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

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• 作者：S ; ra Rinne Dahl ; Charlotte Ramstad Kleivel ; Moustapha Kassem ; Tor Lea ; Elsa Lundanes ; Tyge Greibrokk
• 关键词：Thromboxanes ; Leukotrienes ; Lipoxin ; High-temperature ; Capillary LC– ; MS/MS ; Cell culture ; Human mesenchymal stem cells
• 刊名：Journal of Chromatography A
• 出版年：2009
• 出版时间：29 May 2009
• 年：2009
• 卷：1216
• 期：22
• 页码：4648-4654
• 全文大小：635 K

文摘

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) B₂, TXB₃, leukotriene (LT) B₄, LTD₄ and lipoxin (LX) A₄ in cell culture supernatants was developed and validated. In the present method, a high temperature (70 °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 60 min per sample. Limits of detection in the range of 1–4 ng/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. TXB₂ was found in cultures from both non-differentiated and differentiated hMSCs that were stimulated with a cytokine-containing supernatant obtained from activated T-cells.