Although measurements of plasma F-2-isoprostanes are established markers of oxidative stress, their quantification only reflects acute non-enzymatic lipid peroxidation. In this study, a new approach is described for the rapid isolation and measurement of urinary 8-epi-PGF(2 alpha) and its endogenous beta-oxidation metabolites (2,3-dinor-8-epi-PGF(2 alpha) and 2,3-dinor-5,6-dihydro-PGF(2 alpha)) for use as index of total body oxidative stress. Isoprostanes were partitioned with ethyl acetate and subsequently purified by chromatography on an aminopropyl (NH2) and silica (Si) cartridge. Final analysis of F-2-isoprostanes as trimetilylsilyl-ester/pentafluorobenzyl ester derivatives was carried out by stable isotope dilution mass spectrometry. Overall recovery of F-2-isoprostanes was 80 +/- 4%. Inter- and intra-assay coefficients of variation were 5% and 7%, respectively. In a group of healthy humans, the mean excretion rates expressed as nmol/mmol creatinine for 2,3-dinor-8-epi-PGF(2 alpha), 2,3-dinor-5,6-dihydro-8-epi-PGF(2 alpha), and 8-epi-PGF(2 alpha) were 5.43 +/- 1.93, 2.16 +/- 0.71, and 0.36 +/- 10.16, respectively. Correlations were obtained between 8-epi-PGF(2 alpha), and 2,3-dinor-8-epi-PGF(2 alpha) or 2,3-dinor-5,6-dihydro-8-epi-PGF(2 alpha) (r = 0.998 and r = 0.937, respectively). A strong relationship was also seen between 2,3-dinor-8-epi-PGF(2) and 2,3-dinor-5,6-dihydro-8-epi-PGF(2 alpha) (r = 0.949). The new technique allows for high sample throughput and avoids the need for HPLC and/or other expensive equipment required for the initial sample preparation. Simultaneous analysis of urinary 8-epi-PGF(2 alpha) and its metabolites should provide unique tool in clinical trials exploring the role of oxidant injury in human disease. (c) 2005 Elsevier Inc. All rights reserved.