Quantification of 3-nitrotyrosine in biological tissues and fluids: Generating valid results by eliminating artifactual formation

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 in biological systems we have developed an accurate, sensitive, and specific assay for 3-nitrotyrosine based on combined liquid chromatography tandem mass spectrometry. Our approach allowed simultaneous analysis of both tyrosine and 3-nitrotyrosine and employs isotopomer standards (i.e., [ 15 N 1 , 13 C 9 ]-tyrosine and [ 13 C 6 ]-3-nitrotyrosine). Calibration curves were linear ( r 2 = 0.999) across the range 0.5–100 pg/μL (i.e., 2.2–442 fmol/μL), and the detection limit for standard samples was 0.5 pg/μL (2.2 fmol/μL, or 10 fmol on column; S/N = 5) or 1 pg/μL (4.4 fmol/μL) for extracted (biological) samples. As a component of this study we have undertaken an extensive investigation of artifactual formation of 3-nitrotyrosine under conditions that exist during sample extraction and derivatization. Our studies 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.