Journal of Lipid Research, Vol 30, 607-611, Copyright © 1989 by Lipid Research, Inc.
Determination of phosphatidylcholine and disaturated phosphatidylcholine content in lung surfactant by high performance liquid chromatography
J Scarim, H Ghanbari, V Taylor and G Menon
Abbott Laboratories, Pharmaceutical Products Division, North Chicago, IL 60064.
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.
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