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 spe cies of phosphatidylethanolamine (PE) derivatized with fluorescein-, t hiocarbamoyl-, pyrenesulfonyl-, and dimethylaminonaphthalenesulfonyl ( dansyl)-fluorophores were separated on octadecylsilica with a mobile p hase of acetonitrile-methanol-water in the presence of tetraalkylammon ium phosphates (TAAPs). Under similar HPLC conditions, dansylated phos phatidylserines and PE plasmalogens (ether-linked PLs) were also resol ved. 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 quantifiab le. Effects of the type and concentration of TAAP on capacity factors of PL solutes were indicative of an ion-pair separation processes. HPL C with high-molecular-mass TAAPs favored the separation of the compone nts that remained unresolved with mobile phases containing low-molecul ar-mass TAAPs. The HPLC-fluorescence detection method provided a usefu l approach to quantitative analyses of various PL structures. Composit ions of PL subcomponents were determined directly from peak areas.