Polk Bros. Foundation

Isoprostane F(2alpha) (iPF(2alpha)-III) concentration in plasma and urine is widely accepted as a measure of oxidative stress. Gas chromatography-mass spectrometry (GC/MS) methods for measuring iPF(2alpha)-III involve several steps of sample preparation and are labor-intensive, and ELISA methods, although easier to use, are less reliable. Therefore we developed a simple and sensitive method involving 1-step sample cleanup and HPLC/MS quantification. Samples of plasma or urine were enriched with a deuterated (iPF(2alpha)-III-D4) standard, treated with KOH to liberate the bound isoprostanes, then loaded onto an immunoaffinity column, and the bound isoprostane was eluted with 95% ethanol. The concentrated sample was injected onto a C-18 HPLC column, and the isoprostane was eluted with a gradient of acetonitrile in water and analyzed by electrospray negative ionization, selectively monitoring the ions 353.2 (iPF(2alpha)-III) and 357.2 (iPF(2alpha)-III-D4). The amount of isoprostane in the sample was calculated from the ratio of the intensities of the 2 ions. The described method has a detection limit of 0.5 ng/L, with a linear dynamic range of 1-5000 ng/L. The intra- and interassay imprecisions were 4.68% and 3.88%, respectively. The values obtained correlated strongly with the GC/MS procedure (r = 0.80), but the absolute values were approximately 4- to 5-fold lower, because the present method measures specifically 1 isomer of isoprostane, whereas the GC/MS method measures 4 isomers together. Because of its simplicity and lower limit of quantification, the present method provides a useful noninvasive tool for determining oxidative stress in patients.