Rapid enzymatic hydrolysis of masked deoxynivalenol and zearalenone prior to liquid chromatography mass spectrometry or immuniassay analysis

Recently it has been shown that conjugates (‘masked mycotoxins’) may contribute to the total daily intake of hazardous mycotoxins. Therefore, there is an urgent need for rapid analysis methods that assess the level of both free and masked mycotoxins in food and feed. However, the analysis of masked mycotoxins by either immunoassays or instrumental methods, such as liquid chromatography tandem mass spectrometry (LC-MS/MS), is severely hindered by the lack of standards and the unpredictable cross-reactivity profiles of the available antibodies. In this work, 26 enzymes were explored for rapid hydrolysis of masked mycotoxins using deoxynivalenol-3-glucoside (DON-3G) as model compound. Following initial screening, the most promising enzyme, a fungal 1,3-ß-glucanase (laminarinase), was investigated in detail and found to be fit-for-purpose, providing complete conversions in minutes rather than hours according to LC-MS/MS analyses. As a proof of concept, the enzymatic pretreatment was applied to an extract of beer containing DON-3G. In addition, the feasibility of a fully automated enzymatic pretreatment of masked mycotoxin standards in an autosampler was demonstrated in an imaging surface plasmon resonance immunoassay set-up. Such an automated pretreatment was found to be equally applicable to other mycotoxin conjugates, as shown by the conversion of zearalenone-14-ß-D-glucopyranoside and zearalenone-14-sulphate, in the latter case using a sulphatase enzyme. It is envisaged that laminarinase could be useful for other masked mycotoxins as well.