On-line miniaturized asymmetrical flow field-flow fractionation-electrospray ionization-tandem mass spectrometry with selected reaction monitoring for quantitative analysis of phospholipids in plasma lipoproteins

J Chromatogr A. 2014 Jan 10;1324:224-30. doi: 10.1016/j.chroma.2013.11.035. Epub 2013 Nov 22.


A direct analytical method for high speed quantitative analysis of lipids in human blood plasma using on-line chip-type asymmetrical flow field-flow fractionation-electrospray ionization-tandem mass spectrometry (cAF4-ESI-MS/MS) with selected reaction monitoring (SRM) is described in this study. Utilizing a miniaturized cAF4 channel, high speed size separation of high density lipoproteins (HDL) and low density lipoproteins (LDL) from plasma samples can be accomplished at a microflow rate along with simultaneous desalting of lipoproteins, both of which are conducive to direct ESI of lipids in lipoproteins. This study demonstrates that the SRM method to monitor phospholipids during cAF4-ESI-MS/MS can be successfully applied to the quantitation of lipid molecules in plasma lipoproteins without the need of a separate lipid extraction process. For quantitation of lipids in HDL and LDL during cAF4-ESI-MS/MS runs, a protein standard (carbonic anhydrase, 29 kDa) was added to each plasma sample as an internal standard such that a peak intensity of y67(+5) ions, which are high abundant SRM product ions of CA, could be utilized to calculate the relative intensity of each lipid molecule. The developed method was applied to plasma samples from 10 patients with coronary artery disease (CAD) and 10 healthy control samples, and quantitative analysis of 39 lipid molecules including phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, phosphatidylglycerols, and phosphatidylinositols, resulted in the selection of 13 PL species showing more than 2.5 fold difference in relative abundance (p<0.01) between the groups. The present study demonstrates a high speed analytical method for determining plasma lipid content and distribution without an organic solvent extraction of lipids from plasma.

Keywords: AF4-ESI-MS/MS; Asymmetrical field-flow fractionation; Coronary artery disease; Phospholipids; Selected reaction monitoring; Tandem mass spectrometry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fractionation, Field Flow / methods
  • Humans
  • Lipoproteins, HDL / blood
  • Lipoproteins, HDL / chemistry*
  • Lipoproteins, LDL / blood
  • Lipoproteins, LDL / chemistry*
  • Phospholipids / blood*
  • Phospholipids / chemistry
  • Spectrometry, Mass, Electrospray Ionization / methods*
  • Sphingomyelins / blood
  • Tandem Mass Spectrometry / methods*


  • Lipoproteins, HDL
  • Lipoproteins, LDL
  • Phospholipids
  • Sphingomyelins