Validation according to European Commission Decision 2002/657/EC of a confirmatory method for aflatoxin M_1 in milk based on immunoaffinity columns and high performance liquid chromatography with fluorescence detection

摘要

A high performance liquid chromatographic method with fluorimetric detection for the determination of aflatoxin M_1 (AFM_1) in milk has been optimized and validated according to Commission Decision 2002/657/EC by using the conventional validation approach. The procedure for determining selectivity, recovery, precision, decision limit (CC_(alpha)), detection capability (CC_(beta)) and ruggedness of the method has been reported. The results of the validation process demonstrate the agreement of the method with the provisions of Commission Regulation 401/2006/EC. The mean recovery calculated at three levels of fortification (0.5,1.0, and 1.5-fold the MRL) was 91 % and the maximum relative standard deviation value for the within-laboratory reproducibility was 15%. Limit of detection (LOD) and limit of quantitation (LOQ) values were 0.006 mu g kg~(-1) and 0.015 mu gkg~(-1) while the CC_(alpha) and CC_(beta) values were 0.058 mu g kg~(-1) and 0.065 mu g kg~(-1), respectively. The relative expanded measurement uncertainty of the method was 7%. The method was not affected by slight variations of some critical factors (ruggedness minor changes) as pre-treatment and clean-up of milk samples, thermal treatment and different storage conditions, as well as by major changes valued in terms of milk produced by different species (buffalo, goat and sheep). The method allowed accurate confirmation analyses of milk samples, resulted positive by the screening method. In fact, the Z-score values attained in a proficiency test round were well below the reference value of 1, proving the excellent laboratory performances.