Effect of HepG2 cell 3D cultivation on the metabolism of the anabolic androgenic steroid metandienone.

The elucidation of the metabolic fate of prohibited substances is crucial for the abuse detection. The human hepatocyte cell line HepG2 can be used to study biotransformation. In order to improve this in vitro model system, we compared the HepG2 spheroid generation using three different techniques: a forced floating, a scaffold-free and a scaffold-based method. We characterized the spheroids with regard to the expression levels of the proliferation marker Mki67, the liver-specific marker albumin and biotransformation enzymes. Moreover, the metandienone metabolite pattern was comparatively analysed by high-performance liquid chromatography mass spectrometry. With all three techniques, HepG2 spheroids were generated showing a degree of differentiation. The forced floating method resulted in very large spheroids (1 mm in diameter) showing signs of necrosis in the centre and a very low metandienone conversion rate. The spheroids formed by the two other techniques were comparable in size with 0.5 mm in diameter on average. Among the three different 3D cultivation methods, the HepG2 spheroids formed on Matrigel® as extracellular matrix were the most promising regarding biotransformation studies on anabolic androgenic steroids. Prospectively, HepG2 spheroids are a promising in vitro model system to study multidrug setups, drug-drug interactions and the biotransformation of other substance classes.

Experimental studies on androgen administration in animal models: current and future perspectives.

PURPOSE OF REVIEW: This review aims to report the most recent (2020-2022) experimental scientific studies conducted on animal models, in order to highlight the relevant findings on the adverse effects related to androgen administration. RECENT FINDINGS: Forty-one studies published between January 2020 and July 2022 were selected. The majority of studies investigated the effects of one androgen, whereas only four studies analyzed the effects of two drugs. Nandrolone decanoate was the most investigated drug (20 articles), boldenone was tested in 8 articles, testosterone and stanozolol were used in 7 articles each, 17b-trenbolone, metandienone, and oxandrolone were tested in 1 article each. The articles clarify the adverse effects of androgen administration on the heart, brain, kidney, liver, reproductive and musculoskeletal systems. SUMMARY: The main findings of this review highlight that androgen administration increases inflammatory mediators, altering different biochemical parameters. The results concerning the reversibility of the adverse effects are controversial: on the one hand, several studies suggested that by stopping the androgen administration, the organs return to their initial state; on the other hand, the alteration of different biochemical parameters could generate irreversible organ damage. Moreover, this review highlights the importance of animal studies that should be better organized in order to clarify several important aspects related to androgen abuse to fill the gap in our knowledge in this research field.

Detectability of oxandrolone, metandienone, clostebol and dehydrochloromethyltestosterone in urine after transdermal application.

Situations of both, intentional and inadvertent or accidental doping, necessitate consideration in today's doping controls, especially in the light of the substantial consequences that athletes are facing in case of so-called adverse analytical findings. The aim of this study was to investigate, whether a transdermal uptake of doping substances would be possible. In addition to the period of detectability of the particular substances or respective characteristic metabolites, the possibility of deducing the route of administration by metabolite patterns was also assessed. Twelve male subjects were included in the study. Four common anabolic androgenic steroids (AAS) were dissolved in dimethylsulfoxide to facilitate transdermal administration on different skin regions. One half of the test persons received only oxandrolone (17α-methyl-2-oxa-4,5α-dihydrotestosterone), and the other half were applied a mixture of oxandrolone, metandienone (17ß-hydroxy-17α-methylandrosta-1,4-dien-3-one), clostebol (4-chlorotestosterone-17ß-acetate) and dehydrochloromethyltestosterone (DHCMT). Urine samples were collected 1 h, 6 h and one sample per day for the next 14 consecutive days. Measurements were conducted on a tandem-gas chromatography-mass spectrometry (GC-MS/MS) or tandem-liquid chromatography-MS/MS (LC-MS/MS) system. Substance findings were obtained at least 1 day after application on nearly all skin locations. The results indicated inter-individual variability in detection windows, also varying between the different analytes and possible impact of skin location and skin thickness, respectively. Nevertheless, a rapid and rather long detectability of all substances (or respective metabolites) was given, in some cases within hours after administration and for up to 10-14 days. Hence, the transdermal application or exposure to the investigated AAS is a plausible scenario that warrants consideration in anti-doping.

Medaka embryos as a model for metabolism of anabolic steroids.

In anti-doping science, the knowledge of drug metabolism is a prerequisite to identify analytical targets for the detection of misused prohibited substances. As the most obvious way to study xenobiotic metabolism, the administration to human volunteers, faces ethical concerns, there is a need for model systems. In the present study, we investigated whether Oryzias latipes (medaka) embryos might be an alternative, non-animal test model to study human-like metabolism. In the present study, we exposed medaka embryos at the morula stage to the anabolic steroid metandienone (10 µM or 50 µM) for a period of 2 or 8 days. According to the fish embryo toxicity test (OECD test), we assessed the developmental status of the embryos. We further investigated metandienone metabolites by high-performance liquid chromatography- and gas chromatography-mass spectrometry. Medaka embryos produced three mono-hydroxylated and one reduced metabolite known from human biotransformation. Developmental malformations were observed for the exposition to 50 µM metandienone, while a significant elevation of the heart beat was also present in those individuals exposed to the lower dose for 8 days. The present study demonstrates that the medaka embryo represents a promising model to study human-like metabolism. Moreover, the judgement of developmental parameters of the fish embryos enables for the simultaneous assessment of toxicity.

HepG2 as promising cell-based model for biosynthesis of long-term metabolites: Exemplified for metandienone.

In order to detect the abuse of substances in sports, the knowledge of their metabolism is of undisputable importance. As in vivo administration of compounds faces ethical problems and might even not be applicable for nonapproved compounds, cell-based models might be a versatile tool for biotransformation studies. We coincubated HepG2 cells with metandienone and D3 -epitestosterone for 14 days. Phase I and II metabolites were analyzed by high-performance liquid chromatography (HPLC)-tandem mass spectrometry and confirmed by gas chromatography-mass spectrometry (GC-MS). The metandienone metabolites formed by HepG2 cells were comparable with those renally excreted by humans. HepG2 cells also generated the two long-term metabolites 17ß-hydroxymethyl-17α-methyl-18-nor-androst-1,4,13-trien-3-one and 17α-hydroxymethyl-17ß-methyl-18-nor-androst-1,4,13-trien-3-one used in doping analyses, though in an inverse ratio compared with that observed in human urine. In conclusion, we showed that HepG2 cells are suitable as model for the investigation of biotransformation of androgens, especially for the anabolic androgenic steroid metandienone. They further proved to cover phase I and II metabolic pathways, which combined with a prolonged incubation time with metandienone resulted in the generation of its respective long-term metabolites known from in vivo metabolism. Moreover, we showed the usability of D3 -epitestosterone as internal standard for the incubation. The method used herein appears to be suitable and advantageous compared with other models for the investigation of doping-relevant compounds, probably enabling the discovery of candidate metabolites for doping analyses.

A Highly Sensitive Modified Glassy Carbon Electrode with a Carboxylated Multi-walled Carbon Nanotubes/Nafion Nano Composite for Voltammetric Sensing of Dianabol in Biological Fluid.

The extraordinary prerequisite for the analysis of an anabolic steroid, namely dianabol (DB), has inspired towards the development of a cost-effective and high-performance sensing probe. Thus, a simple and robust electrochemical sensor (c-MWCNTs-Nafion®lGCE) for dianabol (DB), a widely used steroid, was developed using a glassy carbon electrode (GCE) modified with functionalized carboxylated multi-walled carbon nanotubes (c-MWCNT) and Nafion®. At pH 7 - 8, differential pulse-cathodic stripping voltammetry (DP-CSV) displayed two cathodic peaks at -0.85 and -1.35 V that varied linearly over a wide range (9.0 × 10-9 (2.7 µg L-1) - 9.0 × 10-6 (2.7 × 103 µg L-1) mol L-1) and 2.9 × 10-6 (8.7 × 102 µg L-1) - 8.0 × 10-5 (2.4 × 104 µg L-1) mol L-1) of DB concentrations, respectively. The low limits of detection and quantification at peak I (-0.85 V) were 2.7 × 10-9 (8.1 × 10-1 ng mL-1) and 9.0 × 10-9 (2.7 ng mL-1) mol L-1, respectively. The repeatability and reproducibility displayed relative standard deviations lower than 5%. The method was applied for DB analysis in human urine and subsequently compared with the standard HPLC method. Interference of common metabolites in biological fluids samples to DB sensing was insignificant. This method has distinctive advantages e.g. precise, short analytical time, sensitive, economical, reproducible and miniaturized sample preparation for DB analysis in biological samples of human origin.

New Insights into the Metabolism of Methyltestosterone and Metandienone: Detection of Novel A-Ring Reduced Metabolites.

Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a 17α-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. Following the previously reported detection of long-term metabolites with a 17ξ-hydroxymethyl-17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone ("oral turinabol"), in this study we investigated the formation of similar metabolites of metandienone and 17α-methyltestosterone with a rearranged D-ring and a fully reduced A-ring. Using a semi-targeted approach including the synthesis of reference compounds, two diastereomeric substances, viz. 17α-hydroxymethyl-17ß-methyl-18-nor-5ß-androst-13-en-3α-ol and its 5α-analog, were identified following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5ß-metabolite was detected. Additionally, 3α,5ß-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides the classical metabolites included in the screening procedures. Besides their applicability for anti-doping analysis, the results provide new insights into the metabolism of 17α-methyl steroids with respect to the order of reductions in the A-ring, the participation of different enzymes, and alterations to the D-ring.

Anabolic steroids and extreme violence: a case of murder after chronic intake and under acute influence of metandienone and trenbolone.

A 32-year-old male went to the police to claim he just killed his girlfriend by inflicting several stabs with a kitchen knife. He was very nervous and particularly aggressive. About 90 min after the assault, a blood specimen was collected with natrium fluoride as preservative. The blood was free of alcohol, pharmaceuticals and drugs of abuse, but tested positive by LC-MS/MS for metandienone (32 ng/mL) and trenbolone (9 ng/mL). The perpetrator admitted regular consumption of anabolic steroids to enhance his muscular mass, as he was a professional security agent. To document long-term steroid abuse, a hair specimen was collected 3 weeks after the assault, which tested positive for both drugs. Segmental analyses revealed in the proximal 1.5 cm segment, corresponding to the period of the assault, the simultaneous presence of metandienone (11 pg/mg) and trenbolone (14 pg/mg), while only metandienone (3 pg/mg) was identified in the distal 1.5 cm segment. As aggressiveness and violence can be associated with abuse of anabolic steroids, the aetiology of this domestic crime was listed to be due impulsive behaviour in a context of antisocial lifestyle.

Death after misuse of anabolic substances (clenbuterol, stanozolol and metandienone).

A 34-year old male was found breathless and panting at home by his girlfriend three hours after a gym workout. Minutes later, he collapsed and died. Autopsy, histological and chemical analyses were conducted. The examination of the heart showed left ventricular hypertrophy, while the right coronary artery showed only a small vascular lumen (3 mm in diameter), due to its anatomical structure. In femoral blood concentrations of approx. 1 µg/L clenbuterol, approx. 56 µg/L stanozolol and approx. 8 µg/L metandienone, with trenbolone (

Modulation of exercise training related adaptation of body composition and regulatory pathways by anabolic steroids.

Anabolic steroids have a long history of abuse in amateur and professional athletics. However, their interaction with training and the resulting effects on body composition and tissue adaptation, relying on a concert of factors and pathways, remain under investigation. This study aims at investigating the changes of body composition and the expression of selected genes and pathways essential for this adaptation process. Therefore, male wistar rats were treated with the anabolic steroid metandienone in two groups (n = 16; metandienone, metandienone + exercise) alongside with control groups (n = 16; control, exercise). Following a 6-week steep-angle treadmill training protocol, weight of organs, visceral fat and muscles was determined. M. gastrocnemius was histologically assessed by ATPase staining, mRNA and protein levels of factors of regeneration, hypertrophy and myogenesis and selected master regulators and markers were determined. Results show additive effects of anabolic steroids and exercise on body, tibia and reproductive organs weight. Mm. gastrocnemius and soleus weight was increased by training but not anabolic steroids. Muscle fiber diameter and composition remained unchanged. Visceral fat mass and fat cell size was affected by training and anabolic steroids but no additive effects could be observed. Exercise and anabolic steroids result in a complex regulation of the expression of genes in M. Gastrocnemius involved in skeletal muscle metabolism, hypertrophy, inflammation and regeneration. In summary, our data suggests distinct molecular mechanisms involved in the adaptation of the skeletal muscle to anabolic androgenic steroids and exercise. Metandienone treatment neither results in skeletal muscle hypertrophy nor liver-toxic effects but in an induction of skeletal muscle regeneration and an activation of endocrine negative feedback. Moreover our study demonstrates that visceral fat and bone responds with higher sensitivity to ASS and exercise than the skeletal muscle. This apparent plasticity of adipose and bone tissue rather than skeletal muscle could indicate a potentially superior future role of fat rather than muscle related parameters to detect and AAS abuse in a biologic passport strategy in professional athletes.

Isotopically labeled boldenone as a better marker of derivatization efficiency for improved quality control in anti-doping analysis.

At present, anti-doping laboratories use androsterone, a major urinary steroid metabolite, to evaluate completeness of the derivatization step. This is typically done by calculating the ratio of mono-trimethylsilyl (TMS) androsterone to the total mono- and di-TMS androsterone. Certain samples may show an elevated percentage of mono-TMS androsterone indicating a failed derivatization step. In such cases, the laboratory would have to repeat the analysis or perform other remedial actions to ensure that completeness of derivatization is achieved. We have noticed that a poorly derivatized positive control sample spiked with various target analytes has a disproportionally low abundance of the di-TMS derivatives of boldenone and 18-nor-17ß-hydroxymethyl-17α-methylandrosta-1,4,13-trien-3-one (methandienone long-term metabolite). A follow-up investigation confirmed that 1,4-diene-3-one steroids are more likely to fail during the trimethylsilylation step. To better control derivatization efficiency, 13 C3 -boldenone (13C-BLD) was incorporated into our routine procedure as an additional internal standard. Analysis of a large number of urine samples has shown that derivatization of 13C-BLD could be grossly incomplete even in cases when mono-TMS androsterone is well below 1%. In other words, one or both of boldenone and the long-term metabolite of methandienone could remain undetected unless the laboratory has the means to recognize samples where derivatization of 1,4-diene-3-one steroids failed.

Side effects of anabolic steroids used by athletes at Unaizah Gyms, Saudi Arabia: a pilot study.

BACKGROUND: A large number of Saudi athletes are recently shown to use androgenic anabolic steroid (AAS) products to achieve rapid muscle growth without realizing the serious health risks of these drugs. Aim of this study was to elucidate the side effects encountered with prolonged use of AAS products by Saudi athletes. METHODS: A cross-sectional study was conducted, in which 16 regular gym members, 12 of them used AAS, were asked to answer a questionnaire and provide blood samples following current AAS course completion. Hemoglobin, serum proteins, lipid profile and hematological parameters were measured. Meanwhile, the parameters of kidneys, liver, heart, and immune system function were monitored. RESULTS: The subjects reported taking a 3-month course of an AAS comprising three compounds (testosterone enanthate, nandrolone decanoate and methandienone). A two-week gap separated every two courses, during which tamoxifen citrate (40 mg per day) and clomiphene citrate (10 mg per day) were taken to control serum testosterone levels. The intake of AAS one course had remarkable effects on some parameters related to kidney function. However, AAS three courses or more treatments showed abnormal liver and heart enzymes. Moreover, endogenous testosterone levels decreased dramatically with prolonged use of AAS (more than 10 courses). Alpha 2 protein increased by taking more than 10 courses, which might cause acute phase reactant of liver infection or inflammation. CONCLUSIONS: AAS products must be controlled by Saudi ministry of health and should not be taken randomly without the supervision of the healthcare professional.

Drug-drug interaction and doping: Effect of non-prohibited drugs on the urinary excretion profile of methandienone.

The potential consequences of drug-drug interactions on the excretion profile of the anabolic androgenic steroid methandienone (17ß-hydroxy-17α-methylandrosta-1,4-dien-3-one) are discussed. More specifically, we have evaluated by in vitro and in vivo experiments the effects of 7 non-prohibited drugs (fluconazole, ketoconazole, itraconazole, miconazole, fluoxetine, paroxetine, and nefazodone) on the main metabolic pathways of methandienone. These are selected among those most commonly used by the athletes. The in vitro assays were based on the use of human liver microsomes, specific recombinant enzyme isoforms of cytochrome P450 and uridine 5'-diphospho-glucuronosyl-transferase. The in vivo study was performed by analyzing urines collected after the oral administration of methandienone with and without the co-administration of ketoconazole. Methandienone and its metabolites were determined by liquid chromatography-mass spectrometry-based techniques after sample pretreatment including an enzymatic hydrolysis step (performed only for the investigation on phase II metabolism) and liquid/liquid extraction with t-butyl methyl-ether. The results from the in vitro experiments showed that the formation of the hydroxylated and dehydrogenated metabolites was significantly reduced in the presence of itraconazole, ketoconazole, miconazole and nefazodone, whereas the production of the 18-nor-hydroxylated metabolites and glucuronidation reactions was reduced significantly only in the presence of ketoconazole and miconazole. The analysis of the post-administration samples confirmed the in vitro observations, validating the hypothesis that drug-drug interaction may cause significant alterations in the metabolic profile of banned drugs, making their detection during doping control tests more challenging.

Sensitive enzyme immunoassay for screening methandienone in dietary supplements.

Methandienone is a synthetic exogenous steroid which, like other anabolic steroids, is strictly regulated in many countries. In recent years, increasing numbers have been detected of illegal additions into dietary supplements of methandienone and other anabolic androgenic steroids (AAS). In this work, a competitive indirect enzyme-linked immunosorbent assay (ELISA) has been constructed for the detection of methandienone using an antiserum against methandienone. Under optimal experimental conditions, the ELISA achieved a limit of detection of 0.04 ± 0.01 µg.g-1. The obtained intra- and inter-day coefficients of variation were less than 8%. The developed ELISA was applied in the analysis of real dietary supplement samples. To minimise the effect of the sample matrix, the sample extracts were simply diluted before addition into the immunoassay. The achieved recovery values were around 100%. Results obtained from the ELISA correlated well, both in terms of accuracy and precision, with those obtained by UHPLC-MS/MS (reference method). The presented ELISA could be successfully applied for the simple screening of dietary supplements.

Sulfate metabolites as alternative markers for the detection of 4-chlorometandienone misuse in doping control.

Sulfate metabolites have been described as long-term metabolites for some anabolic androgenic steroids (AAS). 4-chlorometandienone (4Cl-MTD) is one of the most frequently detected AAS in sports drug testing and it is commonly detected by monitoring metabolites excreted free or conjugated with glucuronic acid. Sulfation reactions of 4Cl-MTD have not been studied. The aim of this work was to evaluate the sulfate fraction of 4Cl-MTD metabolism by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to establish potential long-term metabolites valuable for doping control purposes. 4Cl-MTD was administered to two healthy male volunteers and urine samples were collected up to 8 days after administration. A theoretical selected reaction monitoring (SRM) method working in negative mode was developed. Ion transitions were based on ionization and fragmentation behaviour of sulfate metabolites as well as specific neutral losses (NL of 15 Da and NL of 36 Da) of compounds with related chemical structure. Six sulfate metabolites were detected after the analysis of excretion study samples. Three of the identified metabolites were characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS). Results showed that five out of the six identified sulfate metabolites were detected in urine up to the last collected samples from both excretion studies. Copyright © 2016 John Wiley & Sons, Ltd.

The binding properties of metandienone and human serum albumin by comparing with other five similar compounds.

Metandienone (MET) is an exogenous anabolic androgenic steroid. The interaction between MET and human serum albumin (HSA) was investigated by molecular modeling and different optical techniques. There was no possibility of energy transfer, and the fluorescence quenching of HSA induced by MET was mainly due to the complex formation. The differences of binding ability between MET and compounds 1-5 were significantly caused by space steric hindrance. The single crystallographic data of two steroids (compounds 4 and 5) were obtained in the methanol at the first time. In addition, the binding ability was slightly affected by -OH, -CH3 , and -COCH3 . The results of displacement experiment demonstrated that the MET binding site was mainly located in site 1 of HSA. H-bonding and van der Waals forces were significant in the MET-HSA binding. MET played an insignificant role on the local conformation change in HSA.

LC-MS/MS detection of unaltered glucuronoconjugated metabolites of metandienone.

The aim of this study was to evaluate the direct detection of glucuronoconjugated metabolites of metandienone (MTD) and their detection times. Metabolites resistant to enzymatic hydrolysis were also evaluated. Based on the common mass spectrometric behaviour of steroid glucuronides, three liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategies were applied for the detection of unpredicted and predicted metabolites: precursor ion scan (PI), neutral loss scan (NL), and theoretical selected reaction monitoring (SRM) methods. Samples from four excretion studies of MTD were analyzed for both the detection of metabolites and the establishment of their detection times. Using PI and NL methods, seven metabolites were observed in post-administration samples. SRM methods allowed for the detection of 13 glucuronide metabolites. The detection times, measured by analysis with an SRM method, were between 1 and 22 days. The metabolite detected for the longest time was 18-nor-17ß-hydroxymethyl-17α-methyl-5ß-androsta-1,4,13-triene-3-one-17-glucuronide. One metabolite was resistant to hydrolysis with ß-glucuronidase; however it was only detected in urine up to four days after administration. The three glucuronide metabolites with the highest retrospectivity were identified by chemical synthesis or mass spectrometric data, and although they were previously reported, this is the first time that analytical data of the intact phase II metabolites are presented for some of them. The LC-MS/MS strategies applied have demonstrated to be useful for detecting glucuronoconjugated metabolites of MTD, including glucuronides resistant to enzymatic hydrolysis which cannot be detected by conventional approaches. Copyright © 2016 John Wiley & Sons, Ltd.

Synthesis of 17ß-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one: A long-term metandienone metabolite.

The goal of this work was a good-yielding chemical synthesis of a metandienone metabolite which is of interest in doping analysis. 20ßOH-NorMD (IUPAC: 17ß-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-triene-3-one) has been identified as a long-term urinary metabolite which can be detected and attributed to metandienone up to almost 3weeks after exposure. The chemical synthesis of its epimer 20αOH-NorMD has been described before, as was an enzymatic synthesis of 20ßOH-NorMD, but no chemical synthesis was published.

C-H Acetoxylation-Based Chemical Synthesis of 17 ß-Hydroxymethyl-17 α-methyl-18-norandrost-13-ene Steroids.

Palladium-catalyzed C-H acetoxylation has been proposed as a key transformation in the first chemical synthesis of steroids bearing a unique 17ß-hydroxymethyl-17α-methyl-18-nor-13-ene D-fragment. This C-H functionalization step was crucial for inverting the configuration at the quaternary stereocenter of a readily available synthetic intermediate. The developed approach was applied to prepare the metandienone metabolite needed as a reference substance in anti-doping analysis to control the abuse of this androgenic anabolic steroid.

Efficient approach for the detection and identification of new androgenic metabolites by applying SRM GC-CI-MS/MS: a methandienone case study.

Identification of anabolic androgenic steroids (AAS) is a vital issue in doping control and toxicology, and searching for metabolites with longer detection times remains an important task. Recently, a gas chromatography chemical ionization triple quadrupole mass spectrometry (GC-CI-MS/MS) method was introduced, and CI, in comparison with electron ionization (EI), proved to be capable of increasing the sensitivity significantly. In addition, correlations between AAS structure and fragmentation behavior could be revealed. This enables the search for previously unknown but expected metabolites by selection of their predicted transitions. The combination of both factors allows the setup of an efficient approach to search for new metabolites. The approach uses selected reaction monitoring which is inherently more sensitive than full scan or precursor ion scan. Additionally, structural information obtained from the structure specific CI fragmentation pattern facilitates metabolite identification. The procedure was demonstrated by a methandienone case study. Its metabolites have been studied extensively in the past, and this allowed an adequate evaluation of the efficiency of the approach. Thirty three metabolites were detected, including all relevant previously discovered metabolites. In our study, the previously reported long-term metabolite (18-nor-17ß-hydroxymethyl,17α-methyl-androst-1,4,13-trien-3-one) could be detected up to 26 days by using GC-CI-MS/MS. The study proves the validity of the approach to search for metabolites of new synthetic AAS and new long-term metabolites of less studied AAS and illustrates the increase in sensitivity by using CI. Copyright © 2016 John Wiley & Sons, Ltd.