Rational selection of reverse phase columns for high throughput LC-MS lipidomics

Area of Science:

• Analytical Chemistry
• Biochemistry
• Mass Spectrometry

Background:

• Natural lipidomes exhibit high complexity and diverse lipid concentrations.
• Analyzing lipidomes requires high-resolution chromatographic and mass spectrometric techniques.
• Reverse-phase chromatography (RPLC) coupled with MS/MS is a common untargeted lipidomics approach.

Purpose of the Study:

• To develop and validate a rapid RPLC-MS/MS workflow for comprehensive lipidome analysis.
• To evaluate the performance of different RPLC columns for lipid separation.
• To identify lipid species in complex biological samples like human blood plasma.

Main Methods:

• A 32-minute RPLC-MS/MS workflow was established using a Q Exactive™ HF Hybrid Quadrupole-Orbitrap™ Mass Spectrometer.
• Lipids were separated on five distinct reverse-phase columns with varying stationary phase properties.
• High mass accuracy MS and data-dependent MS/MS acquisitions were employed for lipid identification.

Main Results:

• The workflow successfully resolved complex lipid mixtures, identifying over 600 lipid species across 18 lipid classes in human plasma.
• Performance evaluation provided insights into the separation capabilities of different RPLC columns.
• The study offers guidance on selecting appropriate reverse-phase columns for specific lipidome analyses.

Conclusions:

• The developed RPLC-MS/MS workflow enables efficient and comprehensive lipidomics profiling.
• Column selection significantly impacts lipid separation and identification in complex samples.
• This method advances the analysis of human plasma lipidomes and supports future lipidomics studies.