Simultaneous determination of masked forms of deoxynivalenol and zearalenone after oral dosing in rats by LC-MS/MS

In vivo metabolism of masked or conjugated mycotoxins is poorly documented as standards are not commercially available and indirect analysis using hydrolytic enzymes is difficult to validate and cumbersome. We synthesised zearalenone-14-glucoside (ZEA-14G) chemically. Deoxynivalenol-3-glucuronide (DON-3GlcA) and glucuronides of 3- and 15-acetyl-deoxynivalenol (3-and 15-ADON-GlcAs), de-epoxydeoxynivalenol, zearalenone (ZEA), alpha- and beta-zearalenol (alpha- and beta-ZOL) were synthesised using rat microsomes. For the first time three ADON-GlcAs were synthesised: two 3-ADON-GlcAs and one 15-ADON-GlcA. After purification, the masked mycotoxin and the metabolites were characterised by NMR (DON-3GlcA, ZEA-14G) or by full scan MS, MS/MS fragmentation, UV-spectra, beta-glucosidase and beta-glucuronidase treatment. In a first experiment, rats were fed orally DON-3-glucoside (DON-3G) and ZEA-14G, together with C-13-DON and C-13-ZEA and were sacrificed after 55 minutes. A total of 21 masked metabolites, metabolites and parent mycotoxins were quantified in rat organs. Whereas DON-3G was hardly hydrolysed in the stomach, ZEA was clearly formed from ZEA-14G. In a second experiment, 3-and 15-ADON were given orally to rats. The acetylated forms of DON were hydrolysed in the stomach, in contrast to DON-3G. Rats can directly glucuronidate ADONs without deacetylation. Neither DOM, alpha-or beta-ZOL nor their glucuronides could be quantified. Glucuronidated 3-ADON accumulated in the small intestines, together with DON-3GlcA in rats fed orally with 3- and 15-ADON. These differences in masked mycotoxins metabolism can be important in risk analysis of masked mycotoxins in food and feed.