F(2)-Isoprostanes (F(2)-IsoPs), regio- and stereoisomers of prostaglandin F(2alpha) (PGF(2alpha)), and urinary F(2)-IsoP metabolites including 2,3-dinor-5,6-dihydro-8-iso-PGF(2alpha) [2,3-dinor-8-iso-PGF(1alpha) (2,3-dinor-F1)] and 2,3 dinor-8-iso-PGF(2alpha) (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(2alpha); 8-iso-PGF(2alpha); 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 F(2)-IsoP concentrations were approximately 0.2-1.5 microg/g creatinine, 2,3-dinor-F1 was approximately1-3 microg/g and 2,3-dinor-F2 was approximately 3-5 microg/g. Additional F(2)-IsoPs metabolites were identified using SRM. The sum of all urinary F(2)-IsoP metabolites was 50-100 microg/g creatinine indicating their greater abundance than F(2)-IsoPs. Women had higher F(2)-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 F(2)-IsoPs and their metabolites for the determination of oxidative stress.