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. 2020 May 6;9(5):388. doi: 10.3390/antiox9050388

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© 2020 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Detection of free 3-NT from nitrated heart proteins by HPLC/ECD. Heart homogenates were nitrated by Sin-1 and generated 3-NT was reduced by dTH. These samples were subjected to pronase digest before HPLC/ECD analysis. (A) Representative chromatogram of authentic 3-NT standard. (B) Representative chromatograms of Sin-1 treated heart proteins after digest with or without dTH. (C) Spiking of potential 3-NT peak with low concentrations of authentic 3-NT standard to proof the identity of this peak. HPLC/ECD analysis was performed with 20 µL of sample at 27 °C with isocratic elution (1.3 mL/min, mobile phase: 26.3 mM sodium citrate and 10.9 mM sodium at pH 4.85 with 3.25 v/v% methanol; 3-NT eluted at 4.39 min).