Reversed-phase high-performance liquid chromatographic separation of molecular species of alkyl ether, vinyl ether, and monoacyl lysophospholipids

An isocratic reversed-phase high-performance liquid chromatographic method was developed which resolved individual molecular species of choline and ethanolamine lysophospholipids utilizing a C₁₈ bonded porous silica stationary phase with a mobile phase comprised of methanol-water-acetonitrile (57:23:20) containing 20 mM choline chloride. Solute retention was primarily determined by hydrophobic interactions with the stationary phase permitting separation of individual molecular species of lysophospholipids according to the composition of the aliphatic chain and the nature of its covalent attachment to the sn-1 hydroxyl group. The elution profile of unsaturated monoacyllsophospholipid or lysoplasmalogen molecular species was readily obtained by measuring UV absorbance at 203 nm. Identification of column eluates containing saturated monoacyl and alkyl ether lysophospholipids was possible utilizing relative retention factors that were obtained for the majority of molecular species present in animal tissues.