Tailoring molecularly imprinted polymer beads for alternariol recognition and analysis by a screening with mycotoxin surrogates

Abstract

Molecularly imprinted porous polymer microspheres have been prepared for selective binding of alternariol (AOH), a phenolic mycotoxin produced by Alternaria fungi. In order to lead the synthesis of recognition materials, four original AOH surrogates have been designed, prepared and characterized. They bear different number of phenol groups in various positions and different degree of O-methylation on the dibenzo[b,d]pyran-6-one skeleton. A comprehensive library of mixtures of basic, acidic or neutral monomers, with divinylbenzene or ethyleneglycol dimethacrylate as cross-linkers, were polymerized at a small scale in the presence of the four molecular mimics of the toxin molecule. This polymer screening has allowed selection of the optimal composition of the microbeads (N-(2-aminoethyl)methacrylamide, EAMA, and ethylene glycol dimethacrylate). The latter are able to bind AOH in water–acetonitrile (80:20, v/v) with an affinity constant of 109±10mM−1 and a total number of binding sites of 35±2μmolg−1, being alternariol monomethylether the only competitor species. Moreover, 1H NMR titrations have unveiled a 1:2 surrogate-to-EAMA stoichiometry, the exact interaction sites and a binding constant of 1.5×104M−2. A molecularly imprinted solid phase extraction (MISPE) method has been optimized for selective isolation of the mycotoxin from aqueous samples upon a discriminating wash with 3mL of acetonitrile/water (20:80, v/v) followed by determination by HPLC with fluorescence detection. The method has been applied, in combination to ultrasound-assisted extraction, to the analysis of AOH in tomato samples fortified with the mycotoxin at five concentration levels (33–110μgkg−1), with recoveries in the range of 81–103% (RSD n=6). To the best of our knowledge, this is the first imprinted material capable of molecularly recognizing this widespread food contaminant.