A metrologically traceable approach for quantifying proteinaceous fining agents by HPLC-HRMS

Residual proteins from fining agents such as egg albumen and animal gelatin may persist in wine, raising food safety and labelling concerns. This study develops a metrologically traceable HPLC-HRMS workflow for the absolute quantification of proteotypic peptides from ovalbumin, ovotransferrin, lysozyme, and collagen α1/α2, using isotope-dilution mass spectrometry in parallel reaction monitoring mode. Method development, calibration hierarchy, and uncertainty evaluation were fully established in ammonium bicarbonate buffer, achieving good calibration linearity (chi-square values below 1) and LOQs of 0.63 to 1.16 mg/L. Wine was evaluated only as a post-extraction matrix effect, through matrix-matched calibration in a reference red wine. Expanded uncertainties were comparable between buffer (28.6-36.6%) and wine extract (27.8-36.2%), indicating that matrix effects had limited impact despite peptide-dependent signal suppression. This framework offers a solid, SI-traceable analytical foundation, established in buffer, for future validation in fortified wine samples, supporting quantitative allergen assessment in winemaking.