Search

Anabolic steroids and β-agonists are commonly prohibited substances found in doping control studies; therefore, the determination of anabolic substances in biological samples is crucial. To analyze the anabolic compounds in urine, an adsorbent, polyethylene glycol (PEG)-grafted magnetic nanoparticle material (Fe 3 O 4 @SiO 2 -PEG), with low toxicity and strong biocompatibility was prepared in this investigation. Compared to those of Fe 3 O 4 and Fe 3 O 4 @SiO 2 , the grafted PEG chains (approximately 5.4 wt.%) on the magnetic nanoparticles improved the extraction efficiencies by factors of 3.9-17.0 and 2.5-2.9, respectively, likely due to the electrostatic attraction and hydrogen bonding. To achieve maximum extraction efficiency, several extraction parameters were optimized, including the kind and volume of desorption solvent, pH, and the extraction and desorption time. The standard curves were linear within the range of 0.5-20 μg/L for methyltestosterone and trenbolone, and 0.02-5 μg/L for clenbuterol. The limits of detection for the three drugs were 0.01-0.12 μg/L. The limits of quantification were 0.02-0.40 μg/L. The levels of precision of the optimized method were assessed based on the respective intra- and inter-day and batch-to-batch relative standard deviations in the ranges of 3.2-5.2 % (n = 5), 5.9-11.3 % (n = 4), and 6.7-9.2 % (n = 3). The Fe 3 O 4 @SiO 2 -PEG nanoparticles could exclude urine matrix interferences (matrix effect of 91.8-98.1 %) and achieve satisfactory recoveries (75.5-116.1 %), affording sensitive and accurate determination of trace anabolic substances in urine., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ostarine (enobasarm) is a selective androgen receptor modulator with great therapeutic potential. However, it is also used by athletes to promote muscle growth and enhance performances without the typical adverse effects of anabolic steroids. Ostarine popularity increased in recent years, and it is currently the most abused "other anabolic agent" (subclass S1.2. of the "anabolic agents" class S1) from the World Anti-Doping Agency's (WADA) prohibited list. Several cases of liver toxicity were recently reported in regular users. Detecting ostarine or markers of intake in biological matrices is essential to document ostarine use in doping. Therefore, we sought to investigate ostarine metabolism to identify optimal markers of consumption. The substance was incubated with human hepatocytes, and urine samples from six ostarine-positive cases were screened. Analyses were performed via liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) and software-assisted data mining, with in silico metabolite predictions. Ten metabolites were identified with hydroxylation, ether cleavage, dealkylation, O -glucuronidation, and/or sulfation. The production of cyanophenol-sulfate might participate in the mechanism of ostarine liver toxicity. We suggest ostarine-glucuronide (C 25 H 22 O 9 N 3 F 3 , diagnostic fragments at m / z 118, 185, and 269) and hydroxybenzonitrile-ostarine-glucuronide (C 25 H 22 O 10 N 3 F 3 , diagnostic fragments at m / z 134, 185, and 269) in non-hydrolyzed urine and ostarine and hydroxybenzonitrile-ostarine (C 19 H 14 O 4 N 3 F 3 , diagnostic fragments at m / z 134, 185, and 269) in hydrolyzed urine as markers to document ostarine intake in doping.

Because of their performance-enhancing effect, anabolic androgenic steroids (AAS) are often misused in sports. Nearly half of the adverse analytical findings (AAF) in 2022 doping controls are correlated to AAS misuse. Metabolites play a crucial role in the bioanalysis of endogenous and exogenous steroids. Therefore, one important field in antidoping research is the investigation on drug metabolizing and steroidogenic enzymes. The introduction of a hydroxy group is the most common reaction, which is catalyzed by cytochrome P450 (CYP) enzymes in phase-I metabolism. Analysis of AAS metabolites is commonly performed using gas chromatography mass spectrometry (GC-MS) systems. Laborious sample preparation and extended run times compared to liquid chromatography (tandem) mass spectrometry (LC-MS/MS) methods are usually correlated with this type of analysis. On the other hand, liquid chromatography (tandem) mass spectrometry (LC-MS[/MS]) methods have a lower separation efficiency than GC-MS methods. Both techniques lack selectivity for hydroxylated 17α-methyltestosterone metabolites. Therefore, as an orthogonal analytical approach, a supercritical fluid chromatography tandem mass spectrometry method was developed to separate four hydroxy metabolites of 17α-methyltestosterone (2α-/2β-/4-/6β-hydroxy-17α-methyltestosterone). This project aimed to get a more in-depth look at the metabolization and analysis of 17α-methyltestosterone and its hydroxylated metabolites. The developed method revealed lower limits of quantitation between 0.6 and 6 ng/ml at an accuracy of 85-115% using a matrix matched calibration. An in vitro study with human liver microsomes shows 6β-hydroxy-17α-methyltestosterone as main metabolite (15.9%) as well as the metabolite 2β-hydroxy-17α-methyltestosterone (0.5%). The results show that the developed method is sensitive and robust. In addition, the method allows a previously missing discrimination of the hydroxylated metabolites in a short analysis time without prior, complex derivatizations., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Adolescents commonly co-abuse many drugs including anabolic androgenic steroids either they are athletes or non-athletes. Stanozolol is the major anabolic used in recent years and was reported grouped with cannabis. The current study aimed at evaluating the biochemical and histopathological changes related to the hypertrophic effects of stanozolol and/or cannabis whether in condition of exercise practice or sedentary conditions. Adult male Wistar albino rats received either stanozolol (5 mg/kg, s.c), cannabis (10 mg/kg, i.p.), and a combination of both once daily for two months. Swimming exercise protocol was applied as a training model. Relative heart weight, oxidative stress biomarkers, cardiac tissue fibrotic markers were evaluated. Left ventricular morphometric analysis and collagen quantification was done. The combined treatment exhibited serious detrimental effects on the heart tissues. It increased heart tissue fibrotic markers (Masson's trichrome stain (p < 0.001), cardiac COL3 (p < 0.0001), and VEGF-A (p < 0.05)), lowered heart glutathione levels (p < 0.05) and dramatically elevated oxidative stress (increased malondialdehyde (p < 0.0001) and 8-OHDG (p < 0.0001)). Training was not ameliorating for the observed effects. Misuse of cannabis and stanozolol resulted in more hypertrophic consequences of the heart than either drug alone, which were at least largely assigned to oxidative stress, heart tissue fibrotic indicators, histological alterations, and morphometric changes., (© 2024. The Author(s).)

The dried blood spot (DBS) is a novel alternative matrix used in 2022 Beijing Winter Olympics and Paralympics. It is capable of distinguishing anabolic androgenic steroid (AAS) esters without the gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) confirmation. In this study, a method for detection of 22 anabolic steroid esters in DBS based on ultra-high liquid chromatography-mass spectrometry (UPLC-MS) with parallel reaction monitoring (PRM) was developed and validated. Methoxylamine was used as the derivatization reagent to improve the sensibility. Specificity, limit of detection (LOD), linearity, stability, robustness, and carryover were evaluated. Steroid esters are nine testosterone esters, six nandrolone esters, five boldenone esters, methenolone enanthate, and trenbolone acetate. UV spectra were determined by HPLC. And density functional theory (DFT) calculation methods could provide theoretical UV spectra data. Three basis set of B3LYP/6-31G(d), B3LYP/6-31+G(d, p), and WB97XD/6-31+G(d, p) were used for the geometry optimizations and TD-DFT calculation. The average deviation (%RD) of B3LYP/6-31+G(d) for all 44 ester oximes are less than 3.0%. This study for the first time provides a method to tentatively identify the 44 E/Z configurations of steroid oxime products., (© 2023 John Wiley & Sons, Ltd.)

Over the past few decades, anabolic androgenic steroids (AASs) have been abused in and out of competition for their performance-enhancing and muscle-building properties. Traditionally, AASs were commonly detected using gas chromatography-mass spectrometry in the initial testing procedure for doping control purposes. Gas chromatography-Orbitrap high-resolution mass spectrometry (GC-Orbitrap-HRMS) is a new technology that has many advantages in comparison with GC-MS (e.g., a maximum resolving power of 240,000 (FWHM at m/z 200), excellent sub-ppm mass accuracy, and retrospective data analysis after data acquisition). Anti-doping practitioners are encouraged to take full advantage of the updated techniques of chromatography-mass spectrometry to develop sensitive, specific, and rapid screening methods for AASs. A new method for screening a wide range of AASs in human urine using GC-Orbitrap-HRMS was developed and validated. The method can qualitatively determine 70 anabolic androgenic steroids according to the minimum required performance limit of the World Anti-Doping Agency. Moreover, the validated method was successfully applied to detect six metabolites in urine after the oral administration of metandienone, and their excretion curves in vivo were studied. Metandienone M6 (17β-hydroxymethyl-17α-methyl-18-nor-androst-1,4,13-trien-3-one) has been identified as a long-term urinary metabolite which can be detected up to 7 weeks, thus providing a longer detection window compared with previous studies. This study provides a rationale for GC-Orbitrap-HRMS in drug metabolism and non-targeted screening., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Ostarine, also known as MK-2866 or enobosarm, is a selective androgen receptor modulator (SARM). It has anabolic properties and as such is widely used in doping, accounting in 2021 for 25 % of the adverse analytical findings (AAF) among the class S1.2 "Other anabolic agents" of products banned by the World Anti-Doping Agency, to which it belongs. But in some cases, it can be responsible for an AAF following contamination. We report the case of an athlete who contaminated herself by exchanging body fluids while kissing her boyfriend, who took 25 mg per day of MK-2866 for 9 days prior to the athlete's AAF (urinary concentration evaluated at 13 ng/mL) without her knowledge. Both subjects came to our lab for hair testing. The athlete's hair was black and slightly frizzy. Six segments of 2 cm then 7 × 3 cm (33 cm) were analysed and showed increasing concentrations, from 2 pg/mg on the first segment to 17.8 pg/mg on the last segment. The boyfriend's hair, light-brown, analyzed on 4 × 2 cm, also showed increasing values, from 65 to 143 pg/mg. These gradients of concentration in the hair's athlete and in her boyfriend were compatible with external contamination of the hair, confirmed by analysis of washing baths, pillowcases (150 pg on each), and the athlete's hairbrush (250 pg). Fingernails were also contaminated, with 21 pg/mg in the athlete and 1041 pg/mg in the boyfriend, with highly contaminated washing baths, and toenails were less contaminated, with 2 pg/mg in the athlete and 17.3 pg/mg in the boyfriend. Urine samples taken 35 days after the start of MK-2866 treatment showed a value of 3690 ng/mL in the boyfriend and 5.7 ng/mL in the athlete. After 6 days off, these concentrations were 3.3 ng/mL and 0.1 ng/mL, respectively. A controlled transfer study was carried out 12 days after discontinuation (urine concentrations returned to negative level). After administration of 17 mg (the 25 mg/mL vial having been controlled at 17 mg/mL), urine samples were taken from the boyfriend and the athlete (n = 10 for each) for more than 25 h after they had been living normally with each other (regular kissing in particular). The boyfriend's urine concentrations ranged from 681 ng/mL to 12822 ng/mL (Tmax = 8:30 hrs), and the athlete's from 0.3 ng/mL to 13 ng/mL with Tmax = 8:30 hrs, i.e. at 22:30 hrs, which corresponded exactly to the time of collection of the urine that showed AAF, with a similar concentration. The dose ingested by the athlete was estimated at 15 µg. These results demonstrate the transfer of ostarine via body fluids between two subjects, with a high risk of AAF in one athlete, as observed in our case., Competing Interests: Conflict of interest JCA declare conflict of interest being the expert witness of the athlete., (Copyright © 2024. Published by Elsevier B.V.)

Excessive use of anabolic-androgenic steroids (AAS) in sport occurs among professional athletes but increasingly also in amateurs. Prevalence of steroid use has been on the rise for a number of years. While the practice involves mostly men, it also occurs in women with an estimated prevalence of 1.6%. Since 2014, a 'steroid passport' has operated for sports people in competition that is based on longitudinal urinary and blood steroid levels, measured by liquid chromatography and mass spectrometry. Androgen excess stimulates muscle growth and improves muscle performance. However, their consumption carries numerous side effects, including myocardial hypertrophy; altered lipid metabolism and pro-thrombotic effects. The excess of AAS is associated with increased risk of atherosclerosis and cardiovascular events. Data for their effects in women is lacking. Perturbations of the menstrual cycle are common in female athletes, with spaniomenorrhea and even amenorrhea. This can be a consequence of gonadotropin insufficiency due to negative caloric balance, but may also be due to endogenous or exogenous hyperandrogenism. The use of AAS is probably underestimated as a public health issue, particularly in women, and thus presents a prevention challenge for healthcare professionals., (Copyright © 2023. Published by Elsevier Masson SAS.)

This article aims to review available literature evidence about the harmful effects of long-term anabolic-androgenic steroid (AAS) abuse on the heart. A review of 11 existing literature articles regarding this association has been used in the development of this review article. There is increasing medical literature documentation of the eventual harmful effect of AAS misuse or abuse on the heart. Individuals who misuse these steroids are susceptible to significant debilitation and loss of productive person-hours, and in severe cases, it can lead to death. Raising awareness about this potentially deleterious effect of anabolic steroids is crucial to prevent its misuse or abuse., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

In comparison to well-known drug-metabolizing organs such as the liver, the metabolic capacity of human skin is still not well elucidated despite the widespread use of topical drug application. To gain a comprehensive insight into anabolic steroid metabolism in the skin, six structurally related anabolic androgenic steroids, testosterone, metandienone, methyltestosterone, clostebol, dehydrochloromethyltestosterone, and methylclostebol, were applied to human keratinocytes and fibroblasts derived from the juvenile foreskin. Phase I metabolites obtained from incubation media were analyzed by gas chromatography-mass spectrometry. The 5α-reductase activity was predominant in the metabolic pathways as supported by the detection of 5α-reduced metabolites after incubation of testosterone, methyltestosterone, clostebol, and methylclostebol. Additionally, the stereochemistry structures of fully reduced metabolites (4α,5α-isomers) of clostebol and methylclostebol were newly confirmed in this study by the help of inhouse synthesized reference materials. The results provide insights into the steroid metabolism in human skin cells with respect to the characteristics of the chemical structures., Competing Interests: Declaration of Competing Interest All authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Boldenone is an anabolic-androgenic steroid (AAS) that is prohibited in equine sports. However, in certain situations, it is endogenous, potentially formed by the microbes in urine. An approach to the differentiation based on the detection of the biomarkers Δ1-progesterone, 20(S)-hydroxy-Δ1-progesterone and 20(S)-hydroxyprogesterone was assessed, and their concentrations were monitored in the urine of untreated female horses (n = 291) alongside boldenone, boldienone, testosterone and androstenedione. Using an ultra-sensitive analytical method, boldenone (256 ± 236 pg/mL, n = 290) and the biomarkers (Δ1-progesterone up to 57.6 pg/mL, n = 8; 20(S)-hydroxy-Δ1-progesterone 85.3 ± 181 pg/mL, n = 130; 20(S)-hydroxyprogesterone 43.5 ± 92.1 pg/mL, n = 158) were detected at low concentrations. The ex vivo production of Δ1-steroids was artificially induced following the storage of urine samples at room temperature for 7 days in order to assess the concentrations and ratios of the monitored steroids. The administration of inappropriately stored feed source also resulted in an increase in 20(S)-hydroxy-Δ1-progesterone concentrations and the biomarker ratios. Using the results from different datasets, an approach to differentiation was developed. In situations where the presence of boldenone exceeds a proposed action limit of 5 ng/mL, the presence of the biomarkers would be investigated. If Δ1-progesterone is above 50 pg/mL or if 20(S)-hydroxy-Δ1-progesterone is above 100 pg/mL with the ratio of 20(S)-hydroxy-Δ1-progesterone:20(S)-hydroxyprogesterone greater than 5:1, then this would indicate ex vivo transformation or consumption of altered feed rather than steroid administration. There remains a (small) possibility of a false negative result, but the model increases confidence that adverse analytical findings reported in female horses are caused by AAS administrations., (© 2023 John Wiley & Sons, Ltd.)

Sulfated metabolites have shown to have potential as long-term markers (LTMs) of anabolic-androgenic steroid (AAS) abuse. The compatibility of gas chromatography-mass spectrometry (GC-MS) with trimethylsilyl (TMS)-derivatives of non-hydrolysed sulfated steroids has been demonstrated, where, after derivatisation, generally, two closely eluting isomers are formed that both have the same molecular ion [M-H 2 SO 4 ] •+ . Sulfated reference standards are in limited commercial availability, and therefore, the current knowledge of the GC-MS behaviour of these compounds is mainly based on sulfating and analysing the available standard reference material. This procedure can unfortunately not cover all of the current known LTMs as these are often not available as pure substance. Therefore, in theory, some metabolites could be missed as they exhibit alternative behaviour. To investigate the matter, in-house sulfated reference materials that bear resemblance to known sulfated LTMs were analysed on GC-MS in their TMS-derivatised non-hydrolysed state. The (alternative) gas chromatographic and mass spectrometric behaviour was mapped, evaluated and linked to the corresponding steroid structures. Afterwards, using fraction collection, known sulfated LTMs were isolated from excretion urine to confirm the observed findings. The categories that were selected were mono-hydroxy-diones, 17-methyl-3,17-diols and 17-keto-3,16-diols as these are commonly encountered AAS conformations. The ability to predict the GC-MS behaviour of non-hydrolysed sulfated AAS metabolites is the corner stone of finding new metabolites. This knowledge is also essential, for example, for understanding AAS detection analyses, for the mass spectrometric characterization of metabolites of new designer steroids or when one needs to characterize an unknown steroid structure., (© 2023 John Wiley & Sons Ltd.)

Objectives: This study aims to evaluate healthcare providers' awareness and experience with anabolic androgenic steroid (AAS) use among gym users in Jordan and their role in mitigating AAS misuse., Methods: A cross-sectional study was conducted between November 2022 and Feb 2023. During the study period, 158 physicians involved in prescribing AAS or treating adverse effects associated with these agents were invited to participate. A questionnaire was created and uploaded on Google Forms and distributed through social media platforms to reach physicians from different medical subspecialties., Results: Of the 158 physicians surveyed, 30.4% were general practitioners, and 63.3% reported having formal education about the risks of bodybuilding with AAS. Among the participants, 48.7% had encountered gym users who were using AAS, with the main reason for referral being the management of side effects associated with AAS use. The median knowledge score regarding AAS and their complications was 10.0 out of 14 (IQR = 7.0), indicating adequate knowledge about most AAS side effects. Around 46% of the physicians (n = 73, 46.2%) believed that gym users might struggle to disclose AAS because they lack trust in them. Finally, the most common advice to gym users was informing them about the health-associated consequences of AAS use, with 92.2% of physicians providing this advice., Conclusion: Although healthcare professionals in Jordan had adequate knowledge about AAS, they needed more to make them confident in dealing with AAS use. This highlights the necessity for specialized teaching and training programs to enhance their knowledge and skills in providing accurate information to AAS users., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Behavioral aspects of organized sports activity for pediatric athletes are considered in a world consumed with winning at all costs. In the first part of this treatise, we deal with a number of themes faced by our children in their sports play. These concepts include the lure of sports, sports attrition, the mental health of pediatric athletes (i.e., effects of stress, anxiety, depression, suicide in athletes, ADHD and stimulants, coping with injuries, drug use, and eating disorders), violence in sports (i.e., concepts of the abused athlete including sexual abuse), dealing with supervisors (i.e., coaches, parents), peers, the talented athlete, early sports specialization and sports clubs. In the second part of this discussion, we cover ergolytic agents consumed by young athletes in attempts to win at all costs. Sports doping agents covered include anabolic steroids (anabolic-androgenic steroids or AAS), androstenedione, dehydroepiandrostenedione (DHEA), human growth hormone (hGH; also its human recombinant homologue: rhGH), clenbuterol, creatine, gamma hydroxybutyrate (GHB), amphetamines, caffeine and ephedrine. Also considered are blood doping that includes erythropoietin (EPO) and concepts of gene doping. In the last section of this discussion, we look at disabled pediatric athletes that include such concepts as athletes with spinal cord injuries (SCIs), myelomeningocele, cerebral palsy, wheelchair athletes, and amputee athletes; also covered are pediatric athletes with visual impairment, deafness, and those with intellectual disability including Down syndrome. In addition, concepts of autonomic dysreflexia, boosting and atlantoaxial instability are emphasized. We conclude that clinicians and society should protect our precious pediatric athletes who face many challenges in their involvement with organized sports in a world obsessed with winning. There is much we can do to help our young athletes find benefit from sports play while avoiding or blunting negative consequences of organized sport activities., (Copyright © 2022. Published by Elsevier Inc.)

The frequent detection of anabolic androgenic steroids (AAS) indicates their popularity among rule-breaking athletes. The so called long-term metabolites play a crucial role in their detection, and non-hydrolysed sulphated metabolites have gained renewed interest, as research has demonstrated their extended detection time compared to the more conventional markers (e.g., for metenolone and mesterolone). Their potential has been investigated using liquid and gas chromatography-mass spectrometry (LC- and GC-MS). However, due to their complementary nature, chances are that the most promising metabolite on one technique does not necessarily exhibit the same behaviour on the other and vice versa. Therefore, a comparison was carried out where as a trial model, metenolone, mesterolone and 17α-methyltestosterone were selected and the most likely long-term sulphated metabolites identified on four mass spectrometric instruments. Additionally, using a modified sample preparation procedure, comparison between conventional and non-hydrolysed sulphated metabolites between different GC-MS instruments was also included. When focusing on each individual marker, no cases were observed where a single metabolite provided a superior detection time on all instruments. Furthermore, for each AAS, there were incidences where a metabolite provided the best detection time on one instrument but could only be detected for a shorter period or not at all on other instruments. This demonstrates that metabolite detection windows and hence their added-value as target substance are unique and dependent on the analytical technique and not only on their pharmacokinetic behaviour. Consequently, in each case, a metabolite versus instrument evaluation is needed to maximise the probabilities of detecting doping offences., (© 2023 John Wiley & Sons Ltd.)

Nonsteroidal selective androgen receptor modulators (SARMs) are a novel class of compounds that have not yet been clinically approved; however, they appear to have a better anabolic/androgenic ratio than steroids and cause slighter side effects. Sports drug testing laboratories are required to maintain continuously updated doping control analytical methods in light of the widespread misuse of SARMs in elite and amateur sports. This paper describes the metabolic conversion of SARM GSK2881078 in thoroughbred horses following oral administration and in vitro with equine liver microsomes. A liquid chromatography-high-resolution mass spectrometry method was used to postulate the plausible structures of the detected metabolites. A total of five (M1-M5) in vivo metabolites and six (M1-M6) in vitro metabolites were detected under experimental conditions. Phase I metabolites mainly result from hydroxylation. Methoxylated and side-chain dissociated metabolites were also detected. Neither sulfonic acid nor glucuronic acid conjugated metabolites were observed in this study. Data reported here could aid in the detection of nonsteroidal SARM GSK2881078 and reveal its illicit use in competitive sports., (© 2023 John Wiley & Sons Ltd.)

Anabolic androgenic steroids are synthetic substances related to the male sex hormones (androgens). These agents promote the growth of skeletal muscle (anabolic effects) and the development of male sexual characteristics (androgenic effects). Anabolic steroids have been illegally used for many years as performance-enhancing drugs in human, equine, and canine sports and as growth promoters in livestock reared to provide meat for human consumption. The analytical challenge to developing effective means of control within these fields has been exacerbated by the reported endogenous nature of some of these steroids. Anabolic steroids have been employed extensively in equine practice over the past 50 years. Their usefulness is largely dependent on subjective opinions, as only minimal studies investigating pharmacodynamics have been carried out in horses. Therefore, their use will vary markedly between practitioners depending on their personal experiences and pressures by trainers to use them. They form part of rational therapy in a variety of conditions. In addition to their use for increasing muscle mass, they are used to varying extents in the raising of yearlings and in the training and racing of horses with the view of improving performance. The use of these agents is prohibited in the horseracing industry by the Association of Racing Commissioners International (ARCI), International Federation of Horseracing Authorities (IFHA), and Fédération Equestre Internationale (FEI)., (© 2023 John Wiley & Sons Ltd.)

Aim: This study aimed to investigate the gas chromatographic properties and mass spectrometric fragmentations of anabolic androgenic steroids (AASs) after trimethylsilylated derivatization. Materials & methods: A total of 113 AASs were analyzed through gas chromatography-mass spectrometry in the full-scan mode. Results: New fragmentation pathways yielding m/z 129, 143 and 169 ions were analyzed. Based on the characteristics of the A-ring, seven classes of drugs were identified and analyzed. Conclusion: The fragmentation pathway of a new classification of 4-en-3-hydroxyl was reported for the first time. The relationship between the chemical structures of AASs and their retention time, along with their molecular ion peak abundance, was also reported herein for the first time.

When testing for anabolic androgenic steroids (AAS) outside sports communities, for example, in healthcare and forensic medicine, urine is the matrix of choice. However, there are drawbacks with urinary sampling, and serum might be useful as a complementary matrix. The aim was to develop an LC-MS/MS method for serum measuring AAS frequently used outside of sport, including testosterone (T), steroid esters, and eight other synthetic AAS. The sample pretreatment included sample precipitation and evaporation. Limit of quantification for the AAS was 0.05-0.5 ng/mL, and linearity was 0.05-20 ng/mL for most of the substances. Generally, the within- and between-day CV results, matrix effect, and process efficiency were <15%. The AAS were stable for at least 6 months at -20°C. Serum samples were obtained from previous studies. A novel finding from an administration study was that T enanthate was present in serum even after 5 years of storage at -20°C. Serum samples from self-reporting AAS individuals, where T esters were detected, were positive for testosterone using the urinary testosterone/epitestosterone criterion >10. Of those identified as positive in traditional urinary doping tests (n = 15), AAS in serum were found in 80% of the subjects. Our results show that serum may be a valid complementary matrix to urine samples for AAS testing., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

From 2012 to 2013, approximately 16 New York residents reported vague, nonspecific adverse health effects which included fatigue, loss of scalp hair, and muscle aches. One patient was hospitalized for liver damage. An epidemiological investigation identified a common factor among these patients; the consumption of B-50 vitamin and multimineral supplements from the same supplier. To investigate whether these nutritional supplements might have been responsible for the adverse health effects observed, comprehensive chemical analyses of marketed lots of the supplements were performed. To determine presence of organic components and contaminants, organic extracts of samples were prepared and analyzed using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS), liquid chromatography high-resolution mass spectrometry (LC-HRMS), and nuclear magnetic resonance (NMR). These analyses revealed the presence of significant levels of methasterone (17β-hydroxy-2α,17α-dimethyl-5α-androstane-3-one), an androgenic steroid and schedule III-controlled substance; dimethazine, an azine-linked dimer of methasterone; and methylstenbolone (2,17α-dimethyl-17β-hydroxy-5α-androst-1-en-3-one), a related androgenic steroid. Methasterone and extracts of certain supplement capsules were identified as highly androgenic in luciferase assays by using an androgen receptor promoter construct. This androgenicity persisted for several days after cell exposure to the compounds. The presence of these components in implicated lots were associated with adverse health effects and the hospitalization of one patient and the presentation of symptoms of severe virilization in a child. These findings underscore the need for more rigorous oversight of the nutritional supplement industry., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Potential scenarios as to the origin of minute amounts of banned substances detected in doping control samples have been a much-discussed problem in anti-doping analysis in recent years. One such debated scenario has been the contamination of female athletes' urine with ejaculate containing doping agents and/or their metabolites. The aim of this work was to obtain complementary information on whether relevant concentration ranges of doping substances are excreted into the ejaculate and which metabolites can be detected in the seminal fluid (sf) and corresponding blood plasma (bp) samples. A method was established to study the concentration and metabolite profiles of stanozolol and LGD-4033-substances listed under anabolic substances (S1) on the World Anti-Doping Agency's Prohibited List-in bp and sf using liquid chromatography high-resolution mass spectrometry (LC-HRMS). For sf and bp, methods for detecting minute amounts of these substances were developed and tested for specificity, recovery, linearity, precision, and reliability. Subsequently, sf and bp samples from an animal administration study, where a boar orally received stanozolol at 0.33 mg/kg and LGD-4033 at 0.11 mg/kg, were measured. The developed assays proved appropriate for the detection of the target substances in both matrices with detection limits between 10 and 40 pg/mL for the unmetabolized drugs in sf and bp, allowing to estimate the concentration of stanozolol in bp (0.02-0.40 ng/mL) and in sf (0.01-0.25 ng/mL) as well as of LGD-4033 in bp (0.21-2.00 ng/mL) and in sf (0.03-0.68 ng/mL) post-administration. In addition, metabolites resulting from different metabolic pathways were identified in sf and bp, with sf resembling a composite of the metabolic profile of bp and urine., (© 2022. The Author(s).)

Δ6-Methyltestosterone was reported as the main active ingredient of the purported "dietary supplement" Jungle Warfare. This compound is structurally similar to 17α-methyltestosterone, containing an additional Δ6 double bond, and is reported to possess notable androgenic activity, raising concerns over the potential for abuse of Jungle Warfare in sport. The in vivo metabolism of Δ6-methyltestosterone in greyhounds was investigated. Urinary phase I (unconjugated) and phase II (glucuronide) metabolites were detected following oral administration using liquid chromatography-mass spectrometry. No phase II sulfate metabolites were detected. The major phase I metabolite was confirmed as 16α,17β-dihydroxy-17α-methylandrosta-4,6-dien-3-one by comparison with a synthetically-derived reference material. Minor amounts of the parent drug were also confirmed. Glucuronide conjugated metabolites were also observed, but were found to be resistant to hydrolysis using the Escherichia coli β-glucuronidase enzyme. Qualitative excretion profiles, limits of detection, and extraction recoveries were determined for the parent drug and the major phase I metabolite. These results provide a method for the detection of Jungle Warfare abuse in greyhounds suitable for incorporation into routine screening methods conducted by anti-doping laboratories., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

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., Competing Interests: PB is the owner of PeterBond.org The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bond, Smit and de Ronde.)

The illicit use of anabolic androgenic steroids (AAS) as performance-enhancing drugs remains a global issue threatening not only the credibility of competitive sports but also public health due to the well-documented adverse effects they elicit. AAS abuse is not restricted only to professional sports, but also extends to recreational athletes and adolescents as well as in livestock production as growth-promoting agents. Testosterone and nandrolone are among the AAS most frequently exploited. Gas chromatography-mass spectrometry is the reference method for AAS detection, but it is strictly laboratory-based and cannot be performed on-site. The great potential of aptamers in bioanalytical applications and specifically for the development of simple analytical tools suitable for on-site analysis has been extensively documented. In this report, we describe the selection and identification of aptamers binding nandrolone, exhibiting affinity dissociation constants in the low nanomolar range. A label-free colorimetric assay based on gold nanoparticles was developed using one of these novel aptamers for the detection of nandrolone and/or its metabolites. The assay could be deployed for the rapid, on-site, facile and cost-effective screening of samples and provide qualitative visual results with a red to purple/blue color change being indicative of a positive result., Competing Interests: Declaration of competing interest None., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Androgenic steroids have been abused by elite athletes for decades. Their performance-enhancing properties for sports that involve strength and power have been confirmed, and this class has been banned from most elite athletic competitions since the mid-1970s. There is a risk of a withdrawal syndrome that includes severe depression, and there seems to be an increased risk of cardiovascular disease associated with chronic abuse. In contrast to high-dosage androgen abuse, androgen therapy at near-physiological dosages is generally safe and effective for male hypogonadism and potential use in sarcopenia and male hormonal contraception., Competing Interests: Disclosure A. Thirumalai and B.D. Anawalt: Nothing to disclose., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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 (17β-hydroxy-17α-methylandrosta-1,4-dien-3-one), clostebol (4-chlorotestosterone-17β-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., (© 2022 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

The purpose of this article is to provide the school nurse with the ability to recognize performance and appearance enhancing substance use and understand treatment and prevention strategies to reduce associated health risks. The use of appearance and performance enhancing drugs and substances, also known as "doping," has been an ethical and health issue in sports for many years. It is vital that school nurses learn to recognize performance and appearance enhancing substance use to ensure student athletes and exercisers receive the care and education they need. Commonly used performance and appearance enhancing substances include anabolic-androgenic steroids, stimulants, diuretics, growth hormone, and supplements. Signs and symptoms that may look similar to other health concerns include palpitations, stunted growth, acne, severe headaches, muscle cramps, dizziness, and dehydration, but some long-term severe complications occur as well. Doping can lead to severe and sometimes permanent organ damage, including liver, kidney, and heart disease. The school nurse can play a key role by working with parents, coaches, school counselors, and educators to foster an anti-doping culture.

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., Competing Interests: Declaration of competing interest None., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Dietary supplements sold for anabolic benefits or performance enhancement often contain substances, which are non-approved and might lack quality controls. With regard to athletes, the inclusion of substances or methods in the prohibited list of the World Anti-Doping Agency is based on medical or scientific evidence. 5α-hydroxy-laxogenin is a synthetic spirostane-type steroid, which is contained in dietary supplements and advertised as anabolic agent. To date, evidence is missing on anabolic or androgenic activity of 5α-hydroxy-laxogenin. We investigated its androgenic potential in two in vitro bioassays. While no activity was observed in the yeast androgen screen, 5α-hydroxy-laxogenin was able to trans-activate the androgen receptor in human prostate cells in a dose-dependent manner. Interestingly, a biphasic response was observed with antagonistic properties at lower concentrations and agonistic effects at higher concentrations tested. The demonstrated androgenic properties of the higher concentrations demonstrate that further investigations should focus on the safety as well as on potential anabolic effects of 5α-hydroxy-laxogenin. This is of interest with regard to abuse for doping purposes., (© 2022. The Author(s).)

Boldenone is an anabolic-androgenic steroid that is prohibited in equine sports. However, in certain situations, it is endogenous or is believed to be formed by microbes in urine, and therefore, an approach for the differentiation is required. Following the identification of Δ1-progesterone and 20(S)-hydroxy-Δ1-progesterone as potential biomarkers of microbial activity, the presence of six steroids was investigated in the postrace urine of castrated male horses (geldings, n = 158). In line with endogenous findings from several other species when ultrasensitive methods are employed, boldenone was detected at low concentrations in all urine samples (27.0-1330 pg/ml). Furthermore, testosterone and androstenedione were detected in 157 samples (≤12,400 and 944 pg/ml, respectively), boldienone in two samples (≤22.0 pg/ml) and 20(S)-hydroxy-Δ1-progesterone in 20 samples (≤66.0 pg/ml). Δ1-Progesterone was not detected in any population samples analysed on arrival at the laboratory. The ex vivo transformation of boldienone, boldenone, androstenedione, Δ1-progesterone and 20(S)-hydroxy-Δ1-progesterone was induced following the storage of urine samples at room temperature for 7 days but not after refrigeration. Because the administration of inappropriately stored feed sources also resulted in an increase in 20(S)-hydroxy-Δ1-progesterone concentrations, a biomarker approach to distinguish steroid administrations was proposed. In situations where the presence of boldenone would exceed a proposed action limit, the presence of Δ1-progesterone and 20(S)-hydroxy-Δ1-progesterone would be investigated. If either Δ1-progesterone or 20(S)-hydroxy-Δ1-progesterone would exceed 50 and 100 pg/ml, respectively, for instance, then this would indicate ex vivo transformation or consumption of altered feed rather than steroid administration., (© 2022 John Wiley & Sons, Ltd.)

Introduction: Endogenous testosterone deficiency or excess anabolic-androgenic steroids (AAS) have been linked to alter the physiology of different organs in the body, more specifically, the vasculature of coronary arteries. Despite the health-related concerns of using synthetic testosterone derivatives, such as AAS, there has been a tremendous increase in the use of AAS among athletes and bodybuilders., Areas Covered: We have highlighted the three main mechanisms that AAS increase the risk of coronary artery disease (CAD): altering the homeostasis of lipid metabolism which results in dyslipidemia and subsequently atherosclerosis, disturbing the function of platelet which results in platelet aggregation and subsequent thrombosis, and increasing the risk of coronary vasospasm by affecting the physiological function of vascular bed., Expert Opinion: Despite the restriction of AAS in specific clinical conditions such as testosterone deficiency and cancer therapy, many amateurs' athletes misuse the AAS. Although there has been a strong association between the AAS misuse and risk of developing CAD, the more valued approach would be a randomized clinical double-blind trial. The suggested primary endpoint would be an occurrence of adverse cardiovascular events, such as myocardial infarction, cerebrovascular accidents, and death. Increasing awareness of the risk of missing AAS among high-risk groups is imperative.

The prevalence of anabolic androgenic steroid (AAS; anabolic steroids) use in recreational sports is underestimated. Due to the influence of social media, an increase in AAS use in recreational sports and in the general population is to be expected. AAS use is associated with significant physical and mental health consequences, and the psychiatric consequences include the risk of developing addictive behaviour. The widespread stigmatization of AAS use also by professionals often undermines users' trust in physicians and drives them into the arms of so-called "gurus." The tightening of anti-doping practices in sports and an exclusively prohibitive stance have so far failed to convincingly curb the problem in recreational sports. Harm reduction strategies could help patients to get the help they need from primary care providers.

Dehydrochloromethyltestosterone (DHCMT) is one of the most detected illicit used anabolic-androgenic steroids in professional sports. Therefore, a fast and accurate analysis of this substance is of great importance for a constructive fight against doping abuse. The conventional method for the analysis of this drug, GC-MSMS, is very sensitive and selective but also very time- and resource-consuming. With the presented work, a new approach for simple detection with LC-HRMSMS without any sample preparation is introduced. The method is based on the direct analysis of two newly described phase-II metabolites of the DHCMT long-term metabolite 4-chloro-18-nor-17β-hydroxymethyl-17α-methyl-5β-androst-13-en-3α-ol (M3). LC-HRMSMS, GC-MSMS, fractionation and derivatization experiments are combined to identify and characterize for the first time two different glucuronide-acid conjugates of this metabolite in positive human urine samples. In addition, a third glucuronide metabolite was identified, however without isomeric structure determination. The detection of these metabolites is particularly interesting for confirmation analyses, as the method is rapid and requires little sample material., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 D 3 -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 17β-hydroxymethyl-17α-methyl-18-nor-androst-1,4,13-trien-3-one and 17α-hydroxymethyl-17β-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 D 3 -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., (© 2021 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Selective androgen receptor modulators, SARMs, are a large class of compounds developed to provide therapeutic anabolic effects with minimal androgenic side effects. A wide range of these compounds are available to purchase online and thus provide the potential for abuse in sports. Knowledge of the metabolism of these compounds is essential to aid their detection in doping control samples. In vitro models allow a quick, cost-effective response where administration studies are yet to be carried out. In this study, the equine phase I metabolism of the non-steroidal SARMs GSK2881078, LGD-2226, LGD-3303, PF-06260414, ACP-105, RAD-140 and S-23 was investigated using equine liver microsomes. Liquid chromatography coupled to a QExactive Orbitrap mass spectrometer allowed identification of metabolites with high resolution and mass accuracy. Three metabolites were identified for both GSK2881078 and LGD-2226, four for LGD-3303 and RAD-140, five for PF-06260414, twelve for ACP-105 and ten for S-23. The equine metabolism of GSK-2881078, LGD-2226, LGD-3303 and PF-06260414 is reported for the first time. Although the equine metabolism of ACP-105, RAD-140 and S-23 has previously been reported, the results obtained in this study have been compared with published data., (© 2021 John Wiley & Sons, Ltd.)

Anabolic-androgenic steroids (AASs), a group of compounds frequently misused by athletes and, unfortunately, also by the general population, have lately attracted global attention; thus, significant demands for more precise, facile, and rapid AAS detection have arisen. The standard methods ordinarily used for AAS determination include liquid and gas chromatography coupled with mass spectrometry. However, good knowledge of steroid metabolism, pretreatment of samples (such as derivatization), and well-trained operators of the instruments are required, making this procedure expensive, complicated, and not routinely applicable. In the drive to meet current AAS detection demands, the scientific focus has shifted to developing novel, tailor-made approaches leading to time- and cost-effective, routine, and field-portable methods for AAS determination in various matrices, such as biological fluids, food supplements, meat, water, or other environmental components. Therefore, herein, we present a comprehensive review article covering recent advances in AAS determination, with a strong emphasis on the increasingly important role of chemically designed artificial sensors, biosensors, and antibody- and fluorescence-based methods.

Synthetic androgenic anabolic steroids (AAS) are banned compounds and considered as major threats by both racing and sports international authorities. Hence, doping control laboratories are continually looking into analytical improvements to increase their detection capabilities, notably by means of emerging technologies. To enhance analytical performances for the detection of synthetic AAS such as stanozolol, specific chromatographic procedures have been developed using recent quaternary liquid chromatography technology originally designed for high-throughput standardized proteomics connected to mass spectrometry. Applying the newly designed elution procedures described in this paper to the analyses of stanozolol and its metabolites in complex matrixes revealed improved sensitivity compared to previously described high-throughput methods. Indeed, we report the consistent and reliable detection of 16β-hydroxy-stanozolol down to 10 pg/mL in equine urine and being detectable up-to 3 months after a microdosing administration. Furthermore, a five months long elimination of stanozolol and its metabolites could be monitored on horse mane sections after a single dose administration. Our work highlights novel solutions to detect AAS with improved sensitivity. The application of such developments constitutes new landmarks for doping control laboratories and could be extended to other targeted compounds in residue analysis, toxicology, and metabolomics. Based on this work, the developed chromatographic method is now freely available within the Evosep Plus program.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A 59-year-old male was found dead at home, with two empty vials of an oily preparation obtained from a manufacturer from East Europe. There was no label on the vial. The subject was a former weightlifter, also known as an anabolic steroids abuser. The local prosecutor ordered a body examination, which was unremarkable, and allowed collecting femoral blood, urine and scalp hair (6 cm, brown). He was treated for cardiac insufficiency with quinidine. Biological specimens were submitted not only to standard toxicological analyses including a screening with liquid chromatography (LC)-quadrupole time of flight, but also to a specific LC-tandem mass spectrometry method for anabolic steroids testing. Ethanol was not found in both blood and urine. Quinidine blood concentration (791 ng/mL) was therapeutic. No drug of abuse was identified. In blood, testosterone was less that 1 ng/mL and no other steroid was identified. In urine, testosterone/epitestosterone was 1.56 and boldenone was present at a concentration of 9 ng/mL. The hair test results, performed on the whole length, demonstrated repetitive steroids abuse, including not only testosterone (140 pg/mg), testosterone propionate (605 pg/mg) and testosterone decanoate (249 pg/mg), but also boldenone (160 pg/mg), trenbolone (143 pg/mg) and metandienone (60 pg/mg). Since forensic laboratories have limited access to steroid urinary metabolite reference material due to specific regulations (to avoid testing athletes before anti-doping verifications), hair analyses seem to be the best approach to document anabolic agents abuse. Indeed, in hair, the target drug is the parent compound; in addition, when compared to blood or urine, this matrix has a much larger window of detection. The pathologist concluded cardiac insufficiency in a context involving repetitive abuse of anabolic drugs. This case indicates that more attention should be paid to anabolic steroids, in a context of sudden cardiac death., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The pressure to gain mass, power, explosiveness, and endurance and to obtain a performance edge continues to a part of sports. Anabolic agents, including selective androgen receptor modulators along with peptides, hormones, and metabolic modulators, continues to evolve. Methods to promote transcription to modify gene expression are a part of the evolution. In order to monitor and improve doping detection, the Athlete Biological Passport has been created. This article provides an up-to-date review of alcohol, anabolic androgens and related agents, stimulants, opioids, and cannabis and related compounds and their effects on athlete health and performance., Competing Interests: Disclosure The authors have no commercial or financial conflicts of interest and any funding sources., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Anti-doping analysis requires an exceptional level of accuracy and precision given the stakes that are at play. Current methods rely on the application of chromatographic techniques linked with mass spectrometry to provide this. However, despite the effectiveness of these techniques in achieving good selectivity and specificity, some issues still exist. In order to reach the minimum required performance level as set by WADA, labs commonly use selective monitoring by quadrupole mass spectrometry. This can be potentially fooled through the use of masking agents or by moving the peaks, as often only a small portion of the spectrum is used for analysis. Further issues exist in the inability to detect new or modified compounds, or to reanalyse samples/spectra. One technique that could overcome these problems is that of comprehensive 2D chromatography. Here a second separation column is employed to generate greater separative power. Compared to conventional separation, GCxGC allows for a greater peak capacity (i.e., number of peaks that can be resolved within a given time) and greater separation of coeluting compounds, which makes the technique promising for the complex task required in anti-doping. When combined with Time of Flight Mass Spectrometry this technique demonstrates vast potential allowing for full mass range datasets to be obtained for retroactive analysis. Similarly, LCxLC provides improvements in resolving power compared to its 1D counterpart and can be used both online as part of the analysis or offline solely as a purification step. In this review we summarise the work in this field so far, how comprehensive chromatography has been applied to anti-doping studies, and discuss the future application for this technique., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Androgenic anabolic steroids are the most misused substances in sports because of their performance-enhancing effects. Often synthetic analogues of endogenously present steroids are administered. To determine their endogenous or exogenous origin, Gas Chromatography Combustion Isotope Ratio Mass Spectrometry (GC-C-IRMS) is used in the field of doping control. Compounds subjected to IRMS analysis must be interference-free, with liquid chromatography fraction collection (HPLC-FC) being the crucial clean-up step. However, this clean-up is challenging, particularly for compounds present at low concentrations in samples with pronounced matrix effects. The compounds of interests for IRMS analyses in doping control are testosterone (T) and its main metabolites (androsterone, etiocholanolone, 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol), epitestosterone, 19-norandrosterone (19-NA), boldenone (B) and its main metabolite (BM), formestane (F) and 6αOH-androstenedione (6aOHADION). Currently, the available methods only deal with a selection of the above-mentioned compounds. Some of these compounds (e.g., 19-NA, B, BM, 6aOHADION) are present in very low concentrations, requiring an extensive and dedicated sample clean-up, and this makes it challenging to develop a universal clean-up procedure. Many of these methods require different and multiple offline HPLC-FC setups, which are labour-intensive and time-consuming. That is problematic during, e.g., large sports events, where reporting time is limited (e.g., 72 h). Therefore, in the current work, we developed a uniform online 2D/3D HPLC-FC method, capable of purifying all relevant target compounds in a single run, leading to the fastest clean-up procedure so far (i.e., 31 min for T and its main metabolites; 46 min for 19-NA, F and 6aOHADION; 48 min for B and BM)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 136 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 21 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Androgens remain abused performance-enhancing drugs in sports. Technologies based on mass spectrometry can detect all forms of androgens but fail if the androgen represents a novel structure. A bioassay detects androgens based on function rather than structure. To date, there has been limited adoption of cell-based in vitro bioassays as a screening tool for nontargeted androgen detection because they require expert personnel and specialized equipment to perform. We now describe the development of a cell-free version of an androgen in vitro bioassay. Stage 1 involved in vitro transcription/translation reactions (IVTT) using a DNA template encoding an enhancer/androgen response element (ARE) regulatory region upstream of a minimal promoter that drives expression of a reporter protein. The assay detected testosterone across the concentration range of 106.7 to 0.0144 ng/ml (3.7 × 10 -7 to 5 × 10 -11 M), with an EC 50 of 6.63 ng/ml (23 nM). To reduce complexity, Stages 2-4 of development included just in vitro transcription (IVT) reactions, whereby the output was an RNA molecule. Stage 2 involved directly labelling the RNA molecule with fluorophore-labelled nucleotide triphosphates, Stage 3 involved reverse transcription-polymerase chain reaction (PCR) of the RNA molecule, and Stage 4 utilized an RNA aptamer, Mango II, as its RNA output. The Stage 4 product detected testosterone across the range of 106.7-0.0001 ng/ml (3.7 × 10 -7 to 5 × 10 -13 M), with an EC 50 of 0.04 ng/ml (0.155 nM). Further to this, we show that the Stage 4 product can detect other androgenic molecules. Relative to cell-based bioassays, the Stage 4 product is easy to perform and could be developed into a routine, high-throughput, nontargeted androgen screen., (© 2021 John Wiley & Sons, Ltd.)

Selective androgen receptor (AR) modulators (SARMs) are potent anabolic agents with a high potential of misuse in horseracing and equestrian sports. In this study, we applied label-free proteomics to discover plasma protein biomarkers in geldings (castrated horses) after administration with a popular SARM named RAD140. Tryptic peptides were prepared from plasma samples and analyzed by nano-flow ultrahigh-performance liquid chromatography-high-resolution tandem mass spectrometry (nano-UHPLC-HRMS/MS) using data-independent acquisition (DIA) method. Orthogonal projection on latent structure-discriminant analysis (OPLS-DA) has led to the development of a predictive model that could discriminate RAD140-administered samples from control samples and could also correctly classify 18 out of 19 in-training horses as control samples. The model comprises 75 proteins with variable importance in projection (VIP) score above 1. Gene Ontology (GO) enrichment analysis and literature review have identified upregulation of AR-regulated clusterin, and proteins associated with inflammation (haptoglobin, cluster of differentiation 14 [CD14], and inter-alpha-trypsin inhibitor heavy chain 4 [ITIH4]) and erythropoiesis (glycosylphosphatidylinositol-specific phospholipase D1 [GPLD1]) after RAD140 administration. Their changes were confirmed by selected reaction monitoring (SRM) experiments. Similar effects have been reported by the use of androgens and other SARMs. This is the first reported study that describes the use of a proteomic biomarker approach to detect horses that have been administered with RAD140 by applying label-free proteomic profiling of plasma samples. These results support the concept of a biomarker-driven approach to enhance the doping control of RAD140 and potentially other SARMs in the future., (© 2020 John Wiley & Sons, Ltd.)

The steroid profile, that is, the urinary concentrations and concentration ratios of selected steroids, is used in sports drug testing to detect the misuse of endogenous steroids such as testosterone. Since several years, not only population-based thresholds are applied but also the steroid profile is monitored via the Athlete Biological Passport whereby the individual reference ranges derived from multiple test results of the same athlete are compared to population-based thresholds. In order to maintain a high probative force of the passport, samples collected or analyzed under suboptimal conditions should not be included in the longitudinal review. This applies to biologically affected or degraded samples and to samples excluded owing to the presence of other substances potentially (or evidently) altering the steroid profile. Nineteen different doping agents comprising anabolic steroids, selective androgen receptor modulators, selective estrogen receptor modulators, ibutamoren, and tibolone were investigated for their effect on the steroid profile using an androgen receptor activation test, an androgen receptor binding assay, an aromatase assay, and a steroidogenesis assay. The in vitro tests were coupled with well-established liquid chromatography/mass spectrometry-based analytical approaches and for a subset of steroidal analytes by gas chromatography/mass spectrometry. The variety of tests employed should produce a comprehensive data set to better understand how a compound under investigation may impact the steroid profile. Although our data set may allow an estimate of whether or not a substance will have an impact on the overall steroid metabolism, predicting which parameter in particular may be influenced remains difficult., (© 2020 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

The widespread abuse of anabolic androgenic steroids by healthy people leads to the risk of major mood disorders and heart failure; thus, the determination of anabolic androgenic steroids is vital. In this study, 17 anabolic androgenic steroids in dietary supplements and external drugs were identified, and their concentration was determined. For this purpose, polyaniline-coated magnetic nanoparticles were prepared and then subjected to magnetic solid-phase extraction combined with high-performance liquid chromatography-tandem mass spectrometry. The experimental parameters of magnetic solid-phase extraction were studied in detail, and the optimal conditions were established. Under the optimal conditions, the limits of detection were in the range of 0.001-0.02 μg/L, with relative standard deviations of 5.52-11.6% (n = 7) for all the steroids, and the enrichment factors were in the range of 20.0-24.8. The developed method was then successfully applied for the determination of 17 anabolic androgenic steroids in real samples, and dehydroepiandrosterone (prasterone) was detected in a commercially available external drug., (© 2021 Wiley-VCH GmbH.)

Androgens, both steroidal and nonsteroidal 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. Although exquisitely sensitive, such approaches may fail to detect novel structures that are developed and marketed. A nontargeted 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 nontargeted detection strategies., (© 2021 John Wiley & Sons, Ltd.)