Reference LC-MS/MS method to detect fresh cheeses adulteration with whey

This study evaluated the potential of a reference method to detect fresh cheeses adulteration with whey by liquid chromatography coupled to mass spectrometry (LC-MS/MS). Qualitative results were expressed as presence or absence of the marker peptide TPEVDDEALEK, obtained by tryptic hydrolysis of β-lactoglobulin. Sample preparation was based on defatting with cold acetone and protein solubilization in ammonium bicarbonate and urea buffer (pH = 8.0). Reversed phase liquid chromatography used a C18 column for separation of the analyte, whose retention time was 4.12 min. Mass spectrometry was carried out with positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode for the precursor ion (m/z 624) and the quantitation (m/z 573) and confirmation transitions (m/z 820; m/z 920) of the peptide. Method validation was carried out in quantitative terms, to set the baseline concentration of the marker peptide in 69 authentic samples, and in qualitative terms, to set the action level that distinguish authentic from adulterated cheeses (350 mg kg−1). Sensitivity was enough to detect cheeses with 10% adulteration and the detection limit was set to 21 mg kg−1. The simple extraction procedure allowed high-throughput analysis of 33 real samples. Results were compared to SDS-PAGE electrophoresis, whose limitations for accurate quantitation were easily overcome by LC-MS/MS. The developed method ensured precision, accuracy, sensitivity, and specificity needed for the unequivocal detection of non-compliant cheeses made with cow or buffalo milk, without dealing with the highly toxic chemical species required for SDS-PAGE. This method can be extended in the future to detect similar adulterations in fresh cheeses prepared with milk from other animal species, as well as in other dairy products.