Validation of a MS Based Proteomics Method for Milk and Egg Quantification in Cookies at the Lowest VITAL Levels: An Alternative to the Use of Precautionary Labeling

Abstract

The prevalence of food allergy has increased over the last decades and consequently the food labeling policies have improved over the time in different countries to regulate allergen presence in foods. In particular, Reg 1169 in EU mandates the labelling of 14 allergens whenever intentionally added to foods, but the inadvertent contamination by allergens still remains an uncovered topic. In order to warn consumers on the risk of cross-contamination occurring in certain categories of foods, a precautionary allergen labelling system has been put in place by food industries on a voluntary basis. In order to reduce the overuse of precautionary allergen labelling (PAL), reference doses and action limits have been proposed by the Voluntary Incidental Trace Allergen Labelling VITAL project representing a guide in this jeopardizing scenario. Development of sensitive and reliable mass spectrometry methods are therefore of paramount importance in this regard to check the contamination levels in foods. In this paper we describe the development of a time-managed multiple reaction monitoring (MRM) method based on a triple quadrupole platform for milk and egg quantification in processed food. The method was in house validated and allowed to achieve levels of proteins lower than 0.2 mg of total milk and egg proteins, respectively, in cookies, challenging the doses recommended by VITAL. The method was finally applied to cookies labeled as milk and egg-free. This method could represent, in perspective, a promising tool to be implemented along the food chain to detect even tiny amounts of allergens contaminating food commodities.

Keywords: PAL; allergens; egg; food; mass spectrometry; milk; multiple reaction monitoring; reference doses.

Figure 1

Experimental workflow for the simultaneous detection of milk and egg allergens in cookie samples.

Figure 2

Flowchart calculation for the conversion of egg and milk synthetic peptides concentration (µg_peptide/mL_extract) into total protein concentration (µg_{tot prot}/g_matrix).

Figure 3

Typical chromatograms recorded for synthetic peptides in cookie matrix (total ion current, peptide concentration level 0.166 µg/mL).

Figure 4

Typical chromatograms acquired for synthetic peptides in the cookie matrix: Extracted Ion Chromatogram XIC of quantifier transitions for most sensitive marker peptides of milk ((A), FFV = m/z 692.9→991.4; (C), TPE = m/z 623.3→572.5) along with their relevant qualifier transition ((B), FFV = m/z 692.9→920.3; (D), TPE = m/z 623.3→819.1) and egg, quantifier transitions ((E), ISQ = m/z 592.1→858.9; (G), NIP = m/z 671.6→557.9); qualifier transitions ((F), ISQ = m/z 592.1→778.5; (H), NIP = m/z 671.6→1114.9) at a level of 0.0125 µg/mL.