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Determination of Nitric Oxide-Derived Nitrite and Nitrate in Biological Samples by HPLC Coupled to Nitrite Oxidation
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...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer Berlin Heidelberg
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3837189/ https://www.ncbi.nlm.nih.gov/pubmed/24293681 http://dx.doi.org/10.1007/s10337-013-2529-0 |
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author | Wu, Anguo Duan, Tingting Tang, Dan Xu, Youhua Feng, Liang Zheng, Zhaoguang Zhu, Jiaxiao Wang, Rushang Zhu, Quan |
author_facet | Wu, Anguo Duan, Tingting Tang, Dan Xu, Youhua Feng, Liang Zheng, Zhaoguang Zhu, Jiaxiao Wang, Rushang Zhu, Quan |
author_sort | Wu, Anguo |
collection | PubMed |
description | 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 C(18) 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. |
format | Online Article Text |
id | pubmed-3837189 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Springer Berlin Heidelberg |
record_format | MEDLINE/PubMed |
spelling | pubmed-38371892013-11-29 Determination of Nitric Oxide-Derived Nitrite and Nitrate in Biological Samples by HPLC Coupled to Nitrite Oxidation Wu, Anguo Duan, Tingting Tang, Dan Xu, Youhua Feng, Liang Zheng, Zhaoguang Zhu, Jiaxiao Wang, Rushang Zhu, Quan Chromatographia Original 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 C(18) 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. Springer Berlin Heidelberg 2013-08-04 2013 /pmc/articles/PMC3837189/ /pubmed/24293681 http://dx.doi.org/10.1007/s10337-013-2529-0 Text en © The Author(s) 2013 https://creativecommons.org/licenses/by/2.0/ Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. |
spellingShingle | Original Wu, Anguo Duan, Tingting Tang, Dan Xu, Youhua Feng, Liang Zheng, Zhaoguang Zhu, Jiaxiao Wang, Rushang Zhu, Quan Determination of Nitric Oxide-Derived Nitrite and Nitrate in Biological Samples by HPLC Coupled to Nitrite Oxidation |
title | Determination of Nitric Oxide-Derived Nitrite and Nitrate in Biological Samples by HPLC Coupled to Nitrite Oxidation |
title_full | Determination of Nitric Oxide-Derived Nitrite and Nitrate in Biological Samples by HPLC Coupled to Nitrite Oxidation |
title_fullStr | Determination of Nitric Oxide-Derived Nitrite and Nitrate in Biological Samples by HPLC Coupled to Nitrite Oxidation |
title_full_unstemmed | Determination of Nitric Oxide-Derived Nitrite and Nitrate in Biological Samples by HPLC Coupled to Nitrite Oxidation |
title_short | Determination of Nitric Oxide-Derived Nitrite and Nitrate in Biological Samples by HPLC Coupled to Nitrite Oxidation |
title_sort | determination of nitric oxide-derived nitrite and nitrate in biological samples by hplc coupled to nitrite oxidation |
topic | Original |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3837189/ https://www.ncbi.nlm.nih.gov/pubmed/24293681 http://dx.doi.org/10.1007/s10337-013-2529-0 |
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