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

Abstract

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 microg L(-1) and 1.5 microg 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 CCalpha, detection capability CCbeta, recovery, repeatability, 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.

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

“…For example, Matraszek-Zuchowska et al [15] and Dusi et al [17] report the analysis of RALs in urine, Wozniak et al [16] the analysis of trenbolones, Kaklamanos et al [18] the analysis of stilbenes and RALs (excepting ␣-zearalenol and ␤-zearalenol) and Schmidt et al [19] the analysis of stilbenes and RALs. Regarding other matrices, several studies describe the determination of several compounds included in our work in meat [21,22], serum [23] and plasma [24,25].…”

“…These compounds can be analyzed directly (e.g. without derivatization) using High Performance Liquid Chromatography/Mass Spectrometry (HPLC/MS) and in recent years most publications in this field use this technique [15][16][17][18][19]. As with most HPLC/MS based methods a potential pitfall is the existence of matrix effects which can be minimized using the higher resolution provided by Ultra High Performance Liquid Chromatography/Mass Spectrometry (UHPLC/MS) [20].…”

A rapid method for simultaneous determination of zearalenone, α-zearalenol, β-zearalenol, zearalanone, α-zearalanol and β-zearalanol in traditional Chinese medicines by ultra-high-performance liquid chromatography–tandem mass spectrometry

“…The availability of ionization sources, i.e., electrospray (ESI) and atmospheric pressure chemical ionization (APCI), has drastically improved the possibilities of employing LC/MS in the mycotoxin analysis [29,30]. In addition, tandem mass spectrometry (MS/MS) results in enhanced performance, providing additional selectivity and generating information with a high degree of structural specificity [15,[31][32][33][34][35]. However, the common perception that utilization of LC-MS/MS practically guarantees selectivity is challenged by several reported examples of lack of selectivity due to ion suppression (or enhancement) caused by the sample matrix.…”

Adverse effects of bisphenol A on the testicular parenchyma of zebrafish revealed using histomorphological methods

“…Samples were processed for their extraction and purification, and finally transferred into vials, using a modification of the technique employed by Schmidt et al (2008). For BPA determination, 20 µl were injected into the LC-MS/MS system with a detection limit of 5 µg/g.…”