GC-MS Analysis of Biological Nitrate and Nitrite Using Pentafluorobenzyl Bromide in Aqueous Acetone: A Dual Role of Carbonate/Bicarbonate as an Enhancer and Inhibitor of Derivatization

Carbon dioxide (CO<sub>2</sub>) and carbonates, which are widely distributed in nature, are constituents of inorganic and organic matter and are essential in vegetable and animal organisms. CO<sub>2</sub> is the principal greenhouse gas in the atmosphere. In human blood, CO&l...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autor principal:	Dimitrios Tsikas
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	acetazolamide carbonic anhydrase derivatization enhancement GC-MS inhibition Organic chemistry QD241-441
Acceso en línea:	https://doaj.org/article/84addc2e79104dd48eeabfe064743163
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:84addc2e79104dd48eeabfe064743163
record_format	dspace
spelling	oai:doaj.org-article:84addc2e79104dd48eeabfe0647431632021-11-25T18:29:04ZGC-MS Analysis of Biological Nitrate and Nitrite Using Pentafluorobenzyl Bromide in Aqueous Acetone: A Dual Role of Carbonate/Bicarbonate as an Enhancer and Inhibitor of Derivatization10.3390/molecules262270031420-3049https://doaj.org/article/84addc2e79104dd48eeabfe0647431632021-11-01T00:00:00Zhttps://www.mdpi.com/1420-3049/26/22/7003https://doaj.org/toc/1420-3049Carbon dioxide (CO<sub>2</sub>) and carbonates, which are widely distributed in nature, are constituents of inorganic and organic matter and are essential in vegetable and animal organisms. CO<sub>2</sub> is the principal greenhouse gas in the atmosphere. In human blood, CO<sub>2</sub>/HCO<sub>3</sub><sup>−</sup> is an important buffering system. Inorganic nitrate (ONO<sub>2</sub><sup>−</sup>) and nitrite (ONO<sup>−</sup>) are major metabolites and abundant reservoirs of nitric oxide (NO), an endogenous multifunctional signaling molecule. Carbonic anhydrase (CA) is involved in the reabsorption of nitrite and nitrate from the primary urine. The measurement of nitrate and nitrite in biological samples is of particular importance. The derivatization of nitrate and nitrite in biological samples alongside their <sup>15</sup>N-labeled analogs, which serve as internal standards, is a prerequisite for their analysis by gas chromatography–mass spectrometry (GC-MS). A suitable derivatization reagent is pentafluorobenzyl bromide (PFB-Br). Nitrate and nitrite are converted in aqueous acetone to PFB-ONO<sub>2</sub> and PFB-NO<sub>2</sub>, respectively. PFB-Br is also useful for the GC-MS analysis of carbonate/bicarbonate. This is of particular importance in conditions of pharmacological CA inhibition, for instance by acetazolamide, which is accompanied by elevated concomitant excretion of nitrate, nitrite and bicarbonate, as well as by urine alkalization. We performed a series of experiments with exogenous bicarbonate (NaHCO<sub>3</sub>) added to human urine samples (range, 0 to 100 mM), as well as with endogenous bicarbonate resulting from the inhibition of CA activity in healthy subjects before and after ingestion of pharmacological acetazolamide. Our results indicate that bicarbonate enhances the derivatization of nitrate with PFB-Br. In contrast, bicarbonate decreases the derivatization of nitrite with PFB-Br. Bicarbonate is not a catalyst, but it enhances PFB-ONO<sub>2</sub> formation and inhibits PFB-NO<sub>2</sub> formation in a concentration-dependent manner. The effects of bicarbonate are likely to result from its reaction with PFB-Br to generate PFB-OCOOH. Nitrate reacts with concomitantly produced PFB-OCOOH to form PFB-ONO<sub>2</sub> in addition to the direct reaction of nitrate with PFB-Br. By contrast, nitrite does not react with PFB-OCOOH to form PFB-NO<sub>2</sub>. Sample acidification by small volumes of 20 wt.% aqueous acetic acid abolishes the effects of exogenous and endogenous bicarbonate on nitrite measurement.Dimitrios TsikasMDPI AGarticleacetazolamidecarbonic anhydrasederivatizationenhancementGC-MSinhibitionOrganic chemistryQD241-441ENMolecules, Vol 26, Iss 7003, p 7003 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	acetazolamide carbonic anhydrase derivatization enhancement GC-MS inhibition Organic chemistry QD241-441
spellingShingle	acetazolamide carbonic anhydrase derivatization enhancement GC-MS inhibition Organic chemistry QD241-441 Dimitrios Tsikas GC-MS Analysis of Biological Nitrate and Nitrite Using Pentafluorobenzyl Bromide in Aqueous Acetone: A Dual Role of Carbonate/Bicarbonate as an Enhancer and Inhibitor of Derivatization
description	Carbon dioxide (CO<sub>2</sub>) and carbonates, which are widely distributed in nature, are constituents of inorganic and organic matter and are essential in vegetable and animal organisms. CO<sub>2</sub> is the principal greenhouse gas in the atmosphere. In human blood, CO<sub>2</sub>/HCO<sub>3</sub><sup>−</sup> is an important buffering system. Inorganic nitrate (ONO<sub>2</sub><sup>−</sup>) and nitrite (ONO<sup>−</sup>) are major metabolites and abundant reservoirs of nitric oxide (NO), an endogenous multifunctional signaling molecule. Carbonic anhydrase (CA) is involved in the reabsorption of nitrite and nitrate from the primary urine. The measurement of nitrate and nitrite in biological samples is of particular importance. The derivatization of nitrate and nitrite in biological samples alongside their <sup>15</sup>N-labeled analogs, which serve as internal standards, is a prerequisite for their analysis by gas chromatography–mass spectrometry (GC-MS). A suitable derivatization reagent is pentafluorobenzyl bromide (PFB-Br). Nitrate and nitrite are converted in aqueous acetone to PFB-ONO<sub>2</sub> and PFB-NO<sub>2</sub>, respectively. PFB-Br is also useful for the GC-MS analysis of carbonate/bicarbonate. This is of particular importance in conditions of pharmacological CA inhibition, for instance by acetazolamide, which is accompanied by elevated concomitant excretion of nitrate, nitrite and bicarbonate, as well as by urine alkalization. We performed a series of experiments with exogenous bicarbonate (NaHCO<sub>3</sub>) added to human urine samples (range, 0 to 100 mM), as well as with endogenous bicarbonate resulting from the inhibition of CA activity in healthy subjects before and after ingestion of pharmacological acetazolamide. Our results indicate that bicarbonate enhances the derivatization of nitrate with PFB-Br. In contrast, bicarbonate decreases the derivatization of nitrite with PFB-Br. Bicarbonate is not a catalyst, but it enhances PFB-ONO<sub>2</sub> formation and inhibits PFB-NO<sub>2</sub> formation in a concentration-dependent manner. The effects of bicarbonate are likely to result from its reaction with PFB-Br to generate PFB-OCOOH. Nitrate reacts with concomitantly produced PFB-OCOOH to form PFB-ONO<sub>2</sub> in addition to the direct reaction of nitrate with PFB-Br. By contrast, nitrite does not react with PFB-OCOOH to form PFB-NO<sub>2</sub>. Sample acidification by small volumes of 20 wt.% aqueous acetic acid abolishes the effects of exogenous and endogenous bicarbonate on nitrite measurement.
format	article
author	Dimitrios Tsikas
author_facet	Dimitrios Tsikas
author_sort	Dimitrios Tsikas
title	GC-MS Analysis of Biological Nitrate and Nitrite Using Pentafluorobenzyl Bromide in Aqueous Acetone: A Dual Role of Carbonate/Bicarbonate as an Enhancer and Inhibitor of Derivatization
title_short	GC-MS Analysis of Biological Nitrate and Nitrite Using Pentafluorobenzyl Bromide in Aqueous Acetone: A Dual Role of Carbonate/Bicarbonate as an Enhancer and Inhibitor of Derivatization
title_full	GC-MS Analysis of Biological Nitrate and Nitrite Using Pentafluorobenzyl Bromide in Aqueous Acetone: A Dual Role of Carbonate/Bicarbonate as an Enhancer and Inhibitor of Derivatization
title_fullStr	GC-MS Analysis of Biological Nitrate and Nitrite Using Pentafluorobenzyl Bromide in Aqueous Acetone: A Dual Role of Carbonate/Bicarbonate as an Enhancer and Inhibitor of Derivatization
title_full_unstemmed	GC-MS Analysis of Biological Nitrate and Nitrite Using Pentafluorobenzyl Bromide in Aqueous Acetone: A Dual Role of Carbonate/Bicarbonate as an Enhancer and Inhibitor of Derivatization
title_sort	gc-ms analysis of biological nitrate and nitrite using pentafluorobenzyl bromide in aqueous acetone: a dual role of carbonate/bicarbonate as an enhancer and inhibitor of derivatization
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/84addc2e79104dd48eeabfe064743163
work_keys_str_mv	AT dimitriostsikas gcmsanalysisofbiologicalnitrateandnitriteusingpentafluorobenzylbromideinaqueousacetoneadualroleofcarbonatebicarbonateasanenhancerandinhibitorofderivatization
_version_	1718411086425751552

GC-MS Analysis of Biological Nitrate and Nitrite Using Pentafluorobenzyl Bromide in Aqueous Acetone: A Dual Role of Carbonate/Bicarbonate as an Enhancer and Inhibitor of Derivatization

Ejemplares similares