A novel on-line two dimensional liquid chromatography (2D LC) based on stop-flow mode coupled with electrospray ionization mass spectrometry (ESI-MS) method was established to separate lipids in human plasma. Hydrophilic interaction liquid chromatography (HILIC) in the first dimension and reversed-phase liquid chromatography (RP LC) in the second dimension were used to separate the lipids into six fractions based on their polar head groups and further into peaks based on aliphatic chains, respectively. A new stop-flow interface with a trap column and an extra make-up flow was designed to construct this system and trap the components eluted from the first dimension. Moreover, the same length of analytical columns and similar flow rates were used in the first and second dimensions. Therefore, the new stop-flow 2D LC system can avoid the sensitivity decrease caused by the dilution effect, which is the shortcoming of comprehensive 2D LC. Three hundred and seventy-two lipids were identified from plasma extract using this 2D LC coupled with ESI-MS in positive mode, and 88 more lipids were detected than one-dimensional RP LC analysis. Peak capacity of this stop-flow 2D LC was 415, which is similar to that of comprehensive 2D LC. The linearity, repeatability and sensitivity of this method were satisfactory, which demonstrated that this method was also suitable for quantitative analysis. All these results indicated that this on-line 2D LC method is powerful for qualitative and quantitative analysis of complex lipids.
Keywords: Lipid; MS; On-line; Stop-flow; Two-dimensional liquid chromatography.
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