Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Receptor-based high-throughput screening and identification of estrogens in dietary supplements using bioaffinity liquid-chromatography ion mobility mass spectrometry
Aqai, P. ; Gómez Blesa, N. ; Major, H. ; Pedotti, P. ; Varani, L. ; Ferrero, V.E.V. ; Haasnoot, W. ; Nielen, M.W.F. - \ 2013
Analytical and Bioanalytical Chemistry 405 (2013)29. - ISSN 1618-2642 - p. 9427 - 9436.
ms-binding assays - multi-residue method - anabolic-steroids - lc-ms - nutritional supplements - chemical derivatization - native marker - contamination - transporter - validation
A high-throughput bioaffinity liquid chromatography-mass spectrometry (BioMS) approach was developed and applied for the screening and identification of recombinant human estrogen receptor a (ERa) ligands in dietary supplements. For screening, a semi-automated mass spectrometric ligand binding assay was developed applying 13C2,15¿N-tamoxifen as non-radioactive label and fast ultra-high-performance–liquid chromatography–electrospray ionisation–triple-quadrupole-MS (UPLC-QqQ-MS), operated in the single reaction monitoring mode, as a readout system. Binding of the label to ERa-coated paramagnetic microbeads was inhibited by competing estrogens in the sample extract yielding decreased levels of the label in UPLC-QqQ-MS. The label showed high ionisation efficiency in positive electrospray ionisation (ESI) mode, so the developed BioMS approach is able to screen for estrogens in dietary supplements despite their poor ionisation efficiency in both positive and negative ESI modes. The assay was performed in a 96-well plate, and all these wells could be measured within 3 h. Estrogens in suspect extracts were identified by full-scan accurate mass and collision-cross section (CCS) values from a UPLC-ion mobility-Q-time-of-flight-MS (UPLC-IM-Q-ToF-MS) equipped with a novel atmospheric pressure ionisation source. Thanks to the novel ion source, this instrument provided picogram sensitivity for estrogens in the negative ion mode and an additional identification point (experimental CCS values) next to retention time, accurate mass and tandem mass spectrometry data. The developed combination of bioaffinity screening with UPLC-QqQ-MS and identification with UPLC-IM-Q-ToF-MS provides an extremely powerful analytical tool for early warning of ERa bioactive compounds in dietary supplements as demonstrated by analysis of selected dietary supplements in which different estrogens were identified.
A review of analytical strategies for the detection of endogenous' steroid abuse in food production
Scarth, J.P. ; Kay, J. ; Teale, P. ; Akre, C. ; Bizec, B. le; Brabander, H.F. de; Vanhaecke, L. ; Ginkel, L.A. van; Points, J. - \ 2012
Drug Testing and Analysis 4 (2012). - ISSN 1942-7603 - p. 40 - 49.
tandem mass-spectrometry - performance liquid-chromatography - carbon-isotope analysis - meat-producing animals - gas-chromatography - anabolic-steroids - residue analysis - doping control - veal calves - pattern-recognition
Detection of the abuse of synthetic steroids in food production is nowadays relatively straightforward using modern techniques such as gas or liquid chromatography coupled to mass spectrometry (GC-MS/MS or LC-MS/MS, respectively). However, proving the abuse of endogenous (or naturally occurring) steroids is more difficult. Despite these difficulties, significant progress in this area has recently been made and a number of methods are now available. The aim of the current review was to systematically review the available analytical approaches, which include threshold concentrations, qualitative marker metabolites, intact steroid esters, gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS), longitudinal testing and omics biomarker profiling. The advantages/disadvantages of these methods are considered in detail, but the choice of which to adopt is dictated by a number of practical, political, and economic factors, which vary in different parts of the world. These include the steroid/species combination requiring analysis, the matrix tested, whether samples are collected from live or slaughtered animals, available analytical instrumentation, sample throughput/cost, and the relevant legal/regulatory frameworks. Furthermore, these approaches could be combined in a range of different parallel and/or sequential screening/confirmatory testing streams, with the final choice being determined by the aforementioned considerations. Despite these advances, more work is required to refine the different techniques and to respond to the ever increasing list of compounds classified as endogenous. At this advanced stage, however, it is now more important than ever for scientists and regulators from across the world to communicate and collaborate in order to harmonize and streamline research efforts. (c) 2012 HFL Sport Science (LGC Ltd) and (c) Her Majesty the Queen in Right of Canada.
Applicability of a yeast bioassay in the detection of steroid esters in hair
Becue, I. ; Bovee, T.F.H. ; Poucke, C. ; Groot, M.J. ; Nielen, M.W.F. ; Peteghem, C. van - \ 2011
Analytical and Bioanalytical Chemistry 399 (2011)3. - ISSN 1618-2642 - p. 1031 - 1039.
green fluorescent protein - tandem mass-spectrometry - estrogenic activity - bovine hair - estradiol benzoate - anabolic-steroids - calf urine - validation - samples - expression
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
The application of reporter gene assays for the detection of endocrine disruptors in sport supplements
Plotan, M. ; Elliot, C.T. ; Scippo, M.L. ; Müller, M. ; Antignac, J.P. ; Malone, E. ; Bovee, T.F.H. ; Mitchell, S. ; Connolly, L. - \ 2011
Analytica Chimica Acta 700 (2011)1-2. - ISSN 0003-2670 - p. 34 - 40.
dietary-supplements - anabolic-steroids - androgen receptor - sex-hormones - chemicals - estrogen - health - urine
The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to ensure the quality and safety of sport supplements for the consumer is essential. The development and validation of two reporter gene assays coupled with solid phase sample preparation enabling the detection of estrogenic and androgenic constituents in sport supplements is reported. Both assays were shown to be of high sensitivity with the estrogen and androgen reporter gene assays having an EC50 of 0.01 ng mL-1 and 0.16 ng mL-1 respectively. The developed assays were applied in a survey of 63 sport supplements samples obtained across the Island of Ireland with an additional seven reference samples previously investigated using LC–MS/MS. Androgen and estrogen bio-activity was found in 71% of the investigated samples. Bio-activity profiling was further broken down into agonists, partial agonists and antagonists. Supplements (13) with the strongest estrogenic bio-activity were chosen for further investigation. LC–MS/MS analysis of these samples determined the presence of phytoestrogens in seven of them. Supplements (38) with androgen bio-activity were also selected for further investigation. Androgen agonist bio-activity was detected in 12 supplements, antagonistic bio-activity was detected in 16 and partial antagonistic bio-activity was detected in 10. A further group of supplements (7) did not present androgenic bio-activity when tested alone but enhanced the androgenic agonist bio-activity of dihydrotestosterone when combined. The developed assays offer advantages in detection of known, unknown and low-level mixtures of endocrine disruptors over existing analytical screening techniques. For the detection and identification of constituent hormonally active compounds the combination of biological and physio-chemical techniques is optimal.
Preventive doping control screening analysis of prohibited substances in human urine using rapid-resolution liquid chromatography/high-resolution time-of-flight mass spectrometry
Vonaparti, A. ; Lyris, E. ; Angelis, Y.S. ; Panderi, I. ; Koupparis, M. ; Tsantili- Kakoulidou, A. ; Peters, R.J.B. ; Nielen, M.W.F. ; Georgakopoulos, C.G. - \ 2010
Rapid Communications in Mass Spectrometry 24 (2010)11. - ISSN 0951-4198 - p. 1595 - 1609.
solid-phase extraction - anabolic-steroids - metabolite identification - high-throughput - olympic games - drugs - stimulants - ionization - validation - diuretics
Unification of the screening protocols for a wide range of doping agents has become an important issue for doping control laboratories. This study presents the development and validation of a generic liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) screening method of 241 small molecule analytes from various categories of prohibited substances (stimulants, narcotics, diuretics, ß2-agonists, ß-blockers, hormone antagonists and modulators, glucocorticosteroids and anabolic agents). It is based on a single-step liquid-liquid extraction of hydrolyzed urine and the use of a rapid-resolution liquid chromatography/high-resolution time-of-flight mass spectrometric system acquiring continuous full scan data. Electrospray ionization in the positive mode was used. Validation parameters consisted of identification capability, limit of detection, specificity, ion suppression, extraction recovery, repeatability and mass accuracy. Detection criteria were established on the basis of retention time reproducibility and mass accuracy. The suitability of the methodology for doping control was demonstrated with positive urine samples. The preventive role of the method was proved by the case where full scan acquisition with accurate mass measurement allowed the retrospective reprocessing of acquired data from past doping control samples for the detection of a designer drug, the stimulant 4-methyl-2-hexanamine, which resulted in re-reporting a number of stored samples as positives for this particular substance, when, initially, they had been reported as negatives.
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.
Detectability of testosterone esters and estradiol benzoate in bovine hair and plasma following pour-on treatment
Stolker, A.A.M. ; Groot, M.J. ; Lasaroms, J.J.P. ; Nijrolder, A.W.J.M. ; Blokland, M.H. ; Riedmaier, I. ; Becker, C. ; Meyer, H.H.D. ; Nielen, M.W.F. - \ 2009
Analytical and Bioanalytical Chemistry 395 (2009)4. - ISSN 1618-2642 - p. 1075 - 1087.
tandem mass-spectrometry - anabolic-steroids - doping control - livestock production - cattle - corticosteroids - stanozolol - nandrolone - samples - misuse
The abuse of synthetic esters of natural steroids such as testosterone and estradiol in cattle fattening and sports is hard to detect via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. An interesting alternative can be provided by the analysis of the administered synthetic steroids themselves, i.e., the analysis of intact steroid esters in hair by liquid chromatography tandem mass spectrometry (LC/MS/MS). However, retrospective estimation of the application date following a non-compliant finding is hindered by the complexity of the kinetics of the incorporation of steroid esters in hair. In this study, the incorporation of intact steroid esters in hair following pour-on treatment has been studied and critically compared with results from intramuscular treatment. To this end animals were pour-on treated with a hormone cocktail containing testosterone cypionate, testosterone decanoate and estradiol benzoate in different carriers. The animals were either treated using injection and pour-on application once or three times having 1 week between treatments using injection and pour-on application. Animals were slaughtered from 10–12 weeks after the last treatment. Both hair and blood plasma samples were collected and analysed by LC/MS/MS. From the results, it is concluded that after single treatment the levels of steroid esters in hair drop to CCß levels (5-20 µg/kg) after 5-7 weeks. When treatment is repeated two times, the CCß levels are reached after 9–11 weeks. Furthermore, in plasma, no steroid esters were detected; not even at the low microgramme per litre level but-in contrast with the pour-on application-after i.m. injection, significant increase of 17ß-testosterone and 17ß-estradiol were observed. These observations suggest that transport of steroid esters after pour-on application is not only performed by blood but also by alternative fluids in the animal so probably the steroid esters are already hydrolysed and epimerized before entering the blood
Validation and application of a yeast bioassay for screening androgenic activity in calf urine and feed
Bovee, T.F.H. ; Bor, G. ; Heskamp, H.H. ; Lasaroms, J.J.P. ; Sanders, M.B. ; Nielen, M.W.F. - \ 2009
Analytica Chimica Acta 637 (2009)1-2. - ISSN 0003-2670 - p. 225 - 234.
tandem mass-spectrometry - in-vitro - liquid-chromatography - estrogenic activity - recombinant assay - anabolic-steroids - receptor ligands - surface waters - binding - dihydrotestosterone
Bioassays are valuable tools for combating the illegal use of steroids in cattle fattening. Previously we described the construction and properties of a rapid and robust yeast androgen bioassay stably expressing the human androgen receptor (hAR) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. In the present study this yeast androgen bioassay was validated as a qualitative screening method for the determination of androgenic activity in calf urine and animal feed. This validation was performed according to EC Decision 2002/657. 20 blank samples were spiked with testosterone, 17¿-methyltestosterone, 19-nortestosterone, 17ß-trenbolone, 17ß-boldenone or 17¿-methylboldenone at 2 or 15 ng mL¿1 in urine and 50 or 100 ng g¿1 in feed. All blank and spiked samples fulfilled the CC¿ and CCß criterions, meaning that all 20 blank samples gave signals below the determined decision limits CC¿ and were thus classified as compliant (¿ = 1%). For each component, at least 19 out of the 20 spiked samples gave a signal above the CC¿ and were thus classified as suspect (ß = 5%). The method was specific, and high amounts of dexamethasone did not interfere with the outcome of the test. Although high levels of 17¿-ethynylestradiol can significantly inhibit the response obtained with low amounts of androgens, that situation is not relevant in veterinary practice. When stored at their specific conditions, the androgens in feed were stable for at least 91 days. Real urine samples from a national control program were screened and a representative part of the compliant and suspect samples were confirmed by gas chromatography¿tandem mass spectrometry
Inter-laboratory comparison of a yeast bioassay for the determination of estrogenic activity in biological samples
Bovee, T.F.H. ; Bor, G. ; Becue, I. ; Daamen, E.J. ; Duursen, M. van; Lehmann, S. ; Vollmer, G. ; Maria, R. De; Fox, E. ; Witters, H. ; Bernhoft, S. ; Schramm, K.W. ; Hoogenboom, L.A.P. ; Nielen, M.W.F. - \ 2009
Analytica Chimica Acta 637 (2009)1-2. - ISSN 0003-2670 - p. 265 - 272.
tandem mass-spectrometry - green fluorescent protein - liquid-chromatography - anabolic-steroids - recombinant assay - surface waters - beta-agonists - urine - validation - abuse
An inter-laboratory exercise was performed with a yeast estrogen bioassay, based on the expression of yeast enhanced green fluorescent protein (yEGFP), for the determination of estrogenic activity in extracts of calf urine samples. Urine samples were spiked with 1 and 5 ng mL¿1 17ß-estradiol and 17¿-ethynylestradiol, 10 and 50 ng mL¿1 mestranol, and 100 ng mL¿1 testosterone and progesterone. Sample extracts of blank and spiked urine samples were prepared at our laboratory and sent to seven laboratories together with a reagent blank, a DMSO blank, and eight 17ß-estradiol stock solutions in DMSO ranging in concentration from 0 to 545 ng mL¿1. Sample extracts and standards were coded and tested blindly. A decision limit (CC¿) was determined based on the response of seven blank urine samples. Signals of the negative controls, e.g. urine samples spiked with 100 ng mL¿1 testosterone or progesterone, were all below the determined CC¿ and were thus screened as compliant. Positive controls, i.e. the urine samples spiked at two levels with 17ß-estradiol, 17¿-ethynylestradiol and mestranol, were almost all screened as suspect, i.e. gave signals above the determined CC¿. Determined EC50 values calculated from the 17ß-estradiol dose¿response curves obtained by the seven laboratories ranged from 0.59 to 0.95 nM
Desorption electrospray ionisation mass spectrometry: A rapid screening tool for veterinary drug preparations and forensic samples from hormone crime investigations
Nielen, M.W.F. ; Hooijerink, H. ; Claassen, F.C. ; Engelen, M.C. ; Beek, T.A. van - \ 2009
Analytica Chimica Acta 637 (2009)1-2. - ISSN 0003-2670 - p. 92 - 100.
growth-promoting agents - anabolic-steroids - injection sites - gas-chromatography - ambient conditions - residue analysis - urine samples - cocktails
Hormone and veterinary drug screening and forensics can benefit from the recent developments in desorption electrospray ionisation (DESI) mass spectrometry (MS). In this work the feasibility of DESI application has been studied. Using a linear ion trap or quadrupole time-of-flight (TOF) MS instrument both full-scan and data-dependent collision-induced dissociation MSn spectra were acquired in seconds without sample preparation. Preliminary data are presented for the rapid screening of (pro)hormone supplement samples, an illegal steroid cocktail and forensic samples from veterinary drug investigations. The potential of this DESI approach is clearly demonstrated since compounds observed could be independently confirmed by liquid chromatography/TOFMS with accurate mass measurement, and/or proton nuclear magnetic resonance spectroscopy. Specific concerns related to false-positive and false-negative findings due to limitations in quantification and memory-effects are briefly discussed. It is envisaged that DESI will achieve a prominent role in hormone and veterinary drug analysis in the near future
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.
Screening for estrogen residues in calf urine: Comparison of a validated yeast estrogen bioassay and gas chromatography-tandem mass spectrometry
Nielen, M.W.F. ; Bovee, T.F.H. ; Heskamp, H.H. ; Lasaroms, J.J.P. ; Sanders, M.B. ; Rhijn, J.A. van; Groot, M.J. ; Hoogenboom, L.A.P. - \ 2006
Food Additives and Contaminants 23 (2006)11. - ISSN 0265-203X - p. 1123 - 1131.
green fluorescent protein - liquid-chromatography - anabolic-steroids - identification - expression - beta
Within the European Union, the control for residues of illegal hormones in food-producing animals is based on urine analysis for a few target analytes using gas chromatography/mass spectrometry and/or liquid chromatography¿tandem mass spectrometry. Recently, we developed a robust yeast bioassay screening tool for estrogens, which was validated as a qualitative screening method in accordance with EC decision 2002/657/EC. In this study, we present long-term performance data and a comparison of urine data obtained with this bioassay, and data from an established gas chromatography¿tandem mass spectrometry (GC/MS/MS) confirmatory analysis method. More than 120 calf urine samples from a controlled reference experiment were analysed using both protocols. According to the GC/MS/MS method, only the natural estrogens 17¿-estradiol and estrone were present in the non-compliant samples. The bioassay was less sensitive than GC/MS/MS for the relatively weak estrogenic compound 17¿-estradiol, in accordance with expectations. Assuming that application of the mass spectrometric method is considered beyond reasonable doubt, the bioassay performed very well: only 5.6% of the calf urine samples found compliant in GC/MS/MS were screened false suspect in the bioassay screening method. The bioassay results of non-compliant urine samples under routine conditions were as predicted, taking into account the relative estrogenicity of the natural estrogens 17¿-estradiol and estrone vs. 17ß-estradiol. Only one sample was screened false negative for 17¿-estradiol and estrone. Application of this fast and simple estrogen bioassay in routine surveillance and control can significantly reduce GC/MS/MS sample workload and allow higher percentages of animals to be screened for potential hormone abuse
Multi residue screening of intact testosterone esters and boldenone undecylenate in bovine hair using liquid chromatography electrospray tandem mass spectrometry
Nielen, M.W.F. ; Lasaroms, J.J.P. ; Mulder, P.P.J. ; Hende, J. van; Rhijn, J.A. van; Groot, M.J. - \ 2006
Journal of Chromatography. B, Analytical technologies in the biomedical and life sciences 830 (2006)1. - ISSN 1570-0232 - p. 126 - 134.
anabolic-steroids - doping control - cattle - corticosteroids - stanozolol - estradiol - samples - urine
The abuse of esters of natural androgenic steroids in cattle fattening and sports is hard to control via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. In veterinary control strange findings of 17ß-testosterone and 17¿-testosterone in urine are often ignored because of the lack of statistically sound reference data of naturally occurring levels. An interesting alternative for inconclusive urine analyses in veterinary control can be provided by the analysis of the administered steroids themselves, i.e. the analysis of intact steroid esters in hair. Unfortunately, the analysis of intact steroid esters is complicated not only by the vulnerability of the esters which precludes alkaline hydrolysis of the hair, but also by the wide polarity range of short and long-chain esters yielding very poor recoveries for either the one or the other. In this study, a multi-steroid esters LC/MS/MS screening method is presented for trace analysis of the synthetic intact esters of 17ß-testosterone and the undecylenate ester of 17ß-boldenone in bovine hair. The method, requiring only 200 mg of pulverised hair, features a mild digestion procedure using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and the use of four deuterium-labelled steroid esters as internal standards covering the wide polarity range of the analytes. In spiked hair samples for most of the analytes the limit of detection and the accuracy using isotope dilution were 2¿5 ng/g and 97¿105%, respectively. The applicability was demonstrated using hair samples from a controlled experiment in which six bovines were injected intramuscularly with two different doses of two commercial mixtures of testosterone esters, and with two different doses of boldenone undecylenate. Depending on the dose all administered testosterone- and boldenone esters were found to be incorporated in bovine hair following a single intramuscular injection, except testosterone propionate which dose might have been too low
Urine testing for designing steroids by liquid chromatography and androgen bioaasay detection and electrospray quadrupole time-of-flight mass spectrometry identification
Nielen, M.W.F. ; Bovee, T.F.H. ; Engelen, M.C. ; Rutgers, P. ; Hamers, A.R.M. ; Rhijn, J.A. van; Hoogenboom, L.A.P. - \ 2006
Analytical Chemistry 78 (2006)2. - ISSN 0003-2700 - p. 424 - 431.
green fluorescent protein - yeast estrogen bioassay - anabolic-steroids - doping control - tetrahydrogestrinone - validation - discovery - testosterone - expression - beta
New anabolic steroids show up occasionally in sports doping and in veterinary control. The discovery of these designer steroids is facilitated by findings of illicit preparations, thus allowing bioactivity testing, structure elucidation using NMR and mass spectrometry, and final incorporation in urine testing. However, as long as these preparations remain undiscovered, new designer steroids are not screened for in routine sports doping or veterinary control urine tests since the established GC/MS and LC/MS/MS methods are set up for the monitoring of a few selected ions or MS/MS transitions of known substances only. In this study, the feasibility of androgen bioactivity testing and mass spectrometric identification is being investigated for trace analysis of designer steroids in urine. Following enzymatic deconjugation and a generic solid-phase extraction, the samples are analyzed by gradient LC with effluent splitting toward two identical 96-well fraction collectors. One well plate is used for androgen bioactivity detection using a novel robust yeast reporter gene bioassay yielding a biogram featuring a 20-s time resolution. The bioactive wells direct the identification efforts to the corresponding well numbers in the duplicate plate. These are subjected to high-resolution LC using a short column packed with 1.7-m C18 material and coupled with electrospray quadrupole time-of-flight mass spectrometry (LC/QTOFMS) with accurate mass measurement. Element compositions are calculated and used to interrogate electronic substance databases. The feasibility of this approach for doping control is demonstrated via the screening of human urine samples spiked with the designer anabolic steroid tetrahydrogestrinone. Application of the proposed methodology, complementary to the established targeted urine screening for known anabolics, will increase the chance of finding unknown emerging designer steroids, rather then being solely dependent on findings of the illicit preparations themselves.
Liquid chromatography-electrospray ionisation-mass spectrometry based method for the determination of estradiol benzoate in hair of cattle
Hooyerink, H. ; Lommen, A. ; Mulder, P.P.J. ; Rhijn, J.A. van; Nielen, M.W.F. - \ 2005
Analytica Chimica Acta 529 (2005)1-2. - ISSN 0003-2670 - p. 167 - 172.
gas-chromatography - anabolic-steroids - doping control - esters - testosterone - ethinylestradiol - nandrolone - residue - plasma
A liquid chromatography (LC)-based method with mass spectrometric (MS/MS) detection was developed for the determination of estradiol benzoate residues in hair of cattle. First hair samples were pulverized with a ball mill followed by treatment with the reducing agent Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). After liquid/liquid extraction samples were further purified by solid-phase extraction. Finally samples were analyzed with LC¿MS/MS using deuterated estradiol benzoate as internal standard. The method was validated following the latest EU guidelines for screening methods using blank hair samples spiked at 5 ng g¿1. The detection capability (CCß) was less than 5 ng g¿1 and the decision limit (CC¿) was 1.6 ng g¿1. The recovery was 27% at the 5 ng g¿1 level and the accuracy was 95%. Confirmation, with two MS/MS transitions, was possible at a concentration of 5 ng g¿1 or higher. Incurred samples obtained from different animal experiments were analyzed and the presence of estradiol benzoate was confirmed. Finally hair samples from different slaughterhouses were analyzed.
Confirmatory analysis of 17B-boldenone, 17a-boldenone and androsta-1, 4-diene-3, 17-dione in bovine urine, faeces, feed and skin swab samples by liquid chromatography-electrospray ionisation tandem mass spectrometry
Nielen, M.W.F. ; Rutgers, P. ; Bennekom, E.O. van; Lasaroms, J.J.P. ; Rhijn, J.A. van - \ 2004
Journal of Chromatography. B, Analytical technologies in the biomedical and life sciences 801 (2004)2. - ISSN 1570-0232 - p. 273 - 283.
nandrolone metabolites - anabolic-steroids - identification - boldenone - testosterone - stanozolol - excretion
The origin, i.e. natural occurrence or illegal treatment, of findings of 17-boldenone (-Bol) and 17-boldenone (-Bol) in urine and faeces of cattle is under debate within the European Union. A liquid chromatographic positive ion electrospray tandem mass spectrometric method is presented for the confirmatory analysis of 17-boldenone, 17-boldenone and an important metabolite/precursor androsta-1,4-diene-3,17-dione (ADD), using deuterium-labelled 17-boldenone (-Bol-d3) as internal standard. Detailed sample preparation procedures were developed for a variety of sample matrices such as bovine urine, faeces, feed and skin swab samples. The method was validated as a quantitative confirmatory method according to the latest EU guidelines and shows good precision, linearity and accuracy data, and CC and CC values of 0.1-0.3 and 0.4-1.0 ng/ml, respectively. Currently, the method has been successfully applied to suspect urine samples for more than a year, and occasionally to faeces, feed and swab samples as well. Results obtained from untreated and treated animals are given and their impact on the debate about the origin of residues of 17-boldenone is critically discussed. Finally, preliminary data about the degree of conjugation of boldenone residues are presented and a simple procedure for discrimination between residues from abuse versus natural origin is proposed.
Value of alternative sample matrices in residue analysis for stanozolol
Nielen, M.W.F. ; Hooijerink, H. ; Essers, M.L. ; Lasaroms, J.J.P. ; Bennekom, E.O. van; Brouwer, L. - \ 2003
Analytica Chimica Acta 483 (2003)1-2. - ISSN 0003-2670 - p. 11 - 17.
anabolic-steroids - doping control - hair
Detection of hormones and veterinary drugs in urine samples is limited to the short-term following illegal administration. Alternative sample matrices such as hair or retina are of interest for prolonged detectability of residues. In this preliminary study, we developed sensitive liquid chromatography¿tandem mass spectrometry methods for the confirmatory analysis of stanozolol in different matrices and applied them to urine, retina and hair samples obtained from 11 suspect bovines at the slaughterhouse. Most of the stanozolol metabolite (16-hydroxystanozolol) levels in urine samples were below 0.5 ng ml-1, but nevertheless in some urines traces of this metabolite could still be positively confirmed. Retina samples were all negative so the value of this matrix for stanozolol might be questioned. The hair samples, on the other hand, yielded many positive stanozolol results, and in some animals levels were up to 100-fold higher than the corresponding metabolite levels in urine thereby underlining the potential for prolonged detectability of stanozolol abuse.
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