Effective control for diethylstilbestrol in cattle in the Netherlands.

In the period from August 1981 to April 1983, 10243 random samples of urine from slaughtered cattle were inspected for the presence of the stilbene derivatives diethylstilbestrol (DES), dienestrol (DE), and hexestrol (HEX). Fast screening of all samples was performed by radioimmunoassay (RIA) with prior clean-up column chromatography. In this screening, 216 samples were indicated for confirmatory analyses by combined high performance liquid chromatography-gas chromatography-mass spectrometry (HPLC-GCMS) on account of an immunochemical response equivalent to 1 microgram/1 DES or more. The presence of DES was confirmed in 184 samples, DE in 5 samples and HEX in 14 samples. In the remaining 13 samples no stilbene derivative could be confirmed. From the results in 1984, after the introduction of the Meat Inspection Act, it is concluded that the use of stilbene derivatives in cattle in the Netherlands has dropped almost to zero. Of 4558 samples investigated, only 6 (0.13%) were indicated as 'stilbene' positive. The presence of DES was confirmed in 2 samples and DE in 4 samples.

Monitoring and identification of residues of anabolic preparations in slaughtered cattle by HPLC with diode array detection.

The new combination of isocratic high performance liquid chromatography (HPLC) with on line UV spectrum detection via a diode array configuration has been applied to the detection and identification of anabolics present in application sites of cattle. Combination of the characteristic retention time in the HPLC chromatogram and a comparison of the full spectrum between 190-400 nm of the anabolic components with that of a standard resulted in a very reliable identification. By means of this method 117 samples of application sites were investigated for the presence of anabolic residues. Of the xenobiotic anabolics , 19-nortestosterone (NT) was found most frequently (in 96 cases), whereas diethylstilbestrol (DES) was found in only 11 cases. In all samples the identification of NT and DES was confirmed by high resolution gas chromatography-mass spectrometry (GCMS).

Development of a chemiluminescent immunoassay for 19-nortestosterone (nandrolone).

An immunoassay for nortestosterone with detection by chemiluminescence is described, and applied to the urine and drug administration site of slaughtered cattle.

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.

Identification of several stilbene derivatives in bovine urine by means of high performance liquid chromatographic fractionation and immunochemical detection.

Radioimmunochemical detection (RIA) following fractionation of urine extracts via high performance liquid chromatography (HPLC) turned out to be a very specific method for the identification of stilbene derivatives in bovine urine. Combination of the high resolution of the HPLC with a specific RIA is a suitable method to discriminate between the presence of different stilbene derivatives like diethylstilboestrol (DES), dienoestrol (DE) and hexoestrol (HEX) or other unknown compounds interfering in the celite-RIA used in the screening. Using this screening method 8200 samples of bovine urine were investigated on the presence of stilbene derivatives of which 133 were classified as 'positive'. In 106 'positive' urines the presence of DES was shown and in 19 'positive' urines the presence of DE or HEX, using the method described in this report whereas in 8 'positive' urines an unknown immunochemical active compound was detected. During 1.5 year of comparative investigation no qualitative discrepancies occurred between the results of the HPLC-immunogram procedure and the final confirmation by high resolution gas chromatography-mass spectometry (GCMS).

Weight increase of the thyroid gland as a tentative screening parameter to detect the illegal use of thyreostatic compounds in slaughter cattle.

A total of 45 normal or enlarged thyroid glands of adult slaughter cattle were weighed and analyzed by thin-layer chromatography for the presence of residues of the following thyreostatic compounds: thiouracil, methylthiouracil, propylthiouracil, phenylthiouracil, and methimazole. In 21 glands, mostly from imported animals, residues of methylthiouracil or methimazole were detected. These thyroids ranged in weight from 68-245 g (mean value 118 g). Glands in which no residues were found, varied in weight between 20 and 124 g (mean value 42 g). Taking an upper limit of 60 g as normal, 4 out of 22 were false-positive' with respect to the 5 thyreostatic compounds screened for, while no false-negative cases were scored. On the basis of these results the weight increase of the thyroid gland is proposed as a simple indirect parameter in the screening for the illegal use of thyreostatic compounds in slaughter cattle.

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.

Occurrence of N-nitrosodiethanolamine (NDELA) in cosmetics from the Dutch market.

N-Nitrosamines as carcinogens in general pose a potential health risk. Since 1992, legal guidelines that restrict the occurrence of N-nitrosamines in cosmetics have been operational in the European Union. Problem issues are NDELA (N-nitrosodiethanolamine) and NMPABAO (2-ethylhexyl 4-(N-nitroso-N-methylamino)benzoate). Data available on the NDELA content of Dutch samples date back to the early 1980s and those for NMPABAO are lacking. For the determination of NDELA in cosmetics, a method based on capillary gas chromatography (GC) without derivatization and online chemiluminescence detection with a Thermal Energy Analyser (TEA) has been developed and validated. The sample is diluted with water, adsorbed onto a kieselgur column and eluted with n-butanol. The extract is transferred to a silica gel column which is eluted with acetone. The eluate is dried and re-dissolved in dichloromethane. The final extract is analysed for NDELA by GC-TEA. The average recovery for NDELA is 99%, range 86-112% (n = 4) and the limit of quantification is 5.3 mug kg(-1). The GC-TEA method was used to determine the NDELA content of 48 cosmetics including gels, shampoos, cremes, milks, conditioners and foams. All determinations were done in duplicate, and for every 10 cosmetics a recovery experiment and a blank determination were performed. The results of these quality assurance experiments were within the performance characteristics of the method developed. In 1996, a content of NDELA above the limit of quantification of 5.3 mug kg(-1) was measured in four out of the 48 cosmetics. Based on these results, a more selective survey was carried out. Now a NDELA content above the limit of quantification was measured in seven out of 25 sampled and analysed cosmetics.

[Diethylstilbestrol in urine of slaughtered cattle]. / Diethylstilbestrol in urine van geslachte runderen.

The results of an investigation during 1981-1984 for the presence of diethylstilbestrol (DES) in 12576 samples of bovine urine in the Netherlands are presented. The screening was performed by radioimmunochemical detection after chromatographic purification on celite. Subsequent final confirmation was performed by combined high pressure liquid chromatography-gas chromatography mass spectrometry. On the average in 1.7% of the 12576 samples the presence of stilbenes , primarily DES, was proven. Probably due to the reintroduction of the meat inspection act regarding the control for DES, a substantial decrease to 0.2% (4 samples) was observed in the first three months of 1984. In these 4 samples out of 2333 samples the presence of dienestrol (DE) was confirmed. No DES or hexestrol (HEX) could be detected.

[A quarter of a century of studies of the abuse of hormonal anabolics in slaughtering animals: no DES-illusion!]. / Een kwart eeuw onderzoek naar misbruik van hormonale anabolica bij slachtdieren: geen DES-illusie!

Over twenty-five years of analytical chemical and analytical biological forensic examination for residues of anabolic agents are reviewed. Up to 1981, this review is not only representative of the research done at the National Institute of Public Health but also of any research in this field in The Netherlands. A more detailed background is presented of the situation and the model of investigation adopted in the analytical-chemical testing of bovine urine for diethylstilbestrol (DES) following legal verdicts in 1981. At the time, examination in accordance with the Meat Regulations as well as a particular method of analysis were condemned. A retrospective study showed the rejected radioimmunochemical method (RIA) to be reliable as regards false-positive results at a level of 2 micrograms or more of DES per litre of urine (2 parts per billion or 2 ppb) rather than the level of 1 ppb adopted in 1981. Extensive and large-scale screening for DES from 1984 up to the present day resulted in the almost complete disappearance of the illegal use of this anabolic compounds in the Netherlands. However, investigations of sites of injection detected in slaughtered cattle during the period from 1983 to 1984, showed that other anabolics were still being used illegally. These studies particularly revealed residues of combined anabolic compounds ('cocktails'). The most common active anabolic ingredients observed were the xenobiotic steroids nortestosterone (nandrolone) and medroxyprogesterone and the exogenous natural steroids oestradiol and testosterone.

[Toxicological aspects of the use of hormones as anabolic agents (author's transl)]. / Toxicologische aspecten van het gebruik van hormonen als anabolica.

The use of anabolic agents may give rise to residues in foods of animal origin and thus to potential effects on public health. Therefore, the physiology, metabolism and toxicity of the three endogenous hormones. 17 beta-oestradiol, progesterone and testosterone are discussed. In addition, the effects of the use of three exogenous hormones, diethylstilboestrol (DES), trenbolone and zeranol are evaluated. As can be expected all six compounds have effects on the endocrine system and reproduction. Endogenous hormones and DES are teratogenic at high-dose levels. With the exception of DES, there is no evidence of a mutagenic action. 17 beta-Oestradiol and testosterone were shown to induce tumours at high-dose levels in experimental animals; probably however, a threshold can be established. The residues in edible tissue resulting from the use of endogenous hormones are much smaller than are the natural levels in cow's milk and butter. In view of the carcinogenicity of DES in man, its use should be strongly discouraged. A definite statement on the acceptability of the use of trenbolone and zeranol cannot be made.

[Anabolic steroid residues in administration sites in slaughtering cattle. October 1983 - January 1985]. / Residuen van anabolica in toedieningsplaatsen bij slachtrunderen. Periode oktober 1983-januari 1985.

Samples from 573 sites of injection or implantation in carcasses of slaughtered cattle and veal calves in the Netherlands were studied by high performance liquid chromatography associated with on-line detection of the total UV spectrum (diode array detector). Anabolic substances were identified in 462 (100%) out of 573 samples. Nortestosterone was most frequently detected (84%) during the period from October 1983 to January 1985. Other xenobiotic steroids observed were medroxyprogesterone (14%), methyltestosterone (2%) and trenbolone (2%). The 'natural' steroids testosterone, oestradiol and progesterone were found in 29%, 66% and 3% of the samples respectively. The stilbene derivatives diethylstilboestrol (DES), dienestrol and hexoestrol were found in 2%, 3% and 0% of the samples. After re-introduction of DES control in the urine of cattle early in 1984, DES residues were no longer detected in the samples from sites of administration. Zeranol was not observed at all. If applicable, all anabolic agents were found to be present in a variety of esterified dosage forms.

[The presence of nortestosterone in edible parts from non-castrated male pigs]. / Het voorkomen van nortestosteron in eetbare delen van niet gecastreerde mannelijke varkens.

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.

Confirmatory analysis of Trenbolone using accurate mass measurement with LC/TOF-MS.

The use of accurate mass measurement as a confirmation tool is examined on a TOF-MS and compared with confirmation using a triple quadrupole mass spectrometer (QqQ-MS). Confirmation of the identity of a substance using mass-spectrometric detection has been described. However, the use of accurate mass measurement for confirmatory analysis has not been taken into account. In this study, criteria for confirmation with accurate mass are proposed and feasibility is demonstrated. Mass accuracy better than 3ppm of the quasi-molecular ion and a fragment and their relative ratios determined with LC/TOF-MS are compared to the criteria of two transition ions and their ratio of LC/QqQ-MS. The results show that these criteria can be met for Trenbolone in samples of bovine urine and that single MS accurate mass measurement is comparable to nominal mass MS/MS for confirmation. The increase in popularity and availability of LC/TOF-MS instruments and the ease, of which exact masses can be measured, make it important to formulate criteria for this type of instrumentation. It is shown in this study that accurate mass measurement can be used for confirmatory analysis. However, more experiments need to be conducted to demonstrate the applicability of accurate mass measurement in general for residue analysis.

Development of a method which discriminates between endogenous and exogenous beta-boldenone.

One potential explanation for the presence of beta-boldenone in calf urine is contamination of the sample with feces containing beta-boldenone. It has been demonstrated that after oral and intramuscular administration of beta-boldenone esters, several metabolites are formed and excreted in urine. One of the (minor) metabolites is 6beta-hydroxy-17alpha-boldenone. This paper describes an analytical method that can discriminate between unconjugated boldenone, its glucuronide- and sulphate-conjugates, 6beta-hydroxy-17alpha/beta-boldenone and coprostanol, a marker for fecal contamination. The method was applied to all samples suspected to contain boldenone within the Dutch National Residue Control Plan. Approximately 10,000 samples of urine were screened (LC-MS) in 2004-2005 by VWA-East, one of the official Dutch control laboratories, from which 261 samples were suspected to contain boldenone. These samples were all analyzed for their conjugation state, 6beta-hydroxy-17alpha/beta-boldenone and for the presence of coprostanol. Alfa-boldenone, the major metabolite in bovine urine after boldenone-ester administration, was found in a large number of these samples. The presence of alpha-boldenone was proven also to be a result of fecal contamination. None of the samples tested contained residues of the metabolite 6beta-hydroxy-17alpha/beta-boldenone. Not finding this metabolite indicates that the origin of alpha-boldenone-conjugates is endogenous. The results confirm that the presence of unconjugated beta-boldenone and alpha-boldenone conjugates next to alpha-boldenone are no indicators for illegal administration of boldenone-esters. No indications were obtained that conjugated beta-boldenone can be of endogenous origin.

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.

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.