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

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.