, Volume 43, Issue 10, pp 925-936
Date: 27 Aug 2008

Women and Smokers Have Elevated Urinary F2-Isoprostane Metabolites: A Novel Extraction and LC–MS Methodology

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F2-Isoprostanes (F2-IsoPs), regio- and stereoisomers of prostaglandin F (PGF), and urinary F2-IsoP metabolites including 2,3-dinor-5,6-dihydro-8-iso-PGF [2,3-dinor-8-iso-PGF (2,3-dinor-F1)] and 2,3 dinor-8-iso-PGF (2,3-dinor-F2), have all been used as biomarkers of oxidative stress. A novel method was developed to measure these biomarkers using a single solid phase extraction (SPE) cartridge, separation by HPLC, and detection by negative mode selected reaction monitoring (SRM) mass spectrometry (MS), using authentic standards of PGF; 8-iso-PGF; 2,3-dinor-F1 and 2,3-dinor-F2 to identify specific chromatographic peaks. The method was validated in a population of healthy, college-aged nonsmokers (n = 6 M/8F) and smokers (n = 6 M/5F). Urinary F2-IsoP concentrations were ~0.2–1.5 μg/g creatinine, 2,3-dinor-F1 was ~1–3 μg/g and 2,3-dinor-F2 was ~3–5 μg/g. Additional F2-IsoPs metabolites were identified using SRM. The sum of all urinary F2-IsoP metabolites was 50–100 μg/g creatinine indicating their greater abundance than F2-IsoPs. Women had higher F2-IsoP metabolite concentrations than did men (MANOVA, main effect P = 0.003); cigarette smokers had higher concentrations than did nonsmokers (main effect P = 0.036). For men or women, respectively, smokers had higher metabolite concentrations than did nonsmokers (P < 0.05). Thus, our method simultaneously allows measurement of urinary F2-IsoPs and their metabolites for the determination of oxidative stress.