Simultaneous quantification of prostaglandins, isoprostane and thromboxane in cell-cultured medium using gas chromatography–mass spectrometry

We have developed a simultaneous quantification method for prostaglandin (PG) E2, PGD2, PGF2α, 8-epi-PGF2α, 6-keto-PGF1α and thromboxane (TX) B2. Using [3,3,4,4-2H4]PGE2, [3,3,4,4-2H4]PGD2, [3,3,4,4-2H4]8-epi-PGF2α, [3,3,4,4-2H4]PGF2α, [3,3,4,4-2H4]6-keto-PGF1α and [18,18,19,19-2H4]TXB2 as internal standards (I.S.), the eicosanoids and their I.S. were simultaneously extracted by solid-phase extraction from cell-cultured medium, derivatized to methyl ester/methoxim/tert.-butyldimethylsilyl ether derivatives and analyzed using gas chromatography–mass spectrometry in the selected ion monitoring mode. The accuracy for the added eicosanoids ranged from 92 to 113%, and coefficients of variation ranged from 0.1 to 12.2%. Increased eicosanoids in RAW264.7 and U937 cells stimulated by lipopolysaccharide were suppressed by NS-398 and indometacin. This simultaneous quantification method can be applied routinely for assaying eicosanoids in vitro.