Multi-detection of mycotoxins by membrane based flow-through immunoassay

•A membrane-based flow-through immunoassay for rapid mycotoxins detection.•Simultaneous multi-detection of ochratoxin A, zearalenone and fumonisin B1.•Visual estimation of the presence of three mycotoxins in cereal grains and silage.•The cut-off limit of test have been set at half of EU legislative limits.

A membrane-based flow-through immunoassay for rapid non-instrumental multi-detection of ochratoxin A (OTA), zearalenone (ZEN) and fumonisin B1 (FB1) in cereal grains and silage was developed. The test is based on a direct competitive enzyme immunoassay performed on a membrane. Alkaline phosphatase was used as enzyme label to decrease matrix interference. The analytical method involved a fast extraction procedure (extraction with a mixture of methanol, water and acetic acid 79:20:1, v/v) followed by a multi-detection. The immunotest allows the visual estimation of the presence of three mycotoxins in less than 15 min. The cut-off limit of this test have been set at a value not higher than half of EU legislative limits (2.5, 50 and 1000 μg kg−1 for OTA, ZEN and FB1 correspondingly in wheat; 2.5, 100 and 1000 μg kg−1 in maize; 25, 125 and 2500 μg kg−1 in silage). Evaluation and validation studies have been performed using spiked and naturally contaminated samples and results were compared with LC-MS/MS. Also the possibility of analyte detection in wheat at two concentration levels by the immunotest was demonstrated with OTA and ZEN as an example. In spite of the semiquantitative-qualitative character of the test, the results demonstrate that the test provides a very good estimation of the mycotoxins concentration in wheat, maize and silage. This test format could be adapted to the multi-detection of other contaminants in food and feed.