Reversed-phase high-performance liquid chromatography of molecular species of phospholipid derivatives

Molecular species of phospholipid (PL) derivatives in two structural series phosphatidic acid (PA) and phosphatidylethanolamine (PE) were separated on octadecylsilica by reversed-phase high-performance liquid chromatography (HPLC)-UV detection. Using a mobile phase of acetonitrile-methanol-water-quaternary ammonium phosphate (QAP), the individual PLs derived from egg phosphatidylcholine were resolved into six major molecular species. The QAP electrolytes served as ion-pair counter-ions or ion-interactants depending on PL structures and on the QAP concentrations. Correlation of logarithmic capacity factors (k') of anionic PLs with the number of carbon atoms in QAP established linear relationships. The ln k' values of compounds in PA and PE series containing respective O-H and N-H functionalities were not linearly proportional to the number of carbon atoms in their corresponding O-alkyls and N-methyls. The k' values of negatively charged PLs increased with increasing QAP concentrations. Conversely, inverse relationships between the QAP concentrations and k' values of PE derivative were observed in HPLC with low QAP concentrations. PA derivatives were useful for trace analyses.