Enzyme-assisted extraction for the HPLC determination of aflatoxin M1 in cheese

The extraction of aflatoxin M1 (AFM1) from cheese is generally carried out using chlorinated organic solvents. In this study, two innovative methods were developed, based on an enzyme-assisted (EA) extraction using proteolytic enzymes (pepsin or pepsin–pancreatin). After purification through an immunoaffinity column, AFM1 is determined by HPLC–FLD. A comparison between the new EA methods and an established chloroform (CH) method was carried out on 24 cheese samples. The results showed that the extraction efficiency of the EA methods was independent of ripening time of cheese, whereas the CH method was not able to fully recover AFM1 from ripened cheeses. The simpler (pepsin) of the two methods has been adopted by our laboratory for routine analysis of AFM1 in cheese. In comparison with the CH method, the pepsin-HCl (P-HCl) method is simpler, avoiding solvent evaporation, dissolution and partition in a separating funnel; moreover, it gives higher recoveries, comparable LOD and LOQ and more accurate results.