Determination of phosphatidylcholine and disaturated phosphatidylcholine content in lung surfactant by high...

A rapid isocratic method for determining the total phosphatidylcholine and disaturated phosphatidylcholine levels in lung surfactant preparations by high performance liquid chromatography (HPLC) is described. The analysis was performed on a 3.9 x 300 mm mu-Porasil column with detection by refractive index. The lipids were eluted with a solvent system of chloroform-acetonitrile-methanol-water-85% phosphoric acid 650:650:500:130:2 (v/v/v/v/v). A 4.6 x 30 mm silica guard column was used in place of an injector loop which served as a sample concentrator and purifier. Phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and phosphatidylglycerol, all known components of lung surfactants, were eluted from the loop column and were prevented from reaching the analytical column. Sphingomyelin and lysophosphatidylcholine elute later than the phosphatidylcholines on the analytical column. The method was developed so that phosphatidylcholines elute as a single peak regardless of the fatty acid chain length (C12-C20). When the sample was first oxidized with a potassium permanganate-potassium metaperiodate solution, and potentially interfering oxidation products were removed by extraction into a basic aqueous phase, then only the disaturated phosphatidylcholines were analyzed.