Flow-Injection Spectrophotometric Determination of Nitrite and Nitrate in Biological Samples

Abstract

Nitrite and nitrate in biological samples can act as an indicator of nitric oxide generated in vivo and have attracted a lot of attention as a messenger of diverse physiological processes. For the determination of such nitrogen oxides, a simple and sensitive flow-injection method coupled with a successful pretreatment method was developed. Nitrite and nitrate in sample solution were injected into a carrier solution containing EDTA and ammonium buffer, and flowed into the copperized cadmium (Cd/Cu) reduction column installed on-line. Here the nitrate was reduced to nitrite, and then nitrite was determined spectrophotometrically on the basis of a diazotization/coupling reaction. The detection limits for nitrite and nitrate were 5×10⁻⁸ M, and the sampling rate was about 40 samples per hour for nitrate determination. The biological samples, such as serum, plasma and cell culture medium, were deproteinized using a batchwise NaOH–ZnSO₄ method before FIA measurement. The NaOH–ZnSO₄ deproteinization method was very effective in term of the recovery of nitrate and the maintenance of the reduction efficiency of the Cd/Cu column. The relative standard deviations were 2.1–3.1%, and the recoveries were 95–100% for the determination of nitrite and nitrate in serum samples through the whole procedure. In the case of cell culture medium, the proposed deproteinization was especially effective for removing the interfering amino acids containing sulfur atom by using the reaction between Zn²⁺ in deproteinization agent and sulfur atom in analytical substances.