Critical Comparison of Analytical Performances of Two Immunoassay Methods for Rapid Detection of Aflatoxin M₁ in Milk

Aflatoxin B₁ (AFB₁) is a secondary metabolite produced by some Aspergillus spp. fungi affecting many crops and feed materials. Aflatoxin M₁ (AFM₁), the 4-hydroxylated metabolite of AFB_1, is the main AFB₁-related compound present in milk, and it is categorized by the International Agency for Research on Cancer (IARC) as a "group 1 human carcinogen". The aim of this work was to evaluate and compare the analytical performances of two commercial immunoassays widely applied for the detection of AFM₁ in milk, namely strip test immunoassay and enzyme linked immunosorbent assay (ELISA). Assay validation included samples at AFM₁ levels of 25, 50, 75 ng/kg and blank samples (AFM₁ < 0.5 ng/kg). With respect to a screening target concentration (STC) of 50 ng/kg the two assays showed cut-off values of 37.7 ng/kg and 47.5 ng/kg for strip test and ELISA, respectively, a false suspect rate for blanks <0.1% (for both assays) and a false negative rate for samples containing AFM₁ at levels higher than STC, of 0.4% (for both assays). The intermediate precision (RSD_ip) was <32% for the strip test and <15% for the ELISA. Method verification through long-term intra-laboratory quality control (QC) measurements confirmed the results from the validation study. Furthermore, a satisfactory correlation of the results obtained with both immunoassays and the AOAC Official Method 2000.08 was obtained for the analysis of cow milk samples naturally contaminated with AFM₁ at levels within "not detected" (< 0.5 ng/kg) and 50 ng/kg. Finally, the extension of the scope of the strip test method to goat and sheep milk was evaluated by applying the experimental design foreseen in the EU regulation.