title: Towards the quantification of residual milk allergens in caseinate-fined white wines using HPLC coupled with single-stage Orbitrap mass spectrometry.
abstract: A method based on LC-ESI-high-resolution (HR)-MS analysis, using a single-stage Orbitrap mass spectrometer, was developed for the quantification of casein allergens potentially present in white wines as a result of fining by caseinate. The method consists of (1) extraction from the matrix by ultrafiltration, (2) digestion with trypsin and (3) detection/quantification of residual caseins, obtained by monitoring the LC-MS response of representative tryptic peptides (peak areas in extracted-ion chromatograms). Method linearity was assessed first on caseinate solutions prepared either in water or in wine matrix (the ultrafiltration residue of a protein-free white wine). Limits of detection (LOD) ranged from 0.1 to 0.3 µg ml(-1) (S/N = 3) in water, and between 0.15 and 0.7 µg ml(-1) in wine matrix, depending on the selected peptide. Method repeatability and reproducibility, measured as response variability (standard deviation) due to LC-MS analysis alone and to both enzymatic digestion and LC-MS analysis, were assessed on caseinate standard solutions in water and ranged from 5 to 12% and from 8 to 20%, respectively. A higher variability was usually observed for the peptide marker response in the case of matrix-matched samples, the only exception being peptide GPFPIIV from β-casein, the marker also providing the highest sensitivity. The method was finally applied to a casein-free white wine ('Greco di Tufo') fined with caseinate at different concentrations, after discarding the precipitate due to casein-wine components aggregation. Minimum detectable added caseinate concentrations (i.e. those corresponding to responses with S/N = 3) were estimated between 39 and 51 µg ml(-1), according to the peptide marker chosen. These limits are compatible with caseinate concentrations typically adopted for wine-fining purposes. Moreover, a cross-check with the calibration performed in wine matrix led to an estimation of the concentration of dissolved caseinate to be in the low ng ml(-1) range.
authors.affiliation: Institute of Sciences of Food Production, National Research Council of Italy (ISPA-CNR), Bari, Italy. Linda.monaci@ispa.cnr.it
authors.forename: L
authors.initials: L
authors.lastname: Monaci
authors.forename: I
authors.initials: I
authors.lastname: Losito
authors.forename: F
authors.initials: F
authors.lastname: Palmisano
authors.forename: M
authors.initials: M
authors.lastname: Godula
authors.forename: A
authors.initials: A
authors.lastname: Visconti
mesh_terms: D000818:Animals; D000941:Antigens / Q000032:analysis*; D002138:Calibration; D002364:Caseins / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D057230:Limit of Detection; D008892:Milk / Q000276:immunology*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014462:Ultrafiltration; D014920:Wine / Q000032:analysis*
chemical_list: D000941:Antigens; D002364:Caseins