Development and in-house validation of an LC-MS/MS method for the determination of stilbenes and resorcylic acid lactones in bovine urine

Stilbenes and zeranol are nonsteroidal estrogenic growth promoters which are banned in the European Union (EU) for use in food-producing animals by Council Directive 96/22/EC. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the screening and confirmation of stilbenes (diethylstilbestrol, dienestrol, hexestrol) and resorcylic acid lactones (zeranol and its metabolites taleranol and zearalanone as well as the mycotoxins alpha-zearalenol, beta-zearalenol and zearalenone) in bovine urine. The method permits the confirmation and quantification of stilbenes and resorcylic acid lactones at levels below 1 mu g L-1 and 1.5 mu g L-1, respectively. The validation was carried out according to Commission Decision 2002/657/EC, Chap. 3.1.3 alternative validation by a matrix-comprehensive in-house validation concept. Decision limit CC alpha, detection capability CC beta, recovery, repeatabiliy, within-laboratory reproducibility and the uncertainty of measurement were calculated. Furthermore, a factorial effect analysis was carried out to identify factors that have a significant influence on the method. Factors considered to be relevant for the method in routine analysis (e.g. operator, matrix condition, storage duration of the extracts before measurement, different cartridge lots, hydrolysis conditions) were systematically varied on two levels. The factorial analysis showed that different cartridge lots, storage durations and matrix conditions can exert a relevant influence on the method.