Separation of pulmonary surfactant phospholipids by high-performance liquid chromatography

The seven major phospholipid classes of pulmonary surfactant were cleanly separated within 45 min by high-performance liquid chromatography (HPLC) on microparticulate silica (5 microns). Phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidyl-ethanolamine, and phosphatidylcholine (PC) were separated with an isocratic mobile phase of acetonitrile--85% phosphoric acid (99:1). Lysophosphatidylcholine and sphingomyelin were subsequently eluted by changing the composition of the mobile phase to acetonitrile--methanol--85% phosphoric acid (97:2:1). Phospholipids were detected by ultraviolet absorbance at 203 nm and were quantitated by integration of peaks. When integrator counts were corrected by using calibration curves obtained from chromatograms of commercial phospholipids and surfactant-derived PC, the HPLC analysis gave results comparable to those obtained by thin-layer chromatography.