We have developed an improved method for the measurement of F-2-isoprostanes using stable isotope dilution capillary gas chromatography/electron capture negative ionization mass spectrometry (GC-ECNI-MS). The F-2-isoprostane family consists of a series of chemically stable prostaglandin F-2 (PGF(2))-like compounds generated during peroxidation of arachidonic acid in phospholipids. There is evidence that measurement of F-2-isoprostanes represents a reliable and useful index of lipid peroxidation and oxidant stress in vivo. Furthermore, 8-epi-PGF(2 alpha), which is one of the more abundant F-2-isaprostanes, is biologically active, being a potent mitogen and vasoconstrictor of rat and rabbit lung and kidney, as well as a partial agonist of platelet aggregation. Measurement of F-2-isoprostanes in biological samples is complex and has involved methods which utilize multiple chromatographic steps, including separation by thin-layer chromatography, leading to poor sample recovery. We now present an improved method for the measurement of plasma and urinary F-2-isoprostanes using a combination of silica and reverse-phase extraction cartridges, high-performance liquid chromatography (HPLC), and GC-ECNI-MS. Different approaches to the derivatization of the F-2-isoprostanes prior to GC-ECNI-MS are also addressed. The overall recovery of F-2-isoprostanes is improved (approx 70% for urine) and the within and between assay reproducibility is 6.7% (n = 23) and 3.7% (n = 3), respectively. The mean urinary excretion of F-2-isoprostanes in eight healthy males was 365 +/- 5 pmol/ mmol creatinine and in three smokers 981 +/- 138 pmol/ mmol creatinine. The mean total (free + esterified) plasma F-2-isoprostane concentration was 952 +/- 38 pmol/liter, with a within and between assay reproducibility of 8% (n = 13) and 5.6% (n = 3), respectively. This improved method for the measurement of F-2-isoprostomes represents a significant advance in terms of the rapidity and yield in the purification of biological samples. The inclusion of HPLC separation enables improved analysis of F-2-isoprostanes by GC-MS. This methodology will assist in defining the role of F-2-isoprostanes as in vivo markers of oxidant stress in clinical and experimental settings. (C) 1999 Academic Press.
