Abstract: We established a method based on liquid  chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) to detect A1 and A2 β-casein in milk. Caseins were extracted from milk by isoelectric point precipitation method,  after trypsin hydrolysis,  the hydrolysates were analyzed by LC-HRMS/MS method. The peptide containing varied amino acid moiety at the 67th position was selected as characteristic species to  distinguish A1 and A2 β-casein. Casein was fully dissolved in Tris-HCl buffer solution. The  acetonitrile was added to the enzymatic hydrolysis system to improve the enzymtic hydrolysis efficiency.  High resolution mass spectrometry and tandem mass spectrometry were used to eliminate the interference signal of impurity. Four kinds of common milk  and two kinds of  A2 milk  were tested by this method. The results show that the content of A1 β-casein is about 2—4 times that of  A2 β-casein in common milk,  while a small amount of  A2 milk samples contain trace A1 β-casein. This method can detect A1 and A2 β-casein in milk with advantages of high reliability and sensitivity,  which provides support for A2 milk source screening and quality control.

Key words: β-casein； , A2 milk； , liquid chromatography； , tandem mass spectrometry