R.W. Stephany

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).

Combination of high-performance liquid chromatography and chemiluminescent immunochemical detection of hormonal anabolics and their metabolites

Abstract A specific identification method is presented for a number of hormonal anabolics, especially 19-nortestosterone, methyltestosterone and zeranol and their metabolites. The method is based on a combination of selective fractionation by h.p.l.c. and immunochemical detection with chemiluminogenic steroid/isoluminol conjugates.

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 RIAs 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.

A chemiluminescent immunoassay for 17α‐methyltestosterone

An immunoassay for the xenobiotic anabolic compound methyltestosterone (MT) is presented. The detection is based on chemiluminescence of a MT-isoluminol conjugate. Applications are presented for detection of methyltestosterone and cross-reacting compounds in bovine urine and tissues of application sites isolated from slaughtered cattle, illegally treated with hormonal anabolics.

A solid-phase chemiluminescence immunoassay for 17α-methyltestosterone

Abstract A solid-phase immunoassay based on chemiluminescence detection is described for the anabolic steroid 17α-methyltestosterone (MT). The reproducibility is increased substantially by introduction of the solid-phase principle and the assay can therefore be used to quantify MT in bovine urine. The detection limit is 10 pg.

Solid-phase chemiluminescence immunoassay for screening of anabolic agents in application sites in slaughtered cattle

Abstract A solid-phase chemiluminescence immunoassay for 19-nortestosterone (NT) is presented; NT-3-carboxymethyloxime/N-(4-aminobutyl)-N-ethylisoluminol serves as the label with an antiserum raised against NT-3-carboxymethyloxime/bovine serum albumin. Some other anabolic compounds (e.g., testosterone and trenbolone) showed substantial cross-reactivity. The assay can be used generally for the detection of anabolic agents in application sites. Because of the high sensitivity (0.1 pg NT/tube at 90% relative binding), only 250 μg of muscle tissue is needed for the assay. Apparent NT contents of 0.4 to 16 000 μg kg−1 tissue can be measured. With a simplified isolation method, about 40 samples can be screened in a working day.

A specific radioimmunoassay for the detection of 19-nortestosterone in urine

The antisera used were raised in rabbits against a 19-nortestosterone-7 alpha-carboxyethylthioether conjugate of bovine serum albumin. Tritium-labeled nortestosterone was used as tracer. Cross reactivities with metabolites of nandrolone, other anabolics and endogenous steroids were very low. To achieve additional specificity a clean up and separation procedure was developed, using isocratic high performance liquid chromatography. So far no qualitative discrepancies have been observed between the assay described here and confirmative determinations by combined high performance liquid chromatography-gas chromatography-mass spectrometry.