Abstract
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.
Highlights
high performance liquid chromatography (HPLC) grade chloroform, acetonitrile, and methanol and certified ACS grade potassium permanganate were obtained from Fisher Scientific Co. (Fairlawn, NJ)
The method was designed so that standard and sample concentrations are approximately equal and a single standard was used for quantification
Results are calculated as total phosphatidylcholines or disaturated phosphatidylcholines versus dipalmitoyl phosphatidylcholine
Summary
The surfactant preparation under investigation was obtained from bovine lung extracts that were further fortified with lipids and prepared as an aqueous suspension. More than 80% of the phospholipids present were phosphatidylcholines and of those approximately 60% were disaturated. The remaining phospholipids were comprised of phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylglycerol (PG), lysophosphatidylcholine(LPC), and sphingomyelin (SPH). The sample contained free fatty acids, triglycerides, cholesterol, protein, and sodium chloride. HPLC grade chloroform, acetonitrile, and methanol and certified ACS grade potassium permanganate were obtained from Fisher Scientific Co. Reagent grade potassium metaperiodate and phosphoric acid were purchased from G.
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