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.
Haschke
Manuel
M
Clinical Research and Development, Department of Anesthesiology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Zhang
Yan Ling
YL
Kahle
Christine
C
Klawitter
Jelena
J
Korecka
Magdalena
M
Shaw
Leslie M
LM
Christians
Uwe
U
D001274:Atmospheric Pressure; D001322:Autoanalysis; D002851:Chromatography, High Pressure Liquid; D015237:Dinoprost / Q000031:analogs & derivatives* / Q000097:blood / Q000652:urine; D006801:Humans; D012016:Reference Values; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization
C075750:8-epi-prostaglandin F2alpha; D015237:Dinoprost