HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ANALYSIS OF PHOSPHOLIPIDS FROM DIFFERENT SOURCES WITH COMBINED FLUORESCENCE AND ULTRAVIOLET DETECTION

An isocratic high-performance liquid chromatographic (HPLC) system was developed for the separation of major phospholipid classes, i.e., pho sphatidylcholine, sphingomyelin, lysophosphatidylcholine, phosphatidyl glycerol, phosphatidylethanolamine, phosphatidylinositol, and phosphat idylserine. Phospholipids were detected with ultraviolet absorption at 205 nm and subsequent fluorescence detection. Fluorescence of the pho spholipids (excitation, 340 nm; emission, 460 nm) was achieved by post column formation of mixed micelles with 1,6-diphenyl-1,3, 5-hexatriene . For ultraviolet absorption there were great differences depending on the saturation of phospholipid fatty acids but for fluorescence the s ensitivity was almost identical for all phospholipids except phosphati dylinositol and lysophosphatidylcholine. Dipalmitoylphosphatidylcholin e showed nearly no ultraviolet but good fluorescence response. Ultravi olet to fluorescence ratio was characteristic for different phospholip ids and for identical phospholipids from different sources. Quantifica tion of phosphatidylcholine and phosphatidylethanolamine with HPLC usi ng N-monomethylphosphatidylethanolamine (dioleoyl) as an internal stan dard gave the same results as phospholipid phosphorus quantification a fter thin-layer chromatography. (c) 1994 Academic Press, Inc.