An Improved Method for the Measurement of Urinary and Plasma F2-Isoprostanes Using Gas Chromatography–Mass Spectrometry

Abstract

We have developed an improved method for the measurement of F2-isoprostanes using stable isotope dilution capillary gas chromatography/electron capture negative ionization mass spectrometry (GC-ECNI-MS). The F2-isoprostane family consists of a series of chemically stable prostaglandin F2(PGF2)-like compounds generated during peroxidation of arachidonic acid in phospholipids. There is evidence that measurement of F2-isoprostanes represents a reliable and useful index of lipid peroxidation and oxidant stressin vivo.Furthermore, 8-epi-PGF2α, which is one of the more abundant F2-isoprostanes, 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 F2-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 F2-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 F2-isoprostanes prior to GC-ECNI-MS are also addressed. The overall recovery of F2-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 F2-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 F2-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 F2-isoprostanes 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 F2-isoprostanes by GC-MS. This methodology will assist in defining the role of F2-isoprostanes asin vivomarkers of oxidant stress in clinical and experimental settings.