Separation of phospholipids and individual molecular species of phospholipids by high-performance liquid chromatography.

Isocratic high-performance liquid chromatography methods are described for separating the major classes of phospholipids and for isolating the individual molecular species of phospholipids. Fractionation of a total lipid extract of rat liver on a silica column resulted in quantitative recoveries of all major phospholipids with preservation of their fatty acid composition. Rat liver phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine were then each chromatographed on a C18 reverse phase column to isolate individual molecular species. Component peaks were identified by their fatty acid composition and quantitated by phosphorus determination. Using this method we found that for each of these phospholipids from 30 to 35 different molecular species can be routinely identified and reproducibly quantitated. A characteristic elution sequence of molecular species permitted their identification based upon their retention times on a reverse phase column.