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Use of hormones for fattening purposes is forbidden in the animal production in Europe (European Commission. 1996. Council Directive EC/96/22 (replacement of 88/146/EC). Off J Eur Commun. L125:3-9; European Commission. 1996. Council Directive EC/96/23. Off J Eur Commun. L125:10-32). Moreover, Regulation (EC) 178/2002 (European Commission. 2002. Regulation EC No 178/2002. Off J Eur Commun. L31:1-24) and Regulation (EC) 882/2004 (European Commission. 2004. Regulation EC No 882/2004. Off J Eur Commun. L165:1-135) oblige the member states to identify emerging risks and use validated and accredited methods for control analysis. Only combinations of bioassay activity screening with chemical identification are suited to uphold all laws. No such combination is described for the detection of (gluco)corticoids. In the present study, the GR-CALUX bioassay was validated as a qualitative screening method for the determination of glucocorticoid activity in feed. This validation was performed according to EC Decision 2002/657/EC (European Commission. 2002. Commission Decision 2002/657/EC from Directive 96/23. Off J Eur Commun. L221:8-36). Twenty-two representative blank feed samples were selected and spiked with 50 ng g(-1) of dexamethasone, 100 ng g(-1) of betamethasone or 500 ng g(-1) of triamcinolone. All blank and spiked feed samples fulfilled the CCα and CCβ criteria; the method was specific and robust and glucocorticoids in feed were stable for at least 88 days.

Zilpaterol is a new powerful beta-agonist, which is officially registered for fattening purposes in cattle in Mexico and South Africa. Its chemical structure is different from the well-known beta-agonists. Therefore, the routinely used screening methods are not likely to be suited for the analysis of zilpaterol. Also gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry (LC-MS) methods need to be adapted to enable detection of zilpaterol. In this study, a LC-MS3 confirmatory method was developed for the simultaneous detection of zilpaterol and di-aromatic beta-agonists in urine samples. A LC-MS2 method was optimised for the detection of zilpaterol in faeces. To study the excretion profile in urine and faeces, a male veal calf was orally treated with daily doses of Zilmax ® during 2 weeks. Zilpaterol was mainly excreted via urine. © 2004 Elsevier B.V. All rights reserved.

Matrix solid-phase dispersion(MSPD) is a new sample pretreatment for solid samples. This technique greatly simplifies sample pretreatment but, nonetheless, the extracts often still require an extra cleanup step that is both laborious and time-consuming. The potential;of combining MSPD with molecularly imprinted solid-phase extraction (MISPE) was investigated in this study. Liver samples were ground in a mortar with Cls sorbent and the homogenized mixture packed into an SPE cartridge and placed on top of a MISPE cartridge. Subsequently, clenbuterol was eluted from the MSPD cartridge onto the MISPE cartridge using acetonitrile containing 1% acetic acid.:The ability of the molecularly imprinted polymer to selectively adsorb analyte in acetonitrile was exploited for re-extracting clenbuterol directly from this acetonitrile extract via the double cartridge tandem system. The analyte was eluted from the MISPE cartridge using acidified methanol, A clear eluate was obtained, which was subsequently evaporated, redissolved, and analyzed by HPLC electrochemical detection (ECD) or ion trap mass spectrometry (LC/IT-MS). The MISPE cartridge used in this study was imprinted using bromoclenbuterol, a structural analogue of clenbuterol, as the template. These MISPE cartridges showed excellent stability. The complete extraction procedure was rapid,and recoveries exceeded 90% for the target analyte. The method detection limit for the LC/IT-MS procedure was

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

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

Previously we described the construction and properties of a rapid yeast bioassay stably expressing human estrogen receptor alpha (hERalpha) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to estrogens. In the present study this yeast estrogen assay was validated as a qualitative screening method for the determination of estrogenic activity in animal feed. This validation was performed according to EC Decision 2002/657. Twenty blank animal feed samples, including milk replacers and wet and dry feed samples, were spiked with 17beta-estradiol (E2beta) at 5 ng g(-1), 17alpha-ethynylestradiol (EE2) at 5 ng g(-1), diethylstilbestrol (DES) at 10 ng g(-1), zearalenone at 1.25 microg g(-1) or equal at 200 microg g(-1). All of these blank and low estrogen spiked feed samples fulfilled the CCalpha and CCbeta criterions, meaning that all 20 blank feed samples gave a signal below the determined decision limit CCalpha and were thus classified as compliant, and at least 19 out of the 20 spiked samples gave a signal above this CCalpha (beta = 5%) and were thus classified as suspect. The method was specific and estrogens in feed were stable for up to 98 days. In this study we also present long-term performance data and several examples of estrogens found in the routine screening of animal feed. This is the first successful example of a developed, validated and applied bioassay for the screening of hormonal substances in feed.

Zilpaterol is a new powerful beta-agonist, which is officially registered for fattening purposes in cattle in Mexico and South Africa. Its chemical structure is different from the well-known beta-agonists. Therefore, the routinely used screening methods are not likely to be suited for the analysis of zilpaterol. Also gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry (LC-MS) methods need to be adapted to enable detection of zilpaterol. In this study, a LC-MS3 confirmatory method was developed for the simultaneous detection of zilpaterol and di-aromatic beta-agonists in urine samples. A LC-MS2 method was optimised for the detection of zilpaterol in faeces. To study the excretion profile in urine and faeces, a male veal calf was orally treated with daily doses of Zilmax ® during 2 weeks. Zilpaterol was mainly excreted via urine. © 2004 Elsevier B.V. All rights reserved.

The simplicity and flexibility of solid-phase microextraction have been combined with the selectivity of molecularly imprinted polymers (MIPs), Silica fibers were coated reproducible with a 75-mum layer of methacrylate polymer either nonimprinted or imprinted with clenbuterol to compare their extraction characteristics under various conditions. Although the template molecule could be removed effectively from the imprinted polymer, structural analogues of clenbuterol were used for evaluation. The influence of pH on the extractability of brombuterol was investigated. Extraction yields up to similar to 80% were obtained when both types of fibers were used to extract brombuterol from phosphate buffer (pH 7.0). In contrast, yields of about 75 and 15% were obtained when extraction was performed from acetonitrile with imprinted and nonimprinted polymers, respectively, which demonstrates the selectivity of the MIP-coated fiber. Time sorption profiles were measured for the extraction of brombuterol from buffer and acetonitrile at the 10 and 100 ng/mL level with both types of fibers in order to compare extraction characteristics. Equilibrium times of about 30 and 90 min were found for the extraction of brombuterol from acetonitrile and buffer, respectively. The MIP-coated fibers were capable of extracting five structural analogues of clenbuterol from both buffer and acetonitrile, which suggests that the amine alcohol part of these molecules is responsible for interaction with the imprinted polymer, To achieve selective extraction of brombuterol from human urine, MIP-coated fibers were washed with acetonitrile after the extraction. Clean extracts and yields of similar to 45% were obtained, demonstrating the suitability of MIP-coated fibers for the analysis of biological samples.

Matrix solid-phase dispersion(MSPD) is a new sample pretreatment for solid samples. This technique greatly simplifies sample pretreatment but, nonetheless, the extracts often still require an extra cleanup step that is both laborious and time-consuming. The potential;of combining MSPD with molecularly imprinted solid-phase extraction (MISPE) was investigated in this study. Liver samples were ground in a mortar with Cls sorbent and the homogenized mixture packed into an SPE cartridge and placed on top of a MISPE cartridge. Subsequently, clenbuterol was eluted from the MSPD cartridge onto the MISPE cartridge using acetonitrile containing 1% acetic acid.:The ability of the molecularly imprinted polymer to selectively adsorb analyte in acetonitrile was exploited for re-extracting clenbuterol directly from this acetonitrile extract via the double cartridge tandem system. The analyte was eluted from the MISPE cartridge using acidified methanol, A clear eluate was obtained, which was subsequently evaporated, redissolved, and analyzed by HPLC electrochemical detection (ECD) or ion trap mass spectrometry (LC/IT-MS). The MISPE cartridge used in this study was imprinted using bromoclenbuterol, a structural analogue of clenbuterol, as the template. These MISPE cartridges showed excellent stability. The complete extraction procedure was rapid,and recoveries exceeded 90% for the target analyte. The method detection limit for the LC/IT-MS procedure was