Anabolic steroids in livestock production: Background and implications for chemical food safety.

The use of steroids in livestock animals is a source of concern for consumers because of the risks associated with the presence of their residues in foodstuffs of animal origin. Technological advances such as mass spectrometry have made it possible to play a fundamental role in controlling such practices, firstly for the discovery of marker metabolites but also for the monitoring of these compounds under the regulatory framework. Current control strategies rely on the monitoring of either the parent drug or its metabolites in various matrices of interest. As some of these steroids also have an endogenous status specific strategies have to be applied for control purposes. This review aims to provide a comprehensive and up-to-date knowledge of analytical strategies, whether targeted or non-targeted, and whether they focus on markers of exposure or effect in the specific context of chemical food safety regarding the use of anabolic steroids in livestock. The role of new approaches in data acquisition (e.g. ion mobility), processing and analysis, (e.g. molecular networking), is also discussed.

The Power of Keratinous Matrices (Head Hair, Body Hair and Nail Clippings) Analysis in a Case of Death Involving Anabolic Agents.

A 29-year-old man with no previous medical history was found dead at home. Anabolic products (tablets and oily solutions) and syringes were found at the scene. The man was known to train regularly at a fitness club and to use anabolic drugs. Following an unremarkable autopsy with normal histology, toxicological analyses were requested by the local prosecutor to provide further information. Blood, head hair (5 cm, black), body hair (axillary and leg) and toe and finger nail clippings were submitted to liquid and gas chromatography coupled to tandem mass spectrometry (LC and GC-MS-MS) methods to test for anabolic steroids. Blood tested positive for testosterone (4 ng/mL), boldenone (26 ng/mL), stanozolol (3 ng/mL) and trenbolone (<1 ng/mL). Segmental head hair tests (2 × 2.5 cm) revealed a repeated consumption of testosterone (65-72 pg/mg), testosterone propionate (930-691 pg/mg), testosterone isocaproate (79 pg/mg to <5 pg/mg), nandrolone decanoate (202-64 pg/mg), boldenone (16 pg/mg), stanozolol (575-670 pg/mg), trenbolone (4 pg/mg-not detected), drostanolone (112-30 pg/mg), drostanolone enanthate (26-5 pg/mg) and drostanolone propionate (15-4 pg/mg). In addition to the substances identified in head hair, testosterone decanoate, testosterone cypionate and nandrolone were identified in both body hair and nails. The experts concluded that the manner of death can be listed as toxic due to massive repetitive use of anabolic steroids during the previous months. For anabolic agents, blood does not seem to be the best matrix to document a fatal intoxication. Indeed, these products are toxics when abused long term and are known to cause cardiac, hepatic and renal diseases. When compared to blood, hair and nails have a much larger window of detection. Therefore, keratinous matrices seem to be the best approach to test for anabolic steroids when a sudden death is observed in the context of possible abuse of steroids.

Sensitive detection of testosterone and testosterone prohormone administrations based on urinary concentrations and carbon isotope ratios of androsterone and etiocholanolone.

The testing strategy for the detection of testosterone (T) or T-prohormones is based on the longitudinal evaluation of urinary steroid concentrations accompanied by subsequent isotope ratio mass spectrometry (IRMS)-based confirmation of samples showing atypical concentrations or concentration ratios. In recent years, the IRMS methodology focussed more and more on T itself and on the metabolites of T, 5α- and 5ß-androstanediol. These target analytes showed the best sensitivity and retrospectivity, but their use has occasionally been challenging due to their comparably low urinary concentrations. Conversely, the carbon isotope ratios (CIR) of the main urinary metabolites of T, androsterone (A) and etiocholanolone (EITO), can readily be measured even from low urine volumes; those however, commonly offer a lower sensitivity and shorter retrospectivity in uncovering T misuse. Within this study, the CIRs of A and ETIO were combined with their urinary concentrations, resulting in a single parameter referred to as 'difference from weighted mean' (DWM). Both glucuronidated and sulfated steroids were investigated, encompassing a reference population (n = 110), longitudinal studies on three individuals, influence of ethanol in two individuals, and re-analysis of several administration studies including T, dihydrotestosterone, androstenedione, epiandrosterone, dehydroepiandrosterone, and T-gel. Especially DWM calculated for the sulfoconjugated steroids significantly prolonged the detection time of steroid hormone administrations when individual reference ranges were applied. Administration studies employing T encompassing CIR common for Europe (-23.8‰ and -24.4‰) were investigated and, even though for a significantly shorter time period and less pronounced, DWM could demonstrate the exogenous source of T metabolites.

Undeclared Doping Substances are Highly Prevalent in Commercial Sports Nutrition Supplements.

Sports nutrition supplements have previously been reported to contain undeclared doping substances. The use of such supplements can lead to general health risks and may give rise to unintentional doping violations in elite sports. To assess the prevalence of doping substances in a range of high-risk sports nutrition supplements available from Dutch web shops. A total of 66 sports nutrition supplements - identified as potentially high-risk products claiming to modulate hormone regulation, stimulate muscle mass gain, increase fat loss, and/or boost energy - were selected from 21 different brands and purchased from 17 web shops. All products were analyzed for doping substances by the UK life sciences testing company LGC, formerly known as the Laboratory of the Government Chemist, using an extended version of their ISO17025 accredited nutritional supplement screen. A total of 25 out of the 66 products (38%) contained undeclared doping substances, which included high levels of the stimulants oxilofrine, ß-methylphenethylamine (BMPEA) and N,ß-dimethylphenethylamine (NBDMPEA), the stimulant 4-methylhexan-2-amine (methylhexaneamine, 1,3-dimethylamylamine, DMAA), the anabolic steroids boldione (1,4-androstadiene-3,17-dione) and 5-androstene-3ß,17α-diol (17α-AED), the beta-2 agonist higenamine and the beta-blocker bisoprolol. Based upon the recommended dose and the potential variability of analyte concentration, the ingestion of some products identified within this study could pose a significant risk of unintentional doping violations. In addition to inadvertent doping risks, the prescribed use of 3 products (4.5%) could likely impose general health risks.

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.

Testing for anabolic steroids in human nail clippings.

Anabolic steroids are synthetic derivatives of testosterone, and their abuse can have numerous health consequences. Identification of this group of drugs has found applications in forensic toxicology, clinical situations, psychiatric disorders, and of course, anti-doping violations. Although anabolic steroids are generally tested in urine and very occasionally in head hair, collectors can face the lack of standard specimens, and therefore, nail clippings can be the unique alternative choice. Although there is no possibility to perform segmental analyses using nail clippings, the window of drug detection is generally much longer in nail when compared to head hair (particularly in male subjects), that is, 3-8 months and 4-12 months for finger and toenail clippings, respectively. A new method was developed, including nail pulverization in a ball mill, sonication for 90 min in methanol, and a combination of liquid-liquid and solid-phase extractions, followed by gas and liquid chromatography coupled to tandem mass spectrometry. To document the application of steroid testing in nail clippings, the authors present 6 authentic cases of abuse, involving stanozolol (7 and 24 pg/mg), nandrolone (6 pg/mg), trenbolone (26, 67, 81, and 89 pg/mg), drostanolone (8 and 11 pg/mg), and testosterone enanthate (14 pg/mg). Given concentrations were always in the low pg/mg range, the use of tandem mass spectrometry appears as a prerequisite.

Simultaneous testing for anabolic steroids in human hair specimens collected from various anatomic locations has several advantages when compared with the standard head hair analysis.

Since the late 90s, hair testing for anabolic steroids in humans has found numerous forensic, clinical, and anti-doping applications. In most cases, analyses were performed on head hair, collected in the vertex regions. However, for various reasons (shaved subject, bald subject, religious belief, cosmetic treatment and aesthetic reason), hair collectors can face the lack of head hair, and therefore, body hair can be the unique alternative choice. Although there is no possibility to perform segmental analyses with body hair, their use has two major advantages: (1) In most cases, anabolic steroids are more concentrated in body hair when compared with head hair, which allows detecting abuse at lower frequency and for lower dosages; and (2) the window of drug detection is generally much longer in body hair when compared with head hair, particularly in male athlete presenting short head hair. To document the relevance of simultaneous collection of head and body hair, the authors present eight authentic cases of anabolic steroids abuse, including clostebol (one case), drostanolone (one case), metandienone (one case), 19-norandrostenedione (one case), stanozolol (two cases) and trenbolone (three cases). In all cases, body hair concentrations were higher than head hair concentrations. Even in three cases, no steroid was identified in head hair, although present in body hair.

Reliable identification and quantification of anabolic androgenic steroids in dietary supplements by using gas chromatography coupled to triple quadrupole mass spectrometry.

The aim of the present research was the identification and quantification of specific anabolic androgenic steroids (AASs) and other sterane structured compounds in dietary supplements (DSs). The adulteration of DSs by these compounds is of a particular concern in athletes, because it might lead to a positive doping result. The research was focused on the optimization of a highly sensitive and selective GC-based analytical strategy using triple quadrupole MS as detector. Chromatographic method and multiple reaction monitoring (MRM) transitions of 28 target compounds were optimized. Sample clean-up was carried out by using a solid phase extraction (SPE) procedure, while the derivatization of AASs was performed by using N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA). The method was validated, and the following parameters were investigated: linearity range, limit of detection, accuracy, and precision expressed in terms of intra-day precision. The calibration curves were evaluated by using regression model and resulting in a good determination coefficients (R2 ≥ 0.9912). The residuals were scattered randomly around zero. The limits of detection (LODs) were lower than 7.0 ng g-1 or ng ml-1 . The accuracy assessment was evaluated in different forms of DSs characterized by high sample-to-sample variability (liquid, powder, tablet, capsule, protein, and herbal-based). Intra-day assay precision was in all cases lower than 20%. The developed analytical method was successfully applied to the analysis of 67 commercially available dietary supplements. In five cases, one or more steroid-type compounds were found in the concentration of 5 ng g-1 -100 µg g-1 , which might result adverse analytical findings in athletes.

Doping in the Ultimate Fighting Championship (UFC): A 4-year epidemiological analysis.

BACKGROUND: Doping is a practice that is present in many sports and organizations, including mixed martial arts and the Ultimate Fighting Championship (UFC). The aim of this study is to explore the epidemiological patterns of doping among UFC athletes. METHODS: We screened the official United-States-Anti-Doping-Agency® (USADA) website, the annual USADA reports and the official UFC website for information on fighters and anti-doping rule violations (ADRVs). Our dataset included gender, age, weight class, testing numbers, date of ADRV, type of ADRV, and duration of suspension. Appropriate statistical tests were conducted to assess for statistical significance. RESULTS: USADA tested 1070 UFC athletes 2624 times as of late 2015 up till the end of 2019 (N = 1070). A total of 209 adverse findings were recorded; out of which, 102 ADRVs were committed by 93 athletes (8.7%) from all weight divisions. This constituted an adverse finding rate of 16.55 per 1000 test and an ADRV rate of 8.08 per1000 test. Mean age of sanctioned athletes was 32 years. Use of anabolic steroids was significantly the most common ADRV recorded (p = 0.018). The men's heavyweight division had an ADRV rate of 19.3 per 1000 tests, significantly higher than that of women's bantamweight division at 2 per 1000 tests (p = 0.03), women's featherweight division at 0 per 1000 tests (p = 0.009), and men's flyweight division at 3 per 1000 tests (p = 0.035). ADRV rate showed a significantly increasing trend among men's weight divisions (p < 0.001). CONCLUSION: Doping is present in mixed martial arts. Increasing testing numbers, raising awareness and education on the risks of doping, and conducting further research on the issue is key to help resolve this problem.