Isoprostane Measurement in Plasma and Urine by Liquid Chromatography–Mass Spectrometry with 1-Step Sample Preparation

Background: Isoprostane F2 (iPF2 -III) concentration in plasma and urine is widely accepted as a measure of oxidative stress. Gas chromatography–mass spectroscopy (GC/MS) methods for measuring iPF2 -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. Methods: Samples of plasma or urine were enriched with a deuterated (iPF2 -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 (iPF2 -III) and 357.2 (iPF2 -III-D4). The amount of isoprostane in the sample was calculated from the ratio of the intensities of the 2 ions. Results: The described method is highly sensitive (detection limit, 0.5 pg/mL), with a linear dynamic range of 1–5000 pg/mL. The intraand 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 4–5-fold lower, because the present method measures specifically 1 isomer of isoprostane, whereas the GC/MS method measures 4 isomers together. Conclusions: Because of its simplicity and sensitivity, the present method provides a useful noninvasive tool for determining oxidative stress in patients. © 2007 American Association for Clinical Chemistry