RP-HPLC/ESI MS determination of acyl chain positions in phospholipids

Since phospholipids are used as excipients in many pharmaceutical products, there is a need for validated strategies to characterize the phospholipid constituents. We describe a reversed phase HPLC/electrospray ionization mass spectrometry method that is an alternative to the laborious phospholipase A2 treatment commonly used to determine the acyl chain positions in phospholipids. This reversed phase HPLC/electrospray mass spectrometry system a) shows good chromatographic resolution for phosphatidylcholine molecular species, b) allows determination of the composition of complex mixtures of phosphatidylcholines, and, most important, c) allows unequivocal assignment of the positions of the acyl chains on the glycerol backbone in mixtures of POPC and OPPC and in mixtures of POPE and OPPE.