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.

Dimitrios Tsikas

doi:10.3390/molecules26227003

Abstract

Carbon dioxide (CO2) and carbonates, which are widely distributed in nature, are constituents of inorganic and organic matter and are essential in vegetable and animal organisms. CO2 is the principal greenhouse gas in the atmosphere. In human blood, CO2/HCO3− is an important buffering system. Inorganic nitrate (ONO2−) and nitrite (ONO−) 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 15N-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-ONO2 and PFB-NO2, 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 (NaHCO3) 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-ONO2 formation and inhibits PFB-NO2 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-ONO2 in addition to the direct reaction of nitrate with PFB-Br. By contrast, nitrite does not react with PFB-OCOOH to form PFB-NO2. Sample acidification by small volumes of 20 wt.% aqueous acetic acid abolishes the effects of exogenous and endogenous bicarbonate on nitrite measurement.

Highlights

Pentafluorobenzyl bromide (2,3,4,5,6-pentafluorobenzyl bromide, PFB-Br) is a useful derivatization reagent for different classes of organic substances including carboxylic acids and amines, as well as inorganic anions, including nitrate and nitrite [1]
The derivatization of inorganic anions such as nitrite, nitrate and carbonate/bicarbonate in biological and environmental samples with PFB-Br using acetone as the organic solvent is performed in the homogenous phase and makes their gas chromatography–mass spectrometry (GC-MS) analysis possible
Our studies indicate that carbonate/bicarbonate exerts diametrically opposed effects on the analysis of nitrite and nitrate by GC-MS when derivatized with PFB-Br

Summary

Introduction

Pentafluorobenzyl bromide (2,3,4,5,6-pentafluorobenzyl bromide, PFB-Br) is a useful derivatization reagent for different classes of organic substances including carboxylic acids and amines, as well as inorganic anions, including nitrate and nitrite [1]. Derivatization with PFB-Br can be performed in water-free organic solvents such as acetonitrile, as well as in aqueous systems in the presence of water-miscible organic solvents such as acetone. Use of an acetone-aqueous sample in a volume ratio of 4:1 enables derivatization in homogenous phase [1]. Reactions with PFB-Br are nucleophilic substitutions of bromide by in homogenous phase [1]. Reactions with PFB-Br are nucleophilic substitutions of bromide by a nucleophile, which can be water, halogenides such as chloride, and other inorganic ions. Due to the fluorine atoms in PFB derivative2so, f 15 their GC-MS analysis in the negative-ion chemical ionization mode revealed the highest sensitivity

Methods

Results

Discussion

Conclusion