Determination of Total and A1-Type β-Casein in Milk and Milk-Derived Ingredients by Liquid Chromatography-Mass Spectrometry Using Characteristic Tryptic Peptides

Background: Gastrointestinal digestion of A1-type beta-casein is conducive to beta-casomorphin-7 with potential adverse digestive health effects. Monitoring of A1-type beta-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 beta-casein in milk and milk-derived ingredients. Data on total and A1-type beta-casein concentrations in milk, nonfat dry milk, and whey protein concentrate was generated. Method: 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, and partial) which are potential sources of beta-casomorphin-7. Results: Total beta-casein was quantified using a neat calibration curve. Recovery and between-day precision RSD were 98% and 5.8%, respectively. A1-type beta-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 beta-casein in the beta-casein standard was 0.444. Samples manufactured from A2-type milk contained 0.26-5.0% A1-type beta-casein relative to total beta-casein. Conclusions: The methods described enable the monitoring of the A1-type beta-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 beta-casein in milk and milk-derived ingredients. The mass fraction of A1-type beta-casein in a commercial beta-casein standard was determined to enable its use as a calibrant.