Gaud Pinel

Evidence that urinary excretion of thiouracil in adult bovine submitted to a cruciferous diet can give erroneous indications of the possible illegal use of thyrostats in meat production

Thyrostats have been banned for use as veterinary drugs in Europe since 1981 because of their carcinogenic and teratogenic properties. Until now, the identification of thiouracil in animal biological matrices has been interpreted as the consequence of an illegal administration. The present paper studies the influence of a cruciferous-based feed on the occurrence of thiouracil as a residue in urine. Urine samples collected from two heifers fed on cabbage or rapeseed cakes were analysed for the presence of thiouracil by 3-iodobenzylbromide derivatization and liquid chromatography-electrospray ionization tandem mass spectroscopy (LC-ESI(−)-MS/MS) analysis. Urine collected after cabbage or rapeseed feeding showed thiouracil concentrations in the range 3–7 and 2–9 µg l−1, respectively, demonstrating a relationship between a diet based on cruciferous vegetables and the occurrence of thiouracil in urine. Thiouracil was excreted in urine in the hours following cruciferous intake. Complete elimination (<0.8 µg l−1) of the compound occurred within 5 days. The precursors in cruciferous vegetables responsible for the thiouracil excretion in urine were proved not to be thiouracil itself.

Generation and processing of urinary and plasmatic metabolomic fingerprints to reveal an illegal administration of recombinant equine growth hormone from LC-HRMS measurements

Growth hormones are proteins produced by the anterior pituitary gland responsible for bone and tissue growth through their effects on carbohydrates, lipids and proteins metabolisms. Despite strict regulations banning the use of recombinant equine growth hormone, this substance is suspected to be misused to improve the horse physical performances. In order to check whether the regulation is fulfilled or not, controls are organized and a new analytical screening tool potentially able to detect such abuse was investigated in this paper. An untargeted metabolomics approach, based on liquid chromatography coupled to high resolution mass spectrometry, was developed and applied to characterize and compare horse urinary and plasmatic metabolic profiles upon reGH administrations. After minimal sample preparation, biological fluids were analyzed by LC-ESI(±)-Q-TOF. Data processing was performed by XCMS software and multivariate data analysis applied to the generated data set allowed building OPLS models to discriminate control versus treated populations. Results demonstrated significant metabolic modifications consecutively to the reGH treatment. A comparative study between urinary and plasmatic signatures was performed to evaluate the resulting metabolomic models and to asses their respective interests in the scope of real application for screening reGH administration.

Analysis of thyreostats: A history of 35 years

Thyreostatic drugs (TS), illegally administrated to livestock for fattening purposes, are banned in the European Union since 1981 (Council Directive 81/602/EC). This paper reviews the trends in the analytical approaches for the determination of TS drugs in biological matrices. After a brief introduction on the different groups of compounds with a thyreostatic action, the most relevant legislation regarding the residue control of these compounds is presented. An overview of the analytical possibilities for the determination of TS in animal matrices, covering sample extraction, purification, separation techniques and detection methods is provided. Additionally, a brief outline of animal experiments is described that illustrates the excretion and distribution profiles of TS residues. Finally, the novel developments in TS analysis are highlighted. Also the possible semi-endogenous status of thiouracil is discussed.

Elimination kinetic of recombinant somatotropin in bovine

Bovine somatotropin (bST), also called growth hormone is a protein hormone produced by the pituitary gland and responsible directly or indirectly for various effects on growth, development and reproductive functions. Its recombinant bovine somatotropin form (rbST) is used in dairy cattle to enhance milk production. Even if the effects of treatment with rbST have been largely studied, until now analytical methods able to detect rbST were limited to immunoassays, which suffer from the impossibility to distinguish between the endogenous and the recombinant form. In this study, a sample preparation procedure based on different precipitation steps, extraction on solid phase and enzymatic digestion was used to purify rbST from serum. The detection was performed by liquid chromatography coupled to tandem mass spectrometry in positive electrospray ionization mode (LC-ESI(+)-MS/MS) allowing the unambiguous identification and quantification of rbST in serum. Samples collected from a cow treated with recombinant bovine somatotropin were analysed and for the first time, the elimination kinetic specific to recombinant somatotropin has been characterized in serum. Detection of rbST was possible from 4h 30min to 4 days after administration and concentration was found up to 10ngmL(-1) during the kinetic.

Elimination kinetic of 17B-estradiol 3-benzoate and 17B-nandrolone laureate ester metabolites in calves' urine

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.

Identification of Recombinant Equine Growth Hormone in Horse Plasma by LC−MS/MS: A Confirmatory Analysis in Doping Control

Equine growth hormone (eGH) has been available since 1998 as an approved drug (EquiGen-5, Bresagen) containing recombinant eGH (reGH). It is suspected of being illegally administered to racehorses in order to improve physical performance and to speed-up wound healing. Thus it may be considered a doping agent which would require a sensitive and reliable method of identification and confirmation in order to regulate its use in racehorses. reGH differs from the native eGH by an additional methionine at the N-terminal (met-eGH) and has never been unambiguously detected in any type of biological matrix at trace concentrations (1-10 microg/L). A plasma sample (4 mL) was treated with ammonium sulfate at the reGH isoelectric point and the pellet was purified by solid-phase extraction. Specific peptides were generated by trypsin digestion and analyzed by LC-MS/MS. The detection limit was 1 microg/L. The method was validated according to European Union regulation (DEC/2002/657/EC) and the Association of Official Racing Chemists (AORC) requirements. Furthermore, it was successfully applied to determining the plasma concentrations of reGH with time using linear ion trap mass analyzer. The presence of this prohibited hormone (reGH) was also successfully detected by triple quadrupole mass spectrometry up to 48 h postadministration of reGH to a horse. The present LC-MS/MS method is the first with adequate sensitivity and specificity for detection of reGH, rbGH, and endogenous eGH. Hence, an efficient analytical tool is proposed as a means to fulfilling the regulation of reGH abuse in the horse racing industry.

Criteria to distinguish between natural situations and illegal use of boldenone, boldenone esters and boldione in cattle. 2. Direct measurement of 17β-boldenone sulpho-conjugate in calf urine by liquid chromatography-high resolution and tandem mass spectrometry.

Boldenone is banned in the European Union (Directive 96/22/EC) as growth promoter for meat producing animals. Boldione (ADD), boldenone and boldenone esters (mainly the undecylenate form) are commercially available as anabolic preparations, either to the destination of human, horse or cattle. Since the late 90s, the natural occurrence of boldenone metabolites has been reported in cattle. According to EU regulation, the unambiguous demonstration of boldenone administration in bovine urine should be provided on the basis of boldenone identification in the corresponding conjugate fraction. An analytical method has been developed and validated according to current standards with main concern to the measurement of intact 17beta-boldenone-sulphate. The analytical procedure included direct extraction-purification of target analyte on octadecylsilyl cartridges and direct detection of phase II metabolite by liquid chromatography (negative electrospray), tandem mass spectrometry (QqQ) or high resolution mass spectrometry (Orbitrap). Decision limit (CCalpha) and detection capability (CCbeta) were respectively 0.2 microg L(-1) and 0.4 microg L(-1) on triple quadrupole and 0.1 microg L(-1) and 0.2 microg L(-1) on hybrid system. The method was successfully applied to the analysis of incurred samples collected in different experiments. 17beta-Boldenone-sulphate was measurable up to 36h after oral administration of boldione, and 30 days after 17beta-boldenone undecylenate intra-muscular injection. This conjugate form was never detected in non-treated animals, confirming its status of definitive candidate marker for boldenone administration in calf.

Direct determination of recombinant bovine somatotropin in plasma from a treated goat by liquid chromatography/high‐resolution mass spectrometry

Recombinant bovine somatotropin (rbST) is used in dairy cattle to enhance milk production. Despite the ban on this hormone in some countries, especially in Europe, there is so far no method available for the direct detection of rbST either in milk or in plasma. An analytical strategy has been developed to analyze rbST in plasma, including a purification procedure based on a precipitation with ammonium sulphate, followed by a solid-phase extraction (SPE)-based clean-up on C4 sorbent and precipitation with cold methanol. The hormone was then digested with trypsin and analyzed by liquid chromatography/high-resolution mass spectrometry (LC/HRMSn) on a linear ion trap coupled with an Orbitrap. The tryptic N-terminal peptide, specific to the difference between the endogenous and recombinant form of the somatotropin, was fragmented and product ions were analyzed at high mass resolution. Applying this approach to goat plasma allowed the direct detection of 10 ng mL(-1) of rbST in fortified samples. It also showed the presence of rbST in plasma collected from a goat treated with the hormone, even 2 days after administration. These results are of a great interest in the field of somatotropin control and undoubtedly constitute a first step in the development of a method for the detection of rbST not only in bovine plasma, but also in other biological matrices such as milk.

Detection of recombinant bovine somatotropin in milk and effect of industrial processes on its stability

A LC-MS/MS method has been developed for the direct detection of recombinant bovine somatotropin (rbST) in milk and dairy products. The sample preparation protocol is based on a solid phase extraction step followed by precipitation with cold methanol and enzymatic digestion. The analysis is focused on the tryptic N-terminal peptide, specific of the recombinant form of the hormone and the detection is performed by LC-ESI(+)-MS/MS. This method has been validated according to the European Union criteria described in the Directive 2002/657/EC. Acceptable performances, with a decision limit (CCalpha) of 1.24 ng mL(-1) and detection capability (CCbeta) of 1.92 ng mL(-1) were obtained. Calculation of repeatability and intermediate reproducibility of the signal at 100 ng mL(-1) lead to relative standard deviations lower than 20%, showing the robustness of the method. Samples subjected to various industrial processes namely, heating, freezing, defatting, pasteurization and spray-drying were then analysed in order to determine the consequences of these treatments on the stability of the hormone. Results showed that temperature related processes, such as pasteurization and spray-drying induce a loss of the hormone up to 95%.

Estranediols profiling in calves' urine after 17β-nandrolone laureate ester administration

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).