Simultaneous determination of resorcylic acid lactones, ß and α trenbolone and stilbenes in bovine urine by UHPLC/MS/MS.

Treatment of cattle with α-zearalanol (zeranol, α-ZAL), a resorcylic acid lactone (RAL), is illegal in European Union countries. Zearalenone, a common contaminant of cattle feed, is also a RAL and there is evidence that it, or its metabolites, can be converted in vivo to α-ZAL (or to ß-zearalanol, ß-ZAL). To determine whether an animal has been treated with α-ZAL it is necessary to quantify separately all the RALs. This work presents the simultaneous determination in urine of RALs, ß-trenbolone (ß-TB) and its metabolite α-trenbolone (α-TB) and the stilbenes diethylstilbestrol (DES), dienestrol (DEN) and hexestrol (HEX) using Ultra High Performance Liquid Chromatography/Mass Spectrometry (UHPLC/MS/MS). Several chromatographic UHPLC columns were tested in order to achieve chromatographic separation of the analytes and the results are shown. Baseline separation of all compounds was not possible, so that careful consideration of the MRM transitions was necessary. The separation chosen for the validation work used a 100mm×2.1mm×1.7µm Phenyl column eluting with a gradient of acetonitrile/methanol/water. The method validation according to EU Decision 657/2002 included linearity, within laboratory reproducibility and trueness, decision limit (CCα) and detection capability (CCß). For all compounds the method was linear in the range 2-12µg/l (1 and 6µg/l for DES) with determination coefficients greater than 0.97 and linear residuals below 20%. Within laboratory reproducibility was lower than 25% and trueness less than 11% for all compounds and concentration levels. CCα ranged from 0.6µg/l (DES) to 1.6 (α-TB) and CCß was 0.8µg/l (α-zearalenol) to 1.9µg/l (α-TB).

External contamination of bovine hair with beta2-agonist compounds: evaluation of decontamination strategies.

Hair analysis has shown great potential in the control of illegal use of veterinary drugs such as beta2-agonists. However, it has been shown that hair can be externally contaminated with drugs which can lead to false positive results. Exposure of bovine hair to aqueous solutions of beta2-agonist compounds results in incorporation of these drugs into the hair. Standard hair washing procedures found in the literature: detergent (Tween-20), phosphate buffer or organic solvents (dichloromethane or methanol) cannot eliminate this external contamination. Beta2-agonists can be extracted from hair very efficiently with 0.1 M HCl, the extraction kinetics of externally and endogenously accumulated clenbuterol at room temperature are different which makes it feasible to discriminate between them. Treatment of hair samples with a 0.1 M HCI solution for 2 h at room temperature results in a ratio of clenbuterol content in the wash solution to clenbuterol content in the washed hair equal to or less than 0.25 for samples from treated cattle; whereas this ratio is equal to or higher than 0.70 for externally contaminated samples. The design of the study was intended to resemble the plausible scenario of hair being sampled a short time after external contamination. A similar study to detect external contamination for hair sampled a long time after exposure is in progress.

Gas chromatographic-mass spectrometric analysis of veterinary tranquillizers in urine: evaluation of method performance.

A method for analysis of veterinary tranquillizers in urine using gas chromatography-mass spectrometry (GC-MS) is described. Detection limits are 5 microg/l for ketamine, azaperone and the phenothiazines (chlor-, aceto- and propionylpromazine), 10 microg/l for haloperidol, 20 microg/l for xylazine and 50 microg/l for azaperol, recoveries for all analytes were higher than 70%. Method performance in terms of within-batch, between-days and between-analysts reproducibility was studied and found to be acceptable. Compliance with European Union criteria for confirmation of GC-MS "positive" results is evaluated and discussed.