Determination of thromboxanes, leukotrienes and lipoxins using high-temperature capillary liquid chromatography-tandem mass spectrometry and on-line sample preparation

J Chromatogr A. 2009 May 29;1216(22):4648-54. doi: 10.1016/j.chroma.2009.03.068. Epub 2009 Mar 28.

Abstract

An on-line strong cation-exchange (SCX)-reversed-phase (RP) capillary liquid chromatographic (cLC) method with ion-trap tandem mass spectrometric (IT-MS/MS) detection for the simultaneous determination of thromboxane (TX) B(2), TXB(3), leukotriene (LT) B(4), LTD(4) and lipoxin (LX) A(4) in cell culture supernatants was developed and validated. In the present method, a high temperature (70 degrees C) was used for the separation on the analytical column to obtain efficient chromatography of the thromboxanes. An on-line sample preparation was performed, where peptides/proteins contained in the matrix were removed by the SCX column. Sample pre-treatment included dilution and filtration, and the analysis time including all sample preparation steps was 60min per sample. Limits of detection in the range of 1-4ng/mL cell culture supernatant, recoveries between 30% and 100%, within day precisions of less than 20% RSD and between day precisions of less than 30% RSD were obtained. Human mesenchymal stem cells (hMSCs) were stimulated with cytokine-containing supernatants derived from activated human T lymphocytes, and thromboxane, leukotriene and lipoxin production was analysed using the developed method. TXB(2) was found in cultures from both non-differentiated and differentiated hMSCs that were stimulated with a cytokine-containing supernatant obtained from activated T-cells.

Publication types

  • Evaluation Study

MeSH terms

  • Cell Line
  • Cells, Cultured
  • Chromatography, Liquid / instrumentation
  • Chromatography, Liquid / methods*
  • Humans
  • Leukocytes, Mononuclear / chemistry
  • Leukocytes, Mononuclear / metabolism
  • Leukotrienes / analysis*
  • Leukotrienes / metabolism
  • Lipoxins / analysis*
  • Lipoxins / metabolism
  • Online Systems
  • Tandem Mass Spectrometry / methods*
  • Thromboxanes / analysis*
  • Thromboxanes / metabolism

Substances

  • Leukotrienes
  • Lipoxins
  • Thromboxanes