Immunoanalytical sample clean-up in mycotoxin analysis

The European Commission has decided to make the improvement of food safety one of its policy priorities (White Paper on Food Safety). Since mycotoxin contamination poses a safety risk, future European legislation will lead to a large increase of the number of samples which have to be analysed for mycotoxin contamination. One of the most important steps in the analysis of mycotoxins is sample preparation prior to analysis by chromatographic methods or enzyme immunoassays. Up to now sample preparation methods, exploiting the selectivity of antibodies, are expensive since commercially available immunoaffinity columns for mycotoxins are one-way-single-use columns applicable to only one mycotoxin analyte. The purpose of this study is to apply three different strategies to reduce the cost of the analysis of mycotoxins. The mycotoxins deoxynivalenol (DON) and zearalenone (ZON) will be used as model substances. One of our strategies will be to prepare immunoaffinity columns by entrapping the antibodies in the pores of a silicate matrix using the sol-gel method. The re-usability of the sol-gel columns is a major advantage compared to the commercially available DON and ZON immunoaffinity columns. The second strategy will use immunofiltration in combination with ultrafiltration for enrichment of DON and ZON. In the past this clean-up method proved to be very simple and rapid, since in general only small amounts of non- purified, highly diluted antisera are necessary. Furthermore, this low cost method will be well suited for the analysis of a large number of samples. The third approach consists of an investigation of the possibilities of both methods to enrich DON and ZON simultaneously. In order to achieve this goal, sol-gel immunoaffinity columns will be prepared by co-immobilising DON and ZON antibodies. Immunofiltration sample extracts will be incubated with small amounts of both DON and ZON antibodies prior to ultrafiltration. Depending on the analyte and the selectivity and performance of the clean-up methods, HPLC will be applied in combination with various detection methods such as UV, fluorescence detection, mass spectrometry and coulometric electrode array detection. After development, optimisation and characterisation of the clean-up methods using DON and ZON standard solutions, the applicability will be investigated for sample pre-treatment of maize samples. Validation of the whole analytical methods will be carried out with maize reference materials. The validated methods will be applied to the analysis of DON and ZON in different food and feed matrices. The The new clean-up methods will be compared with commercially available IACs with regard to reliability of the results and cost effectiveness. Commercialisation of the sample clean-up methods will be taken into account.