On-line two-dimensional capillary strong anion exchange/reversed phase liquid chromatography–tandem mass spectrometry for comprehensive lipid analysis

• On-line 2-dimensional SAX/RP LC–ESI-MS/MS for phospholipid analysis.
• Lipids fractionation in the 1st dimension by SAX using salt step elution followed by nanoflow RPLC in the 2nd dimension.
• A common ionization modifier for mobile phase solutions is employed for both positive and negative ion mode of MS.

An on-line two-dimensional liquid chromatography method was developed for comprehensive lipid profiling by coupling strong anion exchange (SAX) and nanoflow reversed-phase liquid chromatography (nRPLC) prior to electrospray ionization-tandem mass spectrometry (2D-SAX/nRPLC–ESI-MS/MS). Lipids can be classified into four different types according to the electrical propensities of the lipids: anionic, weak anionic, neutral polar, and special lipids. In 2D-SAX/nRPLC, various lipids can be fractionated in the first dimension (SAX: 5 μm to 100 Å, 5.0 cm × 75 μm i.d.) by step elution (methanol and salt solution), followed by the molecular separation of lipids in the second dimension (RP: 3 μm to 100 Å, 7.0 cm × 75 μm i.d.) with binary gradient LC. Since the elution of lipids from SAX can be achieved with a very small volume of eluent delivered from an autosampler, it can be simply implemented with an LC–ESI-MS instrument for full automation, and the salt step elution, including the two-step injection procedure, can be used for the selective analysis of the desired lipid fraction. For nRPLC–ESI-MS/MS run in either positive or negative ion mode, a common ionization modifier (0.05% ammonium hydroxide with 5 mM ammonium formate) was introduced into the binary mobile phase solutions so that 2D-LC–MS could be operated in both ion modes without changing the mobile phase solutions. The developed on-line 2D-SAX/nRPLC–ESI-MS/MS was evaluated with 22 different standard lipids for the optimization of the salt step elution and was applied to a healthy human plasma lipid extract, resulting in the identification of a total of 303 plasma lipids, including 14 different classes.