**Critical Comparison of Analytical Performances of Two Immunoassay Methods for Rapid Detection of Aflatoxin M 1 in Milk**

**Ivan Pecorelli 1, Natascia Guarducci 2, Cristoph von Holst 3, Rita Bibi 1, Michelangelo Pascale 2, Biancamaria Ciasca 2, Antonio F. Logrieco 2 and Veronica M. T. Lattanzio 2,\***


Received: 26 March 2020; Accepted: 21 April 2020; Published: 22 April 2020

**Abstract:** 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 AFB1, 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-o ff 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 (RSDip) 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 O fficial 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 goa<sup>t</sup> and sheep milk was evaluated by applying the experimental design foreseen in the EU regulation.

**Keywords:** Aflatoxin M1; milk; strip test immunoassay; ELISA; method validation

**Key Contribution:** The study provides insights about the process of evaluating and comparing the performance profile of rapid methods currently applied for AFM1 screening in milk. Addressing EU o fficial guidelines, the fitness for purpose of strip test and ELISA based methods was evaluated, including verification of method performances through long-term quality control measurements and comparison with the AOAC reference method.
