Separation and quantitation of phospholipids and their ether analogues by high-performance liquid chromatography

The common mobile phase hexanehsopropanol/water used for separation of phos pholipids on highperformance liquid chromatography silica columns poses sev eral problems, such as incomplete separation and rapid column deterioration . By inclusion of 5 mM ammonium sulfate in the aqueous phase, we were able to substantially improve the chromatographic resolution and obtain complete separation of phosphatidylcholine, phosphatidylethanolamine, lysophosphati dylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidyli nositol, cardiolipin, phosphatidylglycerol, and sphingomyelin. In addition, ammonium sulfate prevented column degeneration and greatly improved reprod ucibility. A new quantitation method for alkenylacyl, alkylacyl, and diacyl forms of phospholipids was also developed based on derivatization with [H- 3]acetic anhydride. Separation and quantitation of the radioactive acetyl d iradylglycerols were performed by straight-phase HPLC coupled to a radioact ive flow detector and enabled detection of the various ether analogues at t he picomole level with high reproducibility. The described methods are mild and nondestructive and can therefore be easily combined with analysis of e ither molecular species or fatty acid and aldehyde composition of the indiv idual phospholipids. (C) 2001 Academic Press.