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.

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.

[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.

Assay for trenbolone and its metabolite 17 alpha-trenbolone in bovine urine based on immunoaffinity chromatographic clean-up and off-line high-performance liquid chromatography-thin-layer chromatography.

An high-performance liquid chromatography (HPLC)-thin-layer chromatography (TLC) method was developed to detect the illegal use of the xenobiotic growth promotor Trenbolone acetate (TBA). Very effective clean-up of bovine urine was achieved by immunoaffinity chromatography (IAC). The active form of TBA, the steroid 17 beta-Trenbolone (17 beta-TB), as well as its major metabolite 17 alpha-Trenbolone (17 alpha-TB), were assayed simultaneously with HPLC and on-line UV detection. The fraction containing 17 alpha-TB and 17 beta-TB (TB-fraction) was collected, and for confirmation 17 beta- and 17 alpha-TB were subsequently separated and identified by TLC. The limit of detection by on-line HPLC-UV (350 nm) was 1-2 micrograms TB/l. Off-line TLC detection was even more sensitive, 0.5 microgram 17 beta- or 17 alpha-TB/1. The assay was validated by investigating urine samples from veal calves implanted with TBA. The presence of 17 beta- and 17 alpha-TB was clearly demonstrated. A survey of the illegal use of TBA in cattle was performed by applying the assay to urine obtained at slaughter. No residues of TBA or its metabolites were found in any of the 144 random samples from the Dutch public health surveillance programme.

A highly specific detection method for diethylstilbestrol in bovine urine by radioimmunoassay following high performance liquid chromatography.

A specific radioimmunoassay (RIA) for diethylstilbestrol (DES) is presented following purification of sample extracts by isocratic reversed phase high performance liquid chromatography. Applications are given of a forensic investigation for the control of DES in bovine urine. Specificity of HPLC-RIA is compared with that of RIA's with other (chromatographic) purification procedures and with gas chromatography-mass spectrometry. Implications of the use of these techniques in practice and the use of various DES-antisera are discussed.

[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.

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).

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).