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Updated: Mar 11, 2026

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis
Published on: November 17, 2011
Hydrophilic interaction and reversed phase mixed-mode liquid chromatography coupled to high resolution tandem mass
Sara Granafei1, Pietro Azzone1, Vito Alessandro Spinelli1
1Dipartimento di Chimica, Università degli Studi di Bari Aldo Moro, Campus Universitario, Via E. Orabona, 4-70126 Bari, Italy.
Abstract:
A hydrophilic interaction liquid chromatography (HILIC) fused-core column (150×2.1mm ID, 2.7μm particle size) and a short reversed-phase liquid chromatography (RPLC) column (20mm×2.1mm ID, 1.9μm) were serially coupled to perform mixed-mode chromatography (MMC) on complex mixtures of phospholipids (PL). Mobile phase composition and gradient elution program were, preliminarily, optimized using a mixture of phosphatidylcholines (PC), phosphatidylethanolamines (PE), their corresponding lyso-forms (LPC and LPE), and sphingomyelins (SM). Thus a mixture of PC extracted from soybean was characterized by MMC coupled to electrospray ionization (ESI) high-resolution Fourier-transform mass spectrometry (FTMS) using an orbital trap analyzer. Several previously undiscovered PC, including positional isomers (i.e. 16:0/19:1 and 19:1/16:0) of PC 35:1 and skeletal isomers (i.e. 18:1/18:2 and 18:0/18:3) of PC 36:3 were identified. Therefore, high-resolution MS/MS spectra unveiled the occurrence of isomers for several overall side chain compositions. The proposed MMC-ESI-FTMS/MS approach revealed an unprecedented capability in disclosing complexity of an actual lipid extract, thus representing a very promising approach to lipidomics.
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