IMPROVED QUANTIFICATION OF 8-EPI-PROSTAGLANDIN F2-ALPHA AND F-2-ISOPROSTANES BY GAS-CHROMATOGRAPHY TRIPLE-STAGE QUADRUPOLE MASS-SPECTROMETRY - PARTIAL CYCLOOXYGENASE-DEPENDENT FORMATION OF 8-EPI-PROSTAGLANDIN F2-ALPHA IN HUMANS

F-2-isoprostanes are considered to be novel markers of lipid peroxidat ion. To study the in vivo formation of F-2-isoprostanes, an improved m ethod was developed for isotope dilution assays involving gas chromato graphy/triple-stage quadrupole mass spectrometry (GC/MS/MS) including thin-layer chromatography (TLC) (sum of all F-2-isoprostanes) and high -performance liquid chromatographic (HPLC) purification (prostaglandin F-2 alpha (PGF(2 alpha)) and 8-epi-PGF(2 alpha)). Following the addit ion of isotopically labeled prostaglandins to urine, the sample was ac idified and applied to a C-18 cartridge. After elution, prostaglandins were derivatized to pentafluorobenzyl esters and subjected to TLC. A broad zone was scratched off, isoprostanes were eluted and after forma tion of their trimethylsilyl ether derivatives the sun of F-2-isoprost anes was determined by GC/MS/MS. For the determination of PGF(2 alpha) and 8-epi-PGF(2 alpha) prior to trimethylsilylation an additional HPL C step was performed and the fractions containing PGF(2 alpha) and 8-e pi-PGF(2 alpha) were analyzed by GC/MS/MS. Using this technique, 8-epi -PGF(2 alpha) concentrations in urine samples as low as 5 pg ml(-1) co uld be determined with high accuracy. The excretion rates of isoprosta nes were studied in comparison with the classical prostaglandins in th ree different groups: healthy adults, healthy children and children wi th hyper-PGE syndrome (HPS), a pathological situation associated with a stimulated PGE(2 alpha) synthesis. F-2-isoprostanes represented the main arachidonic acid metabolites in these groups and 8-epi-PGF(2 alph a) excretion was comparable in its amount to the classical prostanoids . To delineate the cyclooxygenase-catalyzed contribution, the influenc e of indomethacin, an inhibitor of cyclooxygenases, on F-2-isoprostane formation in healthy adults and in HPS children was analyzed. Signifi cantly decreased excretion rates were observed 2 days after indomethac in administration for all prostanoids, including F-2-isoprostanes and 8-epi-PGF(2 alpha). However, the suppression of F-2-isoprostanes and 8 -epi-PGF(2 alpha) excretion rates was less pronounced in comparison wi th the classical prostanoids. An improved and reliable method for the determination of F-2-isoprostanes and especially 8-epi-PGF(2 alpha) ha s been developed. The data obtained on human urine samples indicates a contribution of the cyclooxygenase pathway to the formation of isopro stanes. (C) 1997 John Wiley & Sons, Ltd.