Quantitative Analysis of Phospholipids by HighPerformance Liquid Chromatography

A simple, rapid and reproducible method for the determination of major phospholipids in soybean lecithin and egg yolk lecithin by high performance liquid chromatography (HPLC) was developed. Separation was carried out on a silica (Polygosil 60-7) column, using a mobile phase of acetonitrile/methanol/85% phosphoric acid (780/10/9, by vol.) with detection at 210 nm. The complete separation and determination of the amounts of phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were achieved by this method using stearyldimethyl-benzylammonium chloride as the internal standard (I.S.). Calibration curves between the ratio of peak area (phospholipid/I.S.) in the HPLC and that of weight (phospholipid/I.S.) were obtained with good linearity over a wide range. Correction factors of several phospholipids for I.S. were then determined. As the correction factor of egg yolk PC was influenced by the presence of highly unsaturated fatty acids, another method for determining the correction factor was proposed for unknown egg yolk PC. The present method proved quite useful for assessing the quality and process control of phospholipids.