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

sidered 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 chromatogra-phy/mass spectrometry and immunoassays are imprac-tical for measuring larger numbers of samples. Thus, we developed and validated a semiautomated high-throughput HPLC tandem mass spectrometry assay for the quantification of F2-Isoprostane F2t in human urine and plasma. Methods: After protein precipitation (500 L methanol/ zinc sulfate added to 500 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 spectrome-ter monitoring the deprotonated molecular ions [M-H]– of 15-F2t-IsoP (m/z 3533193) and the internal standard 15-F2t-IsoP-d4 (m/z 3573197). Results: In human urine, the assay was linear from 0.025 to 80 g/L and in human plasma from 0.0025 to 80 g/L (r2>0.99). Interday accuracy and precision for concentra-tions above the lower limit of quantification were <10%. Concentrations of 15-F2t-IsoP in urine of 16 healthy individuals ranged from 55–348 ng/g creatinine. In 16 plasma samples from healthy individuals, free 15-F2t-IsoP was detectable in all samples and concentra-tions were 3–25 ng/L. Conclusions: Our assay meets all predefined method performance criteria, allows for analysis of>80 sam-ples/day, and has sufficient sensitivity for quantifying 15-F2t-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