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

Abstract

A simple, rapid liquid chromatography-tandem mass spectrometry method was developed to identify and quantitate in human urine the isoprostanes iPF2α-III, 15-epi-iPF2α-III, iPF2α-VI, and 8,12-iso-iPF2α-VI along with the prostaglandin PGF2α and 2,3-dinor-iPF2α-III, a metabolite of iPF2α-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 iPF2α-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 iPF2α-III and its metabolite 2,3-dinor-iPF2α-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 iPF2α-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.

Keywords

• liquid chromatography-tandem mass spectrometry
• isoprotanes
• prostaglandins
• quantitation
• validation
• oxidative stress