NADPH Oxidases pp 243-258 | Cite as

HPLC-Based Monitoring of Oxidation of Hydroethidine for the Detection of NADPH Oxidase-Derived Superoxide Radical Anion

  • Jacek ZielonkaEmail author
  • Monika Zielonka
  • Balaraman Kalyanaraman
Part of the Methods in Molecular Biology book series (MIMB, volume 1982)


Hydroethidine is a fluorogenic probe that in the presence of the superoxide radical anion is oxidized to a red fluorescent product, 2-hydroxyethidium. In cells, hydroethidine is also oxidized to other products, including red fluorescent ethidium. Thus, selective monitoring of 2-hydroxyethidium is required for specific detection of the superoxide radical anion. Here, we provide protocols for HPLC- and LC-MS-based quantitation of 2-hydroxyethidium, among other oxidation products. Also, a protocol for continuous sampling for real-time monitoring of superoxide production using rapid HPLC measurements of 2-hydroxyethidium is described.

Key words

Superoxide radical anion Hydroethidine 2-hydroxyethidium HPLC LC-MS 



This work was supported by NIH grants NCI U01 CA178960 and R01 AA022986 to B.K.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Jacek Zielonka
    • 1
    • 2
    • 3
    Email author
  • Monika Zielonka
    • 1
    • 2
  • Balaraman Kalyanaraman
    • 1
    • 2
    • 3
  1. 1.Department of BiophysicsMedical College of WisconsinMilwaukeeUSA
  2. 2.Free Radical Research Center, Medical College of WisconsinMilwaukeeUSA
  3. 3.Cancer Center, Medical College of WisconsinMilwaukeeUSA

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