Separations of molecular species of phosphatidic acid by high-performance liquid chromatography

Molecular species of phosphatidic acid (PA) were separated by reversed-phase high-performance liquid chromatography with mobile phases of acetonitrile-methanol-water containing quaternary ammonium phosphates (OAPs). Effective resolution of PA complexes was achieved by using low-mass OAPs at concentrations ~ 50 mM or high-mass OAPs at concentrations ~ 10 mM. Capacity factor (k') values were found to be dramatically influenced by the type and concentration of OAP, stationary phase specifications, and mobile phase solvent compositions. An increase in the acetonitrile-methanol ratio of the mobile phase tended to enhance the retention and separation of the polar lipid components. Correlation of logarithmic k' values with the total number of carbon atoms in OAP appeared to yield non-linear relationships. Compositions of major molecular species in various PA samples were determined by calibration with synthetic PA standards. Distribution patterns of PA molecular species in samples derived from animal and plant sources were compared.