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) E(2), PGD(2), PGF(2 alpha), 8-epi-PGF(2 alpha), 6-keto-PGF(1 alpha) and thromboxane (TX) B(2). Using [3,3,4,4-(2)H(4)]PGE(2), [3,3,4,4-(2)H(4)]PGD(2), [3,3,4,4-(2)H(4)]8-epi-PGF(2 alpha), [3,3,4,4-(2)H(4)]PGF(2 alpha), [3,3,4,4-(2)H(4)]6-keto-PGF(1 alpha) and [18,18,19,19-(2)H(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.
Tsukamoto
Hiroki
H
Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan.
Hishinuma
Takanori
T
Mikkaichi
Tsuyoshi
T
Nakamura
Hironori
H
Yamazaki
Tohru
T
Tomioka
Yoshihisa
Y
Mizugaki
Michinao
M
D002138:Calibration; D002460:Cell Line; D003470:Culture Media / Q000737:chemistry*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D028421:Isoprostanes / Q000032:analysis*; D011453:Prostaglandins / Q000032:analysis*; D012015:Reference Standards; D013929:Thromboxane B2 / Q000032:analysis*
D003470:Culture Media; D028421:Isoprostanes; D011453:Prostaglandins; D013929:Thromboxane B2