A simple and accurate method to determine nitrite and nitrate in serum based on high-performance liquid chromatography with fluorescence detection

Biomed Chromatogr. 2013 Nov;27(11):1547-53. doi: 10.1002/bmc.2958. Epub 2013 Jun 13.


A simple method for accurate determination of nitrite and nitrate in serum was proposed to avoid the variation of nitrate reduction. For nitrite determination, serum samples were directly precipitated with methanol pre-nitrate conversion, and then the supernatant reacted with 2,3-diaminonaphthalene (DAN) to form 2,3-naphthotriazole (NAT), which was quantitatively analyzed by high-performance liquid chromatography coupled with fluorescence detection (HPLC-FL). For nitrate determination, samples were firstly heated at 70°C for 10 min to inactivate endogenous reductase-inhibiting proteins, then nitrate in the samples was quantitatively reduced to nitrite by reductase added experimentally. The difference in total nitrite concentrations between pre- and post-nitrate conversion was used to calculate the amount of nitrate in the samples. In addition to good specificity, high sensitivity, satisfactory accuracy and reproducibility, our method is simple and suitable for the quantitative determination of nanomolar level of nitrite and nitrate in a large number of serum samples.

Keywords: HPLC-FL; NAT; endogenous protein; nitrate; nitrate reduction; nitrite.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromatography, High Pressure Liquid / methods*
  • Male
  • Nitrates / blood*
  • Nitrites / blood*
  • Rats
  • Rats, Sprague-Dawley
  • Reproducibility of Results
  • Spectrometry, Fluorescence


  • Nitrates
  • Nitrites