Enhanced Automated Online Immunoaffinity Liquid Chromatography-Fluorescence Method for the Determination of Aflatoxin M-1 in Dairy Products

Background: Aflatoxin M-1 (AFM(1)) is found in the milk of cows exposed to feed spoiled by Aspergillus fungi species. These fungi may produce the secondary metabolite aflatoxin B-1, which is converted in the cow liver by hydroxylation to AFM(1) and is then expressed in milk. AFM(1) 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: AFM(1) is extracted using 1% acetic acid in acetonitrile incorporating ionic salts. The AFM(1) in the resulting extract is concentrated using an automated RIDA((R))CREST IMMUNOPREP (R) 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 AFM(1) 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 AFM(1) in whey proteins, milk powders, nutritional products, liquid milk, and yogurt. Allows for high-throughput analysis of AFM(1) with enhanced chromatographic performance. Method applicable to the analysis of AFM(1) in an extended range of milk and milk-based products.