HPLC–Atmospheric Pressure Chemical Ionization MS/MS for Quantification of 15-F2t-Isoprostane in Human Urine and Plasma

BACKGROUND: Quantification of F(2)-Isoprostanes is considered a reliable index of the oxidative stress status in vivo and is valuable in the diagnosis and monitoring of a variety of diseases. Because of complex and lengthy sample preparation procedures, current chromatography/mass spectrometry and immunoassays are impractical for measuring larger numbers of samples. Thus, we developed and validated a semiautomated high-throughput HPLC tandem mass spectrometry assay for the quantification of F(2)-Isoprostane F(2t) in human urine and plasma. METHODS: After protein precipitation (500 microL methanol/zinc sulfate added to 500 microL plasma), samples were injected into the HPLC system and extracted online. The extracts were then back-flushed onto the analytical column and detected with an atmospheric pressure chemical ionization-triple quadrupole mass spectrometer monitoring the deprotonated molecular ions [M-H](-) of 15-F(2t)-IsoP (m/z = 353-->193) and the internal standard 15-F(2t)-IsoP-d(4) (m/z = 357-->197). RESULTS: In human urine, the assay was linear from 0.025 to 80 microg/L and in human plasma from 0.0025 to 80 microg/L (r(2)>0.99). Interday accuracy and precision for concentrations above the lower limit of quantification were <10%. Concentrations of 15-F(2t)-IsoP in urine of 16 healthy individuals ranged from 55-348 ng/g creatinine. In 16 plasma samples from healthy individuals, free 15-F(2t)-IsoP was detectable in all samples and concentrations were 3-25 ng/L. CONCLUSIONS: Our assay meets all predefined method performance criteria, allows for analysis of >80 samples/day, and has sufficient sensitivity for quantifying 15-F(2t)-IsoP concentrations in plasma and urine from healthy individuals. It is, thus, suitable for clinical routine monitoring and the analysis of samples from larger clinical trials.