Analysis of epoxyeicosatrienoic acids by chiral liquid chromatography/electron capture atmospheric pressure chemical ionization mass spectrometry using [13C]-analog internal standards

Rapid Commun Mass Spectrom. 2010 Nov 30;24(22):3237-47. doi: 10.1002/rcm.4760.


The metabolism of arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) is thought to be mediated primarily by the cytochromes P450 (P450s) from the 2 family (2C9, 2C19, 2D6, and 2J2). In contrast, P450s of the 4 family are primarily involved in omega oxidation of AA (4A11 and 4A22). The ability to determine enantioselective formation of the regioisomeric EETs is important in order to establish their potential biological activities and to asses which P450 isoforms are involved in their formation. It has been extremely difficult to analyze individual EET enantiomers in biological fluids because they are present in only trace amounts and they are extremely difficult to separate from each other. In addition, the deuterium-labeled internal standards that are commonly used for stable isotope dilution liquid chromatography/mass spectrometry (LC/MS) analyses have different LC retention times when compared with the corresponding protium forms. Therefore, quantification by LC/MS-based methodology can be compromised by differential suppression of ionization of the closely eluting isomers. We report the preparation of [(13)C(20)]-EET analog internal standards and the use of a validated high-sensitivity chiral LC/electron capture atmospheric pressure chemical ionization (ECAPCI)-MS method for the trace analysis of endogenous EETs as their pentafluorobenzyl (PFB) ester derivatives. The assay was then used to show the exquisite enantioselectivity of P4502C19-, P4502D6-, P4501A1-, and P4501B1-mediated conversion of AA into EETs and to quantify the enantioselective formation of EETs produced by AA metabolism in a mouse epithelial hepatoma (Hepa) cell line.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 8,11,14-Eicosatrienoic Acid / analogs & derivatives*
  • 8,11,14-Eicosatrienoic Acid / chemistry
  • 8,11,14-Eicosatrienoic Acid / metabolism
  • Animals
  • Arachidonic Acid / chemistry
  • Arachidonic Acid / metabolism
  • Aryl Hydrocarbon Hydroxylases / metabolism
  • Carbon Isotopes / chemistry
  • Cell Line, Tumor
  • Chromatography, Liquid / methods*
  • Humans
  • Linear Models
  • Mice
  • Protein Isoforms / chemistry
  • Protein Isoforms / metabolism
  • Rats
  • Reference Standards
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Spectrometry, Mass, Electrospray Ionization / methods*
  • Spectrometry, Mass, Electrospray Ionization / standards
  • Stereoisomerism


  • Carbon Isotopes
  • Protein Isoforms
  • Arachidonic Acid
  • Aryl Hydrocarbon Hydroxylases
  • 8,11,14-Eicosatrienoic Acid