Clinical Chemistry
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Clinical Chemistry 53: 251-258, 2007. First published January 2, 2007; 10.1373/clinchem.2006.074989
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Right arrow Lipids, Lipoproteins, and Cardiovascular Risk Factors
(Clinical Chemistry. 2007;53:251-258.)
© 2007 American Association for Clinical Chemistry, Inc.

Lipids, Lipoproteins, and Cardiovascular Risk Factors

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

Debajit Sircar and Papasani V. Subbaiaha

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

aAddress correspondence to this author at: Department of Medicine, 1819 West Polk St., M/C 797, Chicago, IL 60612. Fax 312-413-0435; e-mail: psubbaia{at}uic.edu.

Background: Isoprostane F2{alpha} (iPF2{alpha}-III) concentration in plasma and urine is widely accepted as a measure of oxidative stress. Gas chromatography–mass spectrometry (GC/MS) methods for measuring iPF2{alpha}-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{alpha}-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{alpha}-III) and 357.2 (iPF2{alpha}-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 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 ~4– to 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 lower limit of quantification, the present method provides a useful noninvasive tool for determining oxidative stress in patients.






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Copyright © 2007 by the American Association for Clinical Chemistry.