Abstract
Endogenous free or protein‐associated 3‐nitrotyrosine (3‐NT) has been proposed as a biomarker of in vivo oxidative damage caused by nitrating agents. Isotopic dilution assay gaschromatographic/mass spectrometric (GC/MS) techniques have been employed to measure endogenous 3‐NT levels. However, the quantitative normal plasma values reported so far are inconsistent. The results vary between the assays; they may have been influenced by in vitro artifactual nitration of tyrosine to 3‐NT. In this study, a simple and artifact‐free derivatization method for quantifying the endogenous 3‐NT content of biological samples by GC/negative chemical ionization MS/MS is presented. The method is based on reduction of the nitro group of the molecule by dithionite, heptafluorobutyric acylation and subsequent methyl derivatization, di‐O‐methyldi‐N‐heptafluorobutyryl being the major derivative. The results showed excellent GC and MS properties, such as low background and a favorable fragmentation pattern. Endogenous 3‐NT was unequivocally quantified using collision‐induced dissociation in the selected reaction monitoring mode, whereas co‐elution of unknown compounds interfered in the selected‐ion monitoring mode. We found that tyrosine was nitrated in the presence of nitrate anions and heptafluorobutyric anhydride, but the product appeared as a di‐O‐methylmono‐N‐heptafluorobutyryl derivative. Therefore, artifactually formed 3‐NT did not contribute to the measured endogenous 3‐NT level owing to its different derivative structure. The method was applied to determine endogenous 3‐NT in human plasma and plasma proteins. A detection limit of 0.03 nM for ^13^C~6~‐labeled 3‐NT in plasma samples was established and the response was linear over a concentration range of 0–50 nM (R^2^ > 0.999). The endogenous free 3‐NT level (mean ± SD) in ultrafiltered plasma samples from 12 healthy adults was 0.74 ± 0.30 nM. The mean concentration of 3‐NT in their plasma total proteins was 0.60 ± 0.40 pmol mg^−1^. Hence, the described method is selective, eliminates the problem of artifactual nitration and is feasible for the quantification of free and protein‐associated 3‐NT in biological samples such as plasma. Copyright © 2003 John Wiley & Sons, Ltd.