Gradient elution reversed-phase chromatographic isolation of individual glycerophospholipid molecular species

We describe a gradient elution reversed-phase high-performance liquid chromatographic approach for isolation of individual glycerophospholipid molecular species which greatly improves resolution and reduces run time compared to isocratic techniques. Separations were optimized and elution order and retention time data established by synthesizing 37 different homogeneous phospholipids comprising the major alkylacyl, diacyl and plasmalogen molecular species in samples derived from mammalian sources. Empirical equations which predict the elution order of individual species were derived. The method was validated with the use of complex mixtures of choline and ethanolamine glycerophospholipid species from isolated rabbit cardiomyocytes and porcine endothelial cells.