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The fate of aflatoxin M1 (AFM1) in cow's milk cheese produced from naturally contaminated raw milk was evaluated. Cow's milk, naturally contaminated by AFM1 at levels ranging from 0.020 to 0.148 μg/kg, was used to manufacture a semihard, ripened Caciotta cheese. The cheeses, produced in eight cheesemaking trials, were ripened for 45 days. The enrichment factor (EF) of cheese did not change during maturation from day 7 to day 45 of ripening. The EF of the aflatoxin was established to evaluate the maximum level of contaminants in cheese, according to Commission Regulation (EC) No. 1881/2006. The EF factor in cheese at 45 days of ripening was between 4.68 and 5.78, with a mean value of 5.16. Moreover, this factor was not affected by the AFM1 level in milk. Pearson's correlation analysis showed a significant negative correlation between EF and cheese yield (r = −0.811). The regression analysis confirmed that the cheese yield and, subsequently, the milk composition, had a marked impact on EF (P < 0.0003). Cheese yield was identified as potentially influencing the presence and concentration of the toxin in cheese, demonstrating that this aspect, along with the cheese hardness category, contributes significantly to an exact definition of the EF. HIGHLIGHTS

Twelve different approaches commonly used for the simultaneous LC tandem MS (MS/MS) determination of mycotoxins (deoxynivalenol, aflatoxins, ochratoxin A, T-2 and HT-2 toxins, fumonisins, and zearalenone) were tested in cereals and feed materials. They comprised different extraction solvents, types of cleanup [solid-phase extraction, QuEChERS, and immunoaffinity (IMA)], and calibration approaches (external or matrix-matched). The percentage of mycotoxins with acceptable recovery, according to Regulation (EC) No. 401/2006, ranged from 9 to 100%. The approach giving the highest percentage of acceptable results was selected and further tested for corn, rice, and feed spiked at three different mycotoxin levels (low, medium, and high). The method is based on extraction with MeOH-water (70 + 30, v/v) and cleanup with two multiantibody IMA columns. For corn and rice spiked at low mycotoxin levels, a significant matrix effect was observed and was compensated by using 13C calibration. At higher mycotoxin levels (medium and high), matrix effects were negligible as no significant differences were observed for the majority of recovery results calculated by 13C calibration and external calibration. Although the proposed method still needs improvement in terms of accuracy and, to a lesser extent, precision, it was successfully tested with four proficiency tests in buckwheat, corn, rice, and feed, giving acceptable z-scores for 97% (34 out of 35) of results.

Aflatoxin M1 (AFM1) is a hepatocarcinogenic and genotoxic derivative of aflatoxin B1 excreted into milk after ingestion of feed contaminated by Aspergillus genus fungi. Because of the important role of dairy products, especially cow cheese, in the human diet, there is great concern about the presence of AFM1 in this food category. EC Regulation No. 1881/2006 establishes the importance of the enrichment factor (EF), an essential parameter that must be defined in order to evaluate the maximum level of the toxin in cheese aiming to ensure that cheese has been produced from compliant milk. The Italian Ministry of Health has established two provisional AFM1 EFs (5.5 and 3.0) to be applied to as many cheese categories (hard and soft), defined according to the moisture content on a fat free basis (MFFB) classification. Two experimental productions of Primosale and Fior di Latte cheese, both belonging to the soft cheese category, showed an EF of 4.1 and 2.9 respectively. Data in literature also suggest that the EF attribution based on the current categorization may need reconsideration.