Elucidation of phosphatidylcholine isomers using two dimensional liquid chromatography coupled in-line with ozonolysis mass spectrometry

• Phosphatidylcholine (PC) molecular species were separated by on-line HILIC × C18 LC.
• Double bond locations in PC fatty acyl chains were assigned by in-line O3-MS.
• 2D-LC was coupled in-line with O3-MS.
• Double bond location was achieved even with PC isomers present in complex mixtures.
• 2D-LC/O3-MS analysis of rat liver phospholipids extract identified 19 PC species.

In this study, two dimensional (2D) liquid chromatography (LC) was coupled in-line with ozonolysis-mass spectrometry (O3-MS) for the elucidation of phosphatidylcholine (PC) structures in phospholipid extracts. In O3-MS, PC molecules carried by mobile phase pass through a length of gas-permeable tubing surrounded by ozone vapor resulting in oxidative cleavage of double bonds. The characteristic aldehyde products are then directly analyzed by eletrospray ionization (ESI)-MS, allowing assignment of the double bond positions within the PC fatty acyl chains. The on-line 2D-LC method employs hydrophilic interaction chromatography as the first dimension to separate phospholipid classes and reversed phase chromatography in the second dimension to further separate PC molecular species. Thus, by combing the separation power of 2D-LC with in-line O3-MS (2D-LC/O3-MS), PC species in complex mixtures can be identified including the detailed structure of their two fatty acid chains. When the 2D-LC/O3-MS method was applied to a rat liver phospholipid extract, 19 PC molecular species were identified with fatty acid isomers unambiguously assigned.