High Resolution Separation of Phospholipids Using a Novel Orthogonal Two-Dimensional UPLC/QTof MS System Configuration

Significance of Topic

Amphiphilic lipids such as phospholipids are crucial in energy storage, membrane integrity and cell signaling. High complexity in biological matrices poses analytical challenges, particularly in resolving low-abundance species and avoiding isotopic interferences.

Study Objectives and Overview

This study describes a novel configuration combining HILIC and reversed phase chemistries on a Waters ACQUITY UPLC System with 2D Technology and Xevo G2 QTof MS. The goal is to achieve orthogonal, high-resolution separation of phospholipids in human plasma, addressing limitations of single-dimension methods.

Methodology and Instrumentation Used

Lipid extraction employed Ostro sample preparation. The 2D LC setup used:

• First dimension: ACQUITY BEH HILIC, 2.1×100 mm, 1.7 µm at 30 °C
• Second dimension: ACQUITY CSH C18, 2.1×100 mm, 1.7 µm at 65 °C
• Trap column: ACQUITY BEH C8 VanGuard, 2.1×5 mm, 1.7 µm
• Waters Xevo G2 QTof MS in ESI positive/negative, MS E mode

Main Results and Discussion

The 2D method overcame co-elution of phosphatidylcholines (PC) and sphingomyelins (SM) seen in 1D reversed phase (1 Da difference) and the limited intra-class resolution in HILIC alone. Peak capacity and dynamic range were notably improved. PC detection increased by 37% over HILIC and 40% over C18 methods. Higher ion intensities and removal of isotopic overlaps enhanced specificity, as demonstrated in targeted heart-cutting experiments.

Benefits and Practical Applications of the Method

• Enhanced peak capacity and resolution for complex lipid mixtures
• Improved identification confidence and quantification accuracy
• Automated, high-throughput workflow compatible with existing UPLC systems
• Flexibility to adapt to diverse two-dimensional LC configurations

Future Trends and Opportunities

Emerging applications include integrating other orthogonal pairs (IEX-RP, SEC-RP) and expanding lipidomics pipelines for biomarker discovery. Continued advances in 2D UPLC hardware and MS detection will further elevate throughput and analytical depth.

Conclusion

The novel orthogonal 2D UPLC/QTof MS configuration significantly improves phospholipid separations, combining the strengths of HILIC and reversed phase chemistries. It enables more confident lipid profiling in complex matrices and offers a versatile platform for advanced lipidomics.

References

• Ritchie M, Mal M, Wong S. Extraction of Phospholipids from Plasma using Ostro Sample Preparation. Waters Application Note 720004201en. 2012 Jan.
• Mal M, Wong S. A HILIC-Based UPLC/MS Method for the Separation of Lipid Classes from Plasma. Waters Application Note 720004048en. 2011 July.
• Ritchie M, Mal M, Wong S. UPLC BEH HILIC: The Preferred Separation Chemistry for Targeted Analysis of Phospholipids. Waters Application Note 720004219en. 2012 Jan.
• Isaac G, McDonald S, Astarita G. Lipid Separation using UPLC with Charged Surface Hybrid Technology. Waters Application Note 720004107en. 2011 Sept.
• Isaac G, McDonald S, Astarita G. Automated Lipid Identification Using UPLC/HDMS E in Combination with SimLipid. Waters Application Note 720004169en. 2011 Dec.