Analysis of oxylipins by high-performance liquid chromatography with evaporative light-scattering detection and particle beam-mass spectrometry

Lipids. 1997 Sep;32(9):1003-10. doi: 10.1007/s11745-997-0130-0.

Abstract

The metabolism of 13S-hydroperoxy-9Z,11E,15Z-octadecatrienoic acid was investigated in a crude enzyme extract from mung bean seedlings (Phaseolus radiatus L.). Hydroperoxide-metabolizing activity was mainly due to a hydroperoxide lyase and, to a lesser extent, to an allene oxide synthase and a peroxygenase. Oxylipins originating from hydrolysis and cyclization of the allene oxide synthase product 12,13-epoxy-9Z,11,15Z-octadecatrienoic acid and from peroxygenase catalysis were identified by high-performance liquid chromatography (HPLC) particle beam-mass spectrometry (PB-MS) and quantified by normal-phase HPLC with an evaporative light-scattering detector (ELSD). An advantage of this methodology was the possibility to avoid extensive derivatization procedures commonly used for the gas chromatographic analysis of oxylipins. Owing to a comparable sample inlet system, the ELSD served an important analytical pilot function for the PB-MS: Qualitatively identical chromatographic patterns were obtained with both detection systems. The HPLC system enabled the separation of methyl 12-oxo-phytodienoate, methyl 11-hydroxy-12-oxo-9Z,15Z-octadecadienoate, methyl 12-oxo-13-hydroxy-9Z,15Z-octadecadienoate, methyl 9-hydroxy-12-oxo-10E,15Z-octadecadienoate, methyl 13-hydroxy-9Z,11E,15Z-octadecatrienoate, methyl 15,16-epoxy-13-hydroxy-9Z,11E-octadecadienoate, and methyl 13-hydroperoxy-9Z,11E,15Z-octadecatrienoate on a Lichrospher DIOL column within 33 min. Compared with a diode array detector, the ELSD proved to be more sensitive, in the case of methyl 12-oxo-13-hydroxy-9Z, 15Z-octadecadienoate by a factor of about 15. In addition, volatile metabolites were analyzed by capillary gas chromatography. The yield of the hydroperoxide lyase product 2E-hexenal was 49%, whereas the sum of oxylipins reached about 15%.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aldehyde-Lyases / metabolism*
  • Aldehydes / analysis
  • Aldehydes / metabolism
  • Chromatography, Gas
  • Chromatography, High Pressure Liquid
  • Cytochrome P-450 Enzyme System / metabolism
  • Fabaceae / enzymology
  • Intramolecular Oxidoreductases / metabolism
  • Linolenic Acids / chemical synthesis
  • Linolenic Acids / metabolism
  • Lipid Peroxides / chemical synthesis
  • Lipid Peroxides / metabolism*
  • Mass Spectrometry
  • Mixed Function Oxygenases / metabolism
  • Molecular Structure
  • Plants, Medicinal
  • Scattering, Radiation
  • Seeds / enzymology

Substances

  • Aldehydes
  • Linolenic Acids
  • Lipid Peroxides
  • 13-hydroperoxy-9,11,15-octadecatrienoic acid
  • 2-hexenal
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • peroxygenase
  • Aldehyde-Lyases
  • hydroperoxide lyase
  • Intramolecular Oxidoreductases
  • hydroperoxide isomerase