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.

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.

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.

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.

Development and application of analytical procedures for the GC-MS/MS analysis of the sulfates metabolites of anabolic androgenic steroids: The pivotal role of chemical hydrolysis.

In this work, we present a gas-chromatography tandem mass spectrometry (GC-MS/MS) method for the identification of the sulfo-conjugate metabolites of pseudo-endogenous steroids (endogenous steroids when administered exogenously). We have preliminarily evaluated the performances of different preparations of sulfatases from Pseudomonas aeruginosa and Helix pomatia, characterized by various origins and catalytic activities, and compared the efficacy of the enzymatic hydrolysis with chemical hydrolysis, performed with a mixture of ethyl acetate, methanol, and sulphuric acid. A procedure for the selective isolation of steroid conjugates from the urine matrix has been designed and optimized, based on the "sequential" extraction of the glucuro-conjugated and of the sulfo-conjugated fractions, performed by two different direct methods, i.e. by ion paired extraction or solid-phase extraction. More specifically, the former method is based on the use of N,N-dimethylephedrinium bromide as the ion paired extraction reagent, while the latter on the use of WAX® (weak anion exchange) cartridges. The performance of the newly developed procedure has been assessed by the analysis of real urine excretion samples collected after the oral intake of a single dose of dehydroepiandrosterone (DHEA) or androstenedione (AED), measuring the concentration of epiandrosterone (EpiA) sulfate. Our results have shown the following: (i) although the yields of chemical hydrolysis and enzymatic hydrolysis are in some cases quite similar, the former is generally preferable since it results in the quantitative cleavage of sulfate moiety; (ii) ion paired extraction has been selected as the most reliable method for direct isolation of sulfate steroids from urine matrices; (iii) EpiA sulfate allows to prolong the detectability of DHEA and AED when compared to routinely used steroidal target compounds.

Analytical approaches applied to the analysis of apprehended formulations of anabolic androgenic steroids.

Anabolic androgenic steroids (AASs) comprise a class of synthetic androgens resulting from chemical modifications of testosterone, known for their illicit consumption, which can result inextensive side effects. Extraction procedures applied to the analysis of their formulations are still limited to a few methodologies, despite the increasing numbers of confiscations of AASs. In this sense, the aims of this work were to evaluate the extraction of active ingredients from formulations of anabolic agents using solid-liquid or liquid-iquid, ultrasonic bath, ultrasonicprobe, and microwave-assisted extraction. The results indicated that the extraction procedures influenced the detected concentration of AASs, as the use of ultrasonic probe and microwave irradiation increased the overall extraction of anabolic agents compared with solid-liquid, liquid-liquid, and ultrasonic bath. Regarding oxymetholone, for instance, the microwave-assisted extraction and ultrasonic probe extracted, respectively, 37.46 ± 1.36 and 35.69 ± 0.98 mg/tablet, while solid-liquid extracted 29.63 ± 0.40 mg/tablet of the activeingredient. Therefore, alternative methods such as microwave-assisted extraction or theultrasonic probe could be used for the analysis of formulations of AASs assisting with the identification of illicit and toxic components.

Uso y abuso de agentes anabolizantes en la actualidad / Current use and abuse of anabolic steroids

El abuso común de esteroides andrógenos anabolizantes (EAA) se ha extendido a la población general y no solamente a atletas de alto rendimiento. Datos epidemiológicos reportan el uso de estas sustancias de manera común en poblaciones jóvenes. La razón del uso de EAA es el deseo de aumentar la masa y la fuerza muscular, y con ello mejorar apariencia física. Debido a la facilidad de adquisición de este tipo de medicamentos por los atletas que abusan de ellos, se ha desarrollado un «sofisticado» conocimiento de la farmacología de los esteroides basado en el análisis subjetivo y anecdótico, sin contar con la información de eventos adversos, lo que lleva a pensar en un problema de salud pública. Desafortunadamente, en el caso de los atletas estas experiencias pueden llegar a influir más que el asesoramiento de su médico. El abuso de EAA por parte de la población de atletas y no atletas hace que el conocimiento de estos sea importante para la práctica médica en el día a día, para la mejora de los cambios y eventos adversos secundarios en empleo de EAA

Common abuse of anabolic androgenic steroids (AAS) is no longer confined to high performance athletes, as it has spread among the general population. Epidemiological data about the abuse of these substances show that it is a common practice in young populations. Its use is based on the desire to increase muscle mass and strength, as well as improving physical performance. The ease of acquisition of this type of substances has developed a "sophisticated" knowledge of steroid pharmacology based on subjective and anecdotal analysis with no adverse event information, which translates into a public health crisis. Unfortunately, athletes seem to be more influenced by these experiences than by their physician's advice. The abuse of AAS by the athlete and non-athlete population and its adverse events ought to be evaluated in order to improve routine clinical practice on this regard

Current use and abuse of anabolic steroids. / Uso y abuso de agentes anabolizantes en la actualidad.

Common abuse of anabolic androgenic steroids (AAS) is no longer confined to high performance athletes, as it has spread among the general population. Epidemiological data about the abuse of these substances show that it is a common practice in young populations. Its use is based on the desire to increase muscle mass and strength, as well as improving physical performance. The ease of acquisition of this type of substances has developed a "sophisticated" knowledge of steroid pharmacology based on subjective and anecdotal analysis with no adverse event information, which translates into a public health crisis. Unfortunately, athletes seem to be more influenced by these experiences than by their physician's advice. The abuse of AAS by the athlete and non-athlete population and its adverse events ought to be evaluated in order to improve routine clinical practice on this regard.

The determination of common anabolic steroid and stimulants in nutritional supplements by HPLC-DAD and LC-MS.

A high-performance liquid chromatography method employing a diode-array detector and mass spectrometry detector was developed, validated and implemented for determining Synephrine, Caffeine, Clenbuterol, Nandrolone, Testosterone and Methylhexaneamine in Nutritional supplements. The use of Nutritional supplements is widespread. Hazards relating to concentration, composition, individual contaminants, supplements interactions as well as positive doping results among athletes present increasing concerns regarding nutritional supplement consuming. The proposed method was validated according to the International Conference on the Harmonization of the Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) standards. The proposed method observed to be accurate, linear, precise, sensitive, required minimal sample preparation and uncomplicated mobile phase. The implementation of the proposed method on nine commercial supplements shows that inaccurate labeling for some supplements regarding the concentration of the ingredients.

Separation of Structurally Similar Anabolic Steroids as Cation Adducts in FAIMS-MS.

Novel synthetic anabolic androgenic steroids have been developed not only to dodge current antidoping tests at the professional sports level, but also for consumption by noncompetitive bodybuilders. These novel anabolic steroids are commonly referred to as "designer steroids" and pose a significant risk to users because of the lack of testing for toxicity and safety in animals or humans. Manufacturers of designer steroids dodge regulation by distributing them as nutritional or dietary supplements. Improving the throughput and accuracy of screening tests would help regulators to stay on top of illicit anabolic steroids. High-field asymmetric-waveform ion mobility spectrometry (FAIMS) utilizes an alternating asymmetric electric field to separate ions by their different mobilities at high- and low-fields as they travel through the separation space. When coupled to mass spectrometry (MS), FAIMS enhances the separation of analytes from other interfering compounds with little to no increase in analysis time. Here we investigate the effects of adding various cation species to sample solutions for the separation of structurally similar or isomeric anabolic androgenic steroids. FAIMS-MS spectra for these cation-modified samples show an increased number of compensation field (CF) peaks, some of which are confirmed to be unique for one steroid isomer over another. The CF peaks observed upon addition of cation species correspond to both monomer steroid-cation adduct ions and larger multimer ion complexes. Notably, the number of CF peaks and their CF shifts do not appear to have a straightforward relationship with cation size or electronegativity. Future directions aim at investigating the structures for these analyte-cation adduct ions for building a predictive model for their FAIMS separations.

Accidental doping. Prevention strategies / Dopaje accidental. Estrategias de prevención

There is growing consumption of nutritional supplements aimed at improving performance because the number of athletes, mainly amateurs, is growing very significantly. This great demand supposes a market of huge proportions, supposing an economic activity that in Spain reached 920 million Euros in the year 2018.This consumption occurs at all levels of sport, from 13% in global numbers, to 100% in some groups of professional sportsmen and women. However, the use of these substances in very few circumstances is done under the advice of a professional, and the athlete takes them on their own. This fact, with the possibility that the product to be taken may contain prohibited substances that do not appear on the labeling, means that an adverse analytical finding can occur in a doping control through so-called accidental doping, which is the use of adulterated or contaminated nutritional supplements containing substances prohibited in sport that have not been declared on the labeling. Between 11.6% and 25.8% of nutritional supplements contaminated with anabolic androgenic steroids have been found to exist. This paper describes the various causes of accidental doping, the substances most frequently used, paying particular attention to the ways of preventing this type of doping based on information and education, product certification and information, the form of prescription, criteria for use and safety of the origin of the products, and precautions followed in case of consumption

Hay un consumo creciente de suplementos nutricionales destinados a mejorar el rendimiento porque el número de deportistas, fundamentalmente aficionados, está creciendo de forma muy importante. Esta gran demanda supone un mercado de proporciones gigantescas, suponiendo un actividad económica que en España alcanzó los 920 millones de euros en el año 2018.Este consume se produce en todos los niveles deportivos, desde el 13 % en cifras globales, hasta el 100 % en algunos grupos de deportistas profesionales. Sin embargo, el uso de estas sustancias en muy pocas circunstancias se realiza bajo al asesoramiento de un profesional y el deportista los toma por su cuenta. Este hecho, junto a la posibilidad de que el producto que se vaya a tomar pueda contener sustancias prohibidas que no figuran en el etiquetado supone que se pueda producir un hallazgo analítico adverso en un control de dopaje a través del denominado dopaje accidental que consiste el que se produce por consumir suplementos nutricionales adulterados o contaminados que contienen sustancias prohibidas en el deporte que no se han declarado en el etiquetado. Se ha comprobado que existe entre el 11,6 y el 25,8% de suplementos nutricionales contaminados con esteroides androgénicos anabolizantes. En este trabajo se describen las diversas causas de dopaje accidental, las sustancias más frecuentemente utilizadas prestando una especial atención a las formas de prevención de este tipo de dopaje que se basan en la información y educación, en la certificación e información de los productos, en la forma de prescripción, en los criterios de uso y seguridad del origen de los productos y en las precauciones que se deben tomar en caso de consumirlos

Stanazolol derived ELISA as a sensitive forensic tool for the detection of multiple 17α-methylated anabolics.

Two valuable forensic tools based on enzyme-linked immunoassays (ELISAs) for the analysis of 17α-methylated steroids were developed using haptens of stanazolol and its conjugates with biotin. Haptens containing terminal carboxylic group were conjugated to bovine serum albumin (BSA), rabbit serum albumin (RSA) or ovalbumin (OVA). Eight batches of antisera (RAbs) obtained by immunization of rabbits were tested in an indirect competitive ELISA system using immobilization of RSA conjugate (RSA/hapten) and competitor immobilization of the biotinylated conjugate (AB-ELISA) to avidin (avidin/hapten). The best results were achieved with the RAb 212 antibodies in RSA/ST-3 and avidin/ST-10 assembled variants. For the RSA/ST-3 system, an IC50 of 0.3 ng/mL and a detection limit of 0.02 ng/mL were measured. In case of avidin/ST-10 variant, IC50 was of 3.9 ng/mL and a detection limit of 0.57 ng/mL were obtained. The effect of solvent was tested as well as the stability of coated microtiter plates over four-month period. The cross-reactivity of the developed assays with other anabolic steroids was tested and high sensitivity towards 17α-methylated steroids was observed. RSA/ST-3 assay showed significant cross-reactivity with 17α-methyltestosterone (81.2%), oxymetholone (30.4%), methandienone (10.0%) and methyl dihydrotestosterone (7.7%). Similarly, in the avidin/ST-10 assay, 17α-methyltestosterone (34.5%), mestanolone (32.1%), oxymetholone (22.7%), methandienone (14.2%), 9-dehydromethyltestosterone (12.5%) and oxandrolone (1.2%) exhibited high cross-reactivity. The functionality of the developed systems was verified by the successful identification of a series of 17α-methylated anabolic steroids in a set of real samples including pharmaceutical preparations seized by the Police of the Czech Republic on the black market.

Fast analysis of 19 anabolic steroids in bovine tissues by high performance liquid chromatography with tandem mass spectrometry.

For the detection of 19 steroid hormones in bovine muscle, a fast and sensitive liquid chromatography with electrospray ionization tandem mass spectrometry method was developed using both positive and negative ionization mode. Chromatographic separation on Poroshell 120-EC C18 column was achieved in less than 10 min using isocratic elution of mobile phase of acetonitrile/methanol/water. The compounds were extracted from muscle tissue using ethyl acetate and quick, easy, cheap, effective, rugged, and safe technique. The purification of the obtained extract was performed by dispersive solid-phase extraction with sorbents C18, primary secondary amine and magnesium sulphate. The method was validated in accordance with the Commission Decision 2002/657/EC. For all steroids tested good recoveries were obtained (from 51.2 to 121.4%) in the concentration range from decision limits until 5 µg/kg. The values of decision limits and the detection capabilities for individual compounds were in the range 0.10-0.48 and 0.17-0.95 µg/kg, respectively. The method was characterized by satisfactory linearity for most compounds (correlation coefficients   > 0.99) and the reproducibility was lower than 35%. The elaborated procedure has met the criteria for confirmatory methods and is currently used in the official control of hormones.

A pilot wastewater-based epidemiology assessment of anabolic steroid use in Queensland, Australia.

Anabolic-androgenic steroids are synthetic compounds prohibited due to their performance-enhancing characteristics. The use of these substances is known to cause health-related issues, which highlights the importance of being able to evaluate the scale of consumption by the general population. However, most available research on the analysis of anabolic steroids is focused on animals and athletes in connection with doping. The potential of wastewater-based epidemiology as an intelligence tool for the assessment of community level use of anabolic steroids is presented herein. A liquid chromatography tandem mass spectrometry method was developed for the analysis of 10 anabolic-androgenic steroids and 14 endogenous hormones in influent wastewater. The validated method was applied to sixteen 24-hour composite wastewater influent samples that were collected over a period of five years from two wastewater treatment plants in Queensland, Australia. Nine investigated compounds were found to be present at concentrations between 14 and 611 ng L-1 which translated into 3-104 mg excreted per 1000 individuals per day. It was concluded that the developed analytical method is suitable for the analysis of AAS in wastewater matrix. Additionally, both the inclusion of metabolites and further investigation into deconjugation by enzymatic hydrolysis would aid in understanding and evaluating community anabolic steroid use. For the first time, this study presents the application of wastewater-based epidemiology on anabolic-androgenic steroids in Australia.