Immunochemical approach for zearalenone-4-glucoside determination.

Zearalenone-4-β-D-glucopyranoside (zearalenone-4-glucoside) detection techniques, based on a combination of acidic or enzymatic hydrolysis of the masked mycotoxin to the parent form (i.e. zearalenone), and immunochemical determination of zearalenone-4-glucoside as a difference between the zearalenone concentration after and before cleavage of the glycosidic bond were developed. The limit of detection for zearalenone-4-glucoside, achieved for the enzyme linked immunosorbent assay, was 3 μg kg(-1); the cut-off level for the sum of zearalenone and zearalenone-4-glucoside determination by a qualitative gel-based immunoassay was 50 μg kg(-1). Trifluoromethanesulfonic acid was checked for acidic hydrolysis and resulted in approximately 70% of glycosidic bond cleavage in optimal conditions. Seven different glycoside hydrolases were tested during the design of the enzymatic hydrolysis technique. Enzymatic hydrolysis combined with enzyme linked immunosorbent assay and gel-based immunoassay determinations was applied for the determination of zearalenone-4-glucoside or the sum of zearalenone and zearalenone-4-glucoside in cereal samples. The chosen enzyme (glucosidase from Aspergillus niger) allowed to cleave 102% of zearalenone-4-glucoside in standard solutions and 85% in cereal samples. Liquid chromatography coupled to tandem mass spectrometry was used as confirmatory method. As a result, good correlations between immunochemical techniques and the chromatographic data were obtained. The developed technique is suitable for simultaneous immunochemical determination of zearalenone and its masked form, zearalenone-4-glucoside.