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

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Journal of the American Society for Mass Spectrometry

Abstract

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., [15N1, 13C9]-tyrosine and [13C6]-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.

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Authors and Affiliations

  1. Ray Williams Biomedical Mass Spectrometry Facility, Wallace Wurth Medical Building, University of New South Wales, Sydney, Australia

    Donghui Yi, Benno A. Ingelse, Mark W. Duncan & George A. Smythe

  2. Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Campus Box C238, 80262, Denver, CO

    Mark W. Duncan

Authors
  1. Donghui Yi
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  2. Benno A. Ingelse
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  3. Mark W. Duncan
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  4. George A. Smythe
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Correspondence to Mark W. Duncan.

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Yi, D., Ingelse, B.A., Duncan, M.W. et al. Quantification of 3-nitrotyrosine in biological tissues and fluids: Generating valid results by eliminating artifactual formation. J Am Soc Mass Spectrom 11, 578–586 (2000). https://doi.org/10.1016/S1044-0305(00)00113-6

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  • DOI: https://doi.org/10.1016/S1044-0305(00)00113-6

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