Applicability of supercritical fluid chromatography - mass spectrometry to metabolomics. I - Optimization of separation conditions for the simultaneous analysis of hydrophilic and lipophilic substances

J Chromatogr A. 2018 Aug 10:1562:96-107. doi: 10.1016/j.chroma.2018.05.055. Epub 2018 May 28.


The aim of this study was to evaluate the suitability of SFC-MS for the analysis of a wide range of compounds including lipophilic and highly hydrophilic substances (log P values comprised between -6 and 11), for its potential application toward human metabolomics. For this purpose, a generic unified chromatography gradient from 2 to 100% organic modifier in CO2 was systematically applied. In terms of chemistry, the best stationary phases for this application were found to be the Agilent Poroshell HILIC (bare silica) and Macherey-Nagel Nucleoshell HILIC (silica bonded with a zwitterionic ligand). To avoid system overpressure at very high organic modifier proportion, columns of 100 × 3 mm I.D. packed with sub-3 μm superficially porous particles were selected. In terms of organic modifier, a mixture of 95% MeOH and 5% water was selected, with 50 mM ammonium formate and 1 mM ammonium fluoride, to afford good solubility of analytes in the mobile phase, limited retention for the most hydrophilic metabolites and suitable peak shapes of ionizable species. A sample diluent containing 50%ACN/50% water was employed as injection solvent. These conditions were applied to a representative set of metabolites belonging to nucleosides, nucleotides, small organic acids, small bases, sulfated/sulfonated metabolites, poly-alcohols, lipid related substances, quaternary ammonium metabolites, phosphate-based substances, carbohydrates and amino acids. Among all these metabolites, 65% of the compounds were adequately analyzed with excellent peak shape, 23% provided distorted peak shapes, while only 12% were not detected (mostly metabolites having several phosphate or several carboxylic acid groups).

Keywords: Metabolomics; Polar compounds; SFC-MS; Supercritical fluid chromatography.

MeSH terms

  • Ammonium Compounds
  • Chemistry Techniques, Analytical / methods*
  • Chromatography, Supercritical Fluid*
  • Fluorides / chemistry
  • Formates / chemistry
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Metabolomics / methods*
  • Nucleosides / chemistry
  • Nucleotides / chemistry
  • Porosity
  • Quaternary Ammonium Compounds / chemistry
  • Silicon Dioxide / chemistry
  • Solvents
  • Tandem Mass Spectrometry*
  • Water / chemistry


  • Ammonium Compounds
  • Formates
  • Nucleosides
  • Nucleotides
  • Quaternary Ammonium Compounds
  • Solvents
  • Water
  • formic acid
  • ammonium fluoride
  • Silicon Dioxide
  • Fluorides