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.