Development and Validation of a Method for the Quantification of Milk Proteins in Food Products Based on Liquid Chromatography with Mass Spectrometric Detection

Abstract

The protection of allergic consumers is crucial to the food industry. Therefore, accurate methods for the detection of food allergens are required. Targeted detection of selected molecules by MS combines high selectivity with accurate quantifcation. A confrmatory method based on LC/selected reaction monitoring (SRM)-MS/MS was established and validated for the quantifcation of milk traces in food. Tryptic peptides of the major milk proteins β-lactoglobulin, β-casein, αS2-casein, and κ-casein were selected as quantitative markers. Precise quantifcation was achieved using internal standard peptides containing isotopically labeled amino acids. For each peptide, qualifer and quantifer fragments were selected according to Commission Decision 2002/657/EC. A simple sample preparation method was established without immunoaffnity or SPE enrichment steps for food matrixes containing different amounts of protein, such as baby food, breakfast cereals, infant formula, and cereals. Intermediate reproducibility, repeatability, accuracy, and measurement uncertainty were determined for each matrix. LOD values of 0.2–0.5 mg/kg, e.g., for β-lactoglobulin, were comparable to those obtained with ELISA kits. An LOQ of approximately 5 mg/kg, expressed as mass fraction skim milk powder, was validated in protein-rich infant cereals. The obtained validation data show that the described LC/SRM-MS/MS approach can serve as a confrmatory method for the determination of milk traces in selected food matrixes.

Proteomics of major bovine milk proteins: Novel insights

“…Up to seven of the major MFGM proteins have been absolutely quantified by MRM (Affolter, Grass, Vanrobaeys, Casado, & Kussmann, 2010;Fong & Norris, 2009;Sénéchal & Kussmann, 2011). Lutter, Parisod, and Weymuth (2011) developed an MRM assay to detect and quantify trace amounts of the four major milk proteins (e.g., α S2 -CN, β-CN, κ-CN and β-Lg) in different food products. Due to its high selectivity, MRM can be considered as a tool to quantify PTMs in milk proteins.…”

Selection of possible signature peptides for the detection of bovine lactoferrin in infant formulas by LC-MS/MS

“…LC-MS/MS as a promising alternative provides wide dynamic range, excellent selectivity, good accuracy and precision compared with conventional protein quantification methods on the analysis of compounds in complex biological matrices. It has been used by other researchers to examine the milk proteome and has resulted in the identification of a number of proteins with surrogate peptides including α-lactalbumin, β-lactoglobulin, casein proteins (α-, β- and κ-casein) and lactoferrin [ 19 – 21 ].…”

Quantification of bovine β-casein allergen in baked foodstuffs based on ultra-performance liquid chromatography with tandem mass spectrometry

“…However, the high cost of stable isotope-labelled protein has limited the scope of application. Lutter et al ( 2011 ) designed a stable isotope-labelled IS homologue of the signature peptide, which was added after the tryptic digestion. This strategy could normalise the variation of the ionisation during the MS detection.…”

“…Although the method had high sensitivity and selectivity during the screening of allergen proteins, it could not be applied for the quantitative determination of allergens in baked foodstuffs. Using the same approach, Lutter et al ( 2011 ) developed a quantitation method for four unheated cow’s milk allergens in food products. They used stable isotope-labelled peptides that were homologues for peptides derived from bovine milk allergens as the internal standards (ISs).…”