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Quantification of Carbonate by Gas Chromatography−Mass Spectrometry

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journal contribution
posted on 01.10.2010, 00:00 by Dimitrios Tsikas, Kristine Chobanyan-Jürgens
Carbon dioxide and carbonates 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. Quantification of bicarbonate and carbonate in inorganic and organic matter and in biological fluids such as blood or blood plasma by means of the GC-MS technology has been impossible so far, presumably because of the lack of suitable derivatization reactions to produce volatile and thermally stable derivatives. Here, a novel derivatization reaction is described for carbonate that allows for its quantification in aqueous alkaline solutions and alkalinized plasma and urine. Carbonate in acetonic solutions of these matrices (1:4 v/v) and added 13C-labeled carbonate for use as the internal standard were heated in the presence of the derivatization agent pentafluorobenzyl (PFB) bromide for 60 min and 50 °C. Investigations with 12CO32−, 13CO32−, (CH3)2CO, and (CD3)2CO in alkaline solutions and GC-MS and GC-MS/MS analyses under negative-ion chemical ionization (NICI) or electron ionization (EI) conditions of toluene extracts of the reactants revealed formation of two minor [i.e., PFB-OCOOH and OCO2−(PFB)2] and two major [i.e., CH3COCH2−C(OH)(OPFB)2 and CH3COCHC(OPFB)2] carbonate derivatives. The latter have different retention times (7.9 and 7.5 min, respectively) but virtually identical EI and NICI mass spectra. It is assumed that CH3COCH2−C(OH)(OPFB)2 is formed from the reaction of the carbonate dianion with two molecules of PFB bromide to form the diPFB ester of carbonic acid, which further reacts with one molecule of acetone. Subsequent loss of water finally generates the major derivative CH3COCHC(OPFB)2. This derivatization reaction was utilized to quantify total CO2/HCO3/CO32− (tCO2) in human plasma and urine by GC-MS in the NICI mode by selected ion monitoring of the anions [M − H] of CH3COCHC(OPFB)2 at m/z 461 for the endogenous species and m/z 462 for the internal standard 13CO32−. Oral intake of the carboanhydrase inhibitor drug acetazolamide by two healthy volunteers resulted in temporary increased excretion of tCO2 in the urine. The method is specific for carbonate, accurate, sensitive and should be applicable to various matrices including human fluids and environmental samples.