This paper describes a rapid and simple technique for the simultaneous quantitative analysis of PGE(2), PGE(3), 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 (r(2)>0.999) for both PGE(2) and PGE(3) in the range tested (1-500 ng/ml, 0.0028-1.4 microM) 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 PGE(2) and PGE(3). Extraction efficiency of PGE(2) and PGE(3) 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 PGE(2) formation was noted when A549 cells were treated with 10 microM of EPA. Concomitantly, EPA increased formation of PGE(3) by 10-fold in A549 cells. This is the first report that unequivocally demonstrates that EPA can be converted to PGE(3) by cyclooxygenase in human cancer cells.