Simultaneous determination of resorcylic acid lactones, β 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 β-zearalanol, β-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, β-trenbolone (β-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 100 mm × 2.1 mm × 1.7 μm 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 (CCβ). For all compounds the method was linear in the range 2-12 μg/l (1 and 6 μg/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 0.6 μg/l (DES) to 1.6 (α-TB) and CCβ was 0.8 μg/l (α-zearalenol) to 1.9 μg/l (α-TB).