A class-selective immunoassay for simultaneous analysis of anilinopyrimidine fungicides using a rationally designed hapten

Abstract

The development of multianalyte immunoassays constitutes a main research issue in the field of bioanalytical techniques. In the present study, class-specific antibodies against the three members of the anilinopyrimidine family of fungicides (pyrimethanil, cyprodinil and mepanipyrim) were raised by using a bioconjugate of a rationally designed hapten [5-(6-methyl-2-(phenylamino)pyrimidin-4-yl)pentanoic acid]. Highly sensitive immunoassays were developed for the generic determination of these compounds, using the competitive enzyme-linked immunosorbent assay (ELISA). Particularly, a direct antibody-coated competitive ELISA afforded identical sensitivity for the three anilinopyrimidines, with IC₅₀ values of 0.26, 0.27 and 0.25 μg L⁻¹ for pyrimethanil, cyprodinil and mepanipyrim, respectively. This immunoassay was fully characterized and applied to the multianalyte determination of anilinopyrimidine fungicides in white and red wines, with a limit of quantification of 1 μg L⁻¹, average recoveries from 93.1 to 114.4%, and relative standard deviations lower than 20%. Commercial wine samples were analyzed and those containing detectable anilinopyrimide residues were verified by a reference chromatographic technique.