Development of a Method for the Quantification of Caseinate Traces in Italian Commercial White Wines Based on Liquid Chromatography-Electrospray Ionization-Ion Trap-Mass Spectrometry Ilario Losito,* ,†,‡ Barbara Introna, † Linda Monaci, § Silvana Minella, † and Francesco Palmisano †,‡ † Department of Chemistry, University of Bari “Aldo Moro”, Via Orabona 4, 70126 Bari, Italy ‡ Department of Chemistry, S.M.A.R.T. Inter-department Research Center, University of Bari, Via Orabona 4, 70126 Bari, Italy § Institute of Sciences of Food Production (ISPA), Italian National Research Council (CNR), Via Amendola 122/O, 70126 Bari, Italy * S Supporting Information ABSTRACT: A method using the combination of size exclusion-solid phase extraction and ultrafiltration, followed by tryptic digestion and analysis of the protein digest by liquid chromatography-electrospray ionization-3D ion trap-mass spectrometry (LC-ESI-3D IT-MS), was developed for the detection and quantification of caseinate traces potentially resulting from fining processes in white wines. In particular, several tryptic peptides generated from the main proteins constituting caseinate (β-, α S1 -, and α S2 -caseins) were used as markers of its presence in the wine matrices; among them, the β-casein peptide GPFPIIV was found to be the best marker for quantification purposes. Method linearity and sensitivity were assessed on a series of Italian commercial white wines, first checked for the absence of any peptide signal attributable to caseins introduced during their production and subsequently spiked with increasing concentrations of caseinate, to provide samples for matrix-matched calibrations. Limits of detection ranging between 0.09 and 0.29 mg/L (S/N = 3), according to the wine, were achieved using a 10 mL sample volume and the MS signal of GPFPIIV as the response related to the caseinate concentration. Such levels are comparable or even lower than the one (0.25 mg/L) recently adopted as a threshold by European Union legislation concerning the indication of milk- and egg-derived fining agents on wine labels, that is, the most restrictive one among those currently proposed in the world. KEYWORDS: milk allergens, wine fining, caseins and caseinate, peptide markers, liquid chromatography-3D ion trap-mass spectrometry ■ INTRODUCTION The risk posed to allergic consumers by the possible presence of milk-, egg-, and fish-derived protein residues in wines has been the object of a very active debate in recent years. In fact, products containing milk- (caseinates or milk powders), egg- (ovalbumin or egg-white powders), or fish-derived (isinglass or fish gelatin) proteins are often used during wine fining. In particular, such proteins are added to wine to promote interactions with undesirable compounds, especially (poly)- phenolic ones, aiming at their partial removal through precipitation. 1 Unfortunately, the presence of protein traces in the final product cannot be excluded. To protect allergic consumers, the Australia and New Zealand Food Standards Code has required wines (as well as all other food products) to carry a declaration on their labels about the presence of renown protein allergens since 2002. 2 After some deferments, the declaration of milk- and egg-derived products on wine labels has become mandatory also in the European Union since July 1, 2012. 3 Similar regulations have also come into force in Canada since August 4, 2012. 4 Despite these legislative developments a remarkable degree of uncertainty still exists on the actual need for indicating potentially allergenic fining agents on wine labels. As an example, one of the key aspects of the new Canadian regulation is represented by the classification of a wine fining agent as either a food additive or a processing aid. In the latter case the final concentration of the agent would be, by definition, negligible; thus, no significant risk would exist for protein- allergic wine consumers and its indication on the product label would not be mandatory. However, no concentration threshold has been indicated by the Canadian authorities to enable such a distinction. On the contrary, the implementing regulation 579/ 2012 issued by the European Commission 3 provides, although not explicitly, a quantitative limit to decide about the indication of fining agents on wine labels. The limit is related to the detection of fining agents in the final product using the analytical methods referred to in Article 120g of Regulation 1234/2007, 5 that is, the methods issued by the Organisation Internationale de la Vigne et du Vin (OIV). Following an interlaboratory trial based on an enzyme-linked immunosorb- ent assay (ELISA), 6 the Criteria for the Methods of Quantif ication of Potentially Allergenic Residues of Fining Agents Proteins in Wines have been published by OIV in 2012. 7 In this Received: August 6, 2013 Revised: October 22, 2013 Accepted: November 26, 2013 Published: November 26, 2013 Article pubs.acs.org/JAFC © 2013 American Chemical Society 12436 dx.doi.org/10.1021/jf4034909 | J. Agric. Food Chem. 2013, 61, 12436-12444