A novel stop-flow two-dimensional liquid chromatography-mass spectrometry method for lipid analysis

Area of Science:

• Analytical Chemistry
• Biochemistry
• Mass Spectrometry

Background:

• Lipid analysis in human plasma is crucial for understanding various physiological and pathological processes.
• Existing methods, such as one-dimensional liquid chromatography (1D LC), often lack the resolution to fully characterize complex lipid profiles.
• Comprehensive two-dimensional liquid chromatography (2D LC) offers higher peak capacity but can suffer from sensitivity loss due to dilution.

Purpose of the Study:

• To develop and validate a novel on-line two-dimensional liquid chromatography (2D LC) system utilizing a stop-flow mode coupled with electrospray ionization mass spectrometry (ESI-MS).
• To improve the separation and identification of lipids in human plasma by combining hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RP LC).
• To overcome the sensitivity limitations associated with conventional comprehensive 2D LC methods.

Main Methods:

• A novel stop-flow interface was designed, incorporating a trap column and make-up flow to manage eluent transfer between the two dimensions.
• Hydrophilic interaction liquid chromatography (HILIC) was employed as the first dimension for separation based on polar head groups.
• Reversed-phase liquid chromatography (RP LC) served as the second dimension for separation based on aliphatic chains, using identical column lengths and similar flow rates to the first dimension.

Main Results:

• The developed 2D LC-ESI-MS method successfully identified 372 lipids in human plasma extracts.
• This approach detected 88 more lipids compared to standard one-dimensional RP LC analysis, demonstrating enhanced coverage.
• The system achieved a peak capacity of 415, comparable to comprehensive 2D LC, while avoiding significant sensitivity reduction.

Conclusions:

• The on-line stop-flow 2D LC-ESI-MS method provides a powerful tool for both qualitative and quantitative analysis of complex lipid mixtures in biological samples.
• This technique significantly enhances lipid identification capabilities and offers satisfactory linearity, repeatability, and sensitivity for quantitative applications.
• The developed system represents a valuable advancement in lipidomics, offering improved performance over existing 1D LC and conventional 2D LC approaches.