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) 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.