HPLC-DAD Determination of Nitrite and Nitrate in Human Saliva Utilizing a Phosphatidylcholine Column.

Małgorzata Tatarczak-Michalewska,Dariusz Majerek,Justyna Kawka,Eliza Blicharska,Wojciech Flieger,Jolanta Flieger,Wojciech Płaziński

doi:10.3390/molecules24091754

Abstract

The aim of this research was to optimize the separation and quantitative determination of nitrites and nitrates in human saliva. HPLC with UV absorption (HPLC/DAD) using a phosphatidylcholine column (IAM.PC.DD2 Regis HPLC) was applied in this assay. Nitrates were detected directly by their absorbance at 210 nm, whereas nitrites were detected after oxidation to nitrates by potassium permanganate at acidic conditions. The kinetics of the permanganate–nitrite reaction was measured chromatographically. The calibration graph for nitrates was linear in the range of 0.5–35 µg mL−1 with a correlation coefficient of 0.9999. The limit of detection was 4.56 ng mL−1. The calibration graph for nitrites (after oxidation to nitrates) was linear in the range of 0.5–15 µg mL−1 with a correlation coefficient of 0.9972. The limit of detection was 4.21 ng mL−1. The nitrate concentrations in the saliva samples were found in the range of 8.98–18.52 μg mL−1, whereas nitrite was in the range of 3.50–5.34 μg mL−1.

Highlights

IntroductionNitrate (NO3 − ) and nitrites (NO2 − ) are interconvertible as the components of the nitrogen cycle in the natural environment as well as the human body via the nitrate–nitrite–nitric oxide pathway [1]
Nitrate (NO3 − ) and nitrites (NO2 − ) are interconvertible as the components of the nitrogen cycle in the natural environment as well as the human body via the nitrate–nitrite–nitric oxide pathway [1].The endogenous source of these ions is the l-arginine–NO pathway, whereas the main dietary source is green leafy vegetables such as celery, lettuce or spinach, accounting for almost 90% of daily nitrate intake [2,3]
It has been proven that phosphatidylcholines (PCs), owing to their interaction with inorganic anions, are able to modify the functioning of membrane processes [4]

Summary

Introduction

Nitrate (NO3 − ) and nitrites (NO2 − ) are interconvertible as the components of the nitrogen cycle in the natural environment as well as the human body via the nitrate–nitrite–nitric oxide pathway [1]. The endogenous source of these ions is the l-arginine–NO pathway, whereas the main dietary source is green leafy vegetables such as celery, lettuce or spinach, accounting for almost 90% of daily nitrate intake [2,3]. Nitrates are biologically inert but could be hazardous after conversion to nitrites. The reduction process is able to proceed in vivo. Salivary bacterial reduction has been recognized as a dominant metabolic conversion process, as a substantial amount of ingested nitrate is concentrated in saliva [7]

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