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Determination and confirmation of selective estrogen receptor modulators (SERMs), anti-estrogens and aromatase inhibitors in bovine and porcine urine using UHPLC-MS/MS
Meijer, Thijs ; Essers, Martien L. ; Kaklamanos, Georgios ; Sterk, Saskia S. ; Ginkel, Leendert A. van - \ 2017
Food Additives & Contaminants. Pt. A, Chemistry, Analysis, Control, Exposure & Risk Assessment 34 (2017)4. - ISSN 1944-0049 - p. 641 - 651.
anti-estrogens - aromatase inhibiters - bovine urine - formestane - porcine urine - SERMs - UHPLC-MS/MS
Selective estrogen receptor modulators (SERMs), anti-estrogens and aromatase inhibitors are prohibited in human sports doping. However, they also present a risk of being used illegally in animal husbandry for fattening purposes. A method was developed and validated using UHPLC-MS/MS for the determination and confirmation of SERMs, anti-estrogens and aromatase inhibiters in bovine and porcine urine. This method was used in a survey of more than 200 bovine and porcine urine samples from Dutch farms. In 18 out of 103 porcine urine samples (17%) and two out of 114 bovine samples (2%) formestane, an aromatase inhibitor, was detected. None of the other compounds was detected. From human doping control it is known that formestane can, in some cases, be of natural origin. Analyses of reference samples from untreated bovine and porcine animals demonstrated the presence of formestane in bovine animals, but not yet in porcine animals. Future research will focus on whether the detected formestane in porcine and bovine urine is from endogenous or exogenous origin, using GC-c-IRMS.
European Analytical Criteria: Past, Present and Future
Vanhaecke, L. ; Gowik, P. ; Bizec, B. Le; Ginkel, L.A. van; Bichon, E. ; Blokland, M.H. ; Brabander, H.F. de - \ 2011
Journal of AOAC International 94 (2011)2. - ISSN 1060-3271 - p. 360 - 372.
ratio mass-spectrometry - carbon-isotope analysis - performance liquid-chromatography - meat-producing animals - veterinary drugs - urinary steroids - residue analysis - bovine urine - cattle urine - lc-ms
In this paper, the past, present, and (possible) future of the European analytical criteria for residues are described. The elaboration of the revision of Commission Decision 93/256/EC was a long process starting in 1996 and ending with the formation of a European Commission (EC) working group in 1998. This working group took account of developments in scientific and technical knowledge at that time and produced a draft version of the revision within 6 months. The revision, finally published in 2002 (2002/657/EC), includes new ideas on the identification of analytes and the criteria for performance assessment as well as validation procedures. Currently (2009), the evolution in analytical equipment and progress in scientific research, accompanied by recent European regulatory changes, demands an update or revision of the 2002/657/EC.
Assessment of two complementary liquid chromatography coupled to high resolution mass spectrometry metabolomics strategies for the screening of anabolic steroid treatment in calves
Dervilly-Pinel, G. ; Weigel, S. ; Lommen, A. ; Chereau, S. ; Rambaud, L. ; Essers, M.L. ; Antignac, J.P. ; Nielen, M.W.F. ; Bizec, B. Le - \ 2011
Analytica Chimica Acta 700 (2011)1-2. - ISSN 0003-2670 - p. 144 - 154.
human urine - bovine urine - nandrolone metabolites - gas - cattle - metabonomics - hair - 19-norandrosterone - 19-nortestosterone - 17-beta-estradiol
Anabolic steroids are banned in food producing livestock in Europe. Efficient methods based on mass spectrometry detection have been developed to ensure the control of such veterinary drug residues. Nevertheless, the use of “cocktails” composed of mixtures of low amounts of several substances as well as the synthesis of new compounds of unknown structure prevent efficient prevention. New analytical tools able to detect such abuse are today mandatory. In this context, metabolomics may represent new emerging strategies for investigating the global physiological effects associated to a family of substances and therefore, to suspect the administration of steroids. The purpose of the present study was to set up, assess and compare two complementary mass spectrometry-based metabolomic strategies as new tools to screen for steroid abuse in cattle and demonstrate the feasibility of such approaches. The protocols were developed in two European laboratories in charge of residues analysis in the field of food safety. Apart from sample preparation, the global process was different in both laboratories from LC-HRMS fingerprinting to multivariate data analysis through data processing and involved both LC-Orbitrap-XCMS and UPLC-ToF-MS-MetAlign strategies. The reproducibility of both sample preparation and MS measurements were assessed in order to guarantee that any differences in the acquired fingerprints were not caused by analytical variability but reflect metabolome modifications upon steroids administration. The protocols were then applied to urine samples collected on a large group of animals consisting of 12 control calves and 12 calves administrated with a mixture of 17ß-estradiol 3-benzoate and 17ß-nandrolone laureate esters according to a protocol reflecting likely illegal practices. The modifications in urine profiles as indicators of steroid administration have been evaluated in this context and proved the suitability of the approach for discriminating anabolic treated animals from control ones. Such an approach may therefore open a new way for the screening of anabolic steroid administration through targeted monitoring of relevant biomarkers highlighted as a result of the metabolomics study.
A RIKILT yeast estrogen bioassay (REA) for estrogen residue detection in urine of calves experimentally treated with 17ß-estradiol
Divari, S. ; Maria, R. De; Cannizzo, F.T. ; Spada, F. ; Mulasso, C. ; Bovee, T.F.H. ; Capra, P. ; Leporati, M. ; Biolatti, B. - \ 2010
Food Additives and Contaminants 27 (2010)1. - ISSN 0265-203X - p. 19 - 28.
tandem mass-spectrometry - green fluorescent protein - liquid-chromatography - growth promoters - beta-agonists - bovine urine - gc-ms - anabolic-steroids - receptor-alpha - muscle-tissues
17ß-Estradiol is one of the most powerful sex steroids illegally used in bovine production. The objective of this study was to evaluate the application and the specificity of the RIKILT yeast estrogen bioassay (REA) for the detection of molecules with estrogenic activities in the urine of calves experimentally treated with anabolics. Four groups of six calves each received an injection of 17ß-estradiol intramuscularly (group B), androsterone and gliburide (group A), and testosterone (group C) molecules at different dosage for 40 days. Group D was the control. The ability of the REA test to detect estrogenic activity in urine samples from all animals was assessed. All estrogen-treated animals (group B) showed as being positive up to 7 days after administration of the highest dosage of 17ß-estradiol, while the other three groups showed as being negative. The identity of estrogenic molecules in the urine of group B (17ß-estradiol, 17-estradiol) was confirmed by gas chromatography-mass spectrometry (GC/MS). This is the first time the REA test has been applied to detect 17ß-estradiol in the urine of calves treated with the hormone in vivo. The technique may offer an advantageous laboratory method for the veterinary surveillance of illegal steroid use.
Development, validation and implementation of a receptor based bioassay capable of detecting a broad range of B-agonist drugs in animal feedingstuffs
Boyd, S. ; Heskamp, H.H. ; Bovee, T.F.H. ; Nielen, M.W.F. ; Elliott, C.T. - \ 2009
Analytica Chimica Acta 637 (2009)1-2. - ISSN 0003-2670 - p. 24 - 32.
tandem mass-spectrometry - immunobiosensor assay - clenbuterol residues - growth promoters - bovine urine - zilpaterol - liver - food
A bioassay was developed for the detection of a broad range of ß-agonist compounds in animal feeds. A solubilised ß2-adrenoceptor was utilised as the binding protein in the assay. This protein was found to be highly stable when stored at 80 °C. The assay was developed and initially validated to determine the sensitivity and relative selectivity against a panel of commonly used ß-agonist compounds. It was also shown that when ß-agonists were present as cocktails in samples a pronounced synergistic effect could be measured. The method was further validated according to EC Decision 2002/657 and proved capable of detecting 250 ng clenbuterol equivalents per gram of sample. This is well below the quantities normally associated with ß-agonist medicated feeds. The ß2-adrenoceptor used in the study only failed to bind the compound zilpaterol, raising doubts as to whether this compound is a true ß2-adrenergic drug
Elimination kinetic of 17B-estradiol 3-benzoate and 17B-nandrolone laureate ester metabolites in calves' urine
Pinel, G. ; Rambaud, L. ; Cacciatore, G. ; Bergwerff, A. ; Elliott, C. ; Nielen, M.W.F. - \ 2008
Journal of Steroid Biochemistry and Molecular Biology 110 (2008)1-2. - ISSN 0960-0760 - p. 30 - 38.
tandem mass-spectrometry - anabolic-steroids - bovine urine - nandrolone metabolites - gas - 19-nortestosterone - confirmation - residues - cattle - horse
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 17 beta-estradiol 3-benzoate and 17 beta-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 (CC alpha) for steroids were below 0.1 mu g L-1, except for 19-norandrosterone (CC alpha = 0.7 mu g L-1) and estrone (CC alpha = 0.3 mu g L-1). Kinetics of elimination of the administered 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate were established by monitoring 17 beta-estradiol, 17 alpha-estradiol, estrone and 17 beta-nandrolone, 17 alpha-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 17 alpha-estradiol and 17 alpha-nandrolone (> 20 and 2 mg L-1, respectively after 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate administration) whereas 17 beta-estradiol, estrone, 17 beta-nandrolone, 19-noretiocholanolone and 19-norandrostenedione were found as secondary metabolites at concentration values up to the mu g 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 17 beta form. (c) 2008 Elsevier Ltd. All rights reserved.
The ultimate veal calf reference experiment: Hormone residue analysis data obtained by gas and liquid chromatography tandem mass spectrometry
Nielen, M.W.F. ; Lasaroms, J.J.P. ; Essers, M.L. ; Sanders, M.B. ; Heskamp, H.H. ; Bovee, T.F.H. ; Rhijn, J.A. van; Groot, M.J. - \ 2007
Analytica Chimica Acta 586 (2007)1-2. - ISSN 0003-2670 - p. 30 - 34.
yeast estrogen bioassay - confirmatory analysis - bovine urine - boldenone - steroids - cattle - calves - feces - androsta-1,4-diene-3,17-dione - 17-alpha-boldenone
A lifetime controlled reference experiment has been performed using 42 veal calves, 21 males and 21 females which were fed and housed according to European regulations and common veterinary practice. During the experiment feed, water, urine and hair were sampled and feed intake and growth were monitored. Thus for the first time residue analysis data were obtained from guaranteed lifetime-untreated animals. The analysis was focused on the natural hormones estradiol and testosterone and their metabolites, on 17 beta- and 17 alpha-nortestosterone, on 17 beta- and 17 alpha-boldenone and androsta-1,4-diene-3,17-dione (ADD), and carried out by gas chromatography tandem mass spectrometry (GC/MS/MS), an estrogen bioassay and liquid chromatography (LC) MS/MS. Feed, water and hair samples were negative for the residues tested. Female calf urines showed occasionally low levels of 17 alpha-estradiol and 17 alpha-testosterone. On one particular sampling day male veal calf urines showed very high levels of 17 alpha-testosterone (up to 1000 ng mL(-1)), accompanied by lower levels of estrone and 17 beta-testosterone. Despite these extreme levels of natural testosterone, 17 beta-boldenone was never detected in the same urine samples; even 17 alpha-boldenone and ADD were only occasionally beyond CC alpha (maximum levels 2.7 ng ng mL(-1)). The data from this unique experiment provide a set of reference values for steroid hormones in calf urine and demonstrate that 17 beta-boldenone is not a naturally occurring compound in urine samples. (c) 2006 Elsevier B.V. All rights reserved.
Validation of a rapid yeast estrogen bioassay, based on the expression of green fluorescent protein, for the screening of estrogenic activity in calf urine
Bovee, T.F.H. ; Heskamp, H.H. ; Hamers, A.R.M. ; Hoogenboom, L.A.P. ; Nielen, M.W.F. - \ 2005
Analytica Chimica Acta 529 (2005). - ISSN 0003-2670 - p. 57 - 64.
tandem mass-spectrometry - beta-agonists - bovine urine - cells - assay - phytoestrogens - extraction - abuse - feed
Previously we described the construction and properties of a rapid yeast bioassay stably expressing human estrogen receptor a (hERa) and yeast enhanced green fluorescent protein (yEGFP) 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 calf urine. This validation was performed, according to EC decision 2002/657, which prescribes the determination of the detection capability (CCß), the specificity/selectivity and the stability/ruggedness/applicability. To determine these performance characteristics, twenty blank urine samples of 19-week-old calves were collected and spiked with 17ß-estradiol (E2ß) at 1 ng ml-1, diethylstilbestrol (DES) at 1 ng ml-1, 17a-ethynylestradiol (EE2) at 1 ng ml-1, a-zearalanol at 30 and 50 ng ml-1 or mestranol at 10 ng ml-1. Following enzymatic deconjugation and solid phase extraction, 100 µl equivalents of these blank and spiked urine samples were screened for estrogenic activity in a 96 well plate using the yeast estrogen bioassay. All of these low estrogen spiked urine samples could be distinguished from the blank samples as all spiked samples gave a signal above the determined decision limit CCa and the mean responses of the spiked samples were higher than the determined detection capability CCß. As this CCß criterion is met, these spiked samples have a lower than 5% probability to be classified as a false negative. The specificity of the method was determined with blank urine samples spiked with a high dose of testosterone or progesterone (1000 ng ml-1). No response to these substances was detected in the yeast estrogen bioassay. There was also no interference of a high dose of testosterone or progesterone on the response of a low dose of the estrogens. Stability of urine samples was checked with spiked urine samples that were kept frozen for up to 90 days, showing that urine samples could be stored at -20 °C for up to 60 days without changing the screening result of the assay. This method has been in routine use at RIKILT for more than one year.
Bioassay-directed identification of estrogen residues in urine by liquid chromatography electrospray quadrupole time-of-flight mass spectrometry
Nielen, M.W.F. ; Bennekom, E.O. van; Heskamp, H.H. ; Rhijn, J.A. van; Bovee, T.F.H. ; Hoogenboom, L.A.P. - \ 2004
Analytical Chemistry 76 (2004)22. - ISSN 0003-2700 - p. 6600 - 6608.
green fluorescent protein - beta-agonists - (geno)toxicity detection - continuous-flow - receptor-alpha - bovine urine - water - extraction - system - abuse
A new approach to the search for residues of known and unknown estrogens in calf urine is presented. Following enzymatic deconjugation and solid-phase extraction, a minor part of the samples is screened for estrogen activity using a recently developed rapid reporter gene bioassay. The remainder of the bioactive extracts is analyzed by gradient liquid chromatography (LC) with, in parallel, bioactivity and mass spectrometric detection via effluent splitting toward a 96-well fraction collector and an electrospray quadrupole time-of-flight mass spectrometer (QTOFMS). The LC fractions in the 96-well plate are used for the detection of estrogen activity using the bioassay. The biogram obtained features a 20-s time resolution, and the suspect well numbers can be easily correlated with the LC/QTOFMS retention time. The mass spectral data from the thus assigned relevant parts of the chromatograms are background subtracted, followed by accurate mass measurement, element composition calculation, and identification. The method allows estrogen activity detection and identification of (un)known estrogens in urine at the 1-2 ng/L level, in compliance with current residue analysis performance for hormone abuse in cattle. The applicability of this LC/bioassay/QTOFMS approach for the identification of estrogens in real-life samples is demonstrated by the analysis of several calf urine samples, and preliminary data from a pig feed sample.