Quantitative high-performance liquid chromatography/electrospray ionization tandem mass spectrometric analysis of 2- and 3-series prostaglandins in cultured tumor cells

This paper describes a rapid and simple technique for the simultaneous quantitative analysis of PGE₂, PGE₃, and other closely related prostaglandins from cultured cells using liquid chromatography/electrospray ionization tandem mass spectrometry. This method permits quantification of selected individual prostaglandins derived either from arachidonic acid (AA) or eicosapentaenoic acid (EPA) from cell extracts without tedious derivatization, lengthy sample preparation, and separation required by GC-MS- or HPLC-UV-based methods. The validation assessment showed that the quantitative determination is linear (r² > 0.999) for both PGE₂ and PGE₃ in the range tested (1-500 ng/ml, 0.0028-1.4 μM) and a coefficient of variation lower than 10% was obtained for samples analyzed on 3 separate days. The detection limit was 2.5 pg for both PGE₂ and PGE₃. Extraction efficiency of PGE₂ and PGE₃ from cell suspensions ranged from 89.4 to 98.2%. As an application of the method, prostaglandins formed by EPA in human lung cancer A549 cells were determined. A 62% reduction of PGE₂ formation was noted when A549 cells were treated with 100 μM of EPA. Concomitantly, EPA increased formation of PGE₃ by 10-fold in A549 cells. This is the first report that unequivocally demonstrates that EPA can be converted to PGE₃ by cyclooxygenase in human cancer cells.