Hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry for the quantification of uridine diphosphate-glucose, uridine diphosphate-glucuronic acid, deoxynivalenol and its glucoside: In-house validation and application to wheat.

Nucleotide sugars, the activated forms of monosaccharides, are important metabolites involved in a multitude of cellular processes including glycosylation of xenobiotics. Especially in plants, UDP-glucose is one of the most prominent members among these nucleotide-sugars, as it is involved in the formation of glucose conjugates of xenobiotics, including mycotoxins, but also holds a central role in the interconversion of energized sugars such as the formation of UDP-glucuronic acid required for cell wall biosynthesis. Here, we present the first HILIC-LC-ESI-TQ-MS/MS method for the quantification of UDP-glucose and UDP-glucuronic acid together with the Fusarium toxin deoxynivalenol (DON) and its major plant detoxification product DON-3-O-glucoside (DON-3-Glc) utilizing a polymer-based column. For sample preparation a time-effective and straightforward 'dilute and shoot' protocol was applied. The chromatographic run time was minimized to 9min including proper column re-equilibration. In-house validation of the method verified its linear range, intra- (1-7%) and interday (8-20%) precision, instrumental LODs between 0.6 and 10ngmL(-1), selectivity and moderate matrix effects with mean recoveries of 85-103%. To prove the methods applicability, we analyzed two sets of wheat extracts obtained from different cultivars grown under standardized greenhouse conditions. The results clearly demonstrated the suitability of the developed method to quantify UDP-glucose, DON and its masked form D3G in diluted wheat extracts. We observed differing concentration levels of UDP-glucose in the two wheat cultivars showing different resistance to the severe plant disease Fusarium head blight. We propose that the higher ability to detoxify DON into DON-3-Glc might be a consequence of the higher cellular UDP-glucose pool in the resistant cultivar.