A Method for Separation and Quantification of Phospholipid Classes in Human Milk

A simple, isocratic method for separating the major phospholipid classes of human milk by HPLC is described. Resolution of phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylcholine, and spingomyelin from human milk phospholipids was achieved in 30 min on a silica column. Total phospholipids were injected in 50 microliter of chloroform:diethyl ether (1:2, vol/vol) and eluted with a solvent mixture of acetonitrile:methanol:sulfuric acid (100:3:.05, vol/vol/vol) at a flow rate of 2.5 ml/min. Fractions were collected and each phospholipid class quantified by analysis of inorganic phosphorus after sulfuric acid digestion. A repeatability study with 19 samples had a coefficient of variation of 5.3%. The analytical recoveries of phospholipid standards averaged 98%. Recoveries varied with phospholipid class; variation was greatest with spingomyelin.