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Clinical Chemistry 53: 489-497, 2007. First published January 26, 2007; 10.1373/clinchem.2006.078972
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(Clinical Chemistry. 2007;53:489-497.)
© 2007 American Association for Clinical Chemistry, Inc.

Endocrinology and Metabolism

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

Manuel Haschke1, Yan Ling Zhang1, Christine Kahle1, Jelena Klawitter1, Magdalena Korecka2, Leslie M. Shaw2 and Uwe Christians1,a

1 Clinical Research and Development, Department of Anesthesiology, University of Colorado Health Sciences Center, Denver, Colorado.
2 Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, PA.

aAddress correspondence to this author at: Clinical Research and Development, Department of Anesthesiology, University of Colorado Health Sciences Center, 4200 East Ninth Ave., Room UH-2122, Campus Box B-113, Denver, Colorado 80262. Fax 303-315-1858; e-mail uwe.christians{at}uchsc.edu.

Background: Quantification of F2-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 F2-Isoprostane F2t in human urine and plasma.

Methods: After protein precipitation (500 µL methanol/zinc sulfate added to 500 µL 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-F2t-IsoP (m/z = 353->193) and the internal standard 15-F2t-IsoP-d4 (m/z = 357->197).

Results: In human urine, the assay was linear from 0.025 to 80 µg/L and in human plasma from 0.0025 to 80 µg/L (r2>0.99). Interday accuracy and precision for concentrations 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 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-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.




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