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.