Production and use of mycotoxins uniformly enriched with stable isotopes for their dosage in biological samples

Abstract

Due to their low concentrations in biological matrices, mycotoxin analyses often encounter detection and quantification problems, especially for toxicokinetic studies. We have developed a strategy to produce in a single process, several fungi secondary metabolites uniformly enriched with 13 C, 15 N stable isotopes in their 'natural' composition. This includes: (1) a plant culture in the presence of 10%, 50% or 100% 13 CO 2 as the only source of carbon, and in the presence or not of 10% 15 N-enriched nitrogen salts – as expected wheat or maize uniformlyincorporate enriched isotopes into their bioproducts; (2) a subsequent solid culture of different filamentous fungi on plant biomass led to the production of a 'natural' mixture of isotopes-enriched mycotoxins – these compounds exhibit a characteristic isotopic cluster, which can be easily detected by mass spectrometry. As an example, we achieved 10% uniformly 13 C-enriched zearalenone, deoxynivalenol and mycophenolic acid by growing Fusarium graminearum or Penicillium brevicompactum on 10% 13 C enriched wheat seeds and 3 to 10% 13 C, 15 N uniformly enriched fumonisins from Fusarium verticillioides cultures on maize seeds or straw. These compounds were used for metabolism and transport studies in mammals either in vitro or in vivo and analysed by MS and MSn spectra of the isotopic cluster but also by 13 C, 15 N NMR. Moreover, such isotopic pattern enrichment can be used for quantitative evaluations of mycotoxins transport across mammalian biological membranes, alone or in their 'natural' conditions in the presence of other fungi secondary metabolites. Finally, we used such enriched compounds with high reliabilityin order to study zearalenone metabolism but these enriched compounds would also be used as internal standards to quantify zearalenone or fumonisins in contaminated food samples.