A Confirmatory Method for Aflatoxin M1 Determination in Milk Based on Immunoaffinity Cleanup and High-Performance Liquid Chromatography with Fluoromet

A sensitive and reliable analytical method based on immunoaffinity chromatography cleanup followed by HPLC separation and fluorimetric detection is described for the quantitative determination of aflatoxin M₁ in milk. The chromatographic separation is accomplished by using a C18 column and a gradient elution with methanol, acetonitrile, and water. No extraction solvent process is required and a minimal milk sample cleanup is performed by a direct loading of the immunoaffinity columns and elution with methanol. The method has been successfully validated according to Decision EC No 657/2002 by using the conventional validation approach. The results of the validation process demonstrate the agreement of the method with the provisions of Regulation EC No 401/2006.