Simultaneous Quantification of Arachidonic Acid Metabolites in Cultured Tumor Cells Using High-Performance Liquid chromatography/electrospray Ionization Tandem Mass Spectrometry

Anal Biochem. 2001 Oct 15;297(2):183-90. doi: 10.1006/abio.2001.5325.

Abstract

A validated method is described for the simultaneous analysis of PGE2, 11-, 12-, and 5-HETEs from cultured cells using HPLC negative electrospray ionization tandem mass spectrometry (LC/MS/MS). This method permits quantification of selected individual arachidonic acid metabolites from cell extracts without derivatization, multiple purification steps, or lengthy separation times required by traditional GC-MS- or HPLC-UV -based methods. Accuracy assessments of values calculated using this method showed deviations from nominal values were < or =15%. An average relative deviation of 7% of mean calculated values was observed for values taken on separate days. The lower limit of detection for all metabolites was 1.3 pg. The method was used to quantify arachidonic acid metabolites present in various cancer cell lines after incubation with arachidonic acid and the selective cyclooxygenase-2 inhibitor celecoxib. Results showed that the presence of celecoxib in lung cancer A549 cells reduced production of both PGE2 and 11-HETE in a concentration-dependent manner.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / analysis*
  • 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / metabolism
  • Animals
  • Arachidonic Acid / metabolism
  • Celecoxib
  • Chromatography, High Pressure Liquid / methods
  • Cyclooxygenase 2
  • Dinoprostone / analysis*
  • Dinoprostone / metabolism
  • Humans
  • Hydroxyeicosatetraenoic Acids / analysis*
  • Hydroxyeicosatetraenoic Acids / metabolism
  • Isoenzymes / antagonists & inhibitors
  • Leukemia / metabolism
  • Lipoxygenase / metabolism
  • Lung Neoplasms / metabolism
  • Membrane Proteins
  • Prostaglandin-Endoperoxide Synthases / metabolism
  • Pyrazoles
  • Rats
  • Spectrometry, Mass, Electrospray Ionization / methods
  • Sulfonamides / metabolism
  • Sulfonamides / pharmacology
  • Tumor Cells, Cultured / cytology
  • Tumor Cells, Cultured / metabolism

Substances

  • Hydroxyeicosatetraenoic Acids
  • Isoenzymes
  • Membrane Proteins
  • Pyrazoles
  • Sulfonamides
  • Arachidonic Acid
  • 5-hydroxy-6,8,11,14-eicosatetraenoic acid
  • 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid
  • 11-hydroxy-5,8,12,14-eicosatetraenoic acid
  • Lipoxygenase
  • Cyclooxygenase 2
  • PTGS2 protein, human
  • Prostaglandin-Endoperoxide Synthases
  • Celecoxib
  • Dinoprostone