In addition to 'traditional' multi-residue and multi-contaminant multiple reaction monitoring (MRM) mass spectrometric techniques devoted to quantifying a list of targeted compounds, the global food industry requires non-targeted methods capable of detecting other possible potentially hazardous compounds. Ultra-high-performance liquid chromatography combined with a single-stage Orbitrap high-resolution mass spectrometer (UHPLC-HRMS Exactive™-Orbitrap Technology) was successfully exploited for the complete selective and quantitative determination of 33 target compounds within three major cross categories (pesticides, antibiotics and mycotoxins) in bakery matrices (specifically milk, wheat flour and mini-cakes). Resolution was set at 50 000 full width at half maximum (FWHM) to achieve the right compromise between an adequate scan speed and selectivity, allowing for the limitations related to the necessary generic sample preparation approach. An exact mass with tolerance of 5 ppm and minimum peak threshold of 10 000 units were fixed as the main identification conditions, including retention time and isotopic pattern as additional criteria devoted to greatly reducing the risk of false-positive findings. The full validation for all the target analytes was performed: linearity, intermediate repeatability and recovery (28 analytes within 70-120%) were positively assessed; furthermore, limits of quantification between 5 and 100 µg kg(-1) (with most of the analytes having a limit of detection below 6 µg kg(-1)) indicate good performance, which is compatible with almost all the regulatory needs. Naturally contaminated and fortified mini-cakes, prepared through combined use of industrial and pilot plant production lines, were analysed at two different concentration levels, obtaining good overall quantitative results and providing preliminary indications of the potential of full-scan HRMS cluster analysis. The effectiveness of this analytical approach was also tested in terms of the formulation of hypotheses for the identification of other analytes not initially targeted which can have toxicological implications (e.g. 3-acetyl-deoxynivalenol and deoxynivalenol-3-glucoside), opening a window on retrospective investigation perspectives in food safety laboratories.
Bovine β-casein is a highly polymorphic protein, with A1 and A2 representing the most frequent variants identified in Western cattle breeds. Depending on the presence of histidine or proline at position 67 of the sequence, β-casein variants can be grouped in two families: those within the A1 family (A1-like), possessing histidine, and those within the A2 family (A2-like), with proline. Upon gastrointestinal digestion, specific peptides endowed with opioid-like activity, called β-casomorphins, may be released from β-casein. Interestingly, the presence of histidine at position 67 seems to favor the release of a peptide called βcasomorphin-7, which has been proposed as a risk factor for the development of some non-communicable diseases. Based on these assumptions, the A2 milk market (i.e., a milk containing only A2 β-casein) is spreading worldwide. In the scientific community, however, the impact of A1 β-casein on human health remains a matter of debate. Aim of the present study was to develop an analytical method based on mass spectrometry to distinguish between bovine A2 milk and commercial milk, typically composed of a mixture of A1-like and A2-like β-casein. By enzymatically hydrolyzing β-casein, a specific peptide containing the critical mutation at position 67 has been identified. This peptide constituted a suitable marker to determine the presence of A1-like and/or A2-like β-casein in bovine milk. The developed method proved appropriate for the detection of βcasein in real milk samples; we estimated that contaminations of A2 milk with up to 1% commercial milk could be successfully detected.
This article was originally published with errors in Figs. 3b, 4a, b and 5, that were introduced during the production process. The correct versions are given below. The original article has been corrected. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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