Fluorescence Polarization Immunoassay for the 3 determination of T-2 and HT-2 toxins and their 4 glucosides in wheat

T-2 and HT-2 toxins and their main modified forms (T-2 glucoside and HT-2 glucoside)may co-occur in cereals and cereal-based products. A fluorescence polarization immunoassay (FPIA)was developed for the simultaneous determination of T-2 toxin, HT-2 toxin and relevant glucosides,expressed as sum. The developed FPIA, using a HT-2-specific antibody, showed high sensitivity (IC50= 2.0 ng/mL) and high cross-reactivity (100% for T-2 toxin and 80% for T-2 and HT-2 glucosides). TheFPIA has been used to develop two rapid and easy-to-use methods using two different extractionprotocols, based on the use of organic (methanol/water, 90:10, v/v) and non-organic (water) solvents,for the determination of these toxins in wheat. The two proposed methods showed analyticalperformances in terms of sensitivity (LOD 10 µg/kg) recovery (92-97%) and precision (relativestandard deviations <=13%), fulfilling the criteria for acceptability of an analytical method for thequantitative determination of T-2 and HT-2 toxins established by the European Union. Furthermore,the methods were then validated in accordance with the harmonized guidelines for the validationof screening methods included in the Regulation (EU) No. 519/2014. The satisfactory analyticalperformances, in terms of intermediate precision (<=25%), cut-off level (80 and 96 µg/kg for the twomethods) and rate of false positives (<0.1%) confirmed the applicability of the proposed methodsas screening method for assessing the content of these toxins in wheat at the EU indicative levelsreported for T-2 and HT-2 toxins.