Co-Occurrence of Aflatoxin B1, Zearalenone and Ochratoxin A in Feed and Feed Materials in Central Italy from 2018 to 2022.

Mycotoxin contamination of feed and feed materials represent a serious health hazard. This study details the occurrence of aflatoxin B1 (AFB1), zearalenone (ZEN) and ochratoxin A (OTA) in 826 feed and 617 feed material samples, collected in two Italian Regions (Umbria and Marche) from 2018 to 2022 analyzed using a UPLC-FLD platform. The developed method was validated and accredited (ISO/IEC 17025) with satisfactory accuracy and precision data obtained in repeatability and intralaboratory reproducibility conditions. Feed had a higher incidence of contaminated samples (26%) with respect to feed materials (6%). AFB1 was found up to 0.1045 mg/kg in cattle feeds and 0.1234 mg/kg in maize; ZEN was detected up to 6.420 mg/kg in sheep feed while OTA was rarely reported and in lower concentrations (up to 0.085 mg/kg). Co-contamination of at least two mycotoxins was reported in 0.8% of the analyzed samples. The incidence of above maximum content/guidance level samples was 2% for feed and feed materials while almost 3-fold-higher for maize (5.8%) suggesting how mycotoxin contamination can affect some matrices more than others. Obtained data can be useful to improve official monitoring plans and therefore further raise awareness of this issue between agriculture stakeholders, healthcare entities and non-professionals.

Monitoring Alternaria toxins in Italian food to support upcoming regulation.

The collection of occurrence data on Alternaria toxins in food and feed across the European countries is required since 2012 by the European Commission, endorsing the relevant scientific opinion by the EFSA CONTAM Panel. Within this framework, occurrence data for Alternaria toxins (Alternariol, Alternariol monomethyl ether, Tenuazonic acid, Tentoxin, and Altenuene) in 97 samples of cereal foods, tomato products, and sunflower seeds have been provided as requested by the Italian national monitoring programme (years 2017-2020). To this purpose, an LC-MS/MS method was set up and validated, obtaining fit for purpose sensitivity, recoveries (70-120%), repeatability (≤20%) and within laboratory reproducibility (≤26%). Occurrence data showed that oilseeds were the most contaminated food group with levels of Tenuazonic acid up to 16752 µg/kg and Tentoxin up to 570 µg/kg, whereas for the other mycotoxin/commodities combinations, the percentage of left censored data (below the limit of quantification) ranged from 74 to 100%.

Evaluation of Aflatoxin M1 enrichment factor in different cow milk cheese hardness category.

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.

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 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-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 (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 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.

Multimycotoxin Analysis by LC-MS/MS in Cereal Food and Feed: Comparison of Different Approaches for Extraction, Purification, and Calibration.

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.

Development and validation of LC-MS/MS method for the determination of Ochratoxin A and its metabolite Ochratoxin α in poultry tissues and eggs.

The objective of this study was to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of Ochratoxin A (OTA) and Ochratoxin α (OTα) in poultry tissues and eggs. The two toxins were extracted by a mixture of acetonitrile/water, purified with a reversed phase C18 solid phase extraction column (SPE) and determined by LC-MS/MS. The LC-MS/MS method performances were evaluated in terms of linearity in solvent and in matrix (ranged from 0.5 to 15.10 µg L-1 for OTA and from 0.60 to 17.85 µg L-1 for OTα), limit of detection (LOD), limit of quantitation (LOQ), specificity, accuracy and precision in repeatability conditions. Recovery experiments were performed by spiking poultry liver, kidney, muscle and eggs around 1 µg kg-1 and 10 µg kg-1. LODs were 0.27 and 0.26 µg kg-1 while LOQs were fixed at 1.0 and 1.2 µg kg-1 for OTA and OTα, respectively. Main recoveries for OTA ranged from 82 to 109% and for OTα ranged from 55 to 89%. The values of within-laboratory relative standard deviation (RSDr) were equal to or below 20%. Considering the results obtained and that all analytical performance criteria were fulfilled, the new extraction and purification method developed for OTA and OTα determination in animal tissues and eggs was found appropriate for control laboratories and research activities designed to ensure food safety.

Validation of a confirmatory method for the determination of sulphonamides in muscle according to the European Union regulation 2002/657/EC.

A simple multiresidue method is described for assaying 10 sulphonamides (SAs) (sulfadiazine, sulfathiazole, sulfapyridine, sulfamerazine, sulfamethazine, sulfamonomethoxine, sulfachlorpyridazine, sulfamethoxazole, sulfaquinoxaline and sulfadimethoxine) in muscle samples. Samples were prepared by homogenizing the tissue, extracting with ethyl acetate and cleaning up with a cation-exchange solid-phase extraction (SPE) column. The detection of analytes was achieved by HPLC-diode array detection (DAD) at 270 nm. The procedure was validated according to the European Union regulation 2002/657/EC determining specificity, decision limit, detection capability, trueness and precision. The results of validation process demonstrate that the method is suitable for application in European Union statutory veterinary drug residue surveillance programmes.