Valeria Vita

The aim of this study was to estimate the aflatoxin M₁ (AFM₁) 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 (AFM₁ concentration higher than 0.042 µg/kg) were then analyzed by high performance liquid chromatographic with fluorimetric detection (HPLC/FLD) confirmation method. AFM₁ concentration ≥ 0.005 µg/kg (ELISA limit of quantitation) was detected in 553 of the 1017 milk samples (54.4%). AFM₁ levels exceeding the European Union maximum limit (ML) of 0.050 µg/kg were detected in 70 samples, 49 of which were determined as non-compliant samples (4.8%). Particularly high concentrations of AFM₁, exceeding 200 µg/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.

Mycotoxin contamination of animal feed is a complex issue in both animal wellness and food safety. The most diffused mycotoxins subject to the official control of animal feed are Aflatoxin B1 (AF), Zearalenone (ZEA), Deoxynivalenol (DON), Ochratoxin A (OCRA), Fumonisins (FUMO), and T-2/HT-2 toxins. This work describes the results of five years of monitoring focused on the evaluation of mycotoxin contamination of animal feed. Analytical determinations were carried out by means of accredited ELISA. The obtained results showed a non-alarming scenario, with several samples resulting as “non-compliant” according to the Maximum Residue Limits (MRLs) set in European Regulation No. 574/2011. Out of 722 analyzed samples coming from 2 Italian regions, Apulia and Basilicata, 14 samples were characterized by mycotoxin concentrations higher than related MRL; in particular, 5, 4, and 5 non-compliant samples for DON, AF, and ZEA, respectively. This study also evaluated the possible correlations between mycotoxin type and feed use with a special focus on animal sensitivity to mycotoxins.

Nitrite and nitrate levels in vegetables are a matter of concern due to their toxicity at high levels and nitrate high accumulation. Moreover, there is a lack of knowledge about their levels in some types of widely consumed vegetables such as chard and rocket. In this study, 124 Swiss chard and wild rocket samples were analyzed for determining nitrite and nitrate using validated and accredited analytical methods by ion chromatography with conductivity detection. High nitrite concentrations, up to 219.5 mg kg⁻¹ f.w., were detected in one Swiss chard and three wild rocket samples. One Margin of Safety (MoS) value was −1) was slightly higher than those reported in the literature for spinach and lettuce. No MoS was <1, but 83% of values were <100. Nitrate concentrations higher than the legal limit were quantified in 11 rucola samples. The verification of 25% of wild rocket samples with nitrate concentration higher than the legal limit confirmed the need for official control. This study also suggests the introduction of legal limits for nitrite/nitrate in Swiss chard and nitrite in wild rocket.

Under the name of lipophilic marine toxins, there are included more than 1000 toxic secondary metabolites, produced by phytoplankton, with the common chemical property of lipophilicity. Due to toxicological effects and geographical distribution, in European legislation relevant compounds are regulated, and their determination is accomplished with the reference liquid chromatography-tandem mass spectrometry method. In this study a modified ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed for the identification and quantification of EU-regulated lipophilic toxins. The method optimization included a refinement of SPE-C18 clean-up, in order to reduce matrix interferences. Improved LC conditions and upgraded chromatographic ammonia-based gradient ensured the best separation of all analytes and, in particular, of the two structural isomers (OA and DTX2). Also, different MS parameters were tested, and confirmation criteria finally established. The validation studies confirmed that all parameters were satisfactory. The requirements for precision (RSD% < 11.8% for each compound), trueness (recoveries from 73 to 101%) and sensitivity (limits of quantification in the range 3–8 µg kg⁻¹) were fulfilled. The matrix effect, ranging from −9 to 19%, allowed the use of a calibration curve in solvent (3–320 µg kg⁻¹ in matrix) for quantification of real samples. Method relative uncertainty ranged from 12 to 20.3%. Additionally, a total of 1000 shellfish samples was analysed, providing a first preliminary surveillance study that may contribute to the knowledge of lipophilic marine toxins contamination. Increase in algae proliferation events and intoxication cases, EFSA suggestions for modification of maximum permitted levels and toxicity equivalency factors, and new studies of important toxic effects underline that implementation of reference methods still represents an important task for health and food safety laboratories.

T-2 and HT-2 toxins are secondary metabolites of various species of Fusarium. These molecules can have high potential toxic effects for human and animal health. In this work, ELISA and ultra performance liquid chromatography with fluorescence detection (UPLC/FLD) were implemented and validated as screening and confirmatory tests for the detection of these two toxins in cereal samples. The developed methods were tested by analyzing 100 samples of cereals by ELISA screening for reducing costs and analysis time and then using UPLC/FLD for confirmation purposes. Both methods met the performance criteria for sensitivity, linearity, selectivity, precision, and ruggedness, as reported in the European Decision No. 2002/657/EC and in Regulation (EC) No. 401/2006. The correlation between ELISA and UPLC/FLD approaches showed good results (r = 0.9056), confirming that these two techniques should be considered to be complementary in the official control activities of cereal and derived products.