the identification of employable and informative metabolites have been developed. In this review, approaches supporting the identification, characterization, and implementation of metabolites exemplified by means of selected doping agents into routine doping controls are presented, and challenges as well as solutions reported and published between 2010 and 2020 are discussed.
Performance- and image-enhancing drugs (PIEDs) misuse is a significant public health issue. Currently, seizure data, surveys, anti-doping testing, and needle service provider data are used to estimate PIED use in populations. These methods are time consuming, single point-in-time measurements that often consist of small sample sizes and do not truly capture PIED prevalence.
Wastewater-based epidemiology (WBE) has been used globally to assess and monitor licit and illicit drug consumption within the general community. This method can objectively cover large populations as well as specific subpopulations (gyms, music festivals, prisons), and has potential as a complementary monitoring method for PIED use. Information obtained through WBE could be used to aid public health authorities in developing targeted prevention and education programmes. Research on PIED analysis in wastewater is limited and presents a significant gap in the literature.
The focus is on anabolic steroids, and one steroid alternative currently growing in popularity; selective androgenic receptor modulators. This encompasses medical uses, addiction, prevalence, user typology and associated public health implications. An overview of wastewater-based epidemiology is described including its benefits, limitations and potential as a monitoring method for PIED use. A summary of previous work in this field is presented. Finally, we summarise gaps in the literature, future perspectives and recommendations for monitoring PIEDs in wastewater.
Within the complex construct of today's anti‐doping work, continuously updated routine doping controls as well as advancements in sampling and analysis have been of particular relevance and importance. New analytes of existing classes of prohibited substances are frequently included into sports drug testing assays, analytical approaches are optimized to allow for better sensitivities, selectivity, and/or faster turnaround times, and research dedicated to addressing analytical issues concerning scenarios of both (potentially) inadvertent doping and new emerging doping agents is constantly conducted. By way of reviewing and summarizing, this annual banned‐substance review evaluates literature published between October 2018 and September 2019 offering an in‐depth evaluation of developments in these arenas and their potential application to substances reported in WADA's 2019 Prohibited List.
Technical innovations are continuously offering new options to research and routine applications in various arenas of analytical chemistry. In combination with a concomitantly increasing knowledge in physiology and pharmacology and a steadily growing supply of new drug candidates and potential therapeutic approaches, sports drug testing laboratories have been urged to develop and refine numerous test methods and strategies in order to provide best-possible levels of information to testing authorities in support of decision-making processes in anti-doping.
The myogenic differentiation of C2C12 myoblast cells is induced by the novel androgen receptor (AR) partial agonist, (17α,20E)-17,20-[(1-methoxyethylidene)bis-(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11), as well as by dihydrotestosterone (DHT). YK11 is a selective androgen receptor modulator (SARM), which activates AR without the N/C interaction. In this study, we further investigated the mechanism by which YK11 induces myogenic differentiation of C2C12 cells. The induction of key myogenic regulatory factors (MRFs), such as myogenic differentiation factor (MyoD), myogenic factor 5 (Myf5) and myogenin, was more significant in the presence of YK11 than in the presence of DHT. YK11 treatment of C2C12 cells, but not DHT, induced the expression of follistatin (Fst), and the YK11-mediated myogenic differentiation was reversed by anti-Fst antibody. These results suggest that the induction of Fst is important for the anabolic effect of YK11.
In sports drug testing, comprehensive studies on the metabolism of therapeutic agents with misuse potential are necessary to identify metabolites that provide utmost retrospectivity and specificity. By commonly employed approaches minor and/or long-term metabolites in urine might remain undetected. Hence, an alternative strategy to unambiguously identify the majority of urinary metabolites including low-abundance representatives is desirable.
Urine samples were collected for 20 days during an elimination study with an oral dose of 5 mg of 17α-C(2) H3 -metandienone. The specimens were processed according to established sample preparation procedures (including fractionation and deconjugation) and subjected to gas chromatography/hydrogen isotope ratio mass spectrometry (GC/IRMS) analysis. Due to the deuteration of the administered drug, urinary metabolites bearing the deuterium label yield abundant and specific signals on the GC/IRMS instrument resulting from the substantially altered (2) H/(1) H ratio. The sample aliquots were measured by gas chromatography/time-of-flight (GC/Q-TOF) mass spectrometry using identical GC conditions, allowing high-resolution/high-accuracy mass data to be obtained on all urinary metabolites previously identified by IRMS.
Within the IRMS chromatograms, labeled metabolites were identified up to 20 days after administration at urinary concentration down to 0.25 ng/mL. More than 50 metabolites were observed with the earlier described long-term metabolite of metandienone, 18-nor-17β-hyroxymethyl,17α-methyl-androst-1,4,13-trien-3-one, being the most prominent glucuronidated metabolite in the studied time window. In the sulfoconjugated steroids fraction, a yet unknown metabolite was observed at m/z 283.1997 comprising the experimentally determined elemental composition of C20 H21 (2) H3 O.
Combining IRMS with high-resolution mass spectrometry considerably facilitates and accelerates metabolite identification of deuterium-labeled compounds in urine. Of particular relevance in doping control, the principle is applicable also to other arenas of drug research, allowing the preparation and administration of e.g. radioactively labeled substances to be omitted. Copyright © 2013 John Wiley & Sons, Ltd.
Analytical chemistry represents a central aspect of doping controls. Routine sports drug testing approaches are primarily designed to address the question whether a prohibited substance is present in a doping control sample and whether prohibited methods (for example, blood transfusion or sample manipulation) have been conducted by an athlete. As some athletes have availed themselves of the substantial breadth of research and development in the pharmaceutical arena, proactive and preventive measures are required such as the early implementation of new drug candidates and corresponding metabolites into routine doping control assays, even though these drug candidates are to date not approved for human use. Beyond this, analytical data are also cornerstones of investigations into atypical or adverse analytical findings, where the overall picture provides ample reason for follow-up studies. Such studies have been of most diverse nature, and tailored approaches have been required to probe hypotheses and scenarios reported by the involved parties concerning the plausibility and consistency of statements and (analytical) facts. In order to outline the variety of challenges that doping control laboratories are facing besides providing optimal detection capabilities and analytical comprehensiveness, selected case vignettes involving the follow-up of unconventional adverse analytical findings, urine sample manipulation, drug/food contamination issues, and unexpected biotransformation reactions are thematized.
Rationale:
Selective androgen receptor modulators (SARMs) represent an emerging class of therapeutics targeting inter alia conditions referred to as cachexia and sarcopenia. Due to their anabolic properties, the use of SARMs is prohibited in sports as regulated by the World Anti-Doping Agency (WADA), and doping control laboratories test for these anabolic agents in blood and urine. In order to accomplish and maintain comprehensive test methods, the characterization of new drug candidates is critical for efficient sports drug testing. Hence, in the present study the mass spectrometric properties of the SARM YK-11 were investigated.
Methods:
YK-11 was synthesized according to literature data and three different stable-isotope-labeled analogs were prepared to support the mass spectrometric studies. Using high-resolution/high-accuracy mass spectrometry following electrospray ionization as well as electron ionization, the dissociation pathways of YK-11 were investigated, and characteristic features of its (product ion) mass spectra were elucidated. These studies were flanked by density functional theory (DFT) computation providing information on proton affinities of selected functional groups of the analyte.
Results and conclusions:
The steroidal SARM YK-11 was found to readily protonate under ESI conditions followed by substantial in-source dissociation processes eliminating methanol, acetic acid methyl ester, and/or ketene. DFT computation yielded energetically favored structures of the protonated species resulting from the aforementioned elimination processes particularly following protonation of the steroidal D-ring substituent. Underlying dissociation pathways were suggested, supported by stable-isotope labeling of the analyte, and diagnostic product ions for the steroidal nucleus and the D-ring substituent were identified. Further, trimethylsilylated YK-11 and its deuterated analogs were subjected to electron ionization high-resolution/high-accuracy mass spectrometry, complementing the dataset characterizing this new SARM. The obtained fragment ions resulted primarily from A/B- and C/D-ring structures of the steroidal nucleus, thus supporting future studies e.g. concerning metabolic pathways of the substance. Copyright © 2017 John Wiley & Sons, Ltd.
Testosterone (T) misuse still represents a major problem in sports drug testing. Many strategies have been developed and applied to routine doping controls within the recent years to enable both identifying suspicious samples in initial testing procedures and to confirm the exogenous origin of urinary T by means of carbon isotope ratio (CIR) determinations. Depending on the tested individual's genotype of UGT2B17, significantly different amounts of T are glucuronidated and excreted, which results in unaffected T/epitestosterone ratios after T misuse in those subjects with the deletion/deletion polymorphism (del/del). The aim of this study was to investigate differences in metabolic pathways of orally administered T between persons of del/del and insertion/insertion (ins/ins) genetic polymorphism. Therefore, a recently established method using hydrogen isotope ratios together with high-resolution and high-accuracy mass spectrometry was applied after administration of deuterated T to n = 4 subjects including both genotypes. Participants collected urine specimens directly before and for up to 8 days after the application. Urine aliquots were prepared to yield unconjugated, glucuronidated, and sulphoconjugated fractions of urinary steroids. Besides the significant difference in the excretion of T-glucuronide, all measured metabolites varied rather on an individual basis than due to a genotype difference. New T metabolites (both methylated and de-methylated) were detected and investigated regarding their potential to enhance the screening for T misuse. Sulphoconjugated epiandrosterone was further identified as the biomarker allowing for a prolonged retrospective detection of T misuse by means of CIR determinations for up to 5 days compared to 1 day if currently applied sports drug testing procedures were used.
The synthetic anabolic androgenic steroid 19-nortestosterone is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA) due to its performance-enhancing effects. Today, doping controls focus predominantly on one main urinary metabolite, 19-norandrosterone glucuronide, which offers the required detection windows for an appropriate retrospectivity of sports drug testing programs. As 19-norandrosterone can also be found in urine at low concentrations originating from in situ demethylation of other abundant steroids or from endogenous production, the exogenous source of 19-norandrosterone needs to be verified, which is commonly accomplished by carbon isotope ratio analyses.
The aim of this study was to re-investigate the metabolism of 19-nortestosterone in order to probe for additional diagnostic long-term metabolites, which might support the unambiguous attribution of an endo- or exogenous source of detected 19-nortestosterone metabolites. Employing a recently introduced strategy for metabolite identification, threefold deuterated 19-nortestosterone (16,16,17-²H3-NT) was administered to one healthy male volunteer and urine samples were collected for 20 days. Samples were prepared with established methods separating unconjugated, glucuronidated and sulfated steroids, and analytes were further purified by means of high-performance liquid chromatography before trimethylsilylation. Deuterated metabolites were identified using gas chromatograph/thermal conversion/isotope ratio mass spectrometer comprising an additional single quadrupole mass spectrometer. Additional structural information was obtained by gas chromatography/time-of-flight mass spectrometry and liquid chromatography/high resolution mass spectrometry.
In general, sulfo-conjugated metabolites were excreted for a longer time period than the corresponding glucuronides. Several unexpected losses of the arguably stable isotope labels were observed and characterized, attributed to metabolic reactions and sample preparation procedures. The detection window of one of the newly detected metabolites was higher than currently used metabolites. The suitability of this metabolite to differentiate between endo- or exogenous sources could however not be verified conclusively.
The three stereoisomers, 11α, 17α-isomer I, 11α, 17β-isomer II and 11β, 17β-isomer III are related substances of the selective progesterone receptor modulator Ulipristal acetate. Herein, we presented an efficient and practical synthesis approach to deliver these three stereoisomers for the first time, and also confirmed the structure of the key intermediate 5a by single-crystal X-ray analysis. Our research will be of immense help for organic chemists to study the impurity profile of Ulipristal acetate.
Copyright © 2014 Elsevier Inc. All rights reserved.
Rationale:
AICAR (5-aminoimidazole-4-carboxamide 1β-D-ribofuranoside) is prohibited in sport according to rules established by the World Anti-Doping Agency. Doping control laboratories identify samples where AICAR abuse is suspected by measuring its urinary concentration and comparing the observed level with naturally occurring concentrations. As the inter-individual variance of urinary AICAR concentrations is large, this approach requires a complementary method to unambiguously prove the exogenous origin of AICAR. Therefore, a method for the determination of carbon isotope ratios (CIRs) of urinary AICAR has been developed and validated.
Methods:
Concentrated urine samples were fractionated by means of liquid chromatography for analyte cleanup. Derivatization of AICAR yielding the trimethylsilylated analog was necessary to enable CIR determinations by gas chromatography/combustion/isotope ratio mass spectrometry. The method was tested for its repeatability and stability over time and a linear mixing model was applied to test for possible isotopic discrimination. A reference population of n = 63 males and females was investigated to calculate appropriate reference limits to differentiate endogenous from exogenous urinary AICAR. These limits were tested by an AICAR elimination study.
Results:
The developed method fulfills all the requirements for adequate sports drug testing and was found to be fit for purpose. The investigated reference population showed a larger variability in the CIR of AICAR than of the endogenous steroids. Nevertheless, the calculated thresholds for differences between AICAR and endogenous steroids can be applied straightforwardly to evaluate suspicious doping control samples with the same statistical confidence as established e.g. for testosterone misuse. These thresholds enabled the detection of a single oral AICAR administration for more than 40 h.
Conclusions:
Determination of thee CIRs is the method of choice to distinguish between an endogenous and an exogenous source of urinary AICAR. The developed method will enable investigations into doping control samples with elevated urinary concentrations of AICAR and clearly differentiate between naturally produced/elevated and illicitly administered AICAR.
Palladium on charcoal hydrogenation of Reichstein's Compound S acetate30 afforded the 5α-dihydro derivative I as the minor, and the 5β-dihydro derivative II as the major, product. Raney nickel hydrogenation of the former gave Reichstein's Compound P acetate (IIIa), which was converted into allo-THS (VIa). Raney nickel hydrogenation of the 5β compound II gave a mixture of THS 21-acetate (Vb) and its 3β epimer IVb. The 5α-dihydro derivative X of DOCA was hydrogenated in a similar fashion to afford the 3β compound XIa which was transformed into allo-tetrahydrodesoxy-corticosterone (XVa), Raney nickel hydrogenation of the 5β-dihydro derivative XII gave a mixture of tetrahydrodesoxycorticosterone 21-acetate (XIIIb) and its 3β epimer XIVb. The presence of a 17α-hydroxyl and of a 16α, 17α-epoxy group in several 5β-3-keto steroids increases the ratio of 3α to 3β alcohols produced by Raney nickel hydrogenation.
Hydrogenation of hydrocortisone acetate and of cortisone acetate in ethyl acetate in the presence of palladium on charcoal leads to the formation of appreciable amounts of 5β isomers. Raney nickel hydrogenation of IIb gives Vila and a low yield of Reichstein's compound C acetate. Similarly hydrogenation of VIb leads to Xa and a small amount of Reichstein's 11-dehydro compound C acetate. Independent syntheses of Reichstein's compound C and 11-dehydro C diacetate are reported. Raney nickel hydrogenation of 3-keto-5β compounds furnishes a mixture of 3α and 3β epimeric derivatives and is the basis of a synthesis of tetrahydro compound F.
The oxidative cyclization–carbonylation of propargylic esters mediated by Pd(II) afforded cyclic orthoesters, which were hydrolyzed into γ-acetoxy-β-ketoesters. Based on the NMR experiments, it was presumed that the cyclization reaction was initiated by a nucleophilic attack of carbonyl oxygen to the alkyne carbon coordinated to palladium(II). When the γ-acetoxy-β-ketoesters were treated with a basic condition, Knoevenagel–Claisen type condensation took place, and spiro furanone derivatives were obtained in good yields. We applied these reactions to steroid derivatives, and steroid derivatives having a spiro furanone fragment were synthesized. Among them, the spiro furanone 4j had vasorelaxant and bradycardiac activities. Compounds 2i–4k had inhibitory effect on CYP3A.
Carbon isotope ratio (CIR) analysis has been routinely and successfully applied to doping control analysis for many years to uncover the misuse of endogenous steroids such as testosterone. Over the years, several challenges and limitations of this approach became apparent, e.g., the influence of inadequate chromatographic separation on CIR values or the emergence of steroid preparations comprising identical CIRs as endogenous steroids. While the latter has been addressed recently by the implementation of hydrogen isotope ratios (HIR), an improved sample preparation for CIR avoiding co-eluting compounds is presented herein together with newly established reference values of those endogenous steroids being relevant for doping controls. From the fraction of glucuronidated steroids 5β-pregnane-3α,20α-diol, 5α-androst-16-en-3α-ol, 3α-Hydroxy-5β-androstane-11,17-dione, 3α-hydroxy-5α-androstan-17-one (ANDRO), 3α-hydroxy-5β-androstan-17-one (ETIO), 3β-hydroxy-androst-5-en-17-one (DHEA), 5α- and 5β-androstane-3α,17β-diol (5aDIOL and 5bDIOL), 17β-hydroxy-androst-4-en-3-one and 17α-hydroxy-androst-4-en-3-one were included. In addition, sulfate conjugates of ANDRO, ETIO, DHEA, 3β-hydroxy-5α-androstan-17-one plus 17α- and androst-5-ene-3β,17β-diol were considered and analyzed after acidic solvolysis. The results obtained for the reference population encompassing n = 67 males and females confirmed earlier findings regarding factors influencing endogenous CIR. Variations in sample preparation influenced CIR measurements especially for 5aDIOL and 5bDIOL, the most valuable steroidal analytes for the detection of testosterone misuse. Earlier investigations on the HIR of the same reference population enabled the evaluation of combined measurements of CIR and HIR and its usefulness regarding both steroid metabolism studies and doping control analysis. The combination of both stable isotopes would allow for lower reference limits providing the same statistical power and certainty to distinguish between the endo- or exogenous origin of a urinary steroid.
Carbon isotope ratio (CIR) analysis has been routinely and successfully used in sports drug testing for many years to uncover the misuse of endogenous steroids. One limitation of the method is the availability of steroid preparations exhibiting CIRs equal to endogenous steroids. To overcome this problem, hydrogen isotope ratios (HIR) of endogenous urinary steroids were investigated as a potential complement; results obtained from a reference population of 67 individuals are presented herein. An established sample preparation method was modified and improved to enable separate measurements of each analyte of interest where possible. From the fraction of glucuronidated steroids; pregnanediol, 16-androstenol, 11-ketoetiocholanolone, androsterone (A), etiocholanolone (E), dehydroepiandrosterone (D), 5α- and 5β-androstanediol, testosterone and epitestosterone were included. In addition, sulfate conjugates of A, E, D, epiandrosterone and 17α- and 17β-androstenediol were considered and analyzed after acidic solvolysis. The obtained results enabled the calculation of the first reference-population-based thresholds for HIR of urinary steroids that can readily be applied to routine doping control samples. Proof-of-concept was accomplished by investigating urine specimens collected after a single oral application of testosterone-undecanoate. The HIR of most testosterone metabolites were found to be significantly influenced by the exogenous steroid beyond the established threshold values. Additionally, one regular doping control sample with an extraordinary testosterone/epitestosterone ratio of 100 without suspicious CIR was subjected to the complementary methodology of HIR analysis. The HIR data eventually provided evidence for the exogenous origin of urinary testosterone metabolites. Despite further investigations on HIR being advisable to corroborate the presented reference-population-based thresholds, the developed method proved to be a new tool supporting modern sports drug testing procedures. Copyright © 2012 John Wiley & Sons, Ltd.
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.
The misuse of anabolic androgenic steroids (AAS) in human sports is controlled by gas chromatography-mass spectrometric analysis of urine specimens obtained from athletes. The analysis is improved with modern high-resolution mass spectrometry (HRMS). The detection and identification of metabolites of stanozolol (I) [3′-hydroxystanozolol (II) and 4β-hydroxystanozolol (III)] and metandienone (IV) [17β-methyl-5β-androst-1-ene-3α,17α-diol (V) and 18-nor-17,17-dimethyl-5β-androsta-1, 13-dien-3α-ol (VI)] with GC-HRMS at 3000 resolution yielded a large increase in the number of positive specimens. A total of 116 anabolic steroid positives were found in this laboratory in 1995 via GC-MS and GC-HRMS screening of 6700 human urine specimens collected at national and international sporting events and at out-of-competition testing. Of the 116 positive cases, 41 were detected using conventional (quadrupole) GC-MS screening. The other 75 positives were identified via GC-HRMS screening. To confirm the HRMS screening result, the urine sample was reanalyzed using a specific sample workup procedure to selectively isolate the metabolites of the identified substance. II and III were selectively isolated via immunoaffinity chromatography (IAC) using an antibody which was prepared for methyltestosterone and shows high cross reactivity to II and III. V and VI were isolated using high-performance liquid chromatography (HPLC) fractionation.
Zeranol ((7R,11S)-7,15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(14),15,17-trien-13-one, also referred to as 7α-zearalanol, Ralone®, Frideron®, Ralgro®, etc.) is a semi-synthetic estrogenic veterinary drug with growth-promoting properties. Its use regarding animal husbandry has been prohibited in the European Union since 1981 and, due to its anabolic effects, it is further recognized as a banned substance in sport. Numerous studies were conducted concerning the identification of the illicit application of zeranol to domestic livestock. These studies also considered the natural occurrence of zeranol as a metabolite of the mycotoxin zearalenone and the issue of differentiating both scenarios, i.e. illegal use or unintended contamination. Human sports drug testing authorities are facing comparable challenges since the deliberate misuse of the (for human application non-approved) drug should be discriminated from adverse analytical findings resulting from the biotransformation of the mycotoxin zearalenone possibly ingested with contaminated food. The active drug (zeranol), its major human metabolites (zearalanone, 7β-zearalanol) and the mycotoxin (zearalenone) plus its major and unique metabolic products (α-zearalenol, β-zearalenol) have been monitored in routine doping controls by means of validated gas chromatography-(tandem) mass spectrometry (GC-(MS/)MS) methods since 1996, and between 2005 and 2010 four samples providing suspicious signals were detected. In agreement with literature data, in vitro metabolism studies demonstrated the metabolic pathway from zearalenone towards zeranol (and common metabolites). In contrast, an administration study urine sample (collected after oral application of 20 mg of zeranol) yielded only ultra-trace amounts of zearalenone and its characteristic metabolites, which supported the assumption that a mycotoxin contamination caused the finding of zeranol in the doping control specimens rather than a misuse of the anabolic agent.
Recently, pharmaceutical industry developed a new class of therapeutics called Selective Androgen Receptor Modulator (SARM) to substitute the synthetic anabolic drugs used in medical treatments. Since the beginning of the anti-doping testing in sports in the 1970s, steroids have been the most frequently detected drugs mainly used for their anabolic properties. The major advantage of SARMs is the reduced androgenic activities which are the main source of side effects following anabolic agents' administration. In 2010, the Swiss laboratory for doping analyses reported the first case of SARMs abuse during in-competition testing. The analytical steps leading to this finding are described in this paper. Screening and confirmation results were obtained based on liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses. Additional information regarding the SARM S-4 metabolism was investigated by ultra high-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS).
A novel steroid compound, (17α,20E)-17,20-[(1-methoxyethylidene)bis(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11), was found to be a partial agonist of the androgen receptor (AR) in an androgen responsive element (ARE)-luciferase reporter assay. YK11 accelerates nuclear translocation of AR. Furthermore, YK11 does not induce amino/carboxyl-terminal (N/C) interaction and prevents 5-α-dihydrotestosterone (DHT)-mediated N/C interaction. Thus, YK11 activates AR without causing N/C interaction, which may in turn be responsible for the partially agonistic nature of YK11 observed in the ARE-luciferase reporter system. YK11 acts as a gene-selective agonist of AR in MDA-MB 453 cells. The effect of YK11 on gene expression relative to that of androgen agonist varies depending on the gene context. YK11 activated the reporter gene by inducing the translocation of the AR into the nuclear compartment, where its amino-terminal domain (NTD) functions as a constitutive activator of AR target genes. Our results suggest that YK11 might act as selective androgen receptor modulator (SARM).
The application of a comprehensive gas chromatography/combustion/isotope ratio mass spectrometry-based method for the measurement of stable carbon isotopes of endogenous urinary steroids excreted as sulphates is presented. The key element in sample preparation is the consecutive cleanup with high-performance liquid chromatography of underivatized and acetylated steroids, which allows the isolation of seven analytes (pregn-5-ene-3β,17α,20α-triol, etiocholanolone, androsterone, epiandrosterone, dehydroepiandrosterone (DHEA), androst-5-ene-3β,17β-diol and androst-5-ene-3β,17α-diol) from a single urine specimen. These steroids are of particular importance to doping controls as they should enable the sensitive and retrospective detection of DHEA abuse by athletes.
Depending on the biological background, the determination limit for all steroids ranges from 5 to 10 ng/mL for a 10 mL specimen. The method is validated by means of linear mixing models for each steroid, which covers the items, repeatability and reproducibility. The specificity was further demonstrated by gas chromatography/mass spectrometry for each analyte, and no influence of the sample preparation or the quantity of analyte on carbon isotope ratios was observed. In order to determine naturally occurring 13C/12C ratios and urinary concentrations of all implemented steroids, a reference population of n = 67 subjects was measured to enable the calculation of reference limits for all relevant steroidal Δ values.
The applicability of the developed method was tested by means of a DHEA excretion study. Despite the fact that orally ingested DHEA is preferentially converted into sulphated metabolites and that the renal clearance of sulphated steroids is slow, only the 13C/12C ratio of EpiA demonstrated the potential to prolong the detection time for DHEA misuse. Copyright
After oral administration of metandienone (17 alpha-methyl-androsta-1,4-dien-17 beta-ol-3-one) to male volunteers conjugated metabolites are isolated from urine via XAD-2-adsorption, enzymatic hydrolysis and preparative high-performance liquid chromatography (HPLC). Four conjugated metabolites are identified by gas chromatography-mass spectrometry (GC/MS) with electron impact (EI)-ionization after derivatization with N-methyl-N-trimethyl-silyl-trifluoroacetamide/trimethylsilyl-imidazole (MSTFA/TMS-Imi) and comparison with synthesized reference compounds: 17 alpha-methyl-5 beta-androst-1-en-17 beta-ol-3-one (II), 17 alpha-methyl-5 beta-androst-1-ene-3 alpha,17 beta-diol (III), 17 beta-methyl-5 beta-androst-1-ene-3 alpha,17 alpha-diol (IV) and 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol (V). After administration of 40 mg of metandienone four bis-hydroxy-metabolites--6 beta,12-dihydroxy-metandienone (IX), 6 beta,16 beta-dihydroxy-metandienone (X), 6 beta,16 alpha-dihydroxy-metandienone (XI) and 6 beta,16 beta-dihydroxy-17-epimetandienone (XII)--were detected in the unconjugated fraction. The metabolites III, IV and V are excreted in a comparable amount to the unconjugated excreted metabolites 17-epimetandienone (VI), 6 beta-hydroxy-metandienone (VII) and 6 beta-hydroxy-17-epimetandienone (VIII). Whereas the unconjugated excreted metabolites show maximum excretion rates between 4 and 12 h after administration the conjugated metabolites III, IV and V are excreted with maximum rates between 12 and 34 h.
Anabolic androgenic steroids (AAS) are misused to a high extent in sports by athletes to improve their physical performance. Sports federations consider the use of these drugs in sports as doping. The misuse of AAS is controlled by detection of the parent AAS (when excreted into urine) and (or) their metabolites in urine of athletes. I present a review of the metabolism of AAS. Testosterone is the principal androgenic steroid and its metabolism is compared with that of AAS. The review is divided into two parts: the general metabolism of AAS, which is separated into phase I and phase II metabolism and includes a systematic discussion of metabolic changes in the steroid molecule according to the regions (A-D rings), and the specific metabolism of AAS, which presents the metabolism of 26 AAS in humans.
The use of anabolic steroids in sports is prohibited by the World Anti-Doping Agency. Until the 1990s, anabolic steroids were solely manufactured by pharmaceutical companies, albeit sometimes on demand from national sports agencies as part of their doping program. Recently the list of prohibited anabolic steroids in sports has grown due to the addition of numerous steroids that have been introduced on the market by non-pharmaceutical companies. Moreover, several designer steroids, specifically developed to circumvent doping control, have also been detected. Because anabolic steroids are most often intensively subjected to phase I metabolism and seldom excreted unchanged, excretion studies need to be performed in order to detect their misuse. This review attempts to summarise the results of excretion studies of recent additions to the list of prohibited steroids in sports. Additionally an update and insight on new aspects for "older" steroids with respect to doping control is given.
The application of a comprehensive gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS)-based method for stable carbon isotopes of endogenous urinary steroids is presented. The key element in sample preparation is the consecutive cleanup with high-performance liquid chromatography (HPLC) of underivatized and acetylated steroids, which allows the isolation of ten analytes (11β-hydroxyandrosterone, 5α-androst-16-en-3β-ol, pregnanediol, androsterone, etiocholanolone, testosterone, epitestosterone, 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol and dehydroepiandrosterone) from a single urine specimen. These steroids are of particular importance to doping controls as they enable the sensitive and retrospective detection of steroid abuse by athletes.
Depending on the biological background, the determination limit for all steroids ranges from 5 to 10 ng/mL for a 10 mL specimen. The method is validated by means of linear mixing models for each steroid, which covers repeatability and reproducibility. Specificity was further demonstrated by gas chromatography/mass spectrometry (GC/MS) for each analyte, and no influence of the sample preparation or the quantity of analyte on carbon isotope ratios was observed. In order to determine naturally occurring 13C/12C ratios of all implemented steroids, a reference population of n = 61 subjects was measured to enable the calculation of reference limits for all relevant steroidal Δ values. Copyright
Steroid profiling is one of the most versatile and informative screening tools for the detection of steroid abuse in sports drug testing. Concentrations and ratios of various endogenously produced steroidal hormones, their precursors and metabolites including testosterone (T), epitestosterone (E), dihydrotestosterone (DHT), androsterone (And), etiocholanolone (Etio), dehydroepiandrosterone (DHEA), 5alpha-androstane-3alpha,17beta-diol (Adiol), and 5beta-androstane-3alpha,17beta-diol (Bdiol) as well as androstenedione, 6alpha-OH-androstenedione, 5beta-androstane-3alpha,17alpha-diol (17-epi-Bdiol), 5alpha-androstane-3alpha,17alpha-diol (17-epi-Adiol), 3alpha,5-cyclo-5alpha-androstan-6beta-ol-17-one (3alpha,5-cyclo), 5alpha-androstanedione (Adion), and 5beta-androstanedione (Bdion) add up to a steroid profile that is highly sensitive to applications of endogenous as well as synthetic anabolic steroids, masking agents, and bacterial activity. Hence, the knowledge of factors that do influence the steroid profile pattern is a central aspect, and pharmaceutical (application of endogenous steroids and various pharmaceutical preparations), technical (hydrolysis, derivatization, matrix), and biological (bacterial activities, enzyme side activities) issues are reviewed.
20-diene −21-carboxylic acid methyl ester (YK11) is a partial agonist of the androgen receptor
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