Separation of the molecular species of intact phosphatidylethanolamines and their N-monomethyl and N,N-dimethyl derivatives by high-performance liquid chromatography on a C8 column

We have developed a gradient reversed-phase C8 high-performance liquid chromatography method for the separation of molecular species of phosphatidylethanolamines (PEs) and their N-monomethyl and N,N-dimethyl derivatives. This method uses a 40-min linear gradient of 88–100% methanol, containing ammonium hydroxide as silanol suppressing agent, and is suitable for metabolic studies using both UV detection at 205 nm and radioactivity flow detection. The elution order of a given PE is inversely related to the polarity of its fatty acid constituents. Lipid classes studied here containing the same fatty acyl chains elute in the order: PE-N,N-dimethyl<PE<PE-N-monomethyl<phosphatidylcholine, indicating that elution order is not simply a function of the numbers of methyl groups on the nitrogen atom of PE.