###### Abstract  
The aim of this study was to estimate the aflatoxin M\({}_{1}\) (AFM\({}_{1}\)) contamination in raw milk and processed milk (pasteurized or UHT) collected from two regions in Italy (Puglia and Basilicata) during a 12-year period: 2012-2023. A total of 1017 milk samples were analyzed first proceeding with screening analysis by enzyme-linked immunosorbent assay (ELISA), and suspected non-compliant samples (AFM\({}_{1}\) concentration higher than 0.042 \(\mu\)g/kg) were then analyzed by high performance liquid chromatographic with fluorimetric detection (HPLC/FLD) confirmation method. AFM\({}_{1}\) concentration \(\geq\) 0.005 \(\mu\)g/kg (ELISA limit of quantitation) was detected in 553 of the 1017 milk samples (54.4%). AFM\({}_{1}\) levels exceeding the European Union maximum limit (ML) of 0.050 \(\mu\)g/kg were detected in 70 samples, 49 of which were determined as non-compliant samples (4.8%). Particularly high concentrations of AFM\({}_{1}\), exceeding 200 \(\mu\)g/kg, were found in four samples, three raw milk and one pasteurized. Regarding this risk exposure study, only the MOE values obtained under \"high exposure scenario\" were lower than 10,000, while those calculated from the overall mean values resulted as not of concern. affatoxin M\({}_{1}\); ELISA; food safety; HPLC/FLD; milk; survey +
Footnote †: journal: Applied Sciences  
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In farm animals, chronic exposure to AFs can reduce performance, alter liver function, compromise immune function, and increase disease susceptibility [9]. The susceptibility of both humans and animals can vary with regard to many factors such as age, exposure length, species, nutrition, etc. It is also worth noting another significant variable which is the possible co-occurrence of different mycotoxins in food and feed (synergistic action) [10]. There are more than 20 known AFs, but regarding food safety the most relevant ones are aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), and aflatoxin M1 (AFM1) [11; 12]. AFM1 is a 4-hydroxylated derivate of aflatoxin B1 (AFB1), formed in the liver by the group of cytochrome P450 (CYP450) enzymes, so it can be found in the milk of cattle fed with AFB1-contaminated feed. A percentage in the range 0.3-6.2% of AFB1 in feed is converted to AFM1 [13]. This percentage depends on the genetic features of the animals, breed, period of lactation, and environmental factors [14; 15]. In dairy cows, the excretion takes 12-24 h after AFB1 intake, and the depruration interval is about 2-3 days from feeding with AFB1-free feed [16; 17; 18]. Regarding human health, the International Agency for Research on Cancer (IARC) has classified AFB1 as a human carcinogen (Group 1), while its metabolite AFM1 is considered less toxic and has been classified as a possible human carcinogen, Group 2B [19; 20]. The liver is the main target organ, as demonstrated by several studies conducted on aflat toxicity [21] which demonstrated that high AFB1 intake can be associated with increased incidence of hepatocellular carcinoma, liver cirrhosis, and human liver cancer, other than immunosuppressive effects [22; 23; 24]. AFM1 occurrence in milk and dairy products poses a concern for the global population, particularly for infants and children who are more susceptible to adverse effects because of high exposure (low body weight) and lower detoxification capability [25]. Taking into account the milk consumption, the AFM1 toxicity, and its heat stability (pasteurization 72-75 \({}^{\circ}\)C or ultra-high temperature processing 120-150 \({}^{\circ}\)C do not significantly reduce or inactivate AFM1) [26], the maximum level (ML) in milk set in Europeis 0.050 \(\mathrm{\SIUnitSymbolMicro g}\)/kg in raw and heat-treated milk and 0.025 \(\mathrm{\SIUnitSymbolMicro g}\)/kg for infant formulae and milk products for infants [27].