Determination of total and A1-Type β-casein in milk and milk-derivedingredients by liquid chromatography – mass spectrometry using characteristic tryptic peptides

Background Gastrointestinal digestion of A1-type β-casein is conducive to β-casomorphin-7 with potential adverse digestive health effects. Monitoring of A1-type β-casein concentration in milk and milk-derived ingredients used in the formulation of A2-type nutritional products with associated health claims is important from a quality standpoint. Objective New analytical methods were developed and validated for total and A1-type β-casein in milk and milk-derived ingredients. Data on total and A1-type β-casein concentrations in milk, nonfat dry milk, and whey protein concentrate was generated. Methods The methods are based on a bottom-up proteomic approach using tryptic marker peptides and stable isotope dilution liquid chromatography – mass spectrometry. The measurement includes all protein sequences (intact, modified, partial) which are potential sources of β-casomorphin-7. Results Total β-casein was quantified using a neat calibration curve. Recovery and between-day precision RSD were 98% and 5.8%, respectively. A1-type β-casein was quantified by the method of standard additions. Between-day precision RSD was 7.2% and limit of quantitation was 0.01% in nonfat dry milk. The mass fraction of A1-type β-casein in β-casein standard was 0.444. Samples manufactured from A2-type milk contained 0.26–5.0% A1-type β-casein relative to total β-casein. Conclusions The methods described enable the monitoring of the A1-type β-casein concentration in milk and milk-derived ingredients destined for the manufacture of A2-type products with associated health claims. Highlights New methods are presented for the analysis of total and A1-type β-casein in milk and milk-derived ingredients. Mass fraction of A1-type β-casein in commercial β-casein standard was determined to enable its use as calibrant.