Characterization of Phospholipids by Two-Dimensional Liquid Chromatography Coupled to In-line Ozonolysis–Mass Spectrometry

In this study, the characterization of phospholipids (PL) was achieved by using a combination of LC/MS/MS and two-dimensional LC/MS. A HILIC LC column was used for PL class separation, while the further molecular species separation of each PL class was achieved by using online HILIC × C18 LC. The double bond positions along the fatty acyl chains of these PL molecular species were also obtained by using the combination of 2D-LC and in-line ozonolysis–MS analysis. The ozonolysis device is composed of a gas-permeable, liquid-impermeable Teflon tube passing through a glass chamber filled with ozone gas, which is then placed in-line between the 2D-LC and the mass spectrometer. The eluting PL molecules in the LC mobile phase passed through the device where they rapidly reacted with the ozone that penetrated through the tubing wall. The ozonolysis products were then detected by MS in real-time, which allowed the localization of the double bonds along the fatty acyl chains in these PL molecular species. This comprehensive method was successfully applied to an egg yolk PL extract, which revealed the detailed structures of the PL molecules.