Separation of intact phosphatidylcholine molecular species by high performance liquid chromatography

Identification of the molecular species of phosphatidylcholines (PCs) is important for studies of lipid metabolism and the structure of cell membranes. The high performance liquid chromatography (HPLC) separations of the intact molecular species of PCs used earlier were mostly on reversed-phase C-18 (octadecyl silica) columns, usually with isocratic elution. The eluent used contained a silanol suppressing agent. Volatile silanol suppressing agents are preferred for the purification of PCs because they can be removed from the HPLC fractions easily by nitrogen stream. They must be used when liquid chromatography-mass spectrometry (LC-MS) and evaporative light scattering detector (ELSD) are used because non-volatile silanol suppressing agents cause high background. For the identification of radioactive metabolites, using both the UV detector at 205 nm and radioactivity flow detector sequentially, we recommend the separation on a C-8 (octyl silica) column, eluted with a gradient of methanol-water containing conc. NH4OH as silanol suppressing agent.