Quantification of the major urinary metabolite of 15-F2t-isoprostane (8-iso-PGF2α) by a stable isotope dilution mass spectrometric assay

The isoprostanes (IsoPs) are a series of novel prostaglandin (PG)-like compounds generated from the free radical-catalyzed peroxidation of arachidonic acid. The first series of IsoPs characterized contained F-type prostane rings analogous to PGF(2 alpha). One F-ring IsoP, 15-F-2t-IsoP (8-iso-PGF(2 alpha)) has been shown to be formed in abundance in vivo and to exert potent biological activity. As a means to assess the endogenous production of this compound, we developed a method to quantify the major urinary metabolite of 15-F-2t-IsoP, 2,3-dinor-5,6-dihydro-15-F-2t-IsoP (2,3-dinor-5,6-dihydro-8-iso-PGF(2 alpha)), by gas chromotography/negative ion chemical ionization mass spectrometry. This metabolite was chemically synthesized and converted to an O-18(2)-labeled derivative for use as an internal standard. After purification, the compound was analyzed as a pentafluorobenzyl ester trimethylsilyl ether. Precision of the assay is +/-4% and accuracy is 97%, The lower limit of sensitivity is approximately 20 pg, Levels of the urinary excretion of this metabolite in 10 normal adults were found to be 0.39 +/- 0.18 ng/mg creatinine (mean +/- 2 SD). Substantial elevations in the urinary excretion of the metabolite were found in situations in which IsoP generation is increased and antioxidants effectively suppressed metabolite excretion. Levels of 2,3-dinor-5,6-dihydro-15-F-2t-IsoP were not affected by cyclooxygenase inhibitors. Thus, this assay provides a sensitive and accurate method to assess endogenous production of 15-F-2t-IsoP as a means to explore the pathophysiological role of this compound in human disease. (C) 1999 Academic Press.