Glucosylation of T-2 and HT-2 toxins using biotransformation and chemical synthesis: Preparation, stereochemistry, and stability.

Plant-derived phase II metabolites of T-2 toxin (T2) and HT-2 toxin (HT2) were first described in 2011 and further characterized in the following years. Since then, some efforts have been made to understand their biosynthesis, occurrence, toxicity, toxicokinetics, and finally relevance for consumers. Thus, the probably most important question is whether and how these metabolites contribute to toxicity upon hydrolysis either during food processing or the gastrointestinal passage. To answer this question, firstly, knowledge on the correct stereochemistry of T2 and HT2 glucosides is important as this affects hydrolysis and chemical behavior. So far, contradictory results have been published concerning the number and anomericity of occurring glucosides. For this reason, we set up different strategies for the synthesis of mg-amounts of T2, HT2, and T2 triol glucosides in both α and ß configuration. All synthesized glucosides were fully characterized by NMR spectroscopy as well as mass spectrometry and used as references for the analysis of naturally contaminated food samples to validate or invalidate their natural occurrence. Generally, 3-O-glucosylation was observed with two anomers of HT2 glucoside being present in contaminated oats. In contrast, only one anomer of T2 glucoside was found. The second aspect of this study addresses the stability of the glucosides during thermal food processing. Oat flour was artificially contaminated with T2 and HT2 glucosides individually and extruded at varying initial moisture content and temperature. All four glucosides appear to be more stable during food extrusion than the parent compounds with the glucosidic bond not being hydrolyzed.

Synthesis and application of a T-2 toxin imprinted polymer.

The synthesis of a T-2 toxin imprinted polymer and its application in food analysis are reported for the first time. A molecularly imprinted polymer (MIP) for the selective recognition of T-2 toxin (T-2) was synthesized by bulk polymerization. Methacrylamide and ethyleneglycol dimethacrylate were applied as functional monomer and cross-linker, respectively. Molecularly imprinted solid-phase extraction (MISPE) procedures were optimized for further application in the analysis of T-2. Scatchard plot analysis revealed that two classes of imprinted binding sites were formed in the imprinted polymer. The dissociation constant (KD) of the higher affinity binding sites was 7.0 micromol/l, while the KD of the lower affinity binding sites was 54.7 micromol/l. The performance of the MIP throughout the clean-up of spiked maize, barley and oat sample extracts was compared with the results obtained when using non-imprinted polymer, OASIS HLB and immunoaffinity columns (IAC). Depending on the food matrix and the spiked concentration, recoveries after MISPE and non-imprinted solid-phase extraction varied respectively from 60% to 73% and from 21% to 57%. Recoveries obtained after clean-up using OASIS HLB and IAC were in the range of 74-104% and 60-85%, respectively. Although highest recoveries were obtained with OASIS HLB sorbents, the designed MIP and the IAC were superior regarding selectivity, cross-reactivity, matrix effect, limits of detection (LOD) and limits of quantification (LOQ). Depending on the matrix, LOD after MISPE ranged from 0.4 microg/kg to 0.6 microg/kg and LOQ from 1.4 microg/kg to 1.9 microg/kg. LOD and LOQ after OASIS HLB clean-up varied from 0.9 microg/kg to 3.5 microg/kg and from 3.1 microg/kg to 11.7 microg/kg, respectively. The LOD and LOQ values obtained with IAC were in the range of 0.3-2.3 microg/kg and 1.0-7.7 microg/kg, respectively. Analysis of 39 naturally contaminated samples (maize, barley and oat) by liquid chromatography tandem mass spectrometry revealed that the MIP could be an excellent alternative for clean-up of contaminated food samples.