Dh. Yi et al., Quantification of 3-nitrotyrosine in biological tissues and fluids: Generating valid results by eliminating artifactual formation, J AM SOC M, 11(6), 2000, pp. 578-586
Citations number
20
Categorie Soggetti
Spectroscopy /Instrumentation/Analytical Sciences
Journal title
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
Reactive nitrogen species such as peroxynitrite can nitrate specific amino
acids, whether free or protein bound, and 3-nitrotyrosine is believed to be
one marker of this reaction. To examine the significance of this pathway i
n biological systems we have developed an accurate, sensitive, and specific
assay for 3-nitrotyrosine based on combined liquid chromatography tandem m
ass spectrometry. Our approach allowed simultaneous analysis of both tyrosi
ne and 3-nitrotyrosine and employs isotopomer standards (i.e., [N-15(1), C-
13(9)]-tyrosine and [C-13(6)]-3-nitrotyrosine). Calibration curves were lin
ear (r(2) = 0.999) across the range 0.5-100 pg/mu L (i.e., 2.2-442 fmol/mu
L), and the detection limit for standard samples was 0.5 pg/mu L (2.2 fmol/
mu L, or 10 fmol on column; S/N = 5) or 1 pg/mu L (4.4 fmol/mu L) for extra
cted (biological) samples. As a component of this study we have undertaken
an extensive investigation of artifactual formation of 3-nitrotyrosine unde
r conditions that exist during sample extraction and derivatization. Our st
udies show that under appropriate conditions (low pH, elevated temperatures
, and in the presence of a vast excess of the two substrates, tyrosine and
the nitrate anion), 3-nitrotyrosine can readily be formed as an artifact. (
J Am Soc Mass Spectrom 2000, 11, 578-586) (C) 2000 American Society for Mas
s Spectrometry.