Aflatoxin M1 in milk: reliability of the immunoenzymatic assay

With the aim to evaluate the reliability of enzyme immunoassays in the determination of Aflatoxin M1 content in raw and pasteurised milk samples, in comparison with the HPLC reference method, the performances of ELISA kits were tested by analysing a large number of both artificially and naturally contaminated samples. The efficiency of the aflatoxins extraction before HPLC analysis was carefully checked in Afaltoxin M1 spiked samples and by using Aflatoxin B2 added as internal standard. Recovery (mean values: 100.1% and 97.8% for aflatoxin M1 and B2, respectively), was lowered only in freezed samples. No effect was detected in case of column overloading, presence of food preservatives or acid pH values. The ELISAs gave the same values of HPLC for the CV (coefficient of variation), recovery, and regression coefficient R2 (0.9-8% and 96.8-108%, and 0.993 respectively) analysing samples containing till 70 ng L-1 of Aflatoxin M1. When the concentration rised till 100ng L-1 a slight overestimation was observed, with CV 7-21%, recovery 96.2-115%, and R2 = 0.876. Analysing a set of 600 real samples, both raw and pasteurised milk, the overestimation by ELISA was confirmed, with a R2=0.788. The evidences in this study confirmed that ELISA is a reliable alternative assay to HPLC in the routine quality control of raw or packaged milk. Regulatory rules require to confirm positive samples by HPLC, but using ELISA no one case of false-negative determination can occur.