High-performance liquid chromatography of phosphatidic acids and related polar lipids

The retention behavior of phosphatidic acids (PA) and phosphatidic acid methyl esters (PM) was studied by reversed-phase ion-pair high-performance liquid chromatography (HPLC). The HPLC systems consisted of mobile phases of acetonitrile-methanol-water containing tetraalkylammonium phosphates (TAAP) and stationary phases of alkyl-bonded silica and polystyrene--<:1iviny1benzene resins. The lipid solutes were more strongly retained when using mobile phases containing larger TAAP at higher concentrations. The results are indicative of an ion-pair retention mechanism. Molecular species of PM were readily resolved despite the complete inseparability of PA under all conditions used. Except for tetrabuty1ammonium phosphate, there was a linear correlation between the logarithmic capacity factors (k') of PM (or PAl and the total number of carbon atoms ofTAAP. The significant concentration dependence of separation factors for certain PM components was related to the size effect of TAAP. A normal-phase HPLC method for the separation of PA from other polar lipids is described.