Liquid chromatographic–mass spectrometric determination of cyclooxygenase metabolites of arachidonic acid in cultured cells

A liquid chromatographic–electrospray ionization-mass spectrometric (LC-ESI-MS) technique was developed to simultaneously determine the cyclooxygenase metabolites of arachidonic acid (6-keto-PGF1α, PGD2, PGE2, PGF2α, and PGJ2) produced by cultured cells. Samples were separated on a C18 column with water–acetonitrile mobile phase, ionized by electrospray, and detected in the positive mode. Selected ion monitoring (SIM) of m/z 353, 335, 335, 319, and 317 were used for quantifying 6-keto-PGF1α, PGD2, PGE2, PGF2α, and PGJ2, respectively. Prostaglandins were detected at concentrations as low as 1 pg (S/N=3) on the column. The method was used to determine the production of PGs from bovine coronary artery endothelial cells (ECs) and human prostate cancer cells (PC-3) with different degree of invasiveness. Bradykinin (10−6 M) stimulated a marked increase in the production of 6-keto-PGF1α, PGE2, and PGF2α and a small increase of PGD2 by ECs. 6-Keto-PGF1α was the major metabolite in these cells. The production of PGE2 was threefold higher and PGD2 was twofold higher in PC-3-S (invasive) cells than in PC-3-U (non-invasive) cells.