Detection of prohibited substances in horses: technical aspects and new prospects

Progress in antidoping control has been closely linked with improvements in analytical methods and equipment. Drug testing became really effective in the sixties with the development of paper chromatography, thin-layer chromatography, gas chromatography, and finally high-performance liquid chromatography. Soon afterwards, mass spectrometry revolutionised the detection of drugs by combining both specificity and sensitivity in a single assay. The different steps of qualitative and quantitative antidoping control are described, as well as its biological and administrative aspects defined by recognised quality standards. More complex molecules, such as peptides hormones (e.g. growth hormone) are now being used as doping substances. Their detection represents a real challenge for laboratories today.Les avancées en matière de contrôle des substances prohibées ont suivi fidèlement l'évolution des méthodes analytiques et de l'instrumentation. Le contrôle est devenu réellement efficace dans les années 1960 avec l'apparition des méthodes de chromatographie, sur papier couches minces, puis en phase gazeuse et enfin en phase liquide à hautes performances. Peu après, l'apparition de la spectrométrie de masse a révolutionné les méthodes de détection en apportant à la fois spécificité et sensibilité. Les différentes étapes du contrôle antidopage qualitatif ou quantitatif sont décrites ainsi que les aspects biologiques, administratifs dans le respect des normes « qualité ». Les nouveaux défis se situent actuellement dans l'introduction de molécules plus complexes telles que les peptides, dans l'arsenal des substances dopantes. Ainsi, les hormones peptidiques telles que l'hormone de croissance par exemple, représentent un réel défi pour les laboratoires

Rôle des contaminants alimentaires dans le contrôle antidopage chez le cheval (étude de l'atropine, la scopolamine, la caféine et la théobromine)

LIMOGES-BU Médecine pharmacie (870852108) / SudocLYON1-BU Santé (693882101) / SudocSudocFranceF

Le contrôle antidopage des animaux, nouvelles perspectives, nouvelles méthodes

Les nouvelles méthodes de détection pouvant être mises en place dans les laboratoires sont principalement axées autour des spectromètres de masse de haute résolution, notamment les appareils de temps de vol (TOF) dont la technologie s'est considérablement améliorée ces dernières années. Ces techniques sont susceptibles d'apporter à la fois une meilleure sensibilité et spécificité. En effet l'accroissement de résolution permet d'augmenter significativement le rapport signal/bruit et dans la mesure où la sensibilité de l'appareil n 'est pas altérée en conséquence, le gain en terme de capacité de détection est donc important. Dans le même esprit, de nouveaux couplages de chromatographie liquide capillaire/ MS/MS, alliant à la fois la résolution chromato graphique des colonnes "micro ou nanobore" aux performances des spectromètres en tandem (MS/MS) récents ou des trappes d'ions permettent d'améliorer significativement les limites de détection des xénobiotiques dopants. Des exemples de détection de stéroïdes anabolisants dans les milieux biologiques équins illustrent ces nouvelles perspectives analytiques. Le second axe de développement s'articule autour de la prise en compte de marqueurs biologiques qui peuvent être altérés par l'administration de biomolécules telles que les hormones de croissance par exemple. Le problème principal posé par l'administration de ces peptides réside dans le fait que ces molécules elles-même sont difficiles à détecter du fait de leur poids moléculaire élevé et surtout par le fait que leur demi-vie est très courte. Ces deux phénomènes ont pour conséquence de rendre aléatoire la mise en évidence de la molécule elle-même, une solution possible à cela consiste à prendre en compte les marqueurs biologiques altérés par l'administration de l'hormone protéique. Ainsi un exemple basé sur le suivi de ces marqueurs et en particulier de l'IGFl, après administration d'hormone de croissance recombinante au cheval est exposé

Feed contaminants and anti doping tests

Pharmacologically active drugs are considered as prohibited at any concentration (with the exception of threshold drugs) in body fluids collected during racing or equestrian events, even if environmental or feeds contamination might occur. This can create unexpected positive cases when feeds or supplements contain unlisted drugs in supplements or in feeds containing alkaloids such as atropine, scopolamine or caffeine. There are several contamination origins depending on drugs involved. Manufacturing or transportation are major factors which may explain the presence of these drugs in the feed. Two studies were carried out to assess the maximum intake of major contaminants absorbed through the diet that would not result in a positive test in urine. Caffeine, theobromine, theophylline, atropine, scopolamine and morphine were administered orally at different dosages, for 2 days before urine was sampled and submitted to analysis by screening methods. Each cocktail of drugs were administered to two horses. Caffeine: a 10 mg daily intake was found to induce urinary concentrations ranged from 23 to 50 ng/ml; theobromine: a 30 mg daily intake was found to produce urinary concentrations from 700 to 900 ng/ml; theophylline: a 15 mg/day intake was found to induce urinary concentrations of 140-500 ng/ml; atropine: a 10 mg/day intake was found to produce 10 to 25 ng/ml in urine; scopolamine: a 10 mg/day intake was found to give urinary concentrations from 25-140 ng/ml and, finally morphine: a 1 mg/day intake was found to produce a range of 10-15 ng/ml in urine. Horse feeds containing contaminants, even at low levels, may produce positive test results in anti-- doping urine analysis. A maximum daily intake or a maximum concentration in feeds must be adopted to avoid inadvertent positive findings in routine tests. The use of feeds or supplements properly tested or guaranteed by manufacturers by an effective quality controlled process is crucial for feeding horses in training or before competition or racing events. The sensitivity of screening tests used to control feeds or feed supplements must at least comply with the concentrations determined in the present study. Nevertheless the lack of international harmonization remains an important issue, a MRL s (Maximum Residue Level) like approach would be the more appropriate as soon as harmonized levels of detection will be adopted.vo

Synthèse de dérivés pyridiniques fonctionnalisés-cétones, cétoamides et cétoesters-par réaction de carbonylation de précurseurs halogènes

La pyridine et ses dérivées jouent un rôle majeur en chimie hétérocyclique de part leur vaste panoplie de potentialités chimiques et biologiques liées à la présence de l'hétéroatome d'azote. A partir de précurseurs halogénés aisément accessibles, deux nouvelles méthodes de synthèse de dérivés pyridiniques fonctionnalisés, catalysés au palladium, ont été développées. Elles conduisent respectivement à des aroylpyridines et des dérivés d'acides pyridylglyoxyliques qui s'avèrent être des intermédiaires potentiels dans la synthèse de nombreuses molécules d'intérêt pharmaceutique ou agrochimique. La première réaction étudiée est une réaction de couplage de substrat pyridinique avec un acide arylboronique sous monoxyde de carbone ; elle dérive de la réaction de Suzuki. L'influence sur la réaction de nombreux facteurs expérimentaux a été examinée. Les plus importants sont la pression de CO, la nature du précurseur catalytique et la nature du motif halogéné présent sur la pyridine. La réaction s'est révélée efficace avec des rendements isolés supérieurs à 80% et des sélectivités en cétones supérieures à 90%. La deuxième méthode de fonctionnalisation met en jeu la réaction de double carbonylation d'iodopyridines conduisant à des composés alpha-cétoamides et alpha-cétoesters.LILLE1-BU (590092102) / SudocSudocFranceF

Unraveling the anabolic-androgenic steroid profile of untreated horses by UHPLC-MS/MS

Clean sport is a priority for the regulatory organs of both the horse racing industry (IFHA, International Federation of Horseracing Authorities) and the Equestrian sports (FEI, Fédération Equestre Internationale). Urinary and blood samples are screened for the presence of hundreds of forbidden substances including anabolic-androgenic steroids (AAS). Because of their importance as ergogenic drugs of abuse and their involvement in equine reproduction, AAS remain an important field of investigation. Consequently, based on the suspected endogenous origin of some forbidden AAS (ß-boldenone as the most elicit one) this study focused on non-treated, out of competition horses, to improve the knowledge on the natural endogenous AAS profile of horses in general. In total, 105 guaranteed non-treated horses (47 geldings, 53 mares and 5 stallions serving as a control) were screened for ß-boldenone and five related steroids: ADD (androstadienedione), AED (androstenedione), αT (alpha-testosterone), ßT (ß-testosterone) and P (progesterone) via UHPLC-MS/MS (Triple Quadripole). The extraction and detection method was validated according to EC guidelines for ß-boldenone, ADD, AED, αT and ßT. Correlations between the occurrences of these AAS were calculated and hypotheses for the alleged endogenous traces were formulated. By using in vitro digestive simulations we were able to test different hypotheses

Does the consumption of moulded feed affect the excretion of anabolic-androgenic steroids in horses?

Background: To ensure fair competition, sport horses have to compete on their own merits, without any unfair advantage that might follow the use of drugs. Therefore, the FEI (Fédération Equestre Internationale) lists all substances that are not allowed in competition. Many steroids are part of this list as the administration of steroids increases nitrogen retention, protein synthesis and the release of erythropoietin in the kidneys, making them very popular as drugs of abuse. Hypothesis/Objectives: Some feed-related moulds can transform plant phytosterols into (anabolic) steroids. What would happen if a horse consumes feed contaminated with these moulds? Due to the strict anti-doping policies applied by the FEI, the excretion of steroids originating from the feed could have major consequences. Methods: A rapid UHPLC-MS/MS analytical method was developed and successfully and thoroughly validated. Multiple spontaneously moulded feed samples were tested for the presence of forbidden and related natural steroids (including boldenone and testosterone). The effect of the consumption of these feeds was tested by in vitro simulation of the horse’s hindgut, in static batch incubations. Samples were taken after 0, 12, 24, 48, and 72h and the incubations were executed in triplicate. The results were compared to a blank in vitro digestion and the digestion of unmoulded feed. Results: In most feed samples no steroids were detected, even when the products were moulded. However, moulded corn contained 1.8 ± 0.5 ng/g AED (4-andostradienedione, a testosterone precursor), and this concentration increased when corn was digested in vitro. The addition of phytosterols to the in vitro digestion led to a further increase of AED (up to 2.9 ± 0.4 ng/g feed). Additionally, one of the phytosterol-rich herbal supplements contained α-testosterone as well (up to 40 ng/g)

In vitro simulation of the hindgut as a tool to study the influence of phytosterol consumption on the excretion of anabolic-androgenic steroids in horses

Introduction: In race horses zero tolerance is held for most anabolic steroids (AAS). Nevertheless, some natural precursors, including phytosterols from grains, have been put forward as a possible explanation for the endogenous prevalence of low concentrations of steroids, with β-Bol as the most illicit candidate. Purpose: This study aimed to evaluate the use of in vitro digestions as a tool to simulate in vivo hindgut fermentation and to study the possible biotransformation from phytosterols or other androgenic precursors to forbidden AAS. Methods: To investigate the possible endogenous biotransformation, in vitro simulations of the horse hindgut were set up, using fecal inocula obtained from eight different horses. In vitro digestion samples were analyzed with a fully validated UHPLC-MS/MS method. Results: The digestive transformation of ADD could be directly linked to the detection of βBol, and the consumption of phytosterols to low levels of AED, a testosterone precursor, but no direct link between phytosterol consumption and βBol detection was observed. Significance: The results are in line with previously described in vivo results, proving that in vitro digestions are a useful tool to study the fermentation reactions that take place in the horse’s hindgut, offering new insights in the detection of, endogenous, anabolic steroids

Pharmacokinetic study of lolitrem B in goat : distribution in milk and plasma

International audienc

Palladium-catalyzed carbonylative cross-coupling reactions of pyridine halides and aryl boronic acids: a convenient access to α-pyridyl ketones

International audienceThe proper choice of solvent, catalyst precursor and CO pressure enables the easy and selective transformation of mono- and dihalopyridines into phenyl pyridyl ketones in 81–95% yields.The proper choice of solvent, catalyst precursor and CO pressure enables the facile and selective transformation of mono- and dihalopyridines into phenyl pyridyl ketones