Article

Monitoring the endogenous steroid profile disruption in urine and blood upon nandrolone administration: An efficient and innovative strategy to screen for nandrolone abuse in entire male horses

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  • Independent Researcher
  • LCH, Verrières le Buisson, France
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Abstract

Nandrolone (17β‐hydroxy‐4‐estren‐3‐one) is amongst the most misused endogenous steroid hormones in entire male horses. The detection of such a substance is challenging with regard to its endogenous presence. The current international threshold level for nandrolone misuse is based on the urinary concentration ratio of 5α‐estrane‐3β,17α‐diol (EAD) to 5(10)‐estrene‐3β,17α‐diol (EED). This ratio, however, can be influenced by a number of factors due to existing intra‐ and inter‐variability standing, respectively, for the variation occurring in endogenous steroids concentration levels in a single subject and the variation in those same concentration levels observed between different subjects. Targeting an efficient detection of nandrolone misuse in entire male horses, an analytical strategy was set up in order to profile a group of endogenous steroids in nandrolone‐treated and non‐treated equines. Experiment plasma and urine samples were steadily collected over more than three months from a stallion administered with nandrolone laurate (1 mg/kg). Control plasma and urine samples were collected monthly from seven non‐treated stallions over a one‐year period. A large panel of steroids of interest (n = 23) were extracted from equine urine and plasma samples using a C 18 cartridge. Following a methanolysis step, liquid‐liquid and solid‐phase extractions purifications were performed before derivatization and analysis on gas chromatography‐tandem mass spectrometry (GC‐MS/MS) for quantification. Statistical processing of the collected data permitted to establish statistical models capable of discriminating control samples from those collected during the three months following administration. Furthermore, these statistical models succeeded in predicting the compliance status of additional samples collected from racing horses. Copyright © 2013 John Wiley & Sons, Ltd.

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... The detection of such a substance is challenging due to its endogenous nature. The current international threshold level for nandrolone misuse is based on the urinary concentration ratio of 5α-estrane-3β,17α-diol (EAD) to 5(10)-estrene-3β,17α-diol (EED) (Kaabia et al., 2014). This ratio, however, can be influenced by several factors due to existing intra-and interanimal variability, meaning, respectively, the variation occurring in endogenous steroid concentration levels in a single subject and the variation in those same concentration levels observed between different subjects. ...
... he 10-200 range for up to 30 days. Following the 5α-estrane-3β,17α-diol metabolite itself as t-butydimethyl oxime derivatives on GC-MS was also found to be specific for uncovering dosing with nandrolone ester derivatives, such as laurate (Laurabolin), phenylpropionate (Nandrolin) and cyclohexylpropionate (Retarbolin) esters(Teale & Houghton, 1991).Kaabia et al. (2014) took the detection of nandrolone usage a step higher by application of GC-MS-MS (GC tandem mass spectrometry) in a steroidomic approach targeting 23 relevant steroidal compounds including hormones and their metabolites. Detailed statistical analysis of results from both serum and urine from seven stallions, including ones treated with L ...
Article
Anabolic androgenic steroids are synthetic substances related to the male sex hormones (androgens). These agents promote the growth of skeletal muscle (anabolic effects) and the development of male sexual characteristics (androgenic effects). Anabolic steroids have been illegally used for many years as performance-enhancing drugs in human, equine, and canine sports and as growth promoters in livestock reared to provide meat for human consumption. The analytical challenge to developing effective means of control within these fields has been exacerbated by the reported endogenous nature of some of these steroids. Anabolic steroids have been employed extensively in equine practice over the past 50 years. Their usefulness is largely dependent on subjective opinions, as only minimal studies investigating pharmacodynamics have been carried out in horses. Therefore, their use will vary markedly between practitioners depending on their personal experiences and pressures by trainers to use them. They form part of rational therapy in a variety of conditions. In addition to their use for increasing muscle mass, they are used to varying extents in the raising of yearlings and in the training and racing of horses with the view of improving performance. The use of these agents is prohibited in the horseracing industry by the Association of Racing Commissioners International (ARCI), International Federation of Horseracing Authorities (IFHA), and Fédération Equestre Internationale (FEI).
... Biomarker monitoring can already be considered a new era in human anti-doping [69] and different biomarker approaches are being developed for equine purposes at the moment. As such, longitudinal monitoring of biomarkers can reveal non-physiological responses independently of the used doping technique or substance and may cause sanctioning of illicit practices [70]. ...
... An example hereof is an efficient strategy that has been developed to screen for abuse of nandrolone, a 'semi'-endogenous steroid in stallions ( Table 3), monitoring the endogenous steroid profile disruption in urine and blood upon nandrolone administration [70]. A panel of (endogenous) steroids of interest was extracted from equine urine and plasma samples and quantified by GC-MS/MS. ...
Chapter
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The higher the pressure to win, the more athletes are inclined to take steps to improve one’s performance through questionable means. To minimize this, strict anti‐doping and medication rules are being enforced. All human and equine athletes are regularly subjected to doping analysis to prevent abuse of forbidden substances from affecting their performance. Anabolic‐androgenic steroids (AASs) have been part of the forbidden substances list for years, because of their muscle building and performance‐enhancing capacities and possible side effects. For most of the AAS, zero‐tolerance is held. However, some AASs can be endogenous to the athletes, such as for example testosterone in males. These endogenous steroids can render it very difficult to reveal steroid abuse. Specific mass spectrometric (MS) methods, including ultra‐high performance liquid chromatography‐MS (UHPLC‐MS/MS), high resolution mass spectrometry (HRMS) and gas chromatography–combustion‐isotope ratio MS (GC‐C‐IRMS), have been put forward to overcome these analytical difficulties. Currently, high‐tech metabolomic methods are being used to build athlete specific biological passports. In the near future, these passports might allow putting a stop to abuse, by staying ahead of the cheats. These are bright prospects, leading towards clean and fair sports competitions worldwide.
... This kind of steroid approach has been previously studied in humans, [6][7][8][9] in cattle [10] and more recently by Kaabia et al. in horse. [11] Thanks to statistical data processing by specific software, it becomes possible to establish models capable of discriminating treated from untreated animals. Moreover, the simple screening of GnRH antibodies in plasma with the follow-up of the steroid profile in urine is supposed to be a reliable complementary analysis to define the status of a male animal with a gelding or female steroid profile. ...
... This approach has been recently used successfully to improve the detection of nandrolone in stallions. [11] In this study anabolic androgenic steroids that have effects on steroid biosynthesis and metabolism are kinetically monitored by a set of known biological molecules. ...
Article
Since the availability on the European market of the vaccine Improvac® dedicated to male pig immunological castration, the risk of misuse of this product in horses is now considered as a threat for the horseracing industry. Immunological castration is not allowed by the racing codes (immune system, Article 6). Indeed, this vaccination against the hypothalamic hormone luteinizing hormone‐releasing hormone or gonadotropin‐releasing hormone (GnRH) will prevent the release from the anterior pituitary of luteinizing hormone and follicle stimulating hormone, which are required for the development and activity of gonads in males (testes) and female (ovaries) and therefore all their subsequent physiological functions. This treatment will induce a strong hormonal variation resulting in a behaviour modification of the animals. In this work, four male standardbreds treated with Improvac® vaccine (two intramuscular injections within 4 weeks) were studied. Monitoring of the total scrotal width showed a decrease of the scrotum size (37%) and production of anti‐GnRH antibodies was detected up to 200 days after the first injection. Anti‐GnRH antibodies were detected in plasma after caprylic acid precipitation followed by an enzyme‐linked immunosorbent assay (ELISA) as a rapid and efficient screening method applicable to routine analysis. These results were correlated to a switch of the sexual status from male group to gelding/female group obtained by a steroidomic approach with urine based on ten endogenous compounds. Copyright © 2017 John Wiley & Sons, Ltd.
... Since the administration of exogenous steroids induces temporal modifications of endogenous steroid profiles, as well as related metabolite levels [26], monitoring for a range of modified endogenous steroid concentrations allows screening for hormone abuse in food-producing animals. Successfully applied for anti-doping purposes in humans, the strategy has also proven its relevance in both equine and bovine, either in urine or plasma [27][28][29][30]. The strategy has also been reported for the investigation of biochemical alterations occurring in human urine as a result of dioxin exposure [31]: metabolic patterns related to dioxin effects in human have, thus, been determined, allowing for the discovery of predictive sets of relevant steroid-related compounds. ...
... Anabolic practice in cattle Targeted and non-targeted metabolomics GC-MS (full scan) GC-MS/MS (SRM) [40] Anabolic steroid animal administration Targeted steroid approach GC-MS/MS (SRM) [41] Intracellular metabolites of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-pdioxin Non-targeted fatty acids metabolomics GC-MS (full scan) [42] Endogenous steroid profile disruption Targeted metabolomics GC-MS/MS (SRM) [27] Aflatoxin B1 hepatotoxicity Non-targeted metabolomics GC-MS (full scan) [43] ...
Article
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This review aims to describe the most significant applications of mass spectrometry-based metabolomics in the field of chemical food safety. A particular discussion of all the different analytical steps involved in the metabolomics workflow (sample preparation, mass spectrometry analytical platform and data processing) will be addressed.
... Finally, targeted steroidomics consists of the quantitative profiling of a selected set of steroids in order to study their concentration levels occurring variations following an anabolic treatment. Such a strategy was for example performed in order to establish suspicion thresholds and identify new biomarkers for DHEA, DiHydroTestosterone (DHT), and testosterone administration in men [64], to discriminate estradiol-treated bovine from the control group [65] and nandrolonetreated equine from control group [66] and served to evidence the evolution of steroid concentrations with regard to the seasonality [67]. ...
Chapter
Full-text available
Testosterone is a key compound of the anabolic androgenic steroids (AAS) family. It has largely been misused in human and animal doping targeting a muscle tissue growth and an enhancement of performances. Such practices constitute a violation against ethical values, food safety, and animal welfare. Consequently, the use of such substance is regulated by WADA and International committees for some animal species such as equine and bovine. Although efficient, the detection of testosterone misuse remains challenging in some cases due to its endogenous origin and its inter- and intra-individual level fluctuation in biological fluids. Novel analytical strategies have been developed and are continuously evolving in order to tackle this issue and to provide a better control of testosterone misuse.
... Despite the regulations in force in human and horses, anabolic steroids have been reported, in human, to be one of the most widely used class of molecules to enhance sport performance (Thevis et al., 2021). In order to meet the needs of doping control laboratories, the benefit of decades of research have led to the implementation of advanced screening methods, generally based on the direct detection of administered drugs or their metabolites (Choi et al., 2018;Delcourt et al., 2021;Kaabia et al., 2014). Yet, the emergence of new doping practices such as the administration of designer drugs (Waller & McLeod, 2017), low dose cocktails (Badoud et al., 2013), endogenous hormones (Scarth et al., 2012) or even genetic doping (Wilkin et al., 2017) are among the main current challenges for doping control. ...
Article
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Introduction Despite their ban, Anabolic Androgenic Steroids (AAS) are considered as the most important threat for equine doping purposes. In the context of controlling such practices in horse racing, metabolomics has emerged as a promising alternative strategy to study the effect of a substance on metabolism and to discover new relevant biomarkers of effect. Based on the monitoring of 4 metabolomics derived candidate biomarkers in urine, a prediction model to screen for testosterone esters abuse was previously developed. The present work focuses on assessing the robustness of the associated method and define its scope of application. Materials and methods Several hundred urine samples were selected from 14 different horses of ethically approved administration studies involving various doping agents’ (AAS, SARMS, β-agonists, SAID, NSAID) (328 urine samples). In addition, 553 urine samples from untreated horses of doping control population were included in the study. Samples were characterized with the previously described LC-HRMS/MS method, with the objective of assessing both its biological and analytical robustness. Results The study concluded that the measurement of the 4 biomarkers involved in the model was fit for purpose. Further, the classification model confirmed its effectiveness in screening for testosterone esters use; and it demonstrated its ability to screen for the misuse of other anabolic agents, allowing the development of a global screening tool dedicated to this class of substances. Finally, the results were compared to a direct screening method targeting anabolic agents demonstrating complementary performances of traditional and omics approaches in the screening of anabolic agents in horses.
... Despite the regulations in force in human and horses, anabolic steroids have been reported, in human, to be one of the most widely used class of molecules to enhance sport performance (Thevis, 2021). In order to meet the needs of doping control laboratories, the bene t of decades of research have led to the implementation of advanced screening methods, generally based on the direct detection of administered drugs or their metabolites (Delcourt, 2021;Choi, 2018;Kaabia, 2014). Yet, the emergence of new doping practices such as the administration of designer drugs (Waller, 2017), low dose cocktails (Badoud, 2013), endogenous hormones (Scarth, 2012) or even genetic doping (Wilkin, 2017) are among the main current challenges for doping control. ...
Preprint
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Despite their ban, Anabolic Androgenic Steroids (AAS) are considered as the most important threat for equine doping purposes. In the context of controlling such practices in horse racing, metabolomics has emerged as a promising alternative strategy to study the effect of a substance on metabolism and to discover new relevant biomarkers of effect. Based on the monitoring of 4 metabolomics derived candidate biomarkers in urine, a prediction model to screen for testosterone esters abuse was previously developed. The present work now focuses on assessing the robustness of the associated method and define its scope of application. Several hundred urine samples were selected from 16 different horses of ethically approved animal experiments involving various doping agents’ applications (AAS, SARMS, β -agonists, SAID, NSAID) (n = 349). In addition, 342 urine samples from untreated animals of general equine populations were included in the study. Samples were characterized with the previously described LC-HRMS/MS developed method, with the objective of assessing both its biological and analytical robustness’s. The study concluded that the measurement of the 4 biomarkers involved in the model was fit for purpose. Further, the classification model confirmed its effectiveness in screening for testosterone esters use; and it demonstrated its ability to screen for the administration of other anabolic agents, allowing the development of a global screening tool dedicated to this class of substances. Finally, the results were compared to a direct screening method targeting anabolic agent’s residues, demonstrating complementarity performances of traditional and omics approaches in the screening of anabolic agents in horses.
... where two matrices, equine plasma and urine, were used to develop a successful statistical model that enabled the extension of the detection period of nandrolone abuse in entire male horses. 143 More intrinsic information was provided beyond the established threshold for nandrolone abuse. Chan et al. used an OPLS-DA model to identify seven biomarkers that were indicative of steroidal aromatase inhibitor administration. ...
Article
Full-text available
Metabolomics is a multidisciplinary field providing workflows for complementary approaches to conventional analytical determinations. It allows for the study of metabolically related groups of compounds or even the study of novel pathways within the biological system. The procedural stages of metabolomics; experimental design, sample preparation, analytical determinations, data processing and statistical analysis, compound identification and validation strategies, are explored in this review. The selected approach will depend on the type of study being conducted. Experimental design influences the whole metabolomics workflow and thus needs to be properly assessed to ensure sufficient sample size, minimal introduced and biological variation and appropriate statistical power. Sample preparation needs to be simple, yet potentially global in order to detect as many compounds as possible. Analytical determinations need to be optimised either for the list of targeted compounds or a universal approach. Data processing and statistical analysis approaches vary widely and need to be better harmonised for review and interpretation. This includes validation strategies which are currently deficient in many presented workflows. Common compound identification approaches have been explored in this review. Metabolomics applications are discussed for clinical and forensic toxicology, human and equine sports anti‐doping and veterinary residues.
... The analysis was performed in blind, and due to privacy, we do not know if samples were bovine or equine. Due to the blind analysis and the thresholds changing between gender, species and age of the animals, we could not compare our results with international guidelines [36]. ...
Article
Full-text available
A simple and fast analytical method able to simultaneously identify and quantify 17 endogenous and exogenous steroidal hormones was developed in bovine and equine blood using UHPLC-MS/MS. A total amount of 500 µL of sample was deproteinized with 500 µL of a mixture of methanol and zinc sulfate and evaporated. The mixture was reconstituted with 50 µL of a solution of 25% methanol and injected in the UHPLC-MS/MS triple quadrupole. The correlation coefficients of the calibration curves of the analyzed compounds were in the range of 0.9932–0.9999, and the limits of detection and quantification were in the range of 0.023–1.833 and 0.069–5.5 ppb, respectively. The developed method showed a high sensitivity and qualitative aspects allowing the detection and quantification of all steroids in equine and bovine blood. Moreover, the detection limit of testosterone (50 ppt) is half of the threshold admitted in plasma (100 ppt). Once validated, the method was used to quantify 17 steroid hormones in both bovine and equine blood samples. The primary endogenous compounds detected were corticosterone (range 0.28–0.60 ppb) and cortisol (range 0.44–10.00 ppb), followed by androstenedione, testosterone and 11-deoxycortisol.
... Beyond the clinical field, the biomarker strategy is also applied to other domains such as the environment 7 or the doping control. 1 For doping control, biomarker developments were highly promoted since the 2000s and succeeded in the establishment of the Athlete's Biological Passport (ABP) for Human 8 and the Equine Biological Passport for the Horse. 9,10 Therefore, in the horse, transcriptomics, 11 proteomics, 12 metabolomics, 13,14 and steroidomics 15,16 were carried out to develop innovative approaches in the fight against doping. ...
Article
Short half‐life doping substances are, quickly eliminated and therefore difficult to control with traditional analytical chemistry methods. Indirect methods targeting biomarkers constitute an alternative to extend detection time frames in doping control analyses. Gene expression analysis (i.e. transcriptomics) has already shown interesting results in both Humans and Equines for erythropoietin (EPO), Growth Hormone (GH) and Anabolic Androgenic Steroid (AAS) misuses. In Humans, circulating cell‐free microRNAs in plasma were described as new potential biomarkers for control of Major Doping Agent (MDA) abuses. The development of a qPCR method allowing the detection of circulating miRNAs was carried out on equine plasma collected on different type of tubes (EDTA, Lithium‐Heparin). Whereas analyzing plasma collected in EDTA tubes is a standard method in molecular biology, analyzing plasma collected in Lithium‐Heparin (LiHep) tubes is challenging, as heparin is a RT‐PCR inhibitor. Different strategies were considered and attention was paid on both miRNAs extraction quality and detection sensitivity. The detection of endogenous circulating miRNAs was performed and compared between the different types of tubes. In parallel, homologs of human miRNAs characterized as potential biomarkers of doping were sought in equine databases. The miRNA eca‐miR‐144, described as potential erythropoiesis stimulating agents (ESAs) administration candidate biomarker was retained and assessed in equine post‐administration samples. The results about the qPCR method development and optimization are exposed as well as the equine miRNAs detection. To our knowledge, this work is the first study and the proof of concept of circulating miRNAs detection in plasma dedicated to equine doping control.
... The decision threshold value was consequently set at T = −1.55. Such a strategy has already been successfully proposed (Gaud Kaabia et al. 2014;Dervilly-Pinel et al. 2015). ...
Article
Within the particular context of controlling chemical residues in food, an alternative to targeted approaches has emerged; it consists in the characterization of physiological perturbations induced upon exposure of animals to a given chemical substance/class of substances to highlight suitable biomarkers addressing safety and/or regulatory issues. Metabolomics in particular has been investigated in the hope of identifying such biomarkers, and a range of studies have demonstrated the efficiency of the strategy. Until very recently, steps remained to be taken toward official or commercial implementation of corresponding tools. In particular, the lack of guidelines and criteria to validate such methods that do not target specific chemical species per se, constituted a bottleneck. In the present work, a metabolomics model dedicated to the detection of β-agonist administration in bovines has been developed and fully validated; criteria (selectivity, robustness, stability, suspicion threshold definition, false positive and false negative rates) have been proposed in agreement with EU expectations (Dec 2002/657), enabling demonstration that performances comply with screening requirements. Although some of the biomarkers involved in the prediction model remain un-elucidated, the corresponding LC-HRMS method has recently been ISO17025 accredited, allowing for the very first official implementation of a metabolomics based strategy within French National Monitoring Plans.
... Ces stratégies ne cherchent pas à identifier directement la présence de molécules suspectées ou de leurs métabolites directs, mais à révéler une signature métabolique ou physiologique spécifique pouvant être associée à une pratique anabolisante. Ces approches dites « indirectes » ou « non ciblées » (Nebbia et al., 2011 ;Pinel et al., 2010) reposent sur des méthodes telles la transcriptomique (Riedmaier, 2015 ;Riedmaier et al., 2009a ;Riedmaier et al., 2012 ;Riedmaier and Pfaffl, 2013 ;Riedmaier et al., 2009b, c), la protéomique (Cacciatore et al., 2009 ;Cunningham et al., 2009 ;Kinkead et al., 2015) ou la métabolomique (Dervilly- Dervilly-Pinel et al., 2012 ;Gallart Ayala et al., 2015 ;Jacob et al., 2014 ;Kouassi Nzoughet et al., 2015b), incluant ses déclinaisons que sont la lipidomique (Kouassi Nzoughet et al., 2015a) et la stéroïdomique (Dervilly-Pinel et al., 2011 ;Kaabia et al., 2014 De plus, et en ce qui concerne l'évolution du contexte réglementaire, il est attendu que la réglementation européenne en matière de contrôle de l'usage des facteurs de croissance intègre de nouveaux paramètres pouvant être utilisés pour organiser encore plus efficacement les plans de contrôle, en l'occurrence l'intégration des progrès techniques relatifs à la détection mais aussi les nouveaux usages ou substances à activité hormonale. ...
... In addition, transcriptomic and metabolomic [22] approaches emerged in horse doping control. Transcriptomic methods for rHuEPO administration detection were developed [23,24] as well as metabolomics for detection of recombinant growth hormone [25,26] or steroids [27,28]. ...
Article
Erythropoiesis Stimulating Agents (ESAs) were developed for therapeutic purposes to stimulate red blood cell (RBC) production. Consequently, tissue oxygenation is enhanced as athlete's endurance and ESAs misuse now benefits doping. Our hypothesis is that most of ESAs should have similar mechanisms and thus have the same effects on metabolism. Studying the metabolome variations could allow suspecting the use of any ESAs with a single method by targeting their effects. In this objective, a metabolomic study was carried out on 3 thoroughbred horses with a single administration of 4.2μg/kg of Mircera(®), also called Continuous Erythropoiesis Receptor Activator (CERA). Blood and urine samples were collected from D-17 to D+74 and haematological parameters were followed throughout the study as plasmatic CERA concentration (ELISA). Urine and plasma metabolic fingerprints were recorded by Liquid Chromatography coupled to High Resolution Mass Spectrometry (LC-HRMS) in positive and negative mode. After preprocessing steps, normalized data were analyzed by multivariate statistics to build OPLS models. Hemoglobin concentration and hematocrit showed a significant increase after CERA administration unlike reticulocytes. CERA concentration showed a high intensity peak and then a slow decrease until becoming undetectable after D+31. Models built with multivariate statistics allow a discrimination between pre and post-administration plasma and urine samples until 74days after administration, i.e. 43days longer than ELISA method. By reducing and studying variables (ions), some potential candidate biomarkers were found.
... Ces stratégies ne cherchent pas à identifier directement la présence de molécules suspectées ou de leurs métabolites directs, mais à révéler une signature métabolique ou physiologique spécifique pouvant être associée à une pratique anabolisante. Ces approches dites « indirectes » ou « non ciblées » (Nebbia et al., 2011 ;Pinel et al., 2010) reposent sur des méthodes telles la transcriptomique (Riedmaier, 2015 ;Riedmaier et al., 2009a ;Riedmaier et al., 2012 ;Riedmaier and Pfaffl, 2013 ;Riedmaier et al., 2009b, c), la protéomique (Cacciatore et al., 2009 ;Cunningham et al., 2009 ;Kinkead et al., 2015) ou la métabolomique (Dervilly- Dervilly-Pinel et al., 2012 ;Gallart Ayala et al., 2015 ;Jacob et al., 2014 ;Kouassi Nzoughet et al., 2015b), incluant ses déclinaisons que sont la lipidomique (Kouassi Nzoughet et al., 2015a) et la stéroïdomique (Dervilly-Pinel et al., 2011 ;Kaabia et al., 2014 De plus, et en ce qui concerne l'évolution du contexte réglementaire, il est attendu que la réglementation européenne en matière de contrôle de l'usage des facteurs de croissance intègre de nouveaux paramètres pouvant être utilisés pour organiser encore plus efficacement les plans de contrôle, en l'occurrence l'intégration des progrès techniques relatifs à la détection mais aussi les nouveaux usages ou substances à activité hormonale. ...
Article
Full-text available
Surveillance of trace metals in foods of animal origin - focus on the exploratory plan to test for methylmercury in fish The surveillance of trace metals such as arsenic, lead, cadmium, nickel and mercury in foodstuffs of animal origin is ensured by an operational plan aiming at risk identification and the quantification and characterisation of the hazards related to trace metals found in foods. In 2014, several surveillance and control plans (targeted sampling) as well as an exploratory plan were implemented to monitor trace metals (lead, cadmium, mercury and methylmercury) in foodstuffs. These plans generated 6,908 analyses in various matrices (fish products, livestock products, milk, game, poultry, rabbits and honey). Processing of the results showed a completion rate of 99.3% and a rate of non-compliance (with the regulatory maximum levels or national alert thresholds) ranging from 0.7% to 16% across all sectors, excluding the equine industry. The identified non- compliances were managed based on the identified risk. They also helped to maintain or strengthen the surveillance of certain analyte/matrix pairs, such as lead in game meat and cadmium in equine liver. In general, the surveillance system in place has contributed to estimating consumer exposure to trace metals as well as to populating databases (Methylmercury exploratory plan) for enhanced risk assessment. The analysis of the monitoring system was an opportunity to present prospects for improvement including the need to define more suitable sample targeting criteria that are easier to implement. Another area for improvement would be the implementation of a tool for improving the quality of data generated by monitoring and control plans.
... Determination of high concentrations of 17β-19-nortestosterone in female pigs may indicate that the sex of the animal from which the sample was taken has been incorrectly noted, that the individual is a cryptorchid animal, or has one of the various defects of the reproductive system defined as the intersex phenomenon. In the literature, there are also reports on the natural occurrence of 17β-19-nortestosterone in horses (stallions) and 17α-19-nortestosterone in pregnant cows and newborn calves (18,22,27,29,35). ...
Article
Full-text available
Studies of anabolic hormone residues in the tissues of slaughter animals have been carried out in Poland for more than 25 years. During the period of 2011 to 2015, a total of 35 387 samples from different animal species were tested in the National Residue Control Programme for the presence of residues of compounds that cause hormonal effects, as listed in Annex 1 of Directive 96/23/EC. The research was conducted in the National Reference Laboratory and eight regional laboratories in departments of veterinary hygiene located throughout the country. Urine, muscle tissue, serum, kidney fat, and drinking water were the targeted matrices. Test methods based on instrumental techniques such as gas and liquid chromatography coupled with mass spectrometry were applied, as well as enzyme-linked immunosorbent assays (ELISA). The concentration of detected hormones exceeded the decision limits in 30 samples, the consequence of which was 41 non-compliances with current applicable criteria. The hormones found present pseudo-endogenous (nortestosterone and boldenone) only, while synthetic hormones were not identified. The non-compliant findings constitute a small percentage (0.085%) of the five-year analysis compilation. On this basis the related food produced in Poland can be accepted as safe for human consumption with regard to the hormone residues tested.
... Ces stratégies ne cherchent pas à identifier directement la présence de molécules suspectées ou de leurs métabolites directs, mais à révéler une signature métabolique ou physiologique spécifique pouvant être associée à une pratique anabolisante. Ces approches dites « indirectes » ou « non ciblées » (Nebbia et al., 2011;Pinel et al., 2010) reposent sur des méthodes telles la transcriptomique (Riedmaier, 2015;Riedmaier et al., 2009a;Riedmaier et al., 2012;Riedmaier and Pfaffl, 2013;Riedmaier et al., 2009b, c), la protéomique (Cacciatore et al., 2009;Cunningham et al., 2009;Kinkead et al., 2015) ou la métabolomique Dervilly-Pinel et al., 2012;Gallart Ayala et al., 2015;Jacob et al., 2014;Kouassi Nzoughet et al., 2015b), incluant ses déclinaisons que sont la lipidomique (Kouassi Nzoughet et al., 2015a) et la stéroïdomique (Dervilly-Pinel et al., 2011;Kaabia et al., 2014). Ces nouvelles approches permettent ainsi de découvrir des marqueurs moléculaires d'effets, qui peuvent ensuite faire l'objet d'un suivi ciblé dans un contexte de dépistage de pratiques anabolisantes. ...
Article
L’utilisation des promoteurs de croissance est interdite en élevage au sein de l’Union européenne depuis 1988. Afin de garantir au consommateur des denrées exemptes de résidus de ce type de substances, un dispositif européen de surveillance et de contrôle accompagne cette mesure, qui en France est organisé depuis 1988 dans le cadre des plans de surveillance et de contrôle mis en place par la direction générale de l’Alimentation. Le présent article décrit le cadre réglementaire, les modalités de mise en oeuvre en termes de composés d’intérêt, d’espèces animales concernées, de matrices biologiques pertinentes et de stratégies analytiques adaptées. Les données issues des plans 2014 illustrent l’ensemble du dispositif.
... Usually, nandrolone is distributed in the ester form as phenylpropionate, [7] laureate, [8][9][10] or decaonate. [11] This type of administration decreases the rate of absorption of the drug, leading to a prolonged anabolic effect. ...
Article
Under European legislation, the use of growth promoters is forbidden in food-producing livestock. The application of unofficial protocols with diverse combinations of veterinary drugs, administered in very low concentrations, hinders reliable detection and subsequent operative prevention. It was observed that nandrolone (anabolic steroid) and ractopamine (β-adrenergic agonist) are occasionally administered to animals, but little is known about their synergic action when they are administered together. Two specific analytical methods based on liquid chromatography-tandem mass spectrometry have been developed, both of which include hydrolysis of the corresponding conjugates. For the nandrolone method, solid-phase extraction was necessary for the complete elimination of the interferences, while employment of the Quantitation Enhanced Data-Dependent scan mode during MS acquisition of ractopamine enabled the utilization of simple liquid-liquid extraction. The nandrolone method was linear in the range of 0.5–25 ng/mL, while the ractopamine calibration curve was constructed from 0.5 to 1000 ng/mL. The corresponding coefficients of correlations were >0.9907. The lower limit of quantification for both methods was 0.5 ng/mL, followed by overall recoveries >81%. Precisions expressed as relative standard deviations were <17%, while matrix effects were minimal. Urine samples taken at the slaughterhouse from veal calves enrolled in an experimental treatment consisting of intramuscular administration of β-nandrolone-phenylpropionate accompanied with a ractopamine-enriched diet were analysed. Those methods might be useful for studying the elimination patterns of the administered compounds along with characterization of the main metabolic pathways. Copyright
... uronides and sulphates determined separately, was determined. Multivariate statistical analyses showed that the model could be used to classify animals into a treated and untreated group. Both studies show the potential of steroid profiling as a promising strategy to determine whether bovine animals have been treated with (natural) hormones or not.Kaabia et al. (2013) published a profiling study to detect the administration of Nandrolone in horses. ...
Article
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... Today, routine screening of AAS misuse is mainly implemented using GC-MS platforms after a deconjugation and derivatization step [3]. Thanks to proper quantitation of parameters from the steroid profile, suspicious urine samples exhibiting unexpected patterns of steroid metabolites can be distinguished from normal ones [4,5]. For confirmation purpose, the former require additional targeted testing by isotope ratio MS (IRMS). ...
Article
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Antidoping procedures are expected to greatly benefit from untargeted metabolomic approaches through the discovery of new biomarkers of prohibited substances abuse. Endogenous steroid metabolites were monitored in urine samples from a controlled elimination study of testosterone undecanoate after ingestion. A platform coupling ultra-high pressure LC with high-resolution quadrupole TOF MS was used and high between-subject metabolic variability was successfully handled using a multiblock data analysis strategy. Links between specific subsets of metabolites and influential genetic polymorphisms of the UGT2B17 enzyme were highlighted. This exploratory metabolomic strategy constitutes a first step toward a better understanding of the underlying patterns driving the high interindividual variability of steroid metabolism. Promising biomarkers were selected for further targeted study.
... 3r) which corresponds to the 95th and 99th percentile level of confidence, respectively (Fig. 2). Such a strategy as already been successfully proposed (Kaabia et al. 2014) for the implementation of screening criteria in the context of steroid abuse in horses, based on targeted steroid profiling. ...
Article
A study was performed to determine if untargeted metabolomics of urine could be used to establish a predictive tool for identifying -agonists misuse in cattle. Although prohibited for more than 20 years within the EU, growth promoting practices for livestock fattening purposes are still suspected. Methods based on gas- or liquid chromatography coupled to (tandem) mass spectrometry are today considered as the state-of the-art to monitor, in a targeted approach, residues of known drugs. To overcome the detection of anabolic practices, new synthetic xenobiotic growth promoters have been designed and new ways of applications, such as the administration of low dose cocktails, have been developed. In this context, innovative screening strategies are urgently needed to enable efficient control of such practices. Omic technologies have recently shown their relevance in highlighting physiological response resulting from anabolic compounds administration. LC-HRMS based metabolomics is one of the approaches allowing profiling biological matrices to reveal biological effects of a drug. In the present work, a metabolomics study performed on urine samples collected in the frame of several independent experiments involving different animals, different -agonists treatments and different parameters (doses, compounds mixture, treatment length), allowed highlighting biomarkers of interest and implementing a robust statistical model to predict for -agonists treated bovines. Performances of the proposed model fit with EU requirements for screening methods.
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Detection of doping with steroids that are also endogenous in the horse can be challenging, and a variety of approaches to distinguish exogenous administration from their natural presence are employed. Knowledge of endogenous concentrations of various steroids in different genders of horses (intact male, castrated male and female) and factors that could naturally affect them is beneficial for establishing ways for detection of their use. The current internationally adopted approaches include concentration‐based thresholds in urine and plasma, steroid ratios in urine and targeting the administered intact steroid esters in plasma and hair. However, these have their limitations, and therefore, other strategies, such as additional biomarkers and steroid profiling based on longitudinal testing and multivariate analysis, have been investigated and could potentially improve detection of the use of endogenous steroids in horses. This paper aims to provide a comprehensive overview of the steroids (androgens, oestrogens and progestogens) that have been reported to be endogenous to horses in literature, their concentration ranges in different genders and factors potentially affecting them as well as current and possible future approaches to detect their use.
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In order to overcome the challenge associated with the screening of Anabolic-Androgenic Steroids (AAS) abuses in animal competitions, a non-targeted LC-HRMS based metabolomics approach was implemented on equine urine samples to highlight potential biomarkers associated with the administration of such compounds, using testosterone esters as model steroids. A statistical model relying on 4 potential biomarkers intensity could be defined to predict the status of the samples. With a routine application perspective, the monitoring of the highlighted potential biomarkers was first transferred into high-throughput LC-SRM. The model's performances and robustness of the approach were preserved and providing a first demonstration of metabolomics-based biomarkers integration within a targeted workflow using common benchtop MS instrumentation. In addition, with a view to the widespread implementation of such biomarker-based tools, we have transferred the method to a second laboratory with similar instrumentation. This proof of concept allows the development and application of biomarker-based strategies to meet current doping control needs.
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In this work multivariate strategies were employed in order to highlight new potential biomarkers of interest to detect the exogenous treatment of steers intramuscularly treated with boldenone undecylenate. Serum samples collected from treated (n = 4) and control (n = 8) crossbred animals of varying ages and weights were extracted using a simple sample preparation procedure based on salt assisted liquid‐liquid extraction. Data acquisition was performed using liquid chromatography and Q‐ExactiveTM Orbitrap mass spectrometry. Data processing and treatment were performed using two non‐targeted workflows: 1) Compound Discoverer software and 2) XCMS package on the open‐source R software combined with MetaboAnalyst. Three potential biomarkers were highlighted taking into account the chromatographic shapes, the feature location on the generated s‐plots, the fold change, the adjusted p‐values, the coefficient of variation in the QC samples and the area under the ROC curves. Predicted formulas based on mass accuracy, structural composition and spectra similarity were proposed. A robust statistical model to predict the boldenone treatment was further developed based on the weighted abundances of the selected biomarkers. The requirements for screening methods were successfully fulfilled, together with a wider detection window in comparison with the monitoring of the deconjugated metabolite boldenone, although biomarker identification studies are still ongoing.
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Despite the impressive innate physical abilities of horses, camels, greyhounds or pigeons, doping agents might be administered to these animals to improve their performance. To control these illegal practices, anti‐doping analytical methodologies have been developed. This review compiles the analytical methods that have been published for the detection of prohibited substances administered to animals involved in sports, over 30 years. Relevant papers meeting the search criteria that discussed analytical methods aiming to detect and/or quantify doping substances in animal biological matrices, published from 1990 to 2019 were considered. A total of 317 studies were included, of which 298 were related to horses, demonstrating significant advances towards the development of doping detection methods for equine sports. However, analytical methods for the detection of doping agents in sports involving other species are lacking. Due to enhanced accuracy and specificity, chromatographic analysis coupled to mass spectrometry detection is preferred over immunoassays. Regarding biological matrices, blood and urine remain the first choice, although alternative biological matrices, such as hair and faeces, have been considered. With the increasing number and type of drugs used as doping agents, the analytes addressed in the published papers are diverse. It is very important to continue to detect and quantify these drugs, recognizing those that are most frequently used, in order to punish the abusers, protect animals’ health, and ensure a healthier and genuine competition.
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The evidence of continuous rise of novel doping agents and novel doping strategies calls for the development of more accurate multi‐target screening methods. Direct multi‐target screening approaches are restricted to the targeted substances and their turnover. The development of effective “indirect” screening methods requires a priori deep‐understanding of the substance metabolism. The biological passport has been demonstrated to be very effective, but it is limited to about 20 indirect parameters. The standard anti‐doping analytical methods are hence targeted and does not aim to directly identify unknown substances. Also, the detection of doping agents is limited by the substances excretion. We propose to consider metabolomics for screening of abuse of performance enhancing hormones by athletes, with the basis on the following pieces of evidence: 1) Hormones have a strong influence on human metabolism, changing several parameters in many tissues, organs, and bio‐fluids. 2) Metabolomics has been demonstrated to be a very accurate tool to depict the metabolic status of several organisms, tissues and for several human diseases, including hormonal deficiencies. 3) Metabolomics has been demonstrated to be able to distinguish hormone‐treated animals from controls in many species, without the need of a priori knowledge of the metabolism for the specific substance. The literature shows that metabolomics could be an appropriate tool to detect hormonal abuse, keeping in mind the strength and the limitation of such an approach.
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Recent advances in label-free quantitative proteomics may support its application in identifying and monitoring biomarkers for the purpose of doping control in equine sports. In this study, we developed a workflow of label-free quantitative proteomics to propose plasma protein biomarkers in horses after administration with krypton (Kr), a potential erythropoiesis-stimulating agent. Plasma proteomes were profiled by using nano-liquid chromatography-high resolution mass spectrometry. An in-house mass spectral library consisting of 1,121 proteins was compiled using samples collected from geldings (castrated horses) in the administration trial and geldings in training. A data-independent acquisition (DIA) method was used to quantify an array of plasma proteins across plasma samples from the administration trial. Statistical analyses proposed a profile of 83 biomarker candidates that successfully differentiated Kr-administered samples from control samples, with the ability to detect Kr exposure for up to 13 days (the last sample collected in the administration trial). The model also correctly classified 32 in-training geldings as untreated controls. This is significantly longer than the one hour detection time of plasma Kr using headspace gas chromatography-tandem mass spectrometry. Bioinformatic analyses enriched biomarker candidates relevant to complement activation and iron metabolism. The upregulation of transferrin receptor protein 1 (TFRC), one of the candidates related to iron metabolism, in plasma after Kr administration was validated by selected reaction monitoring (SRM) of corresponding peptides. These results have demonstrated label-free quantitative proteomics as a promising approach to propose plasma protein biomarkers to enhance doping control. Data are available via ProteomeXchange with identifier PXD017262.
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A steroidomics workflow has been developed in the objective of monitoring a wide range (n >150) of steroids in urine. The proposed workflow relies on the optimization of an adequate SPE extraction step followed by an UHPLC-HRMS/MS simultaneous analysis of both free and conjugated forms of C18, C19 and C21 steroid hormones. On the basis of 44 selected steroids, representative of main classes of steroids constituting the steroidome, the performances of the developed workflow were evaluated in terms of selectivity, repeatability (< 13%) and linearity (R2> 0.985 on in the concentration range [0.01 – 10 ng/mL]). As metabolites identification and characterization constitute the bottleneck of such profiling approaches, a homemade database was created encompassing a large number of characterized free and conjugated steroids (n> 150) for putative steroid-like biomarkers identification purposes. The efficiency of the workflow in highlighting fine modifications within the urinary steroidome was assessed in the frame of an anabolic treatment involving an intra-muscular administration of boldenone undecylenate (2 mg/kg) to veals (n=6) and the investigation of potential steroid biomarkers. Besides monitoring known phase II metabolites of boldenone in the bovine specie, namely, boldenone glucuronide and sulfate, the applied strategy also permitted to observe, upon boldenone administration, a modified profile of epiboldenone glucuronide. Furthermore, 31 signals corresponding to non-identified steroid species could also be highlighted as impacted upon the exogenous steroid treatment. This study is the first to simultaneously investigate both free and conjugated C18, C19 and C21 steroid hormones in their native form using UHPLC-HRMS/MS and allowing their comprehensive profiling. This strategy was probed in-vivo.
Chapter
This chapter provides some updated information on contemporary methods for hormone and &;#x003B2;&;#x02010;agonist analyses. It deals with the classical approaches for the effective detection and identification of exogenous hormones. The chapter examines specific problems related to control strategies for natural hormones. These include both the traditional and generally recognized natural hormones as well as a series of androgenic steroids that can be present in biological samples obtained from a series of species. The fact that natural background concentrations can be present strongly complicates the analyses. The application of mass spectrometry (MS) in combination with gas chromatography (GC) or liquid chromatography (LC) is considered the &;#x00022;gold standard&;#x00022; for analytical methods in residue analysis. The chapter presents an overview of the available bio&;#x02010;based screening methods for the detection of hormones and &;#x000DF;&;#x02010;agonists, focusing on estrogens, androgens, progestogens, corticosteroids, thyroids, &;#x000DF;2&;#x02010;agonists, and growth hormones (GH).
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The use of anabolic androgenic steroids (AAS) is prohibited in both human and equine sports. Conventional approach in doping control testing for AAS (as well as other prohibited substances) is accomplished by the direct detection of target AAS or their characteristic metabolites in biological samples using hyphenated techniques such as gas-chromatography or liquid-chromatography coupled with mass-spectrometry. Such approach, however, falls short when dealing with unknown designer steroids where reference materials and their pharmacokinetics are not available. In addition, AASs with fast elimination times renders the direct detection approach ineffective as the detection window is short. Targeted metabolomics approach is a plausible alternative to the conventional direct detection approach for controlling the misuse of AAS in sports. Since the administration of AAS of the same class may trigger similar physiological responses or effects in the body, it may be possible to detect such administrations by monitoring changes in the endogenous steroidal expression profile. This study attempts to evaluate the viability of using the targeted metabolomic approach to detect the administration of steroidal aromatase inhibitors, namely androst-4-ene-3,6,17-trione (6-OXO) and androsta-1,4,6-triene-3,17-dione (ATD), in horses. Total (free and conjugated) urinary concentrations of 31 endogenous steroids were determined by gas chromatography-tandem mass spectrometry (GC-MS/MS) for a group of 2 resting and 2 in-training thoroughbred geldings having been treated with either 6-OXO or ATD. Similar data were also obtained from a control (untreated) group of in-training thoroughbred geldings (n = 28). Statistical processing and chemometric procedures using principle component analysis (PCA) and orthogonal projection of latent structures-discriminant analysis (OPLS-DA) have highlighted 7 potential biomarkers that could be used to differentiate urine samples obtained from the control and the treated groups. Based on this targeted metabolomic approach, the administration of 6-OXO and ATD could be detected much longer as compared with the conventional direct detection approach.
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The use of glucocorticoids as growth promoters for meat-producing animals is strictly regulated within the European Union. However, in the past few years, a higher frequency of non-compliant bovine urine samples for prednisolone has been noticed, which could not be directly related to fraudulent use of prednisolone. As such, questions have risen about the origin of this compound. Unfortunately, at present, no decisive strategy has been established to discriminate between endogenous and exogenous prednisolone. In this study, an untargeted metabolomics strategy, based on Orbitrap and QqTOF mass spectrometry, was deployed to reveal urinary biomarkers, which are indicative for the exogenous administration of the synthetic glucocorticoid prednisolone. For this purpose, prednisolone was administered intramuscularly and per os to 12 bovines and a total of 2700 urine samples were collected before, during and after treatment. Multivariate statistical data analysis (i.e. OPLS-DA) revealed four differentiating metabolites that allowed discrimination between urine samples collected before and during prednisolone administration. None of these compounds were present in urine containing endogenous prednisolone, of which the formation was induced by the administration of a synthetic analogue of adrenocorticotropic hormone. Only one metabolite was retained as a highly suitable biomarker during growth-promoting and therapeutic prednisolone treatment, with 93.4 % sensitivity and 96.3 % specificity. Besides, this compound could be detected up to 4 days after a single therapeutic per os prednisolone administration. Based on accurate mass, isotope pattern, and MS/MS spectra, this compound was putatively annotated and is suggested as an actionable biomarker for exogenous prednisolone administration.
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The present study was conducted to elucidate the profile of circulating gonadotropins and gonadal hormones from birth to puberty and relationship between gonadal seasonality and hormonal secretion in both sexes of Thoroughbred horses. Spring-born colts (n=6) and fillies (n=9) were blood sampled weekly from jugular vein from birth to 60 weeks of age. Circulating FSH, LH, prolactin, testosterone, progesterone, estradiol-17β, and immunoreactive (ir)-inhibin were measured by radioimmunoassay. In both sexes, the steroid hormones levels were remarkably high at birth, rapidly dropped within a week and remained at the lower levels until the start of second spring after birth. Ir-inhibin was also high during the birth, remaining lowest during winter and again increasing towards the second summer. There was an increase in FSH concentration in foals during the first summer months after birth and in the next summer, the FSH concentration along with that of LH increased significantly. The seasonal increase in circulating prolactin was remarkable even in the first year, and no differences were noted between the two summers. These results clearly demonstrated that the hypothalamo-pituitary axis is already responsive to changes in photoperiod and secrete prolactin similar to adult horses, but pituitary gonadotrophs for FSH and LH secretion is less sensitive. When the values of these hormones in the second breeding season after birth were compared with adult values of the respective sex in the breeding season, no significant differences were observed, indicating that spring-born fillies and colts have already attained the stage of puberty at the second breeding season after birth.
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During last decades, the use of natural steroids in racing and food producing animals for doping purposes has been flourishing. The endogenous or exogenous origin of these naturally occurring steroids has since remained a challenge for the different anti-doping laboratories. The administration of these substances to animals is usually made through an intra-muscular pathway with the steroid under its ester form for a higher bioavailability and a longer lasting effect. Detecting these steroid esters would provide an unequivocal proof of an exogenous administration of the considered naturally occurring steroids. A quick analytical method able to detect at trace level (below 50pg/mL) a large panel of more than 20 steroid esters in serum and plasma potentially used for doping purposes in bovine and equine has been developed. Following a pre-treatment step, the sample is submitted to a solid phase extraction (SPE) before analysis with UPLC-MS/MS. The analytical method's efficiency has been probed through three different in vivo experiments involving testosterone propionate intra-muscular administration to three heifers, 17-estradiol benzoate intra-muscular administration to a bull and a heifer and nandrolone laurate intra-muscular administration to a stallion. The results enabled detecting the injected testosterone propionate and 17-estradiol benzoate 2 and 17 days, respectively, post-administration in bovine and nandrolone laurate up to 14 days post-administration in equine. The corresponding elimination profiles in bovine serum and equine plasma have been established. The first bovine experiment exhibited a maximal testosterone propionate concentration of 400pg/mL in one of the three heifer serum within 5h post-administration. The second bovine experiment reported a maximal 17-estradiol benzoate concentration of 480pg/mL in the same matrix recorded 9 days after its administration. The last equine experiment resulted in a maximal nandrolone laurate concentration of 440pg/mL in horse plasma 24h after administration.
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Due to their performance enhancing properties, use of anabolic steroids (e.g. testosterone, nandrolone, etc.) is banned in elite sports. Therefore, doping control laboratories accredited by the World Anti-Doping Agency (WADA) screen among others for these prohibited substances in urine. It is particularly challenging to detect misuse with naturally occurring anabolic steroids such as testosterone (T), which is a popular ergogenic agent in sports and society. To screen for misuse with these compounds, drug testing laboratories monitor the urinary concentrations of endogenous steroid metabolites and their ratios, which constitute the steroid profile and compare them with reference ranges to detect unnaturally high values. However, the interpretation of the steroid profile is difficult due to large inter-individual variances, various confounding factors and different endogenous steroids marketed that influence the steroid profile in various ways. A support vector machine (SVM) algorithm was developed to statistically evaluate urinary steroid profiles composed of an extended range of steroid profile metabolites. This model makes the interpretation of the analytical data in the quest for deviating steroid profiles feasible and shows its versatility towards different kinds of misused endogenous steroids. The SVM model outperforms the current biomarkers with respect to detection sensitivity and accuracy, particularly when it is coupled to individual data as stored in the Athlete Biological Passport.
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Δ(13) C and δ(13) C values of endogenous urinary steroids represent physiological random variables. Measurement uncertainty and biological scatter likewise contribute to the variances. The statistical distributions of negative controls are well investigated, but there is little knowledge about the corresponding distributions of steroid-users. For these reasons valid discrimination of steroid users from non-users by (13) C/(12) C analysis of endogenous steroids requires elaborate statistical treatment. Corresponding Bayesian approaches are presented following an introduction to the rationale. The use of mixture models appears appropriate. The distribution of routine data has been deconvolved and characterized accordingly. The mixture components, which presumably represent steroid users and non-users, exhibit considerable overlap. The validity of a given result depends on both the analytical uncertainty and the prior probability of doping offenses. Low analytical uncertainties but high prior probabilities facilitate valid detection of doping offenses. Two recommendations can be deduced. First, before starting an (13) C/(12) C analysis, any initial suspicion should be well-substantiated. This precludes use of permissive criteria derived from the steroid profile. Secondly, knowledge of relevant (13) C/(12) C distributions is required. This must cover representative numbers of authentic steroid users. Finally, it is desirable that the conditional probability for steroid administration rather than the measurement uncertainty is calculated and reported. This quantity possesses superior validity and it is largely independent of laboratory bias. The findings suggest and facilitate flexible handling of decision limits. Proposals for the evaluation of stable isotope data are presented. Copyright © 2012 John Wiley & Sons, Ltd.
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A method for the detection and quantitation of 35 endogenous steroids in equine serum was developed and validated. Androgens, estrogens, progestins and their metabolites potentially present in serum were simultaneously monitored in one method using on-line sample extraction by turbulent flow chromatography (TFC) on a 2-dimensional liquid chromatography system and detected on a triple-stage quadrupole mass spectrometer by electrospray ionization. Analytes were detected and quantitated by single-reaction monitoring or selected-ion monitoring. Limits of detection (range 0.025-10ngmL(-1)) and quantitation (range 0.125-25ngmL(-1)) along with recovery and matrix effects were determined for each analyte. Inter- and intra-day accuracy and precision was assessed for with the majority of analytes having %CV less than 20% and accuracy within 20% of the expected concentrations. Eight of the 35 analytes were unable to meet these guidelines across all of the quality control concentrations monitored for each analyte. This method was used to determine the endogenous steroid profiles of Thoroughbred horses and has been modified for use in non-human primates and cell culture.
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This report describes significance testing for PLS and OPLS® (orthogonal PLS) models. The testing is applicable to single-Y cases and is based on ANOVA of the cross-validated residuals (CV-ANOVA). Two variants of the CV-ANOVA are introduced. The first is based on the cross-validated predictive residuals of the PLS or OPLS model while the second works with the cross-validated predictive score values of the OPLS model. The two CV-ANOVA diagnostics are shown to work well in those cases where PLS and OPLS work well, that is, for data with many and correlated variables, missing data, etc. The utility of the CV-ANOVA diagnostic is demonstrated using three datasets related to (i) the monitoring of an industrial de-inking process; (ii) a pharmaceutical QSAR problem and (iii) a multivariate calibration application from a sugar refinery. Copyright © 2008 John Wiley & Sons, Ltd.
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For years it has been suspected that natural hormones are illegally used as growth promoters in cattle in the European Union. Unfortunately there is a lack of methods and criteria that can be used to detect the abuse of natural hormones and distinguish treated from non-treated animals. Pattern recognition of steroid profiles is a promising approach for tracing/detecting the abuse of natural hormones administered to cattle. Traditionally steroids are analysed in urine as free steroid after deconjugation of the glucuronide (and sulphate) conjugates. The disadvantage of this deconjugation is that valuable information about the steroid profile in the sample is lost. In this study we develop a method to analyse steroids at very low concentration levels (ng l(-1)) for the free steroid, glucuronide and sulphate conjugates in urine samples. This method was used to determine concentrations of natural (pro)hormones in a large population (n = 620) of samples from male and female bovine animals and from bovine animals treated with testosterone-cypionate, estradiol-benzoate, dihydroepiandrosterone and pregnenolone. The data acquired were used to build a statistical model applying the multivariate technique 'Soft Independent Modeling of Class Analogy' (SIMCA). It is demonstrated that by using this model the results of the urine analysis can indicate which animal may have had illegal treatment with natural (pro)hormones.
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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.
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Androstenedione (4-androstene-3,17-dione) is banned by the World Anti-Doping Agency (WADA) as an endogenous steroid. The official method to confirm androstenedione abuse is isotope ratio mass spectrometry (IRMS). According to the guidance published by WADA, atypical steroid profiles are required to trigger IRMS analysis. However, in some situations, steroid profile parameters are not effective enough to suspect the misuse of endogenous steroids. The aim of this study was to investigate the atypical steroid profile induced by androstenedione administration and the detection of androstenedione doping using IRMS. Ingestion of androstenedione resulted in changes in urinary steroid profile, including increased concentrations of androsterone (An), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (5α-diol), and 5β-androstane-3α,17β-diol (5β-diol) in all of the subjects. Nevertheless, the testosterone/epitestosterone (T/E) ratio was elevated only in some of the subjects. The rapid increases in the concentrations of An and Etio, as well as in T/E ratio for some subjects could provide indicators for initiating IRMS analysis only for a short time period, 2-22h post-administration. However, IRMS could provide positive determinations for up to 55h post-administration. This study demonstrated that, 5β-diol concentration or Etio/An ratio could be utilized as useful indicators for initiating IRMS analysis during 2-36h post-administration. Lastly, Etio, with slower clearance, could be more effectively used than An for the confirmation of androstenedione doping using IRMS.
Article
17β-Nandrolone (17β-NT) is one of the most frequently misused anabolic steroids in meat producing animals. As a result of its extensive metabolism combined with the possibility of interferences with other endogenous compounds, detection of its illegal use often turns out to be a difficult issue. In recent years, proving the illegal administration of 17β-NT became even more challenging since the presence of endogenous presence of 17β-NT or some of its metabolite in different species was demonstrated. In bovines, 17α-NT can occur naturally in the urine of pregnant cows and recent findings reported that both forms can be detected in injured animals. Because efficient control must both take into account metabolic patterns and associated kinetics of elimination, the purpose of the present study was to investigate further some estranediols (5α-estrane-3β,17β-diol (abb), 5β-estrane-3α,17β-diol (bab), 5α-estrane-3β,17α-diol (aba), 5α-estrane-3α,17β-diol (aab) and 5β-estrane-3α,17α-diol (baa)) as particular metabolites of 17β-NT on a large number of injured (n=65) or pregnant (n=40) bovines. Whereas the metabolites abb, bab, aba and baa have previously been detected in urine up to several days after 17β-NT administration, the present study showed that some of the isomers abb (5α-estrane-3β,17β-diol) and bab (5β-estrane-3α,17β-diol) could not be detected in injured or pregnant animals, even at very low levels. This result may open a new way for the screening of anabolic steroid administration considering these 2 estranediols as biomarkers to indicate nandrolone abuse in cattle.
Article
Soma, L. R., Uboh, C. E., You, Y., Guan, F., McDonnell, S. Plasma concentrations of testosterone and nandrolone in racing and nonracing intact male horses. J. vet. Pharmacol. Therap. 35, 132–138. Pennsylvania (PA) State Racing Commissions regulate the endogenous androgenic steroid, testosterone (TES), in racing intact males (RIM) by quantification of TES in post-race samples. Post-race plasma samples (2209) collected between March 2008 and November 2010 were analyzed for TES, nandrolone (NAN), and other anabolic steroids (ABS). Of the 2209 plasma samples, 2098 had quantifiable TES ≥25 pg/mL. Plasma (mean ± SD) concentrations of TES and NAN in RIM were 329.2 ± 266.4 and 96.0 ± 67.8 pg/mL, respectively. Only 64.6% of RIM had quantifiable concentration of NAN, and there was no relationship between TES and NAN. Plasma TES concentrations were significantly (P < 0.0001) higher during the months of April, May, June, July, and August. A significantly higher (P < 0.006) plasma TES was observed in Thoroughbred (TB) (347.6 ± 288.5 pg/mL) vs. that in Standardbred (STB) (315.4 ± 247.7 pg/mL). Plasma concentrations of TES from breeding stallions (BS) were 601.6 ± 356.5 pg/mL. Statistically significant (P < 0.0001) lower plasma concentrations of the two steroids were observed in RIM horses. Based on quantile distribution of TES in the RIM and BS populations, 99.5% were at or below 1546.1 and 1778.0 pg/mL, respectively. Based on this population of RIM, the suggested upper threshold plasma concentration of endogenous TES in horses competing in PA should remain at 2000 pg/mL.
Article
Analysis of equine plasma samples to detect the abuse of anabolic steroids can be complicated when the parent steroid is endogenous to the animal. Anabolic steroids are usually administered intramuscularly as synthetic esters and therefore detection of the exogenous esters provides unequivocal proof of illegal administration. An ultra high performance liquid chromatography tandem mass spectrometric (UPLC-MSMS) method for the analysis of esters of testosterone (propionate, phenylpropionate, isocaproate, and decanoate) and boldenone (undecylenate) in equine plasma has been developed. Esters were extracted from equine plasma using a mixture of hexane and ethyl acetate and treated with methoxyamine hydrochloride to form methyloxime derivatives. Metenolone enanthate was used as an internal standard. After chromatographic separation, the derivatized steroid esters were quantified using selected reaction monitoring (SRM). The limit of detection for all of the steroid esters, based on a signal to noise ratio (S/N) of 3:1, was 1-3 pg/mL. The lower limit of quantification (LLOQ) for the all of the steroid esters was 5 pg/mL when 2 mL of plasma was extracted. Recovery of the steroid esters was 85-97% for all esters except for testosterone decanoate which was recovered at 62%. The intra-day coefficient of variation (CV) for the analysis of plasma quality control (QC) samples was less than 9.2% at 40 pg/mL and less than 6.0% at 400 pg/mL. The developed assay was used to successfully confirm the presence of intact testosterone esters in equine plasma samples following intramuscular injection of Durateston® (mixed testosterone esters).
Article
Changes in the endogenous profile of androgenic anabolic steroids (AAS) may be interpreted as markers of doping. The objective of this study was to evaluate the endogenous profile of AAS in male athletes of the 2009 Brazilian National Soccer Championship, in normal conditions, particularly in the light of the revision of World Anti-Doping Agency's (WADA) Technical Document on the Interpretation of Endogenous AAS in athletes for doping control drafted in that year, as well as comparing these results to profiles already published in the literature. The upper limit of the 95% central reference interval of the following parameters for the studied population were estimated to be significantly higher than WADA's criteria, with a confidence of 90%: DHEA (about 2.3 times higher), Adiol (1.2 times higher), Bdiol (2.7 times higher), and Adiol/E (6 times higher). These findings seem to imply that WADA's criteria proposed in 2009 for DHEA, Adiol, Bdiol, and Adiol/E may not have been applicable to the studied population. Moreover, their comparison to previously published studies pointed to the need to evaluate in detail the appropriateness of adopting these criteria as universal, since there seems to be variations among different populations of athletes.
Article
Doping with natural steroids can be detected by evaluating the urinary concentrations and ratios of several endogenous steroids. Since these biomarkers of steroid doping are known to present large inter-individual variations, monitoring of individual steroid profiles over time allows switching from population-based towards subject-based reference ranges for improved detection. In an Athlete Biological Passport (ABP), biomarkers data are collated throughout the athlete's sporting career and individual thresholds defined adaptively. For now, this approach has been validated on a limited number of markers of steroid doping, such as the testosterone (T) over epitestosterone (E) ratio to detect T misuse in athletes. Additional markers are required for other endogenous steroids like dihydrotestosterone (DHT) and dehydroepiandrosterone (DHEA). By combining comprehensive steroid profiles composed of 24 steroid concentrations with Bayesian inference techniques for longitudinal profiling, a selection was made for the detection of DHT and DHEA misuse. The biomarkers found were rated according to relative response, parameter stability, discriminative power, and maximal detection time. This analysis revealed DHT/E, DHT/5β-androstane-3α,17β-diol and 5α-androstane-3α,17β-diol/5β-androstane-3α,17β-diol as best biomarkers for DHT administration and DHEA/E, 16α-hydroxydehydroepiandrosterone/E, 7β-hydroxydehydroepiandrosterone/E and 5β-androstane-3α,17β-diol/5α-androstane-3α,17β-diol for DHEA. The selected biomarkers were found suitable for individual referencing. A drastic overall increase in sensitivity was obtained. The use of multiple markers as formalized in an Athlete Steroidal Passport (ASP) can provide firm evidence of doping with endogenous steroids. Copyright
Article
Estradiol benzoate (EB) has been one of the most widely used estrogenic agents in animal husbandry, as a way of exogenously introducing the natural hormone estradiol-17β into the animal organism. Estradiol was previously employed to induce anabolic effects or reproductive improvements in cattle. However, the employment of EB in European countries has been permanently forbidden by Directive 2008/97/EC to guarantee consumers' health. Despite this prohibition, the control of estradiol-17β and its esters continues to be a difficult task for residue-monitoring plans in European Communities because official analyses of natural thresholds for hormones in cattle have not yet been established, leading to a lack of confirmation for any exogenous administration of natural hormones. Several researchers have worked on excretion profiles of metabolites, variation in specific hormonal ratios and metabolomic fingerprints after hormonal treatments. This research focuses on the possible existence of disturbances in the serum profile of animals treated with EB in terms of steroid sex hormones (androgens, oestrogens and progestogens), by investigating the serum levels of several of these hormones. The serum samples were collected from three groups of cows: one treated with an intramuscular injection of EB, one treated with a combination of intravaginal EB and progesterone and a control (non-treated) group. The samples have been analysed by a validated high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method, and 17 natural hormones were identified and quantified. Subsequently, data from the serum profiles were submitted for statistic and multivariate analysis, and it was possible to observe a manifest variation between animal groups. The obtained results can help in the development of a viable screening tool for monitoring purposes in cattle.
Article
The recent discovery of a novel, membrane localized progestin receptor (mPR) unrelated to the classical progesterone receptor (PR) in fishes and its subsequent identification in mammals suggests a potential mediator of non-traditional progestin actions, particularly in tissues where PR is absent. While early studies on mPR focused on final oocyte maturation in fishes, more current studies have examined mPRs in multiple mammalian systems in both reproductive and non-reproductive tissues as well as in diseased tissues. Here we review the current data on mPR in mammalian systems including male and female reproductive tracts, liver, neuroendocrine tissues, the immune system and breast and ovarian cancer. We also provide new data demonstrating mPR expression in the RAW 264.7 immune cell line and bone marrow-derived macrophages as well as mPR expression and downstream gene regulation in ovarian cancer cells.
Article
Steroid profiling provides valuable information to detect doping with endogenous steroids. Apart from the traditionally monitored steroids, minor metabolites can play an important role to increase the specificity and efficiency of current detection methods. The applicability of several minor steroid metabolites was tested on administration studies with low doses of oral testosterone (T), T gel, dihydrotestosterone (DHT) gel and oral dehydroepiandrosterone (DHEA). The collected data for all monitored parameters were evaluated with the respective population based reference ranges. Besides the traditional markers T/E, T and DHT, minor metabolites 4-OH-Adion and 6α-OH-Adion were found as most sensitive metabolites to detect oral T administration. The most sensitive metabolites for the detection of DHEA were identified as 16α-OH-DHEA and 7β-OH-DHEA but longest detection up to three days (after oral administration of 50 mg) was obtained with non-specific 5β-steroids and its ratios. Steroids applied as a gel had longer effects on the metabolism but were generally not detectable with universal decision criteria. It can be concluded that population based reference ranges show limited overall performance in detecting misuse of small doses of natural androgens. Although some minor metabolites provide additional information for the oral testosterone and DHEA formulations, the topical administered steroids could not be detected for all volunteers using universal reference limits. Application of other population based threshold limits did not lead to longer detection times.
Article
17beta-Nandrolone (17beta-NT) is one of the most recurrent forbidden anabolic steroid used in meat producing animals breeding. Because efficient control must both take into account metabolic patterns and associated kinetics of elimination, the metabolism of 17beta-NT in bovines has already been investigated and is well documented, but only focussing on its main metabolites (i.e. 17alpha-nandrolone, 19-noretiocholanolone and 19-norandrostenedione). The goal of the present study was to enlarge this panel of 17beta-NT metabolites, especially through the urinary estranediols fraction in order to perform a more global steroid profiling upon 17beta-nortestosterone laureate ester administration in calves. A GC-MS/MS method has been developed to monitor and quantify 5 estranediols isomers including 5alpha-estrane-3beta,17beta-diol (abb), 5beta-estrane-3alpha,17beta-diol (bab), 5alpha-estrane-3beta,17alpha-diol (aba), 5alpha-estrane-3alpha,17beta-diol (aab) and 5beta-estrane-3alpha,17alpha-diol (baa). Their urinary elimination kinetics have been established allowing detection of 4 estranediols up to several days after administration. All animals demonstrated homogeneous patterns of elimination both from a qualitative (metabolite profile) and quantitative point of view (elimination kinetics in urine). 5alpha-Estrane-3beta,17alpha-diol (aba) was found as the major metabolite with concentrations up to 100microgL(-1).
Article
The Athlete Biological Passport (ABP) is an individual electronic document that collects data regarding a specific athlete that is useful in differentiating between natural physiologic variations of selected biomarkers and deviations caused by artificial manipulations. A subsidiary of the endocrine module of the ABP, that which here is called Athlete Steroidal Passport (ASP), collects data on markers of an altered metabolism of endogenous steroidal hormones measured in urine samples. The ASP aims to identify not only doping with anabolic-androgenic steroids, but also most indirect steroid doping strategies such as doping with estrogen receptor antagonists and aromatase inhibitors. Development of specific markers of steroid doping, use of the athlete's previous measurements to define individual limits, with the athlete becoming his or her own reference, the inclusion of heterogeneous factors such as the UDPglucuronosyltransferase B17 genotype of the athlete, the knowledge of potentially confounding effects such as heavy alcohol consumption, the development of an external quality control system to control analytical uncertainty, and finally the use of Bayesian inferential methods to evaluate the value of indirect evidence have made the ASP a valuable alternative to deter steroid doping in elite sports. The ASP can be used to target athletes for gas chromatography/combustion/ isotope ratio mass spectrometry (GC/C/IRMS) testing, to withdraw temporarily the athlete from competing when an abnormality has been detected, and ultimately to lead to an antidoping infraction if that abnormality cannot be explained by a medical condition. Although the ASP has been developed primarily to ensure fairness in elite sports, its application in endocrinology for clinical purposes is straightforward in an evidence-based medicine paradigm.
Article
Measurement of steroid esters in bovine hair samples, using sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS), provides a powerful tool for identifying animals treated illicitly with growth promoters. The successful application of such testing requires appropriate sampling of hair from treated animals. This paper describes the results of hair analysis by LC-MS/MS for two animal studies in which animals were treated with estradiol-3-benzoate and nortestosterone decanoate. The results from the first animal study indicate that animals treated with these anabolic steroids may not always be identified from analysis of hair samples; positive test results occur sporadically and only for some of the treated animals. The results from the second animal study identify conditions attaching to positive hair samples, such as, that concentrations of steroid esters in hair are related to distance of sampling from point of injection and to time post-treatment, that concentrations of steroid esters in hair are related to dose given to the animal but that this relationship may vary over time post-treatment, and that steroid esters may be measured in regrowth hair taken some weeks after treatment. Steroid esters are determined along the length of the hair, confirming that accumulation of steroid esters into hair occurs from various sources, including blood, sweat and sebum. The reported research provides some useful insights into the mechanisms governing the persistence of steroid esters in bovine hair following illicit treatment with growth promoters.
Article
The detection of steroid residues in hair is a powerful strategy to demonstrate long-term administration of these growth promoters in meat production animals. Analysis of the ester form of administered steroids is an unambiguous approach to prove the illegal use of natural hormones. For detection, gas chromatography-mass spectrometry (GC-MS/MS) was generally used. However, recent advances in liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology have improved the robustness and potential sensitivity of this method. This paper describes development and validation, according to Commission Decision 2002/657/EC, of LC-MS/MS and GC-MS/MS methods, in two separate laboratories, for determination of steroid esters in bovine hair. Bovine hair samples taken from animals treated with estradiol-3-benzoate and nortestosterone decanoate, as well as from an untreated animal, were used to evaluate the comparability of the results of the two validated methods. The results of the inter-comparison demonstrate that both the LC-MS/MS and the GC-MS/MS methods are fit for purpose and capable of determining steroid esters in hair samples from treated bovine animals.
Article
Following administration of the anabolic steroid 19-nortestosterone or its esters to the horse, a major urinary metabolite is 19-nortestosterone-17beta-sulphate. The detection of 19-nortestosterone in urine from untreated animals has led to it being considered a naturally occurring steroid in the male horse. Recently, we have demonstrated that the majority of the 19-nortestosterone found in extracts of 'normal' urine from male horses arises as an artefact through decarboxylation of the 19-carboxylic acid of testosterone. The aim of this investigation was to establish if direct analysis of 19-nortestosterone-17beta-sulphate by liquid chromatography/tandem mass spectrometry (LC/MS/MS) had potential for the detection of 19-nortestosterone misuse in the male horse. The high concentrations of sulphate conjugates of the female sex hormones naturally present in male equine urine were overcome by selective hydrolysis of the aryl sulphates using glucuronidase from Helix pomatia; this was shown to have little or no activity for alkyl sulphates such as 19-nortestosterone-17beta-sulphate. The 'free' phenolic steroids were removed by solid-phase extraction (SPE) prior to LC/MS/MS analysis. The method also allowed for the quantification of the sulphate conjugate of boldenone, a further anabolic steroid endogenous in the male equine with potential for abuse in sports. The method was applied to the quantification of these analytes in a population of samples. This paper reports the results of that study along with the development and validation of the LC/MS/MS method. The results indicate that while 19-nortestosterone-17beta-sulphate is present at low levels as an endogenous substance in urine from 'normal' male horses, its use as an effective threshold substance may be viable.
Article
Ovarian function was monitored for 33 mo in captive feral mares (Equus caballus) by following serum progesterone (P) levels. A P level greater than 2.0 ng/ml was considered indicative of ovulation. Feral mares were seasonally polyestrus with the majority of animals ovulating between May and October. During the first year after capture, none of the mares ovulated during the anestrous season. However, in subsequent years, approximately 10% of mares ovulated during the months of November, January and February. P levels during the luteal phase of the cycle ranged from 2.0 to 21.0 ng/ml which were similar to levels in domestic breeds of mares. The pattern of P concentrations during pregnancy was also similar to the pattern in domestic mares. These data confirmed the seasonality of ovulation in feral mares but indicated that this seasonality was not as rigid as previously believed. Captive feral mares were similar to domestic breeds in the percentage of mares ovulating all year and in the P levels achieved during the estrous cycle and pregnancy.
Article
As part of a continuing research program associated with the detection of anabolic steroid residues in horse urine, normal samples from entire male horses have now been investigated. Isomers of three C-18 neutral steroids; 4-estren-17-ol-3-one (1), estrane-3,17-diol (2) and an unsaturated estranediol having a possible structure (3), have been identified in urine samples from two male horses aged 8 and 14 years. Of these three steroids, compound (2) was not detected in the urine of a 2.5 yr old entire male nor in the majority of post-race urine samples from entire male horses average age 3.8 yrs (n = 34). Ten of these samples showed tentative indications of this compound. Although the isolation of isomers of estrane-3,17-diol from human non-pregnancy urine has been reported previously, analysis of non-pregnancy urine samples in the present study did not reveal the presence of these compounds.
Article
Validations of analytical methods are important for the generation of data for bioavailability, bioequivalence and pharmacokinetic studies. It is essential to use well defined and fully validated analytical methods to obtain reliable results that can be satisfactorily interpreted. This manuscript is intended to provide guiding principles for the evaluation of a method's overall performance. For this purpose, all of the variables of the method are considered, including sampling procedure, sample preparation, chromatographic separation, detection and data evaluation. The criteria considered are as follows: stability, selectivity, limits of quantification and of detection, accuracy, precision, linearity, recovery and ruggedness. Models used for analytical calibration curves are explained in term of validity and limitations, along with a presentation of the most common statistical considerations used to validate the model. Appropriate means of testing precision and accuracy, the most important factors in assessing method quality, are presented. Other issues, such as re-validation, cross-validation, partial sample volume, endogenous drugs and biological matrix of limited availability, are also discussed.
Article
To gain further information on gonadal function of the stallion, concentrations of testicular steroids in blood plasma (bpl) and seminal plasma (spl) and their distribution in the ejaculate were determined. Blood and semen samples from a total of 11 stallions were collected from November to July. Estrone (E1), estrone sulfate (E1S), estradiol-17beta (E2beta) and testosterone (T) were determined in bpl and spl, and in addition androstenedione (A), dehydroepiandrosterone (DHEA) and 5alpha-dihydrotestosterone (5alpha-DHT) were measured in spl. At certain points of time, aliquots of an ejaculate were centrifuged, washed and the distribution of E1, E1S, E2beta and T into seminal plasma and the sperm fraction was assessed. Hormone assay was by RIA, partly after prior separation by HPLC. Mean concentrations (X(g) x DF) were as follows: E2beta (bpl) 31.1 (1.16), (spl) 24.2 (1.42) pg ml(-1); E1 (bpl) 143.3 (1.21), (spl) 117.7 (1.53) pg ml(-1); E1S (bpl) 157.3 (1.44), (spl) 2.92 (1.42) ng ml(-1); T (bpl) 570.6 (1.43), (spl) 23.1 (1.68) pg ml(-1); A (spl) 17.9 (1.39) pg ml(-1); DHEH (spl) 12.4 (1.51) pg ml(-1); 5alpha-DHT (spl) 9.7 (1.29) pg ml(-1). Except for E2beta and A in seminal plasma, a seasonal pattern was established for all other steroids with lowest mean values occurring from November to April. From the semen parameters determined, only motility was correlated to season. There was a higher correlation among oestrogen in blp than in spl and the only correlation identified between oestrogenic and androgenic steroids was between T and E2beta in blp. In spl, T was correlated with A and 5alpha-DHT. T was the dominant free steroid in bpl while it was E1 in spl; T and E1S concentrations were about 23- and 54-fold lower in spl compared to bpl with E1S, however, showing the highest absolute values in both fluids. In the fractionated ejaculate an association of free oestrogens, particularly E2beta, with spermatozoa was observed.
Article
A high-performance liquid chromatographic-tandem mass spectrometric (HPLC/MS/MS) method for the determination of 19-nortestosterone and its esters (cyclopentanepropionate, phenylpropionate, and decanoate) in equine plasma is achieved using an atmospheric pressure chemical ionization (APCI) interface in selected reaction monitoring (SRM) mode. The two internal standards used were 16,16, 17-(2)H(3)-19-nortestosterone for 19-nortestosterone and methenolone acetate for its esters. The steroids studied were extracted from plasma samples with a mixture of diethyl ether/n-hexane (9:1, v/v). The quantification limits for 19-nortestosterone, 19-nortestosterone cyclopentanepropionate, 19-nortestosterone phenylpropionate, and 19-nortestosterone decanoate were 0.16, 5.0, 0.1, and 2.0 ng/mL, respectively, when 2 mL of plasma were used. The recoveries of most of the steroids were 71.6-101.0% except for the decanoate, which could be recovered to about 39.8%. The responses were linear, with correlation coefficients varying from 0.9897 to 0.9999 in the concentration range of 0.1 to 50.0 ng/mL for the steroids studied. When applied to equine (mare) plasma samples, the present method allowed detection of 19-nortestosterone up to 23 days after an intra-muscular injection of 400 mg as the decanoate.
Article
Determination of whether the major metabolite of nandrolone in urine, 19-norandrosterone (19-NA), is exogenous or endogenous in origin is one of the most exciting challenges for antidoping laboratories. Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) can be used to differentiate these two origins by carbon isotopic ratio analysis. A complete method for purification of 19-NA in urine has been established. Acetylated ketosteroids, and in particular 19-NA, are isolated from the urine matrix before analysis after hydrolysis and purification of urine by reversed-phase and normal solid-phase extraction. The limit of detection for 19-NA was about 60 ng with recoveries of 54-60%. Evidence of exogenous administration of 19-NA may be established from isotope ratio determination from the 13C/12C ratios of several synthetic 19-norsteroids compared to those obtained for endogenous steroids.
Article
Contents Very little information is available regarding the physiological mechanisms involved in the normal sexual activity in the stallion and, in particular, the endocrine control of reproduction is still not clearly understood. This experiment was designed to determine the short‐term effect of sexual stimulation on plasma concentrations of testosterone, cortisol, oestrone sulphate and 15‐ketodihydro‐PGF 2 α in stallions. Semen samples were collected from 10 lighthorse stallions of proven fertility using a Missouri model artificial vagina. At the same time, blood samples were collected from the jugular vein with heparinized tubes, 20 and 10 min before oestrous mare exposure, at exposure and 10, 20, 30 min after dismounting. Testosterone concentrations showed a sharp rise 10 min after mating (p < 0.001), reached a plateau, and then showed a further increase 30 min after mating (p < 0.001). Cortisol concentrations increased 10 min after mating (p < 0.001) and remained at high levels in the subsequent samples taken. A peak of oestrone sulphate was observed 10 min after mating (p < 0.001). 15‐Ketodihydro‐PGF 2 α concentrations decreased rapidly at the moment of the exposure of the stallions to an oestrous mare (p < 0.05), returned to pre‐mating concentrations and then decreased again 30 min after mating (p < 0.05).
Article
A sensitive and specific method has been developed to detect semi-quantitatively testosterone in horse hair samples. The method involved a washing step with sodium dodecylsulfate aqueous solution. The mane and tail hair samples (100mg) were dissolved in 1 mL of sodium hydroxide for 15 min at 95 degrees C in the presence of d3-boldenone used as internal standard. The next three steps involved diethyl ether extraction and a solid phase extraction on Isolute C18 (EC) cartridges eluted with methanol. The residue was derivatized by adding 100 microL of acetonitrile and 30 microL of PFPA then incubating for 15 min at 60 degrees C. After evaporation, 30 microL of hexane was added and 2.5 microL was injected into the column (a bonded phase fused silica capillary column DB5MS, 30 m x 0.25 mm i.d. x 0.25 microm film thickness) of a Trace GC chromatograph. In order to improve the sensitivity of the method, damping gas flow has been optimized. Testosterone was identified in MS(2) full scan mode on the Polaris Q instrument. The assay was capable of detecting less than 1 pg mg(-1). The recovery was close to 90%. The analysis of tail and mane samples collected from a gelding horse having received a single dose of testosterone propionate (1 mg kg(-1)) showed the presence of testosterone in the range of 1-6 pg mg(-1) in hair collected during 5 months after administration.
Article
For almost two decades we have known that enzymatic hydrolysis of "normal" urine samples from the entire male horse using Escherichia coli (E. coli) followed by solvolysis (ethyl acetate:methanol:sulphuric acid) results in the detection of significant amounts of estr-4-ene-3,17-dione (19-norandrost-4-ene-3,17-dione) along with estr-4-en-17beta-ol-3-one (19-nortestosterone, nandrolone) in extracts of the hydrolysed urine and that both steroids are isolated from the solvolysis fraction. This solvolysis process is targeted at the steroid sulphates. Also we have shown that 19-norandrost-4-ene-3,17-dione and 19-nortestosterone are isolated from testicular tissue extracts. Subsequently, evidence was obtained that 19-nortestosterone detected in extracts of "normal" urine from male horses may not be derived from the 17beta-sulphate conjugate. However, following administration of 19-nortestosterone based proprietary anabolic steroids to all horses (males, females and castrates), the urinary 19-nortestosterone arising from the administration is excreted primarily as the 17beta-sulphate conjugate. Thus, if the 19-nortestosterone-17beta-sulphate conjugate arises only following administration this has interesting implications for drug surveillance programmes to control administration of 19-nortestosterone based anabolic preparations to male horses. These results have led us to consider that the precursors to 19-nortestosterone and 19-norandrost-4-ene-3,17-dione, present in the urine prior to the hydrolysis steps, have the same basic structure except for the functionality at the 17-position. We have used preparative high pressure liquid chromatography (LC) and LC fractionation to separate these precursors from the high amounts of oestrogenic sulphates present in "normal" urine from the entire male horse. Purified fractions have then been studied by liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) to identify the precursors.
Article
A method to quantify metabolites of 17beta-nandrolone (17betaN) in boar and horse urine has been optimized and validated. Metabolites excreted in free form were extracted at pH 9.5 with tert-butylmethylether. The aqueous phases were applied to Sep Pak C18 cartridges and conjugated steroids were eluted with methanol. After evaporation to dryness, either enzymatic hydrolysis with beta-glucuronidase from Escherichia coli or solvolysis with a mixture of ethylacetate:methanol:concentrated sulphuric acid were applied to the extract. Deconjugated steroids were then extracted at alkaline pH with tert-butylmethylether. The dried organic extracts were derivatized with MSTFA:NH4I:2-mercaptoethanol to obtain the TMS derivatives, and were subjected to analysis by gas chromatography mass spectrometry (GC/MS). The procedure was validated in boar and horse urine for the following metabolites: norandrosterone, noretiocholanolone, norepiandrosterone, 5beta-estran-3alpha, 17beta-diol, 5alpha-estran-3beta, 17beta-diol, 5alpha-estran-3beta, 17alpha-diol, 17alpha-nandrolone, 17betaN, 5(10)-estrene-3alpha, 17alpha-diol, 17alpha-estradiol and 17beta-estradiol in the different metabolic fractions. Extraction recoveries were higher than 90% for all analytes in the free fraction, and better than 80% in the glucuronide and sulphate fractions, except for 17alpha-estradiol in the glucuronide fraction (74%), and 5alpha-estran-3beta, 17alpha-diol and 17betaN in the sulphate fraction (close to 70%). Limits of quantitation ranged from 0.05 to 2.1 ng mL(-1) in the free fraction, from 0.3 to 1.7 ng mL(-1) in the glucuronide fraction, and from 0.2 to 2.6 ng mL(-1) in the sulphate fraction. Intra- and inter-assay values for precision, measured as relative standard deviation, and accuracy, measured as relative standard error, were below 15% for most of the analytes and below 25%, for the rest of analytes. The method was applied to the analysis of urine samples collected after administration of 17betaN laureate to boars and horses, and its suitability for the quantitation of the metabolites in the three fractions has been demonstrated.
Article
In elite sports, indirect testing of testosterone abuse is mainly based on the testosterone over epitestosterone (T/E) ratio. Since this marker is characterized by a small ratio of intra- to inter-individual variation, it is surprising that current anti-doping strategy uses a screening test based on a population-based limit. From a database of more than 15,000 steroid profiles obtained from routine controls, the collection of steroids profiles of 11 elite athletes followed during 2 years, and a longitudinal study involving 17 amateur athletes, 8 of which were orally administrated testosterone undecanoate pills, we selected 12 case studies to represent the possible scenarios to which the anti-doping laboratories are confronted. Various detection strategies at the disposal of the laboratories are employed and discussed, including isotope ratio mass spectrometry (IRMS) analysis and a Bayesian interpretation of the T/E-time profile. The weak sensitivity versus specificity relation of a population-based limit for the T/E ratio is outlined. As a result, we propose a Bayesian screening test whose T/E threshold progressively evolves from a population basis to a subject basis as the number of individual test results increases. We found that this screening test heightens drastically the capacity to detect testosterone abuse, at no additional financial and administrative expenses for anti-doping authorities.
Article
Nandrolone (17beta-hydroxy-4-estren-3-one, NAD) is an endogenous steroid hormone; thus, the detection of its metabolites is not conclusive of NAD doping in racehorses. NAD doping control in male horses is based on the threshold, namely, the concentration ratio of 5alpha-estran-3beta,17alpha-diol (ETA) to 5(10)-estren-3beta,17alpha-diol (ETE). The ETA/ETE ratio of 1/1 was determined based on statistical data of authentic horses in International Federation of Horseracing Authorities. To individuals with complex metabolic disorders, however, such a threshold might not be applicable. The aim of this study was to establish an analytical method that discriminates endogenous steroids from exogenous ones in horse urine after NAD administration using gas chromatography/combustion/carbon isotope ratio mass spectrometry (GC/C/IRMS). Urine was sampled from NAD-administered and authentic horses. Ten millilitres of urine was hydrolyzed and subjected to liquid-liquid extraction and solid phase extraction. The residue of the extracts purified by HPLC was derivatized by acetylation. As a result of measurement of the (13)C/(12)C ratio (delta(13)C) by GC/C/IRMS, the delta(13)C values of ETA for NAD-administered and authentic horses were -32.20+/-0.35 per thousand and -27.85+/-0.75 per thousand (n=60), respectively. The detection limit of ETA in this GC/C/IRMS analysis was approximately 25 ng/ml. This study indicates that the measurement of delta(13)C by GC/C/IRMS enables us to discriminate exogenous ETA derived from NAD administration from endogenous ETA, proving that GC/C/IRMS is a useful technique to complement the ETA/ETE ratio.
Article
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 17beta-estradiol 3-benzoate and 17beta-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 (CCalpha) for steroids were below 0.1 microg L(-1), except for 19-norandrosterone (CCalpha=0.7 microg L(-1)) and estrone (CCalpha=0.3 microg L(-1)). Kinetics of elimination of the administered 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate were established by monitoring 17beta-estradiol, 17alpha-estradiol, estrone and 17beta-nandrolone, 17alpha-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 17alpha-estradiol and 17alpha-nandrolone (>20 and 2 mg L(-1), respectively after 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate administration) whereas 17beta-estradiol, estrone, 17beta-nandrolone, 19-noretiocholanolone and 19-norandrostenedione were found as secondary metabolites at concentration values up to the microg 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 17beta form.
Nandrolone in the colt: A review of the science relating to the threshold value
  • E Houghton
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A steroidomic based analytical strategy has been set to screen for nandrolone misuse in equine Drug Testing and Analysis Drug Test
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