Rapid simultaneous extraction and magnetic particle-based enzyme immunoassay for the parallel determination of ochratoxin A, fumonisin B1 and deoxynivalenol mycotoxins in cereal samples

This work describes the development of a rapid method for the extraction and the immunochemical determination of three mycotoxins, deoxynivalenol, fumonisin B1, and ochratoxin A, which are frequently found together naturally in cereal samples (wheat and corn flours). The 3 mycotoxins were extracted simultaneously in dichloromethane, and then three parallel spectrophotometric enzyme immunoassays were carried out in conventional microtiter plates using magnetic beads. Three specific monoclonal mouse antibodies for the three mycotoxins were immobilized in an oriented way using protein G functionalized magnetic particles. The magnetic immunoassay schemes decrease the incubation times, improve greatly the efficiency of the separation and washing steps, and also allow the easy elimination of interfering matrix components from the extracted samples. The method has limits of detection of 5.0 ± 1.4 ng mL−1 for FB1, 4.3 ± 1.8 ng mL−1 for DON, and 0.1 ± 0.05 ng mL−1 for OTA (n = 5), and the relative standard deviations of the determinations of the three mycotoxins are less than about 10% RSD. The method allows us to measure concentrations of these mycotoxins well below the limits of the European Union legislation in cereal samples. The developed multiplex magnetic particle-based enzyme immunoassay (mpEIA) method was validated with certified reference materials (wheat and maize flour samples) and three official AOAC (Association of Analytical Chemists) chromatographic analytical methods for each of the three mycotoxins. This method is high-throughput and accurate for the rapid determination of FB1, DON, and OTA in commercial cereal and feedstuff samples.