Lipidomic Analysis of Human Plasma Using Bond Elut Lipid Extraction with the Agilent 6545 LC/Q-TOF

Significance of the Topic

Lipidomics provides comprehensive profiling of lipid species in human plasma, enabling the discovery of biomarkers for metabolic disorders, cardiovascular disease, and other health conditions. Advances in sample preparation and high-resolution mass spectrometry have driven the need for reproducible, high-throughput workflows that minimize solvent use and sample handling.

Study Objectives and Overview

This study introduces a novel solid-phase extraction (SPE) workflow using the Agilent Bond Elut Lipid Extraction 1 mL cartridge combined with an Agilent 6545 LC/Q-TOF system. The goals were to optimize lipid recovery from human plasma, evaluate method reproducibility, and compare performance against traditional liquid-liquid extraction (LLE) techniques.

Methodology

100 µL of human plasma (SRM 1950) was precipitated with ice-cold acetonitrile/methanol (99:1, v/v) to remove proteins. The homogenate was loaded onto Bond Elut Lipid Extraction cartridges under gravity or low pressure. After washing with 90:10 acetonitrile/water, lipids were eluted with 1:1 chloroform/methanol. Eluates were dried under nitrogen, reconstituted in n-butanol/methanol (1:1, v/v), and analyzed by LC/Q-TOF in positive and negative ion modes. Data processing employed Agilent MassHunter Lipid Annotator, Profinder, and Mass Profiler Professional for lipid identification and statistical analysis.

Used Instrumentation

• Agilent 6545 LC/Q-TOF with Jet Stream ESI
• Agilent 1290 Infinity II LC (binary pump, autosampler, column compartment)
• Agilent Bond Elut Lipid Extraction SPE cartridges (1 mL)
• Eppendorf pipettes, PPM-48 positive pressure manifold
• Agilent Poroshell HPH-C18 analytical and guard columns
• Vortexer, sonicator, nitrogen evaporator

Main Results and Discussion

A total of 595 lipids were annotated in plasma, comprising 347 compounds across 13 classes in positive mode and 248 compounds across nine classes in negative mode. Key classes included phosphatidylcholines, triacylglycerides, sphingomyelins, and fatty acids. Method reproducibility was excellent, with instrument RSD 6.4% and overall workflow RSD 9.4%. On-cartridge recovery of total phospholipids was 102% compared to precipitation alone, indicating minimal lipid loss and efficient cleanup of salts and other interferences.

Benefits and Practical Applications

The SPE approach reduces solvent consumption, sample transfers, and processing time relative to LLE. The simplified workflow supports automation in 96-well format, enhances reproducibility, and is suitable for large-scale clinical and research studies requiring robust lipid profiling.

Future Trends and Opportunities

Integration of SPE workflows with robotic platforms and ultra-high-throughput chromatography will accelerate lipidomics applications in precision medicine. Coupling improved informatics pipelines, spectral libraries, and machine learning will further refine lipid identification and quantitation, expanding the utility of lipidomics in diagnostics and therapeutic monitoring.

Conclusion

The Agilent Bond Elut Lipid Extraction SPE method combined with high-resolution LC/Q-TOF offers a streamlined, reproducible, and high-throughput solution for human plasma lipidomics. It outperforms traditional LLE in efficiency and automation potential, supporting advanced lipid profiling in clinical and industrial laboratories.

References

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