Determination of thromboxanes, leukotrienes and lipoxins using high-temperature capillary liquid chromatography-tandem mass spectrometry and on-line sample preparation.
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.
Dahl
Sandra Rinne
SR
Department of Chemistry, University of Oslo, Norway. sandrari@kjemi.uio.no
Kleiveland
Charlotte Ramstad
CR
Kassem
Moustapha
M
Lea
Tor
T
Lundanes
Elsa
E
Greibrokk
Tyge
T
D002460:Cell Line; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000378:metabolism; D015289:Leukotrienes / Q000032:analysis* / Q000378:metabolism; D044045:Lipoxins / Q000032:analysis* / Q000378:metabolism; D009862:Online Systems; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013931:Thromboxanes / Q000032:analysis* / Q000378:metabolism
D015289:Leukotrienes; D044045:Lipoxins; D013931:Thromboxanes