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Identifying the mechanisms that compel prosocial and antisocial behaviors is essential when seeking to develop effective social policies. While selfishness is ever present, in many situations cooperation is the norm. Testosterone increases a variety of antisocial behaviors but can also induce cooperation and honesty. We tested whether testosterone influences taking money from others, a measure of proactive aggression. We administered synthetic testosterone to participants (N = 163) in a double-blind placebo-controlled study before they made decisions in the power-to-take (PTT) game in which participants can take resources controlled by another while the other person can destroy resources so they are not taken. Three measures of testosterone were obtained from blood samples before and after drug administration to assess parametric effects. The data showed that an average 62% increase in total testosterone had no effect on average taking or destroying decisions in the PTT game. But taking money from others increased linearly in participants with supraphysiologic levels of testosterone. At the same time, testosterone improved participants' moods and those with positive affect destroyed fewer resources. Our results show that testosterone should be viewed as a nuanced modulator of social interactions rather than the cause of antisocial behaviors.

BACKGROUND: The prevalence of doping in competitive sport, and the methods for assessing prevalence, remain poorly understood. This reduces the ability of researchers, governments, and sporting organizations to determine the extent of doping behavior and the impacts of anti-doping strategies. OBJECTIVES: The primary aim of this subject-wide systematic review was to collate and synthesize evidence on doping prevalence from published scientific papers. Secondary aims involved reviewing the reporting accuracy and data quality as evidence for doping behavior to (1) develop quality and bias assessment criteria to facilitate future systematic reviews; and (2) establish recommendations for reporting future research on doping behavior in competitive sports to facilitate better meta-analyses of doping behavior. METHODS: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were used to identify relevant studies. Articles were included if they contained information on doping prevalence of any kind in competitive sport, regardless of the methodology and without time limit. Through an iterative process, we simultaneously developed a set of assessment criteria; and used these to assess the studies for data quality on doping prevalence, potential bias and reporting. RESULTS: One-hundred and five studies, published between 1975 and 2019,were included. Doping prevalence rates in competitive sport ranged from 0 to 73% for doping behavior with most falling under 5%. To determine prevalence, 89 studies used self-reported survey data (SRP) and 17 used sample analysis data (SAP) to produce evidence for doping prevalence (one study used both SRP and SAP). In total, studies reporting athletes totaled 102,515 participants, (72.8% men and 27.2% women). Studies surveyed athletes in 35 countries with 26 involving athletes in the United States, while 12 studies examined an international population. Studies also surveyed athletes from most international sport federations and major professional sports and examined international, national, and sub-elite level athletes, including youth, masters, amateur, club, and university level athletes. However, inconsistencies in data reporting prevented meta-analysis for sport, gender, region, or competition level. Qualitative syntheses were possible and provided for study type, gender, and geographical region. The quality assessment of prevalence evidence in the studies identified 20 as "High", 60 as "Moderate", and 25 as "Low." Of the 89 studies using SRP, 17 rated as "High", 52 rated as "Moderate", and 20 rated as "Low." Of the 17 studies using SAP, 3 rated as "High", 9 rated as "Moderate", and 5 rated as "Low." Examining ratings by year suggests that both the quality and quantity of the evidence for doping prevalence in published studies are increasing. CONCLUSIONS: Current knowledge about doping prevalence in competitive sport relies upon weak and disparate evidence. To address this, we offer a comprehensive set of assessment criteria for studies examining doping behavior data as evidence for doping prevalence. To facilitate future evidence syntheses and meta-analyses, we also put forward "best practice" recommendations and reporting guidelines that will improve evidence quality. ispartof: SPORTS MEDICINE vol:51 issue:9 pages:1909-1934 ispartof: location:New Zealand status: published

Pulmonary rehabilitation (PR) is a comprehensive intervention based on a thorough patient assessment followed by personalised interventions designed to improve the physical and psychological condition of patients with chronic respiratory diseases and to promote the long-term adherence to health-enhancing behaviours [1]. While the clinical importance of physical activity is recognised across all stages of disease, the Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2019 strategy for chronic obstructive pulmonary disease (COPD) states that patients that remain highly symptomatic and/or those with a history of moderate or severe exacerbations despite optimal pharmacotherapy are indicated for PR [2]. Improvements in symptoms, increases in quality of life and gains in functional capacity after PR are independent of age, sex or the baseline degree of airflow limitation [3, 4]. However, it is known that patients with higher symptoms of dyspnoea, worse functional capacity and poor health status at baseline are more likely to be good responders to PR [5]. While PR is traditionally applied in clinically stable patients, there is increasing evidence for its beneficial effects following hospitalisations [6] and in those with frequent exacerbations [5]. In patients with very severe disease awaiting lung transplantation significant improvements in exercise capacity and health status were reported after short-term comprehensive PR [7]. Moreover, an increasing number of specific (non-)­pharmacological interventions are available and can be combined with PR in the subgroup of patients with very advanced disease, including neuromuscular electrical stimulation (NMES), noninvasive ventilatory support and anabolic agents. Finally, PR may be an appropriate setting to introduce advance care planning (ACP) [8]. The role of these personalised and targeted interventions will be highlighted in this case report., A cachectic patient with very severe COPD and chronic respiratory failure may benefit from comprehensive and personalised pulmonary rehabilitation including neuromuscular electrical stimulation, noninvasive ventilation and anabolic steroids http://bit.ly/31Ss7WZ

BackgroundA few days of bed rest or immobilization following injury, disease, or surgery can lead to considerable loss of skeletal muscle mass and strength. It has been speculated that such short, successive periods of muscle disuse may be largely responsible for the age-related loss of muscle mass throughout the lifespan.ObjectiveTo assess whether a single intramuscular injection of nandrolone decanoate prior to immobilization can attenuate the loss of muscle mass and strength in vivo in humans.Design, setting and participantsThirty healthy (22 +/- 1 years) men were subjected to 7 days of one-legged knee immobilization by means of a full leg cast with (NAD, n = 15) or without (CON, n = 15) prior intramuscular nandrolone decanoate injection (200 mg).MeasuresBefore and immediately after immobilization, quadriceps muscle cross-sectional area (CSA) (by means of single-slice computed tomography (CT) scans of the upper leg) and one-legged knee extension strength (one-repetition maximum [1-RM]) were assessed for both legs. Furthermore, muscle biopsies from the immobilized leg were taken before and after immobilization to assess type I and type II muscle fiber cross-sectional area.ResultsQuadriceps muscle CSA decreased during immobilization in both CON and NAD (-6 +/- 1% and -6 +/- 1%, respectively; main effect of time PConclusionsThis is the first study to report that nandrolone decanoate administration does not preserve skeletal muscle mass and strength during a short period of leg immobilization in vivo in humans.

Bimagrumab is a fully human monoclonal antibody that blocks the activin type II receptors, preventing the activity of myostatin and other negative skeletal muscle regulators. Objectives: To assess the effects of bimagrumab on skeletal muscle mass and function in patients with COPD and reduced skeletal muscle mass. Methods: Sixty-seven COPD patients (mean FEV1 1.05 L [41.6% predicted]; aged 40–80 years; body mass index

Steroid hormones are intensively studied from environmental water systems. In environmental samples their concentrations are very low, but their quantities are not meaningless since steroids belong to the group of endocrine disruptors, causing cancer and developmental disorders in humans. On the contrary, only a few recent articles deal with the research of plant steroid alcohols (sterols) in biological and environmental water systems. The reason may be that sterols are not categorized as high-risk contaminants, although their chemical structures are similar with the human steroids. Mostly, steroids are analyzed with chromatography and mass spectrometry. In addition, capillary electrophoresis has shown to be a promising separation technique in metabolic studies because of the possibility to obtain simultaneous differentiation of glucosides and the corresponding native steroids. That is not easily processed in chromatography. This review highlights the determination of human and plant steroids detected in biological samples and environmental water systems with capillary electrophoresis.

Prior to the start of cross-sex hormone therapy (CSH), androgenic progestins are often used to induce amenorrhea in female to male (FtM) pubertal adolescents with gender dysphoria (GD). The aim of this single-center study is to report changes in anthropometry, side effects, safety parameters, and hormone levels in a relatively large cohort of FtM adolescents with a diagnosis of GD at Tanner stage B4 or further, who were treated with lynestrenol (Orgametril®) monotherapy and in combination with testosterone esters (Sustanon®). A retrospective analysis of clinical and biochemical data obtained during at least 6 months of hormonal treatment in FtM adolescents followed at our adolescent gender clinic since 2010 (n = 45) was conducted. McNemar’s test to analyze reported side effects over time was performed. A paired Student’s t test or a Wilcoxon signed-ranks test was performed, as appropriate, on anthropometric and biochemical data. For biochemical analyses, all statistical tests were done in comparison with baseline parameters. Patients who were using oral contraceptives (OC) at intake were excluded if a Mann-Whitney U test indicated influence of OC. Metrorrhagia and acne were most pronounced during the first months of monotherapy and combination therapy respectively and decreased thereafter. Headaches, hot flushes, and fatigue were the most reported side effects. Over the course of treatment, an increase in musculature, hemoglobin, hematocrit, creatinine, and liver enzymes was seen, progressively sliding into male reference ranges. Lipid metabolism shifted to an unfavorable high-density lipoprotein (HDL)/low-density lipoprotein (LDL) ratio; glucose metabolism was not affected. Sex hormone-binding globulin (SHBG), total testosterone, and estradiol levels decreased, and free testosterone slightly increased during monotherapy; total and free testosterone increased significantly during combination therapy. Gonadotropins were only fully suppressed during combination therapy. Anti-Mullerian hormone (AMH) remained stable throughout the treatment. Changes occurred in the first 6 months of treatment and remained mostly stable thereafter. Treatment of FtM gender dysphoric adolescents with lynestrenol monotherapy and in combination with testosterone esters is effective, safe, and inexpensive; however, suppression of gonadotropins is incomplete. Regular blood controls allow screening for unphysiological changes in safety parameters or hormonal levels and for medication abuse.

Detection of the abuse of synthetic steroids in food production is nowadays relatively straightforward using modern techniques such as gas or liquid chromatography coupled to mass spectrometry (GC-MS/MS or LC-MS/MS, respectively). However, proving the abuse of endogenous (or naturally occurring) steroids is more difficult. Despite these difficulties, significant progress in this area has recently been made and a number of methods are now available. The aim of the current review was to systematically review the available analytical approaches, which include threshold concentrations, qualitative marker metabolites, intact steroid esters, gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS), longitudinal testing and omics biomarker profiling. The advantages/disadvantages of these methods are considered in detail, but the choice of which to adopt is dictated by a number of practical, political, and economic factors, which vary in different parts of the world. These include the steroid/species combination requiring analysis, the matrix tested, whether samples are collected from live or slaughtered animals, available analytical instrumentation, sample throughput/cost, and the relevant legal/regulatory frameworks. Furthermore, these approaches could be combined in a range of different parallel and/or sequential screening/confirmatory testing streams, with the final choice being determined by the aforementioned considerations. Despite these advances, more work is required to refine the different techniques and to respond to the ever increasing list of compounds classified as endogenous. At this advanced stage, however, it is now more important than ever for scientists and regulators from across the world to communicate and collaborate in order to harmonize and streamline research efforts. (c) 2012 HFL Sport Science (LGC Ltd) and (c) Her Majesty the Queen in Right of Canada.

In patients with more severe chronic obstructive pulmonary disease ( COPD), the benefits of rehabilitation might not be clear and, therefore, new treatment options have been developed to increase the benefits of rehabilitation. This review provides an overview of new approaches being developed as an addition to exercise training. In turn, the benefits of adding ventilatory support, oxygen, anabolics or neuromuscular stimulation to a rehabilitation programme will be discussed. While positive benefits for a number of these approaches have been found, many questions remain unsolved. Therefore, at present, we cannot recommend these new tools as part of the routine management of patients with COPD who start a rehabilitation programme.

Unification of the screening protocols for a wide range of doping agents has become an important issue for doping control laboratories. This study presents the development and validation of a generic liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) screening method of 241 small molecule analytes from various categories of prohibited substances (stimulants, narcotics, diuretics, ß2-agonists, ß-blockers, hormone antagonists and modulators, glucocorticosteroids and anabolic agents). It is based on a single-step liquid-liquid extraction of hydrolyzed urine and the use of a rapid-resolution liquid chromatography/high-resolution time-of-flight mass spectrometric system acquiring continuous full scan data. Electrospray ionization in the positive mode was used. Validation parameters consisted of identification capability, limit of detection, specificity, ion suppression, extraction recovery, repeatability and mass accuracy. Detection criteria were established on the basis of retention time reproducibility and mass accuracy. The suitability of the methodology for doping control was demonstrated with positive urine samples. The preventive role of the method was proved by the case where full scan acquisition with accurate mass measurement allowed the retrospective reprocessing of acquired data from past doping control samples for the detection of a designer drug, the stimulant 4-methyl-2-hexanamine, which resulted in re-reporting a number of stored samples as positives for this particular substance, when, initially, they had been reported as negatives.

The origin, i.e. natural occurrence or illegal treatment, of findings of 17alpha-boldenone (alpha-Bol) and 17beta-boldenone (beta-Bol) in urine and faeces of cattle is under debate within the European Union. A liquid chromatographic positive ion electrospray tandem mass spectrometric method is presented for the confirmatory analysis of 17beta-boldenone, 17alpha-boldenone and an important metabolite/precursor androsta-1,4-diene-3,17-dione (ADD), using deuterium-labelled 17beta-boldenone (beta-Bol-d3) as internal standard. Detailed sample preparation procedures were developed for a variety of sample matrices such as bovine urine, faeces, feed and skin swab samples. The method was validated as a quantitative confirmatory method according to the latest EU guidelines and shows good precision, linearity and accuracy data, and CCalpha and CCbeta values of 0.1-0.3 and 0.4-1.0 ng/ml, respectively. Currently, the method has been successfully applied to suspect urine samples for more than a year, and occasionally to faeces, feed and swab samples as well. Results obtained from untreated and treated animals are given and their impact on the debate about the origin of residues of 17beta-boldenone is critically discussed. Finally, preliminary data about the degree of conjugation of boldenone residues are presented and a simple procedure for discrimination between residues from abuse versus natural origin is proposed.

A high-throughput bioaffinity liquid chromatography-mass spectrometry (BioMS) approach was developed and applied for the screening and identification of recombinant human estrogen receptor α (ERα) ligands in dietary supplements. For screening, a semi-automated mass spectrometric ligand binding assay was developed applying (13)C2, (15) N-tamoxifen as non-radioactive label and fast ultra-high-performance-liquid chromatography-electrospray ionisation-triple-quadrupole-MS (UPLC-QqQ-MS), operated in the single reaction monitoring mode, as a readout system. Binding of the label to ERα-coated paramagnetic microbeads was inhibited by competing estrogens in the sample extract yielding decreased levels of the label in UPLC-QqQ-MS. The label showed high ionisation efficiency in positive electrospray ionisation (ESI) mode, so the developed BioMS approach is able to screen for estrogens in dietary supplements despite their poor ionisation efficiency in both positive and negative ESI modes. The assay was performed in a 96-well plate, and all these wells could be measured within 3 h. Estrogens in suspect extracts were identified by full-scan accurate mass and collision-cross section (CCS) values from a UPLC-ion mobility-Q-time-of-flight-MS (UPLC-IM-Q-ToF-MS) equipped with a novel atmospheric pressure ionisation source. Thanks to the novel ion source, this instrument provided picogram sensitivity for estrogens in the negative ion mode and an additional identification point (experimental CCS values) next to retention time, accurate mass and tandem mass spectrometry data. The developed combination of bioaffinity screening with UPLC-QqQ-MS and identification with UPLC-IM-Q-ToF-MS provides an extremely powerful analytical tool for early warning of ERα bioactive compounds in dietary supplements as demonstrated by analysis of selected dietary supplements in which different estrogens were identified.

International audience; Metabolomics is a science of interest in food analysis to describe and predict properties of food products and processes. It includes the development of analytical methods with the ultimate goal being the identification of so-called quality markers, (i.e. sets of metabolites that correlate with, for example, quality, safety, taste, or fragrance of foodstuffs). In turn, these metabolites are influenced by factors as genetic differences of the raw food ingredients (such as animal breed or crop species differences), growth conditions (such as climate, irrigation strategy, or feeding) or production conditions (such as temperature, acidity, or pressure). In cases where the routine-based measurement of a food property faces some limitations such as the lack of knowledge regarding the target compounds to monitor, monitoring based on a limited set of crucial biomarkers is a good alternative, which is of great interest for food safety purposes regarding growth promoting practices. Such an approach may be more efficient than using a classic approach based on a limited set of known metabolites of anabolic compounds. In this context, screening strategies allowing detection of the physiological response resulting from anabolic compound administration are promising approaches to detect their misuse. The global metabolomics workflow implemented for such studies is presented and illustrated through various examples of biological matrices profiling (tissue, blood, urine) and for different classes of anabolic compounds (steroids, beta-agonists and somatotropin). (c) 2012 John Wiley & Sons, Ltd.

Detection of the abuse of synthetic steroids in food production is nowadays relatively straightforward using modern techniques such as gas or liquid chromatography coupled to mass spectrometry (GC-MS/MS or LC-MS/MS, respectively). However, proving the abuse of endogenous (or naturally occurring) steroids is more difficult. Despite these difficulties, significant progress in this area has recently been made and a number of methods are now available. The aim of the current review was to systematically review the available analytical approaches, which include threshold concentrations, qualitative marker metabolites, intact steroid esters, gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS), longitudinal testing and omics biomarker profiling. The advantages/disadvantages of these methods are considered in detail, but the choice of which to adopt is dictated by a number of practical, political, and economic factors, which vary in different parts of the world. These include the steroid/species combination requiring analysis, the matrix tested, whether samples are collected from live or slaughtered animals, available analytical instrumentation, sample throughput/cost, and the relevant legal/regulatory frameworks. Furthermore, these approaches could be combined in a range of different parallel and/or sequential screening/confirmatory testing streams, with the final choice being determined by the aforementioned considerations. Despite these advances, more work is required to refine the different techniques and to respond to the ever increasing list of compounds classified as endogenous. At this advanced stage, however, it is now more important than ever for scientists and regulators from across the world to communicate and collaborate in order to harmonize and streamline research efforts. (c) 2012 HFL Sport Science (LGC Ltd) and (c) Her Majesty the Queen in Right of Canada.

International audience; A multiresidue method was developed for screening, quantification, and confirmation of nine natural androgen steroids and their metabolites in urine. Steroids were first extracted from urine by solid phase extraction, enzymatically deglucuronated, re-extracted using a liquid/liquid extraction for purification, and finally acetylated for GC/MS and GC/FID analysis. Each step of sample preparation, as well as analysis, was optimized: solid phase extraction, liquid/liquid extraction, and derivatization reaction ... Therefore, a rugged sample preparation procedure was developed leading to extracts of sufficient purity (recoveries >66% and few matrix compounds). The whole methodology allowed reliable detection and quantification of the nine steroids at low concentration levels. Linearity and repeatability were established and were found to be satisfactory (R-2 > 0.996, RSD < 11%). Finally, the method was applied to quantify compounds of interest in real samples collected from healthy volunteers and patients treated with 4-androstenedione or dehydroepiandrosterone.

International audience; The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to ensure the quality and safety of sport supplements for the consumer is essential. The development and validation of two reporter gene assays coupled with solid phase sample preparation enabling the detection of estrogenic and androgenic constituents in sport supplements is reported. Both assays were shown to be of high sensitivity with the estrogen and androgen reporter gene assays having an EC(50) of 0.01 ng mL(-1) and 0.16 ng mL(-1) respectively. The developed assays were applied in a survey of 63 sport supplements samples obtained across the Island of Ireland with an additional seven reference samples previously investigated using LC-MS/MS. Androgen and estrogen bio-activity was found in 71% of the investigated samples. Bio-activity profiling was further broken down into agonists, partial agonists and antagonists. Supplements (13) with the strongest estrogenic bio-activity were chosen for further investigation. LC-MS/MS analysis of these samples determined the presence of phytoestrogens in seven of them. Supplements (38) with androgen bio-activity were also selected for further investigation. Androgen agonist bio-activity was detected in 12 supplements, antagonistic bio-activity was detected in 16 and partial antagonistic bio-activity was detected in 10. A further group of supplements (7) did not present androgenic bio-activity when tested alone but enhanced the androgenic agonist bio-activity of dihydrotestosterone when combined. The developed assays offer advantages in detection of known, unknown and low-level mixtures of endocrine disruptors over existing analytical screening techniques. For the detection and identification of constituent hormonally active compounds the combination of biological and physio-chemical techniques is optimal. (C) 2010 Elsevier B.V. All rights reserved.

International audience; The use of anabolic agents in food producing animals is prohibited within the European Union since 1988. The illegal use of natural steroid hormones control is however still a current challenge, especially regarding the limitations of existing screening methods. In this context, the present study aimed to develop a new screening approach based on the emerging 'untargeted profiling' concept, but with a special emphasis on steroids phase II conjugated metabolites, in the scope of revealing potential biomarkers signing a fraudulent administration of 4-androstenedione. After extraction and separation of the urinary glucuronide and sulfate steroid fractions, each one was analyzed separately by UPLC-MS/MS using the precursor ion scan acquisition mode. This approach was carried out in order to monitor product ion characteristic of sulfate (m/z 97) and glucuronide (m/z 113) functional groups, and then to fish for any potential conjugated steroid leading to these ionic species after fragmentation. After statistical analysis, 86 metabolites (33 from steroid compounds and 53 from other unknown substances) were highlighted as potential biomarkers of 4-androstenedione abuse. After application of several robustness criteria, 26 metabolites (whom 5 were unambiguously structurally identified), were finally selected to build a statistical model which could be used as new diagnostic tool for screening purposes. (C) 2011 Elsevier B.V. All rights reserved.

Forensic Sci.Int. ISI Document Delivery No.: 844PF Times Cited: 11 Cited Reference Count: 43 Boccard, Julien Badoud, Flavia Grata, Elia Ouertani, Samia Hanafi, Mohamed Mazerolles, Gerard Lanteri, Pierre Veuthey, Jean-Luc Saugy, Martial Rudaz, Serge Elsevier ireland ltd Clare; International audience; Anti-doping authorities have high expectations of the athlete steroidal passport (ASP) for anabolic-androgenic steroids misuse detection. However, it is still limited to the monitoring of known well-established compounds and might greatly benefit from the discovery of new relevant biomarkers candidates. In this context, steroidomics opens the way to the untargeted simultaneous evaluation of a high number of compounds. Analytical platforms associating the performance of ultra-high pressure liquid chromatography (UHPLC) and the high mass-resolving power of quadrupole time-of-flight (QTOF) mass spectrometers are particularly adapted for such purpose. An untargeted steroidomic approach was proposed to analyse urine samples from a clinical trial for the discovery of relevant biomarkers of testosterone undecanoate oral intake. Automatic peak detection was performed and a filter of reference steroid metabolites mass-to-charge ratio (m/z) values was applied to the raw data to ensure the selection of a subset of steroid-related features. Chemometric tools were applied for the filtering and the analysis of UHPLC-QTOF-MS(E) data. Time kinetics could be assessed with N-way projections to latent structures discriminant analysis (N-PLS-DA) and a detection window was confirmed. Orthogonal projections to latent structures discriminant analysis (O-PLS-DA) classification models were evaluated in a second step to assess the predictive power of both known metabolites and unknown compounds. A shared and unique structure plot (SUS-plot) analysis was performed to select the most promising unknown candidates and receiver operating characteristic (ROC) curves were computed to assess specificity criteria applied in routine doping control. This approach underlined the pertinence to monitor both glucuronide and sulphate steroid conjugates and include them in the athletes passport, while promising biomarkers were also highlighted.

The aim of the present study was to demonstrate the applicability of a yeast androgen and estrogen bioassay in the detection of steroid esters in hair samples of animals treated with a hormone ester cocktail. The outcome of the activity screenings was critically compared with the results previously obtained with LC-MS/MS analysis. Hair samples of one pour-on treated animal, 10 ml DMSO containing 25 mg estradiol benzoate (EB), 60 mg testosterone decanoate (TD) and 60 mg testosterone cypionate (TC), were selected and analyzed with the androgen and estrogen yeast bioassay. Results showed that by the introduction of a hydrolysis step, bioassays can be used to screen for the presence of hormone esters in hair samples. Based on the difference in fluorescence responses between the nonhydrolyzed and the hydrolyzed hair samples, it was possible to detect the presence of EB up to at least 56 days after a single pour-on treatment and to detect the presence of TC and TD up to at least 14 days after the treatment. Although the LC-MS/MS analysis could detect TC and TD up to 49 days after treatment, bioassays have the advantage that they can also detect any (un)known steroid ester. Keywords Testosterone ester . Estradiol benzoate . Yeast bioassay . Untargeted analysis . Hair

International audience; Corticosteroids are hormonal substances widely used in human and veterinary medicine for their anti-inflammatory properties. Among the numerous existing artificial corticosteroids, dexamethasone remains the most commonly used, mainly throughout esterified forms such as acetate or phosphate. An experimental study was designed to assess its drug residue levels in urine and feces, as well as its fixation in bovine hair following a single administration of 0.15 mg/kg b.w. dexamethasone acetate and 0.12 mg/kg b.w. dexamethasone sodium phosphate. Different analytical methods based on GC-MS or LC-MS/MS were used for measuring dexamethasone and its esterified forms, which were implemented in 3 different European laboratories in the field that collaborated for this study. The obtained results confirmed the high and rapid urinary excretion rate of dexamethasone, with a maximal concentration (267 mu g/L) measured one day after administration and 98% elimination within 3 days. The concentrations obtained with the GC-NCI-MS procedure (using chemical oxidation as derivatization) were found significantly higher than the ones obtained with LC-ESI-MS/MS, indicating a possible contribution of dexamethasone phase I and/or II metabolites to the monitored signal. Fecal elimination was also found rapid (95% elimination within 3 days) with a maximum concentration level (28.5 mu g/kg) observed one day after administration. Detectable levels of dexamethasone in hair appeared on day 2 (11.5 mu g/kg), reached a maximum around one week, and could be identified until 22 days upon treatment, establishing the suitability of hair as a biological matrix for medium to long-term residue controls of dexamethasone. (C) 2010 Elsevier Inc. All rights reserved.

International audience; The use of steroid hormones as growth promoters in cattle is banned within the European Union since 1988 but can still be fraudulently employed in animal breeding farms for anabolic purposes. While efficient targeted confirmatory methods have been implemented in control laboratories for many years, fast and reliable screening methods are still required, especially in the case of natural hormones abuse, but more globally for new "fishing" strategies allowing to reveal the use of even unknown anabolic agents. The development of focused profiling or untargeted metabolomic approaches is thus emerging in this context. The present study was a focused profiling study using steroids phase II metabolites, with the aim to get a better understanding of the steroid metabolism disruptions after exogenous administration of androstenedione and finally reveal potential biomarkers signing its administration. A sample preparation procedure was first developed, based on a separation of 31 glucuronide and sulphate conjugate compounds using an anion exchange SPE system. Each fraction was then analysed by UPLC-MS/MS in MRM mode showing a rapid (between 4 h and 4 days after treatment) and huge excretion of several direct metabolites of androstenedione such as etiocholanolone-glucuronide or epiandrosterone-sulphate. (C) 2010 Elsevier B.V. All rights reserved.

Unification of the screening protocols for a wide range of doping agents has become an important issue for doping control laboratories. This study presents the development and validation of a generic liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) screening method of 241 small molecule analytes from various categories of prohibited substances (stimulants, narcotics, diuretics, ß2-agonists, ß-blockers, hormone antagonists and modulators, glucocorticosteroids and anabolic agents). It is based on a single-step liquid-liquid extraction of hydrolyzed urine and the use of a rapid-resolution liquid chromatography/high-resolution time-of-flight mass spectrometric system acquiring continuous full scan data. Electrospray ionization in the positive mode was used. Validation parameters consisted of identification capability, limit of detection, specificity, ion suppression, extraction recovery, repeatability and mass accuracy. Detection criteria were established on the basis of retention time reproducibility and mass accuracy. The suitability of the methodology for doping control was demonstrated with positive urine samples. The preventive role of the method was proved by the case where full scan acquisition with accurate mass measurement allowed the retrospective reprocessing of acquired data from past doping control samples for the detection of a designer drug, the stimulant 4-methyl-2-hexanamine, which resulted in re-reporting a number of stored samples as positives for this particular substance, when, initially, they had been reported as negatives.

17beta-Estradiol is one of the most powerful sex steroids illegally used in bovine production. The objective of this study was to evaluate the application and the specificity of the RIKILT yeast estrogen bioassay (REA) for the detection of molecules with estrogenic activities in the urine of calves experimentally treated with anabolics. Four groups of six calves each received an injection of 17beta-estradiol intramuscularly (group B), androsterone and gliburide (group A), and testosterone (group C) molecules at different dosage for 40 days. Group D was the control. The ability of the REA test to detect estrogenic activity in urine samples from all animals was assessed. All estrogen-treated animals (group B) showed as being positive up to 7 days after administration of the highest dosage of 17beta-estradiol, while the other three groups showed as being negative. The identity of estrogenic molecules in the urine of group B (17beta-estradiol, 17alpha-estradiol) was confirmed by gas chromatography-mass spectrometry (GC/MS). This is the first time the REA test has been applied to detect 17beta-estradiol in the urine of calves treated with the hormone in vivo. The technique may offer an advantageous laboratory method for the veterinary surveillance of illegal steroid use.

The abuse of synthetic esters of natural steroids such as testosterone and estradiol in cattle fattening and sports is hard to detect via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. An interesting alternative can be provided by the analysis of the administered synthetic steroids themselves, i.e., the analysis of intact steroid esters in hair by liquid chromatography tandem mass spectrometry (LC/MS/MS). However, retrospective estimation of the application date following a non-compliant finding is hindered by the complexity of the kinetics of the incorporation of steroid esters in hair. In this study, the incorporation of intact steroid esters in hair following pour-on treatment has been studied and critically compared with results from intramuscular treatment. To this end animals were pour-on treated with a hormone cocktail containing testosterone cypionate, testosterone decanoate and estradiol benzoate in different carriers. The animals were either treated using injection and pour-on application once or three times having 1 week between treatments using injection and pour-on application. Animals were slaughtered from 10-12 weeks after the last treatment. Both hair and blood plasma samples were collected and analysed by LC/MS/MS. From the results, it is concluded that after single treatment the levels of steroid esters in hair drop to CCbeta levels (5-20 microg/kg) after 5-7 weeks. When treatment is repeated two times, the CCbeta levels are reached after 9-11 weeks. Furthermore, in plasma, no steroid esters were detected; not even at the low microgramme per litre level but--in contrast with the pour-on application--after i.m. injection, significant increase of 17beta-testosterone and 17beta-estradiol were observed. These observations suggest that transport of steroid esters after pour-on application is not only performed by blood but also by alternative fluids in the animal so probably the steroid esters are already hydrolysed and epimerized before entering the blood.

Hormone and veterinary drug screening and forensics can benefit from the recent developments in desorption electrospray ionisation (DESI) mass spectrometry (MS). In this work the feasibility of DESI application has been studied. Using a linear ion trap or quadrupole time-of-flight (TOF) MS instrument both full-scan and data-dependent collision-induced dissociation MS n spectra were acquired in seconds without sample preparation. Preliminary data are presented for the rapid screening of (pro)hormone supplement samples, an illegal steroid cocktail and forensic samples from veterinary drug investigations. The potential of this DESI approach is clearly demonstrated since compounds observed could be independently confirmed by liquid chromatography/TOFMS with accurate mass measurement, and/or proton nuclear magnetic resonance spectroscopy. Specific concerns related to false-positive and false-negative findings due to limitations in quantification and memory-effects are briefly discussed. It is envisaged that DESI will achieve a prominent role in hormone and veterinary drug analysis in the near future.

An inter-laboratory exercise was performed with a yeast estrogen bioassay, based on the expression of yeast enhanced green fluorescent protein (yEGFP), for the determination of estrogenic activity in extracts of calf urine samples. Urine samples were spiked with 1 and 5 ngmL(-1) 17beta-estradiol and 17alpha-ethynylestradiol, 10 and 50 ngmL(-1) mestranol, and 100 ngmL(-1) testosterone and progesterone. Sample extracts of blank and spiked urine samples were prepared at our laboratory and sent to seven laboratories together with a reagent blank, a DMSO blank, and eight 17beta-estradiol stock solutions in DMSO ranging in concentration from 0 to 545 ngmL(-1). Sample extracts and standards were coded and tested blindly. A decision limit (CCalpha) was determined based on the response of seven blank urine samples. Signals of the negative controls, e.g. urine samples spiked with 100 ngmL(-1) testosterone or progesterone, were all below the determined CCalpha and were thus screened as compliant. Positive controls, i.e. the urine samples spiked at two levels with 17beta-estradiol, 17alpha-ethynylestradiol and mestranol, were almost all screened as suspect, i.e. gave signals above the determined CCalpha. Determined EC(50) values calculated from the 17beta-estradiol dose-response curves obtained by the seven laboratories ranged from 0.59 to 0.95 nM.

Bioassays are valuable tools for combating the illegal use of steroids in cattle fattening. Previously we described the construction and properties of a rapid and robust yeast androgen bioassay stably expressing the human androgen receptor (hAR) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. In the present study this yeast androgen bioassay was validated as a qualitative screening method for the determination of androgenic activity in calf urine and animal feed. This validation was performed according to EC Decision 2002/657. 20 blank samples were spiked with testosterone, 17alpha-methyltestosterone, 19-nortestosterone, 17beta-trenbolone, 17beta-boldenone or 17alpha-methylboldenone at 2 or 15 ngmL(-1) in urine and 50 or 100 ngg(-1) in feed. All blank and spiked samples fulfilled the CCalpha and CCbeta criterions, meaning that all 20 blank samples gave signals below the determined decision limits CCalpha and were thus classified as compliant (alpha=1%). For each component, at least 19 out of the 20 spiked samples gave a signal above the CCalpha and were thus classified as suspect (beta=5%). The method was specific, and high amounts of dexamethasone did not interfere with the outcome of the test. Although high levels of 17alpha-ethynylestradiol can significantly inhibit the response obtained with low amounts of androgens, that situation is not relevant in veterinary practice. When stored at their specific conditions, the androgens in feed were stable for at least 91 days. Real urine samples from a national control program were screened and a representative part of the compliant and suspect samples were confirmed by gas chromatography-tandem mass spectrometry.

Efficient control of the illegal use of anabolic steroids must both take into account metabolic patterns and associated kinetics of elimination; in this context, an extensive animal experiment involving 24 calves and consisting of three administrations of 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate esters was carried out over 50 days. Urine samples were regularly collected during the experiment from all treated and non-treated calves. For sample preparation, a single step high throughput protocol based on 96-well C-18 SPE was developed and validated according to the European Decision 2002/657/EC requirements. Decision limits (CC alpha) for steroids were below 0.1 mu g L-1, except for 19-norandrosterone (CC alpha = 0.7 mu g L-1) and estrone (CC alpha = 0.3 mu g L-1). Kinetics of elimination of the administered 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate were established by monitoring 17 beta-estradiol, 17 alpha-estradiol, estrone and 17 beta-nandrolone, 17 alpha-nandrolone, 19-noretiocholanolone, 19-norandrostenedione, respectively. All animals demonstrated homogeneous patterns of elimination both from a qualitative (metabolite profile) and quantitative point of view (elimination kinetics in urine). Most abundant metabolites were 17 alpha-estradiol and 17 alpha-nandrolone (> 20 and 2 mg L-1, respectively after 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate administration) whereas 17 beta-estradiol, estrone, 17 beta-nandrolone, 19-noretiocholanolone and 19-norandrostenedione were found as secondary metabolites at concentration values up to the mu g L-1 level. No significant difference was observed between male and female animals. The effect of several consecutive injections on elimination profiles was studied and revealed a tendency toward a decrease in the biotransformation of administered steroid 17 beta form. (c) 2008 Elsevier Ltd. All rights reserved.

Within the European Union, the control for residues of illegal hormones in food-producing animals is based on urine analysis for a few target analytes using gas chromatography/mass spectrometry and/or liquid chromatography-tandem mass spectrometry. Recently, we developed a robust yeast bioassay screening tool for estrogens, which was validated as a qualitative screening method in accordance with EC decision 2002/657/EC. In this study, we present long-term performance data and a comparison of urine data obtained with this bioassay, and data from an established gas chromatography-tandem mass spectrometry (GC/MS/MS) confirmatory analysis method. More than 120 calf urine samples from a controlled reference experiment were analysed using both protocols. According to the GC/MS/MS method, only the natural estrogens 17alpha-estradiol and estrone were present in the non-compliant samples. The bioassay was less sensitive than GC/MS/MS for the relatively weak estrogenic compound 17alpha-estradiol, in accordance with expectations. Assuming that application of the mass spectrometric method is considered beyond reasonable doubt, the bioassay performed very well: only 5.6% of the calf urine samples found compliant in GC/MS/MS were screened false suspect in the bioassay screening method. The bioassay results of non-compliant urine samples under routine conditions were as predicted, taking into account the relative estrogenicity of the natural estrogens 17alpha-estradiol and estrone vs. 17beta-estradiol. Only one sample was screened false negative for 17alpha-estradiol and estrone. Application of this fast and simple estrogen bioassay in routine surveillance and control can significantly reduce GC/MS/MS sample workload and allow higher percentages of animals to be screened for potential hormone abuse.

The abuse of esters of natural androgenic steroids in cattle fattening and sports is hard to control via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. In veterinary control strange findings of 17beta-testosterone and 17alpha-testosterone in urine are often ignored because of the lack of statistically sound reference data of naturally occurring levels. An interesting alternative for inconclusive urine analyses in veterinary control can be provided by the analysis of the administered steroids themselves, i.e. the analysis of intact steroid esters in hair. Unfortunately, the analysis of intact steroid esters is complicated not only by the vulnerability of the esters which precludes alkaline hydrolysis of the hair, but also by the wide polarity range of short and long-chain esters yielding very poor recoveries for either the one or the other. In this study, a multi-steroid esters LC/MS/MS screening method is presented for trace analysis of the synthetic intact esters of 17beta-testosterone and the undecylenate ester of 17beta-boldenone in bovine hair. The method, requiring only 200 mg of pulverised hair, features a mild digestion procedure using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and the use of four deuterium-labelled steroid esters as internal standards covering the wide polarity range of the analytes. In spiked hair samples for most of the analytes the limit of detection and the accuracy using isotope dilution were 2-5 ng/g and 97-105%, respectively. The applicability was demonstrated using hair samples from a controlled experiment in which six bovines were injected intramuscularly with two different doses of two commercial mixtures of testosterone esters, and with two different doses of boldenone undecylenate. Depending on the dose all administered testosterone- and boldenone esters were found to be incorporated in bovine hair following a single intramuscular injection, except testosterone propionate which dose might have been too low.

New anabolic steroids show up occasionally in sports doping and in veterinary control. The discovery of these designer steroids is facilitated by findings of illicit preparations, thus allowing bioactivity testing, structure elucidation using NMR and mass spectrometry, and final incorporation in urine testing. However, as long as these preparations remain undiscovered, new designer steroids are not screened for in routine sports doping or veterinary control urine tests since the established GC/MS and LC/MS/MS methods are set up for the monitoring of a few selected ions or MS/MS transitions of known substances only. In this study, the feasibility of androgen bioactivity testing and mass spectrometric identification is being investigated for trace analysis of designer steroids in urine. Following enzymatic deconjugation and a generic solid-phase extraction, the samples are analyzed by gradient LC with effluent splitting toward two identical 96-well fraction collectors. One well plate is used for androgen bioactivity detection using a novel robust yeast reporter gene bioassay yielding a biogram featuring a 20-s time resolution. The bioactive wells direct the identification efforts to the corresponding well numbers in the duplicate plate. These are subjected to high-resolution LC using a short column packed with 1.7-microm C18 material and coupled with electrospray quadrupole time-of-flight mass spectrometry (LC/QTOFMS) with accurate mass measurement. Element compositions are calculated and used to interrogate electronic substance databases. The feasibility of this approach for doping control is demonstrated via the screening of human urine samples spiked with the designer anabolic steroid tetrahydrogestrinone. Application of the proposed methodology, complementary to the established targeted urine screening for known anabolics, will increase the chance of finding unknown emerging designer steroids, rather then being solely dependent on findings of the illicit preparations themselves.

A liquid chromatography (LC)-based method with mass spectrometric (MS/MS) detection was developed for the determination of estradiol benzoate residues in hair of cattle. First hair samples were pulverized with a ball mill followed by treatment with the reducing agent Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). After liquid/liquid extraction samples were further purified by solid-phase extraction. Finally samples were analyzed with LC–MS/MS using deuterated estradiol benzoate as internal standard. The method was validated following the latest EU guidelines for screening methods using blank hair samples spiked at 5 ng g−1. The detection capability (CCβ) was less than 5 ng g−1 and the decision limit (CCα) was 1.6 ng g−1. The recovery was 27% at the 5 ng g−1 level and the accuracy was 95%. Confirmation, with two MS/MS transitions, was possible at a concentration of 5 ng g−1 or higher. Incurred samples obtained from different animal experiments were analyzed and the presence of estradiol benzoate was confirmed. Finally hair samples from different slaughterhouses were analyzed.

Detection of hormones and veterinary drugs in urine samples is limited to the short-term following illegal administration. Alternative sample matrices such as hair or retina are of interest for prolonged detectability of residues. In this preliminary study, we developed sensitive liquid chromatography–tandem mass spectrometry methods for the confirmatory analysis of stanozolol in different matrices and applied them to urine, retina and hair samples obtained from 11 suspect bovines at the slaughterhouse. Most of the stanozolol metabolite (16-hydroxystanozolol) levels in urine samples were below 0.5 ng ml −1 , but nevertheless in some urines traces of this metabolite could still be positively confirmed. Retina samples were all negative so the value of this matrix for stanozolol might be questioned. The hair samples, on the other hand, yielded many positive stanozolol results, and in some animals levels were up to 100-fold higher than the corresponding metabolite levels in urine thereby underlining the potential for prolonged detectability of stanozolol abuse. © 2002 Elsevier Science B.V. All rights reserved.