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 mu L methanol/Zinc sulfate added to 500 mu L 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 mu g/L and in human plasma from 0.0025 to 80 mu g/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. (c) 2007 American Association for Clinical Chemistry.