High-performance liquid chromatographic separation of molecular species of neutral phospholipids

Molecular species of neutral phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), were resolved by reversed-phase high-performance liquid chromatography (HPLC) using mobile phases of acetonitrile-methanol-water containing tetraalkylammonium phosphates (TAAPs). Competitive interactions of TAAPs and analyte solutes with a reversed-phase HPLC column resulted in reduced retention of PC or PE with concomitant increase in detection sensitivity. The chromatographic data for PC and PE were distinctly different from those for negatively charged phospholipids where ion-pair retention mechanisms prevailed. While PC (or PE) components eluted at longer retention times with a larger size of TAAP, an increase in the TAAP concentration invariably caused a decrease in phospholipid retention times. Optimization of HPLC conditions by using high concentrations (25-100 mM) of tetramethylammonium phosphate in acetonitrile-methanol-water (70:22:8) facilitated elution of components with improved peak symmetry. HPLC separations of neutral phospholipids derived from animal sources were more complex than those from soybeans.