Verrières-le-Buisson, France
Verrières-le-Buisson, France

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PubMed | Montpellier SupAgro, UK Institute of Food Research, European Commission - Joint Research Center Ispra, Bruker and 9 more.
Type: Journal Article | Journal: Metabolomics : Official journal of the Metabolomic Society | Year: 2015

The metabo-ring initiative brought together five nuclear magnetic resonance instruments (NMR) and 11 different mass spectrometers with the objective of assessing the reliability of untargeted metabolomics approaches in obtaining comparable metabolomics profiles. This was estimated by measuring the proportion of common spectral information extracted from the different LCMS and NMR platforms. Biological samples obtained from 2 different conditions were analysed by the partners using their own in-house protocols. Test #1 examined urine samples from adult volunteers either spiked or not spiked with 32 metabolite standards. Test #2 involved a low biological contrast situation comparing the plasma of rats fed a diet either supplemented or not with vitamin D. The spectral information from each instrument was assembled into separate statistical blocks. Correlations between blocks (e.g., instruments) were examined (RV coefficients) along with the structure of the common spectral information (common components and specific weights analysis). In addition, in Test #1, an outlier individual was blindly introduced, and its identification by the various platforms was evaluated. Despite large differences in the number of spectral features produced after post-processing and the heterogeneity of the analytical conditions and the data treatment, the spectral information both within (NMR and LCMS) and across methods (NMR vs. LCMS) was highly convergent (from 64 to 91% on average). No effect of the LCMS instrumentation (TOF, QTOF, LTQ-Orbitrap) was noted. The outlier individual was best detected and characterised by LCMS instruments. In conclusion, untargeted metabolomics analyses report consistent information within and across instruments of various technologies, even without prior standardisation.


Boyard-Kieken F.,Laboratoire des Courses Hippiques | Boyard-Kieken F.,National School of Engineering in Agricultural and Food Industries | Dervilly-Pinel G.,National School of Engineering in Agricultural and Food Industries | Garcia P.,Laboratoire des Courses Hippiques | And 4 more authors.
Journal of Separation Science | Year: 2011

Growth hormone (GH) is a polypeptide suspected of being used in horse racing to speed up physical performances. Despite scientific advances in the recent years, the control of its administration remains difficult. In order to improve it, a metabolomics study through LC-high resolution mass spectrometry measurements was recently initiated to assess the metabolic perturbations caused by recombinant equine growth hormone administration. Few tens of ions not identified structurally were highlighted as compounds responsible for the modification of metabolic profiling observed in treated animals. This previous work was based on the use of Uptisphere Strategy NEC as the chromatographic column. In parallel, more and more metabolomics studies showed the interest of the use of new chromatographic supports such as hydrophilic interaction chromatography for the analysis of polar compounds. It is in this context that an investigation was conducted on Uptisphere HDO and Luna hydrophilic interaction chromatography stationary phases to generate and process urinary metabolomics fingerprints, which could allow to establish a comparison with Uptisphere Strategy NEC. The chromatographic column the most adapted for the detection of new biomarkers of GH administration has been used to set up a relevant statistical model based on the analysis of more than hundred biological samples. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Decloedt A.I.,Ghent University | Bailly-Chouriberry L.,Laboratoire des Courses Hippiques | Vanden Bussche J.,Ghent University | Garcia P.,Laboratoire des Courses Hippiques | And 3 more authors.
Drug Testing and Analysis | Year: 2016

To ensure fair competition and to protect the horse's welfare, horses have to compete on their own merits, without any unfair advantage that might follow the use of drugs. Therefore, regulatory authorities list all substances that are not allowed in competition, including most anabolic-androgenic steroids. As zero-tolerance is retained, the question arose whether the consumption of mouldy feed could lead to the excretion of steroids, due to the biotransformation of plant phytosterols to steroids. A rapid ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analytical method, previously validated according to AORC (Association of Official Racing Chemists) and EC (European Commission) guidelines, was used to measure steroids in different sample types. Multiple mouldy feed samples were tested for the presence of steroids. The effect of digestion was tested by in vitro simulation of the horse's hindgut in batch incubations. In most feed samples no steroids were detected, even when the products were mouldy. Mouldy corn however showed to contain up to 3.0 ± 0.4 µg/kg AED (4-androstenedione), the main testosterone precursor. This concentration increased when mouldy corn (with added phytosterols) was digested in vitro. An herbal phytosupplement also showed to contain α-testosterone. These results demonstrate that it is important to caution against the consumption of any feed or (herbal) supplement of which the detailed ingredients and quantitative analysis are unknown. The consumption of mouldy corn should especially be avoided, not only from a horse health and welfare point of view, but also to avoid possible inadvertent positive doping results. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.


Decloedt A.,Ghent University | Bailly-Chouriberry L.,Laboratoire des Courses Hippiques | Bussche J.V.,Ghent University | Garcia P.,Laboratoire des Courses Hippiques | And 3 more authors.
Analytical and Bioanalytical Chemistry | Year: 2015

Doping control is a main priority for regulatory bodies of both the horse racing industry and the equestrian sports. Urine and blood samples are screened for the presence of hundreds of forbidden substances including anabolicandrogenic steroids (AASs). Based on the suspected endogenous origin of some AASs, with β-boldenone as the most illicit candidate, this study aimed to improve the knowledge of the naturally present AAS in horse urine. To this extent, a novel ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated according to the Association of Official Racing Chemists (AORC) and European Commission (EC) guidelines, proving the power of this new method. Low limits of detection (0.2 ng/mL), good reproducibility (percentage of standard deviation (%RSD)<10 %), high recovery (94.6 to 117.1 %), selectivity and specificity, and a linear response (confirmed with R2>0.99 and lack-of-fit analysis) were obtained for all included AASs. With this method, urine samples of 105 guaranteed untreated horses (47 geldings, 53 mares, and 5 stallions serving as a control) were screened for β-boldenone and five related natural steroids : androstadienedione (ADD), androstenedione (AED), alphatestosterone (αT), beta-testosterone (βT), and progesterone (P). Progesterone, β-testosterone, and α-testosterone were detected in more than half of the horses at low concentrations (<2 ng/mL). Occasionally, not only testosterone and progesterone but also low concentrations of AED, ADD, and boldenone (Bol) were found (0.5-5 ng/mL). © Springer-Verlag Berlin Heidelberg 2015.


PubMed | Ghent University and Laboratoire des Courses Hippiques
Type: | Journal: The Journal of steroid biochemistry and molecular biology | Year: 2015

Traditionally, steroids other than testosterone are considered to be synthetic, anabolic steroids. Nevertheless, in stallions, it has been shown that -Bol can originate from naturally present testosterone. Other precursors, including phytosterols from feed, have been put forward to explain the prevalence of low levels of steroids (including -Bol and ADD) in urine of mares and geldings. However, the possible biotransformation and identification of the precursors has thus far not been investigated in horses. To study the possible endogenous digestive transformation, in vitro simulations of the horse hindgut were set up, using fecal inocula obtained from eight different horses. The functionality of the in vitro model was confirmed by monitoring the formation of short-chain fatty acids and the consumption of amino acids and carbohydrates throughout the digestion process. In vitro digestion samples were analyzed with a validated UHPLC-MS/MS method. The addition of -Bol gave rise to the formation of ADD (androsta-1,4-diene-3,17-dione) or T. Upon addition of ADD to the in vitro digestions, the transformation of ADD to -Bol was observed and this for all eight horses inocula, in line with previously obtained in vivo results, again confirming the functionality of the in vitro model. The transformation ratio proved to be inoculum and thus horse dependent. The addition of pure phytosterols (50% -sitosterol) or phytosterol-rich herbal supplements on the other hand, did not induce the detection of -Bol, only low concentrations of AED, a testosterone precursor, could be found (0.1 ng/mL). As such, the digestive transformation of ADD could be linked to the detection of -Bol, and the consumption of phytosterols to low concentrations of AED, but there is no direct link between phytosterols and -Bol.


PubMed | Ghent University and Laboratoire des Courses Hippiques
Type: Journal Article | Journal: Drug testing and analysis | Year: 2016

To ensure fair competition and to protect the horses welfare, horses have to compete on their own merits, without any unfair advantage that might follow the use of drugs. Therefore, regulatory authorities list all substances that are not allowed in competition, including most anabolic-androgenic steroids. As zero-tolerance is retained, the question arose whether the consumption of mouldy feed could lead to the excretion of steroids, due to the biotransformation of plant phytosterols to steroids. A rapid ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analytical method, previously validated according to AORC (Association of Official Racing Chemists) and EC (European Commission) guidelines, was used to measure steroids in different sample types. Multiple mouldy feed samples were tested for the presence of steroids. The effect of digestion was tested by in vitro simulation of the horses hindgut in batch incubations. In most feed samples no steroids were detected, even when the products were mouldy. Mouldy corn however showed to contain up to 3.00.4g/kg AED (4-androstenedione), the main testosterone precursor. This concentration increased when mouldy corn (with added phytosterols) was digested in vitro. An herbal phytosupplement also showed to contain -testosterone. These results demonstrate that it is important to caution against the consumption of any feed or (herbal) supplement of which the detailed ingredients and quantitative analysis are unknown. The consumption of mouldy corn should especially be avoided, not only from a horse health and welfare point of view, but also to avoid possible inadvertent positive doping results. Copyright 2016 John Wiley & Sons, Ltd.


Garcia P.,Laboratoire des Courses Hippiques | Paris A.-C.,Laboratoire des Courses Hippiques | Gil J.,Laboratoire des Courses Hippiques | Popot M.-A.,Laboratoire des Courses Hippiques | Bonnaire Y.,Laboratoire des Courses Hippiques
Biomedical Chromatography | Year: 2011

A sensitive method using LC/ESI-MSn has been developed on a quadrupole linear ion trap mass analyser for the detection of nine β2 agonists (cimaterol, clenbuterol, fenoterol, formoterol, mabuterol, terbutaline, ractopamine, salbutamol and salmeterol) in horse urine. The method consists of solid-phase extraction on CSDAU cartridges before analysis by LC/ESI-MSn. The efficiency of extraction combined with the sensitivity and the selectivity of MSn allowed the detection of these compounds at pg/mL levels. Administration studies of fenoterol and formoterol are reported and show their possible detection after inhalation. The method is applicable for screening and confirmatory analysis. © 2010 John Wiley & Sons, Ltd.


Balssa F.,Laboratoire des Courses Hippiques | Fischer M.,Laboratoire des Courses Hippiques | Bonnaire Y.,Laboratoire des Courses Hippiques
Steroids | Year: 2011

5α-Estrane-3β,17α-diol is the major metabolite of nandrolone in horse urine. The presence of 5α-estrane-3β,17α- diol in female and gelding urines is prohibited by Racing Rules and its natural presence in male urine led regulation authorities to establish a concentration threshold of 45 ng/mL. This paper describes a rapid, simple and stereoselective synthesis of 5α-estrane-3β,17α-diol, providing horseracing laboratories with an essential reference material for their antidoping performance. © 2011 Elsevier Inc. All rights reserved.


Balssa F.,Laboratoire des Courses Hippiques | Fischer M.,Laboratoire des Courses Hippiques | Bonnaire Y.,Laboratoire des Courses Hippiques
Steroids | Year: 2014

5(10)-Estrene-3β,17α-diol is an essential reference material for doping analysis in horse-racing laboratories. It is used to detect misuse, for doping purpose, of the pregnancy status in the mare. Its stereoselective synthesis from 17β-estradiol-3-methyl ether (prepared from estrone or 17β-estradiol) was performed in four steps: (1) Mitsunobu inversion of the 17β-alcohol; (2) Birch reduction of the aromatic ring; (3) stereoselective reduction of the 3-ketone via Noyori asymmetric transfer hydrogenation; (4) chemoenzymatic purification. © 2014 Elsevier Inc. All rights reserved.


Moulard Y.,Laboratoire des Courses Hippiques | Bailly-Chouriberry L.,Laboratoire des Courses Hippiques | Boyer S.,Laboratoire des Courses Hippiques | Garcia P.,Laboratoire des Courses Hippiques | And 2 more authors.
Analytica Chimica Acta | Year: 2011

Liquid chromatography-mass spectrometry (LC-MS) has been widely used in doping control laboratories over the last two decades. Currently, simple quadrupole, triple quadrupole and ion trap are the most commonly employed analyzers in toxicological analysis. Nevertheless, the main lack of these technologies is the restricted number of target compounds simultaneously screened without loss of sensitivity. In this article we present an innovative screening approach routinely applied in the French horse doping control laboratory based on high resolution (50. 000) and high mass accuracy (<5. ppm) in full scan MS mode for more than 235 target analytes screened from an initial volume of 5. mL of urine. The sample preparation was classically founded on solid phase extraction by means of reverse phase C18 cartridges. LC-MS analyses were carried out on a Shimadzu binary HPLC pumps linked to a C18 Sunfire column associated with the high resolution exactive benchtop orbitrap mass spectrometer. This screening was performed alternatively in positive-negative ionization mode during the same run. Thus, the identification of compounds of interest was made using their exact mass in positive-negative ionization mode at their expected retention time. All data obtained were processed by ToxID software (ThermoFisherScientific) which is able to identify a molecule by theoretical mass and retention time. In order to illustrate this innovative technology applied in our laboratory, sample preparation, validation data performed on 20 target compounds from 16 different horse urine samples, chromatograms and spectra will be discussed in this paper. © 2011 Elsevier B.V.

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