Enhanced Automated Online Immunoaffinity Liquid Chromatography–Fluorescence Method for the Determination of Aflatoxin M1 in Dairy Products

Abstract

Abstract Background Aflatoxin M1 (AFM1) is found in the milk of cows exposed to feed spoiled by Aspergillus fungi species. These fungi may produce the secondary metabolite aflatoxin B1, which is converted in the cow liver by hydroxylation to AFM1 and is then expressed in milk. AFM1 is regulated in milk and other dairy products because it can cause serious health issues, such as liver and kidney cancers, in humans and is an immunosuppressant. Objective To optimize the chromatographic protocol and to extend the matrix scope to include a wider range of dairy products: whey powder, whey protein concentrate, whey protein isolate, liquid milk, skim milk powder, whole milk powder, adult nutritional products, and yogurt. Methods AFM1 is extracted using 1% acetic acid in acetonitrile incorporating ionic salts. The AFM1 in the resulting extract is concentrated using an automated RIDA®CREST IMMUNOPREP® online cartridge coupled to quantification by HPLC–fluorescence. Results The method was shown to be accurate, with acceptable recovery (81.2–97.1%) from spiked samples. Acceptable precision was confirmed, with a relative standard deviation (RSD) for repeatability of 6.6–11.2% and an RSD for intermediate precision of 7.5–16.7%. Method LOD and robustness experiments further demonstrated the suitability of this method for routine compliance testing. Analysis of an international proficiency trial sample generated results that were comparable with the value assigned from alternative independent methods. Conclusion A method with improved chromatography for high-throughput, routine testing of AFM1 in an extended range of dairy products is described. The method was subjected to single-laboratory validation and was found to be accurate, precise, and fit for purpose. Highlights Single-laboratory validation of an automated online immunoaffinity cleanup fluorescence HPLC method for AFM1 in whey proteins, milk powders, nutritional products, liquid milk, and yogurt. Allows for high-throughput analysis of AFM1 with enhanced chromatographic performance. Method applicable to the analysis of AFM1 in an extended range of milk and milk-based products.