Development and validation of HPLC-FLD method for aflatoxin M1 determination in milk and dairy products

Abstract

Abstract Milk and dairy products are the most consumed foods in human diet and their safety is in the attention centre of control authorities. Aflatoxin M1 (AFM1) is a dangerous toxin that can occur in milk and dairy products as a metabolite formed from aflatoxin B1 contained in contaminated animal feed. Therefore, the aim of this study was to develop and validate a reliable method for the determination of AFM1 content in milk and dairy products based on HPLC with fluorescence detection employing immunoaffinity columns (IAC) pre-treatment. Optimal chromatographic separation of AFM1 was achieved using a water/acetonitrile mixture (80/20, v/v) as a mobile phase, column with C18 stationary phase maintained at 25 °C, and fluorescence detection at excitation wavelengths of 360 nm and emission of 440 nm. Efficacy of AFM1 extraction from the samples was found to be influenced by the elution agent composition. The best results were obtained using 1.25 mL of acetonitrile/methanol (3/2, v/v) and 1.25 mL of water. Validation parameters of the proposed method met the criteria set by the European legislation with the limits of detection and quantification at 0.002 and 0.007 µg/kg, respectively. Also, suitability of the method was confirmed by its application for AFM1 determination in certified reference material. Finally, the method was applied for AFM1 determination in 25 milk and dairy products collected in Slovakia; the AFM1 content was below the limit of quantification. It was concluded that the method is suitable for AFM1 content monitoring in milk and dairy products.