A glassy carbon electrode modified with silver nanoparticles and functionalized multi-walled carbon nanotubes for voltammetric determination of the illicit growth promoter dienestrol in animal urine.

An electroanalytical method for determining dienestrol (DNL) in bovine urine samples is described. A glassy carbon electrode (GCE) modified with silver nanoparticles and functionalized multi-walled carbon nanotubes was used as working sensor. The modified GCE displays substantial analytical improvements including an amplified signal, fast electron transfer kinetics, and resistance to fouling. The irreversible oxidation signal of DNL is pH-dependent. Best reactivity is found at pH 3.0, where a typical anodic peak is recorded at 0.8 V (vs. Ag/AgCl). Square-wave voltammetry revealed a 8.4 nM detection limit (1.9 µg L-1), good repeatability and reproducibility (RSDs <5.0%), and good accuracy (93.2-99.4% recovery from spiked samples). The modified electrode is highly stable even in the presence of ions (Na+ and K+), urea and uric acid. The electrochemical sensor fulfills all requisites to be used as forensic device in surveillance of illegal livestock practices. Graphical abstract Schematic presentation of the construction of a glassy carbon electrode modified with silver nanoparticles and functionalized multi-walled carbon nanotubes. This sensor exhibited a remarkable performance for voltammetric detection of the illicit growth promoter dienestrol in animal urine.

Validation of screening method for residues of diethylstilbestrol, dienestrol, hexestrol, and zeranol in bovine urine using immunoaffinity chromatography and gas chromatography/mass spectrometry.

A method was developed, using commercially available immunoaffinity chromatography cleanup cartridges, followed by detection by gas chromatography/mass spectrometry, to screen for residues of the hormone growth promotants diethylstilbestrol, dienestrol, hexestrol, and zeranol in bovine urine. The single-laboratory, in-house validation included assessment of recoveries, repeatability, linearity of response, detection capability, and specificity (cross-reactivity) with a suite of antibiotics and other hormonal growth promotants. The method was validated for screening at a target concentration of 2.0 microg/L in urine. The detection capabilities for the analytes were diethylstilbestrol, 0.24; dienestrol, 0.15; hexestrol, 0.84; and zeranol, 0.28 microg/L.

Determination of three anabolic compounds in calf urine by liquid chromatography with photodiode-array detection.

A method for the determination of three anabolic hormones (diethylstilbestrol, dienestrol and trenbolone) in calf urine is described. After enzymatic hydrolysis, the samples were cleaned up by C18 solid-phase extraction. Drugs were extracted with hexane and analyzed by isocratic elution on a Discovery RP-Amide C16 5 microns column with photodiode-array detection at 240 and 347 nm. Both retention time and UV spectra were used for identification. Detection limits for the HPLC system were calculated to be 0.3 ng injected for all analytes in the standard mixture. However, for urine samples these limits increased because of the presence of unidentified matrix components. After extraction from urine, the limits of detection for the whole analytical procedure were 5 and 10 ng injected for trenbolone and stilbenes, respectively. The average recoveries of the hormones from spiked samples were in the range 53.1-56.7% with RSD between 11.3 and 14.5% for the whole procedure in the concentration range 25-2.5 ng ml-1.

Urinary excretion of diethylstilbestrol in the ostrich.

Stilboestrol tablets (20 x 1 mg) were given to 4 ostriches. Urine was collected over a period of 8 days and stored frozen at-20 degrees C pending analysis. Analyses were performed on a gas chromatograph-mass selective detector for the presence of parent compound and/or metabolites. Diethylstilbestrol and its metabolite, dienestrol, were detected in urine; dienestrol only for 1 day but diethylstilbestrol for 8 days after administration. Residue analysis for the use of diethylstilbestrol as growth promoter can be performed on the urine of ostriches by scanning for parent compound only since it can be detected longer than the metabolite.

Analysis of diethylstilbestrol, dienestrol and hexestrol in biological samples by immunoaffinity extraction and gas chromatography-negative-ion chemical ionization mass spectrometry.

A method has been developed for the detection of diethylstilbestrol, together with dienestrol and hexestrol, using extraction with a single immunoaffinity column containing antibodies raised against diethylstilbestrol, followed by gas chromatography-negative-ion chemical ionization mass spectrometry. Immunoaffinity columns were prepared by coupling immunoglobulin G fractions obtained from rabbit antisera with a Sepharose matrix. The immunizing agent was synthesized by introducing a carboxyl group into the diethylstilbestrol molecule and coupling this product to bovine serum albumin. The columns were used for immunoadsorption of diethylstilbestrol and other estrogens, after dilution of samples with phosphate buffer, and were eluted with acetone-water (95:5 v/v). A derivatization method suitable for gas chromatographic-mass spectrometric analysis of diethylstilbestrol and other estrogens was developed using pentafluorobenzyl bromide and ethanolic potassium hydroxide as reagents. The derivatives obtained were detectable at the sub-picogram level using gas chromatography with negative-ion chemical ionization mass spectrometry. Recoveries of cis- and trans-diethylstilbestrol, dienestrol and hexestrol from the immunoaffinity columns, determined after extraction from urine, plasma and buffer, ranged from 28 to 96%. The sensitivity for diethylstilbestrol in urine samples was ca. 10 ppt. The method was applied to the analysis of urine from calves given a single dose of 10 mg of diethylstilbestrol. Free and glucuronic acid conjugated diethylstilbestrol decreased with time, but their ratio was variable.

Oestrogen radioreceptor assay for multi-residue screening of bovine urine for oestrogenic anabolic compounds.

An improved radioreceptor assay (RRA), based on the method originally described by Korenman (1968), was used for the screening of urine samples of cattle for the presence of exogenous oestrogenic anabolic compounds, i.e. stilbenes and zeranol. The method includes extraction of the hormones from urine samples with a simultaneous purification using reversed-phase C18 cartridges. HPLC is used to isolate the anabolics from the naturally occurring oestrogens. Fractions containing the stilbenes and zeranol are collected and subsequently quantified using the RRA with the oestrogen receptor, isolated from immature calf uteri, as binder and tritiated 17 beta-oestradiol as tracer. Urine samples from untreated calves and cows, as well as samples from calves treated with zeranol/trenbolon acetate, dienoestrol or hexoestrol or samples containing diethylstilboestrol, were analysed using this RRA method. Sensitivity, specificity and predictive values were calculated at different classification levels (0.4, 0.5, 0.6 and 1.0 ng 'apparent' oestradiol per ml urine). An optimum sensitivity (89%) with a maximum specificity (95%) was reached at a classification level of 0.6 ng/ml. At this level the detection limits in urine samples are 0.5 ng/ml for hexoestrol, 0.6 ng/ml for diethylstilboestrol, 0.9 ng/ml for dienoestrol and 5.0 ng/ml for zeranol.

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.

[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.