Received on June 14, 2006
Accepted on November 30, 2006

Lipids, Lipoproteins, and Cardiovascular Risk Factors

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

¹ Departments of Medicine and Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL
² Department of Medicine and Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL

^* To whom correspondence should be addressed. E-mail: psubbaiah{at}uic.edu.

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 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 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.