title: A derivatization assay using gaschromatography/negative chemical ionization tandem mass spectrometry to quantify 3-nitrotyrosine in human plasma.
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.
authors.affiliation: Department of Clinical Physiology, Göteborg University, Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden.
authors.forename: Ann-Sofi
authors.initials: AS
authors.lastname: Söderling
authors.forename: Henrik
authors.initials: H
authors.lastname: Ryberg
authors.forename: Anders
authors.initials: A
authors.lastname: Gabrielsson
authors.forename: Mona
authors.initials: M
authors.lastname: Lärstad
authors.forename: Kjell
authors.initials: K
authors.lastname: Torén
authors.forename: Sohbat
authors.initials: S
authors.lastname: Niari
authors.forename: Kenneth
authors.initials: K
authors.lastname: Caidahl
mesh_terms: D000328:Adult; D016477:Artifacts; D002138:Calibration; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D015394:Molecular Structure; D012680:Sensitivity and Specificity; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000097:blood*
chemical_list: C002744:3-nitrotyrosine; D014443:Tyrosine