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Four-Dimensional Lipidomic Analysis Using Comprehensive Online UHPLC × UHPSFC/Tandem Mass Spectrometry.

Zuzana Lásko1, Tomáš Hájek1, Robert Jirásko1

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This study introduces a novel multidimensional chromatography method for lipidomic analysis, significantly increasing peak capacity for complex biological samples. The advanced technique enables detailed lipidome characterization and identification of numerous lipid species.

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Area of Science:

  • Analytical Chemistry
  • Biochemistry
  • Chromatography

Background:

  • Complex biological samples require advanced separation techniques for comprehensive analysis.
  • Multidimensional chromatography enhances resolution and peak capacity, crucial for lipidomics.
  • Existing methods may lack the resolution needed for detailed lipidome profiling.

Purpose of the Study:

  • To develop a novel comprehensive online multidimensional chromatography method for lipidomic analysis.
  • To combine reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) and ultrahigh-performance supercritical fluid chromatography (UHPSFC) for enhanced separation.
  • To significantly increase peak capacity for detailed characterization of lipid species in biological samples.

Main Methods:

  • A two-dimensional chromatography system combining RP-UHPLC (150 mm C18 column) and UHPSFC (10 mm silica column) with sub-2 μm particles.
  • Coupling the system with a high-resolution quadrupole-time-of-flight (QTOF) mass analyzer using electrospray ionization (ESI).
  • Utilizing both full-scan and tandem mass spectrometry (MS/MS) with positive and negative ion polarities, including MSE and fast data-dependent acquisition.

Main Results:

  • Achieved a peak capacity 10 times higher than RP-UHPLC and 18 times higher than UHPSFC alone.
  • Enabled ultrafast analysis in the second dimension (UHPSFC) with a sampling time of 0.55 min.
  • Successfully characterized the lipidomic profile of human plasma, identifying 298 lipid species from 16 subclasses with high confidence.

Conclusions:

  • The developed multidimensional chromatography method offers superior peak capacity and resolution for lipidomic analysis.
  • This approach allows for detailed characterization and confident identification of a wide range of lipid species.
  • The method is effective for analyzing complex biological matrices like human plasma.