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Journal of Lipid Research, Vol. 48, 1607-1617, July 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Quantitation of isoprostane isomers in human urine from smokers and nonsmokers by LC-MS/MS¹

Weiying Yan^*, Gary D. Byrd^2, and Michael W. Ogden

^* Department of Physiology and Pharmacology, Wake Forest University Medical Center, Winston-Salem, NC
Human Studies Division, R. J. Reynolds Tobacco Company, Winston-Salem, NC

¹ Part of this work was presented at the 54th Annual Conference of the American Society for Mass Spectrometry (Seattle, WA, June, 2006).

Published, JLR Papers in Press, April 24, 2007.

² To whom correspondence should be addressed. e-mail: byrdg{at}rjrt.com

A simple, rapid liquid chromatography-tandem mass spectrometry method was developed to identify and quantitate in human urine the isoprostanes iPF₂-III, 15-epi-iPF₂-III, iPF₂-VI, and 8,12-iso-iPF₂-VI along with the prostaglandin PGF₂ and 2,3-dinor-iPF₂-III, a metabolite of iPF₂-III. Assay specificity, linearity, precision, and accuracy met the required criteria for most analytes. The urine sample storage stability and standard solution stability were also tested. The methodology was applied to analyze 24 h urine samples collected from smokers and nonsmokers on controlled diets. The results for iPF₂-III obtained by our method were significantly correlated with results by an ELISA, although an 2-fold high bias was observed for the ELISA data. For iPF₂-III and its metabolite 2,3-dinor-iPF₂-III, smokers had significantly higher concentrations than nonsmokers (513 ± 275 vs. 294 ± 104 pg/mg creatinine; 3,030 ± 1,546 vs. 2,046 ± 836 pg/mg creatinine, respectively). The concentration of iPF₂-VI tended to be higher in smokers than in nonsmokers; however, the increase was not statistically significant in this sample set. Concentrations of the other three isoprostane isomers showed no trends toward differences between smokers and nonsmokers. Among smokers, the daily output of two type VI isoprostanes showed a weak correlation with the amount of tobacco smoke exposure, as determined by urinary excretion of total nicotine equivalents.

Supplementary key words liquid chromatography-tandem mass spectrometry • isoprotanes • prostaglandins • quantitation • validation • oxidative stress • cigarette smoking

Abbreviations: CID, collision-induced dissociation; ESI, electrospray ionization; FDA/CDER, Food and Drug Administration Center for Drug Evaluation and Research; HCl, hydrochloric acid; LC-MS, liquid chromatography-mass spectrometry; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LOQ, limit of quantitation; MRM, multiple reaction monitoring; NH₄OH, ammonium hydroxide; QC, quality control; RSD, relative standard deviation