Determination of Nitric Oxide-Derived Nitrite and Nitrate in Biological Samples by HPLC Coupled to Nitrite Oxidation

Abstract

Nitrite and nitrate are main stable products of nitric oxide, a pivotal cellular signaling molecule, in biological fluids. Therefore, accurate measurement of the two ions is profoundly important. Nitrite is difficult to be determined for a larger number of interferences and unstable in the presence of oxygen. In this paper, a simple, cost-effective and accurate HPLC method for the determination of nitrite and nitrate was developed. On the basis of the reaction that nitrite is oxidized rapidly to nitrate with the addition of acidic potassium permanganate, the determination of nitrite and nitrate was achieved by the following strategy: each sample was injected twice for HPLC analysis, i.e. the first injection was to measure nitrate, and the second injection was to measure total nitrate including initial nitrate and the nitrate from the conversion of nitrite with the addition of acid potassium permanganate in the sample. The amount of nitrite can be calculated as difference between injections 2 and 1. The HPLC separation was performed on a reversed phase C18 column for 15 min. The mobile phase consisted of methanol–water (2:98 by volume); the water in the mobile phase contained 0.60 mM phosphate salt (potassium dihydrogen and disodium hydrogen phosphate) and 2.5 mM tetrabutylammonium perchlorate (TBAP). The UV wavelength was set at 210 nm. Additionally, we systemically investigated the effects of the concentration of phosphate salt and TBAP in the mobile phase, the pH of the mobile phase, and the amount of acidic potassium permanganate added to the sample on the separation efficacy. The results showed that the limits of detection (LOD) and the limit of quantitation (LOQ) were 0.075 and 0.25 μM for nitrate (containing the oxidized nitrite), respectively. The linear range was 1–800 μM. This developed approach was successfully applied to assay nitrite/nitrate levels in cell culture medium, cell lysate, rat plasma and urine.

Irisin Lowers Blood Pressure by Improvement of Endothelial Dysfunction via AMPK‐Akt‐eNOS‐NO Pathway in the Spontaneously Hypertensive Rat

“…Besides, NO has an extremely short half‐life therefore, it is difficult to detect NO production in so short a time. Because of the above‐mentioned limitation, scientists realize that measurement of NO metabolites (ie, nitrite and nitrate) might be an alternative method, determined by the Griess reaction method, chemiluminescence, or high‐performance liquid chromatography (HPLC) . Among these methods, HPLC, with the advantages of high sensitivity, was widely applied recently.…”

“…Because of the above-mentioned limitation, scientists realize that measurement of NO metabolites (ie, nitrite and nitrate) might be an alternative method, determined by the Griess reaction method, 54 chemiluminescence, 55 or high-performance liquid chromatography (HPLC). 56 Among these methods, HPLC, with the advantages of high sensitivity, was widely applied recently. Because of the lack of HPLC equipment, we used DAF-2 DA fluorescent probes and the Griess reaction method to determine NO production instead in the present study.…”

Hemin‐Graphene Derivatives with Increased Peroxidase Activities Restrain Protein Tyrosine Nitration

“…[33] As shown in Figure 6B,t he retention time of nitrite was around 8.0 min, and that of nitrate was about 11.1 min. [33] As shown in Figure 6B,t he retention time of nitrite was around 8.0 min, and that of nitrate was about 11.1 min.…”

“…[33] The mobile phase consisted of 2% methanol and 98 %w ater containing 0.60 mmol L À1 phosphate salt (KH 2 PO 4 and Na 2 HPO 4 )a nd 2.5 mmol L À1 tetrabutylammonium perchlorate (TBAP) at 1mLmin À1 . The reaction mixtures contained 1mmol L À1 NaNO 2 ,1mmol L À1 H 2 O 2 ,3 mmol L À1 hemin and p-hemin predissolved in ammonia, 78.2 mgmL À1 rGO or GO, and the equivalent of 3 mmol L À1 hemin in hemin-rGO or hemin-GO composites.…”

Quantification of nitric oxide by high‑performance liquid chromatography‑fluorometric method in subgenomic hepatitis C virus‑replicon expressing Huh7 cells upon treatment with acetylsalicylic acid

“…High-performance liquid chromatography (HPLC) has been used previously to quantify NO (15) due to its high sensitivity (16) and was identified as a potentially more useful method to quantify NO in hepatocyte cell culture, based on the reaction of NO 2 with 2, 3-diaminonaphthalene (DAN) as an indicator of NO formation, under acidic conditions to obtain the fluorescent product 2, 3-naphthotriazole (NAT) (13). DAN has excitation and emission wavelengths of 375 and 415 nm, respectively, and a 10-30 nM detection limit (14).…”