Analysis of Bovine Immunoglobulin G in Milk-Based Commodities: Liquid Chromatography-Tandem Mass Spectrometry vs. Affinity Liquid Chromatography.

Abstract

Background: Current analytical methods for bovine immunoglobulin G (IgG) have certain limitations in terms of selectivity, reproducibility, and applicability to complex matrices. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) using peptide-based quantitation and a suitable calibrant can potentially overcome some of these limitations.

Objective: Commercial bovine IgG standards and colostrum powder were evaluated for suitability as calibrants. The feasibility of using a single peptide marker for quantitation of total IgG by LC-MS/MS was assessed in a range of milk-based commodities. The LC-MS/MS method was compared to AOAC method 2010.01, based on affinity liquid chromatography, with and without the casein precipitation step. Heat stability of bovine IgG in a model nutritional product was evaluated.

Method: After dilution in buffer, reduction, alkylation, and tryptic digestion, a single IgG1 + 3 isoform-specific peptide was used for quantitation of total IgG. The corresponding isotopically labeled peptide was used for internal standardization. Colostrum powder was used as matrix-matched calibrant.

Results: Linearity was demonstrated in the 5-500 mg/L total IgG range. Within-day and intermediate precision were <4% and <6%, respectively, in two milk-based matrices and recovery was 95-99%. Major differences were noted between the LC-MS/MS method and AOAC method 2010.01 with and without casein precipitation, caused by factors such as loss of IgG during casein precipitation and inclusion of partially denatured IgG in the result. Heat processing loss of bovine IgG in a nutritional product was ca. 25% by the LC-MS/MS method.

Conclusions: The LC-MS/MS method is applicable to milk-based commodities and nutritional products as well. A higher fraction of IgG (including denatured IgG) is accessible to measurement by LC-MS/MS than by AOAC method 2010.01. Casein precipitation in whey-based matrices causes loss of IgG.

Highlights: A robust and widely applicable LC-MS/MS method using colostrum powder as calibrant and a single peptide marker for total IgG is being proposed.