ANALYSIS OF F-2-ISOPROSTANES AS INDICATORS OF NONENZYMATIC LIPID-PEROXIDATION IN-VIVO BY GAS-CHROMATOGRAPHY MASS-SPECTROMETRY - DEVELOPMENT OF A SOLID-PHASE EXTRACTION PROCEDURE

Recently, it has been reported that a series of prostaglandin F-2-like compounds (F-2-isoprostanes) are produced in vivo during peroxidation of arachidonic acid by a mechanism independent of the cyclooxygenase pathway. Of these, 8-epi-PGF(alpha)(2) is shown to be a potent vasoconstrictor. We describe an improved method for analysing F-2-isoprostanes in biological fluids. The method involves solid-phase extraction on an octadecylsilane (C-18) and an aminopropyl (NH2) cartridge. After conversion to pentafluorobenzyl ester and trimethylsilyl ether derivatives, F-2-isoprostanes are analysed by negative-ion chemical ionization mass spectrometry using tetradeuterated PGF(alpha)(2) as the internal standard. The limit of detection of the assay was 10 pg/ml, with a coefficient of variation ranging from 9.4 to 15.1%. Analysis of plasma samples from healthy volunteers (n = 7) revealed no quantifiable levels of free (unesterified) 8-epi-PGF(alpha)(2). However, the plasma samples contained 58 to 166 pg/ml of 8-epi-PGF(alpha)(2) when analyzed for the total (sum of free and esterified) F-2-isoprostanes. The main advantages of the method lie in the improved recovery, gas chromatographic separation and speed compared to existing techniques.