Reversed-phase high-performance liquid chromatography of phospholipids with fluorescence detection

Reversed-phase high-performance liquid chromatographic (HPLC) behavior of fluorescent labeled phospholipids (PLs) was studied. Molecular species of phosphatidylethanolamine (PE) derivatized with fluorescein-, thiocarbamoyl-, pyrenesulfonyl-, and dimethylaminonaphthalenesulfonyl (dansyl)-fluorophores were separated on octadecylsilica with a mobile phase of acetonitrilemethanol-water in the presence of tetraalkylammonium phosphates (TAAPs). Under similar HPLC conditions, dansylated phosphatidylserines and PE plasmalogens (ether-linked PLs) were also resolved. Incorporation of the fluorescein moiety to the parent PE appeared to facilitate further resolution of its subcomponents. Subcomponents of dansylated PE derived from egg phosphatidylcholines were quantifiable. Effects of the type and concentration of TAAP on capacity factors of PL solutes were indicative of an ion-pair separation processes. HPLC with high-molecular-mass TAAPs favored the separation of the components that remained unresolved with mobile phases containing low-molecular-mass TAAPs. The HPLC-fluorescence detection method provided a useful approach to quantitative analyses of various PL structures. Compositions of PL subcomponents were determined directly from peak areas.