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

Figure 6.

Figure 6

Detection of free 3-NT from control and diabetic animals with and without combined nitration by Sin-1. Kidney homogenates of healthy control and diabetic (ZDF) rats (10 mg/mL protein) were subjected to pronase digest before HPLC/ECD analysis or LC-MS/MS analysis. A special digestion protocol was used (see Methods for kidney samples). HPLC/ECD analysis was performed with 40 µL of sample at 27 °C with isocratic elution (0.75 mL/min, mobile phase: 26.3 mM sodium citrate and 10.9 mM sodium at pH 3.75; 3-NT eluted at 7.03 min). Representative chromatograms are shown for the HPLC/ECD quantification (A) or LC-MS/MS analysis (B). LC-MS/MS analysis was performed after adding an identical amount of deuterated 3-NT (D3-3-NT) to compensate for matrix effects. Mass transitions monitored at 227 → 117 (3-NT) and 230 → 119 (D3-NT) are shown. Representative MS/MS spectra of precursor ion at m/z = 227 are shown for the 3-NT standard (100 nM) and the sample from ZDF rat with 1 mM Sin-1 treatment (C).