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

摘要

We have developed a simultaneous quantification method for prostaglandin (PG) E_2, PGD_2, PGF_(2α), 8-epi-PGF_(2α), 6-keto-PGF_(1α) and thromboxane (TX) B_2. Using [3,3,4,4-~2H_4]PGE_2, [3,3,4,4-~2H_4]PGD_2, [3,3,4,4-~2H_4]8-epi-PGF_(2α), [3,3,4,4-~2H_4]PGF_(2α), [3,3,4,4-~2H_4]6-keto-PGF_(1α) and [18,18,19,19-~2H_4]TXB_2 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.