Abstract
Zearalenone (ZON) and its four metabolites, α-zearalenol (α-ZOL), .β-zearalenol (β-ZOL), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL), have been shown to competitively bind to estrogen receptors (ERs) in a number of in-vitro tests. As the binding affinities of these substances to ERs are different, a simultaneous quantitative determination is necessary for the accurate risk assessment. Therefore liquid chromatography/tandem mass spectrometry using electrospray ionization (LC-ESI-MS/MS) was evaluated for the applications to beer and related raw materials. In this method, Zearalanone (ZAN), which doesn't occur in nature, was used as an internal standard for quantification. Sample preparation for malt was performed by the extraction of the commodities with a mixture of acetonitrile and water, followed by solid phase extraction (SPE) with C-18 cartridge. As for beer products, samples were degassed and applied directly to the same cartridge. After separation on a phenyl column, quantification limits of 1.1-1.6 μg/kg in malt and 0.2-2.0 μg/kg in beer were achieved with the negative ionization mode.
To further characterize the ZON metabolisms in brewing, ZON was added to wort at the level of 100 μg/kg and fermentation tests were conducted for up to 10 days using two commercial strains of Saccharomyces pastrianus. After fermentation, ZON levels were decreased to 1.0 μg/kg in both experiments. Instead of ZON, β-ZOL, which shows lower estrogenic activity than ZON, was detected mainly at levels of 78.3 ng/g and 88.2 μg/kg. α-ZAL and β-ZAL which show higher estrogenic activity than ZON were not detected in both cases.
