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R W Stephany

Publications and source records attributed to R W Stephany.

At least 19 recordsLinked to original sources
PubMed2006

Determination of resorcylic acid lactones in biological samples by GC-MS. Discrimination between illegal use and contamination with fusarium toxins.

An EU project, FAIR5-CT-1997-3443, has been undertaken to distinguish illegal use of zeranol from consumption of food contaminated with Fusarium spp. toxin. One of the tasks was development of screening and confirmatory methods of analysis. This paper describes a new method based on two-step clean-up and GC-MS analysis. The first clean-up step is matrix-dependant; the second is applicable to both urine and meat. The MS is operated in negative chemical ionisation mode. The method is quantitative for zeranol and taleranol, alpha- and beta-zearalenol, and zearalenone and qualitative for zearalanone. Validation was performed according to the latest EU performance criteria (Commission Decision 2002/657). For analysis of urine CC(alpha) and CC(beta) for the method (microg L(-1)) were 0.06-0.11 for zeranol, 0.07-0.12 for taleranol, 0.07-0.11 for alpha-zearalenol, 0.21-0.36 for beta-zearalenol, 0.35-0.60 for zearalenone, and 0.19-0.33 zearalanone. Within-laboratory reproducibility was 16.2, 11.2, 31.9, 30.1, 26.6, and 54.2% for zeranol, taleranol, alpha-zearalenol, beta-zearalenol, zearalenone, and zearalanone, respectively. It was found that all the compounds are stable in urine at -20 degrees C for at least a year. Part of the validation program was organisation of a small proficiency study (ringtest) and a correlation study with an LC-MS-MS method developed by the Veterinary Science Division (VSD; Belfast, UK-NI). This study showed there was good correlation between results from both laboratories. The method can be used for quantitative analysis discriminating illegal use of zeranol from consumption of zearalenone-contaminated food.

Animals↗
PubMed2004

Presence and metabolism of the anabolic steroid boldenone in various animal species: a review.

The review summarizes current knowledge on the possible illegal use of the anabolic steroid boldenone. The presence of' boldenone and metabolites in different animal species and the possibility of the occurrence of endogenous boldenone and metabolites is assessed, as are the methods of analysis used for detection. Different laboratories in the European Union have examined the occurrence of boldenone and its metabolites. The results were discussed at different meetings of a European Commission DG-SANCO Working Party) and summarized in an expert report. The situation of the different laboratories at this time is also covered herein. The overall conclusion of the Working Party was that there was a necessity for further research to distinguish between naturally occurring and illegally used boldenone forms. The confirmation of the presence of boldenone metabolites (free and conjugated forms) in certain matrices of animals is proposed as a marker for the illegal treatment with boldenone.

Anabolic Agents↗
PubMed2001

Hormones in meat: different approaches in the EU and in the USA.

The use of hormonal active growth promoters ("hormones") in farm animals can increase the production of veal and beef significantly up to 15%. However, in the different parts of the world the regulation regarding the use of such hormones differs sharply. In the European Union there exists a total ban on such use in contrast to the United States of American where the use of some hormones is authorized under strict conditions. An overview is given of the different opposing aspects and their consequences are discussed. It has to be concluded that in some EU Member States an extended black market exists. For the USA no experimental evidence is available for such a black market. In the EU the number of ascertained different illegal "hormones" ranges between about 35 and 55. In the USA the number of legal hormones in total is six. The levels of hormone residues found in beef originating from the USA are in the fast majority of cases below the Maximum Residue Limit as recommended by the FAO/WHO Joint Expert Committee of Food Additives. No comparable experimental data are available for the EU. Finally other food commodities have to be taken into account to assess potential risks of the dietary intake of "hormones". Eggs, example given, contribute more to the dietary intake of estradiol than beef, whether the animal is legally treated with hormones or not.

Animals↗
PubMed1999

Supercritical fluid extraction of methyltestosterone, nortestosterone and testosterone at low ppb levels from fortified bovine urine.

A multi-residue supercritical fluid extraction (SFE) method is proposed for the isolation of nortestosterone, testosterone and methyltestosterone from bovine urine. Prior to SFE, bovine urine was hydrolyzed and then fortified with the three steroids at 100 ng/ml and 50 ng/ml each for HPLC analysis and 25 ng/ml and 12.5 ng/ml each for GC-MS analysis. The samples then were mixed with an adsorbent material, placed in an SFE extraction vessel prepacked with a 3-ml SPE column containing neutral alumina and the testosterones were extracted from the urine matrix using unmodified supercritical CO2 at 27.2 MPa and 40 degrees C. The steroids were retained in-line on the neutral alumina sorbent in the SPE column while co-extracted artifactial material was trapped off-line after CO2 decompression. After SFE, the SPE column was removed from the extraction vessel, and the trapped steroids were eluted from the neutral alumina sorbent with 3 ml of a methanol-water mixture. Eluates were used directly without post-SFE clean-up either for HPLC analysis (detection limit 50 ng/ml) or for GC-MS analysis (detection limit 5 ng/ml after steroid derivatization). The multi-residue SFE recoveries (n=6) for nortestosterone, testosterone and methyltestosterone from hydrolyzed bovine urine by GC-MS analysis were 90.8+/-6%, 93.9+/-3% and 92.5+/-5%, respectively for each steroid at the 12.5 ng fortification level.

Animals↗
PubMed1998

Results of the second inventory on quality assurance/quality control (QA/QC) and good laboratory practice (GLP) for European Union National Reference Laboratories for residue analyses.

In February 1996 the "Second inventory on QA/QC and GLP for EU-NRLs" was submitted to all European Union National Reference Laboratories (EU-NRLs) for residue analyses. This second inventory was a follow up on the "Inventory on QA/QC" (1993) which was used for the organisation of the first workshop on "GLP for EC-NRLs". In February 1997 a response on the second inventory of 100% was achieved. From the completed inventories it became evident that almost all EU-NRLs for residue analyses now have a described quality program based on an international standard. However only 33% of the EU-NRLs are officially accredited, certified or in compliance with good laboratory practice (GLP) principles. Most of the accredited EU-NRLs have a quality program based on the European Standard EN45001. Seven of the EU-NRLs still do not have a described quality program or have not appointed a quality officer and for 12 of the EU-NRLs the independence of the QA officer is not formally arranged. Only about 50% of the EU-NRLs have a standard operating protocol (SOP) to handle complaints. Almost all of the EU-NRLs have SOPs available and a system to control them. In comparison with the results of the first inventory it is evident that most of the EU-NRLs have made considerable progress in the implementation of quality systems. However it is also evident that a substantial number of EU-NRLs still lack some critical QA facilities. The new deadline for the full implementation of all relevant QA facilities is January 2002.

Animals↗
PubMed1994

Endogenic nortestosterone in cattle?

When residues of nortestosterone (NT) were found in the urine of cattle, racehorses or bodybuilders, exogenic administration was thought to be proven. In previous literature, no records were found of the endogenic presence of this molecule. In the horse-racing world, Houghton and Courthot found that NT is normally present in the urine of the stallion. Belgian and Dutch researchers found that NT is also present in the urine and edible parts of the intact boar. Vandenbroeck et al. (1991) suggested the endogenous presence of NT (in the beta form) in the pregnant cow. Meyer (1992) reported the presence of NT (in the alpha form) in relatively high amounts in the urine of the cow peri-partum and the neo-natal calf. These observations may have important consequences for veterinary meat inspection in the EU. Therefore, in Belgium a large scale experiment was set up in co-operation with the EU Community Reference Laboratory (RIVM). In this paper the present state of the results in this area is presented. A large number of urine samples (> 50) of pregnant non-treated cows were collected and analysed by gas chromatography-mass spectrometry (GC-MS) in 4 different laboratories. Further samples (> 100) were taken, but only analysed in one laboratory. The results proved clearly that NT may indeed be detectable in the alpha form in the urine of pregnant cows, from at least 2 months, but most probably from 4-5 months before partus.

Animals↗
PubMed1994

Confirmational analysis of beta-agonists by cryotrapping gas chromatography-Fourier transform infrared spectrometry.

Cryotrapping gas chromatography-Fourier transform infrared spectrometry has been used for confirmation analysis of the beta-agonists clenbuterol, salbutamol, mabuterol, bromobuterol, cimaterol, cimbuterol and mapenterol in urine and liver samples of veal calves, subsequent to selected ion detection gas chromatography-mass spectrometry. Samples have been analysed as their trimethylsilyl and methylboronate derivatives. Methylboronate derivatives yielded strongly diminished chemical background and interference levels in the infrared chromatograms of standards and samples. The limit of identification for methylboronate derivatives was at the low ppb level in incurred samples. The similarity of analyte and reference spectra, together with the retention time, was found to be a useful criterion for confirmation of unknown compounds.

Adrenergic beta-Agonists↗
PubMed1992

Liquid chromatographic purification and detection of anabolic compounds.

The role of liquid chromatography within methods of analysis for steroids, related compounds and beta-agonists in biological samples is discussed. Special attention is given to the application of liquid chromatography in sample preparation and extract clean-up. Different forms of liquid chromatography, including immunoaffinity chromatography, are compared and evaluated. Methods for confirmation based on gas chromatography-mass spectrometry and cryotrapping Fourier transform infrared spectrometry are discussed.

Adrenergic beta-Antagonists↗
PubMed1992

High-performance liquid chromatographic separation and detection methods for anabolic compounds.

The role of high-performance liquid chromatography (HPLC) in methods of analysis for anabolic compounds in biological samples is reviewed. Special attention is given to both the separation and detection of anabolic compounds. A distinction is made between on-line detection systems, such as ultraviolet detection and diode-array detection, and off-line detection methods with special emphasis on immunochemical detection methods using non-isotopic labels. A number of applications are given to elucidate the possibilities of HPLC in the analysis of anabolic compounds.

Anabolic Agents↗
PubMed1989

Molecular spectroscopy in forensic residue analysis: a general overview of reliability, applicability and cost effectiveness.

Classification of an analytical result for forensic purposes is briefly discussed, in addition to mandatory specificity and limits of appropriate analytical methods. Direct information on the molecular structure of the analyte is in general more reliable than indirect information. Direct information is obtained from molecular spectroscopic methods, in contrasts to chromatographic or immunochemical methods, which provide only indirect information. The cost effectiveness ratio as calculated per analyte is indicated for various analytical techniques.

Animals↗
PubMed1989

Quality criteria for the detection of analytes in test samples with special reference to anabolic agents and related compounds.

In order to create a system for qualitative analysis, in which well defined limits of ambiguity can be set, criteria have been formulated for application to the identification method(s) used. Criteria are presented for separation techniques (thin-layer chromatography, gas chromatography, high-performance liquid chromatography) and for spectrometric methods (ultraviolet-visible spectroscopy via diode array, mass spectrometry, infrared spectroscopy), as well as general considerations for the whole procedure.

Anabolic Agents↗
PubMed1989

Effective monitoring of residues of nortestosterone and its major metabolite in bovine urine and bile.

The results of a newly developed method for the detection and identification of residues of nortestosterone (NT) and one of its major metabolites, 17 alpha-nortestosterone (epiNT) are described. The method is based on sample clean-up by immunoaffinity chromatography and detection by high-performance liquid chromatography and/or gas chromatography-mass spectrometry (selected-ion monitoring). All samples of bile from calves that had been treated with NT contained significant amounts of epiNT (6-18 micrograms/l). The NT content of these samples, if detectable, was below 1 microgram/l. Urine contained, with one exception, less than 1 microgram/l epiNT. NT itself if detectable, was, present in urine or bile at levels below 0.1 microgram/l. The results corresponds well with results obtained with a radioimmunoassay procedure.

Animals↗
PubMed1989

Multi-immunoaffinity chromatography: a simple and highly selective clean-up method for multi-anabolic residue analysis of meat.

A method for the detection of nortestosterone (NT) in bovine muscle at levels below 1 microgram/kg is described, based on enzymatic digestion of the sample, clean-up by immunoaffinity chromatography after defatting and detection by gas chromatography-mass spectrometry (selected-ion monitoring). The immunoaffinity matrix was prepared after combining the isolated immunoglobulin G fractions from a rabbit antiserum raised against NT and methyltestosterone (MT). Its capacity per millilitre of gel was approximately 10 ng for each of the two steroids. Results for samples containing 0.1 microgram/kg NT and above are described. It is concluded that for multi-residue analysis of samples of muscle at levels as low as 0.1 microgram/kg, multi-immunoaffinity chromatography is a very suitable method of sample clean-up. For purposes of quantification the trideuterated internal standard [16,16,17 alpha-2H3] nortestosterone was synthesized.

Anabolic Agents↗
PubMed1989

Quantitative analysis of anabolics on thin-layer chromatographic plates using image analysis techniques.

A new detection system is introduced for the quantitative analysis of thin-layer chromatographic plates, which is based on a relatively simple, cheap but advanced image analysis system. Both one- and two-dimensional plates can be analysed. Recording and analysis can also be performed from photographs or even slides. Applications are shown for a number of samples containing anabolic compounds.

Anabolic Agents↗
PubMed1989

[The presence of nortestosterone in edible parts from non-castrated male pigs].

Nortestosterone is a major growth-promoting agent in Europe which is often used illegally in various species of meat animal. Recent studies showed that this compound was also present in the urine of young male pigs (boars) to which nortestosterone had not been administered. To determine to which extent nortestosterone may also be present in liver and muscle tissues, samples of the urine, bile, liver and muscle of twenty five boars were analysed. The mean and highest concentrations, detected respectively in muscle were 1.1 and 13 micrograms/kg and were 23 and 200 micrograms/kg in liver. The corresponding concentrations in urine were 55 and 132 micrograms/l and 88 and 212 micrograms/l in bile.

Animals↗
PubMed1989

Application of diethylstilbestrol dipropionate in bulls. I. Excretion of residues in urine and faeces and histological and immunohistochemical changes in the prostate.

In this experiment 20 one year old bulls received a single intramuscular injection of the anabolic preparation diethylstilbestrol dipropionate (DES-DP) (an oil preparation or an emulsion). Four animals received a corresponding placebo. The application of DES-DP to bulls caused characteristic histological alterations in the peripheral glandular epithelium of the prostate, which could be observed until four weeks after treatment. The value of histological investigation as a screening method was, however, limited by the occurrence of only few metaplastic lesions and a rapid recovery. By contrast, immunohistochemistry using a polyclonal cytokeratin antiserum K40 appeared to be a specific and very sensitive method to detect oestrogen-induced lesions in the prostate. In only two animals, six weeks after injection with the DES-emulsion, false-negative results were obtained, demonstrating the potential value of this screening method. The excretion of DES in the urine and faeces was monitored using radioimmunoassay following chromatographic purification of the urine and faeces extracts. The excretion of DES in urine was faster for animals of the oil group. The DES content in urine decreased to the 1 microgram/l level after 42 days (emulsion group) or 70 days (oil group). The excretion in faeces was comparable to that in urine. After day 21 the excretion patterns of the two excreta were indistinguishable.

Animals↗
PubMed1989

Application of diethylstilbestrol dipropionate in bulls. II. Residues in bile, liver, kidney, muscle tissues and application site.

The second part of an experiment is described in which 20 one year old bulls were injected with diethylstilbestrol (DES) dipropionate containing preparations. Analysis of DES content was performed in several tissues, such as the injection site, diaphragm muscle, psoas muscle, liver, kidney and bile. In the injection site appreciable amounts of DES were found. Measurable amounts of DES were also found in liver and kidney until 4 weeks after injection. In bile, DES concentrations were even higher than those in urine, and were well correlated with DES concentrations in urine. Implications for screening purposes are discussed.

Animals↗