The development and characterisation of an immunoaffinity column used for the simultaneous selective extraction of Fusarium toxins from grain products

In the present study, a multifunctional immunoaffinity column (IAC) with the binding of three antibodies against four target analytes was developed. The aim of this work was to optimise the IAC method regarding the selective and effective extraction of Fusarium toxins from grain products and the method used to couple to antibodies. The IAC was constructed by covalently coupling the three different highly specific antibodies (to deoxynivalenol (DON), T-2 toxin/HT-2 toxin (T-2/HT-2) and zearalenone (ZEN)) onto CNBr-activated Sepharose 4B and packing them into a solid-phase extraction cartridge. The optimal amount bound to each column was selected: 1.25 mg DON per column, 0.2 mg T-2/HT-2 per column and 0.3 mg ZEN per column. The purification conditions, including the loading, washing and eluting solutions, were optimised. The values obtained for the maximum binding capacity of the IAC for DON, T-2, HT-2 and ZEN were 261, 233, 198 and 281 ng, respectively. To obtain the quantitative results for four Fusarium toxins extracted from different matrices, a liquid chromatography-mass spectrometry method was utilised. The recoveries of the four Fusarium toxins at two different spiked concentrations were in the range of 93.3-104% with a relative standard deviation (RSD) within the range of 2.41-4.84%, indicating the high purification efficiency of the columns. After eight cycles of use, the column recovery rate still remained over 85%. The method was accurate and precise (recoveries of 89.7-105% with an RSD within 3.18-10.4%), and it was applied to analyse actual flour samples.