Microanalysis of Phospholipids of Mammalian Cells by Liquid Chromatography/Electrospray Ionization-Mass Spectrometry

A fast and sensitive analytical method of phospholipids by capillary liquid chromatography/electrospray ionization-mass spectrometry was applied to the analysis of individual molecular species of phospholipids in mammalian cells with or without previous treatment with phospholipases. Using a quadrupole mass spectrometer, we selected a simultaneous analysis method under four different functions for detection of positive and negative molecular related ions as well as fragment ions. In the analysis of the elution patterns of the phospholipids, a two-dimensional map revealed to be very useful. By the treatment with phospholipases A or phospholipases C, significant decrease of substrate phospholipids in extracted lipid mixture or cells. Increase of products, such as lyso-phospholipids, and diglycerides or ceramides were effectively determined. Especially by the treatment with PLA1 or PLA2, accumulation of specific molecular species of lyso-phospholipids, either unsaturated or saturated, were respectively detected. Application of anion exchange column was also examined for effective separation and identification of acidic phospholipids, such as phosphatidylinositol, phosphatidyl-glycerol, phosphatidylserine, phosphatidic acid, and their lyso-forms. Analyses of these acidic lyso-phospholipids are very important to elucidate their function as lipid mediators.