Anal. Chem.

2010, 82, 7897–7905

Quantification of Carbonate by Gas

Chromatography-Mass Spectrometry†
Dimitrios Tsikas* and Kristine Chobanyan-Jürgens

Institute of Clinical Pharmacology, Hannover Medical School, D-30623 Hannover, Germany

Carbon dioxide and carbonates are widely distributed in increased excretion of tCO2 in the urine. The method is
nature, are constituents of inorganic and organic matter, and specific for carbonate, accurate, sensitive and should be
are essential in vegetable and animal organisms. CO2 is the applicable to various matrices including human fluids
principal greenhouse gas in the atmosphere. In human and environmental samples.
blood, CO2/HCO3- is an important buffering system.
Quantification of bicarbonate and carbonate in inorganic Entire oxidation of carbon and organic material produces
and organic matter and in biological fluids such as blood carbon dioxide (carbonic acid gas, carbonic anhydride). In the
or blood plasma by means of the GC-MS technology has temperate zones of the Earth, the atmosphere contains about 0.04%
been impossible so far, presumably because of the lack (v/v) of CO2. CO2 is constituent of carbonate type of minerals
of suitable derivatization reactions to produce volatile and and products of animal metabolism. CO2 is necessary for the
thermally stable derivatives. Here, a novel derivatization respiration cycle of plants and animals. CO2 is soluble in water
reaction is described for carbonate that allows for its (88 mL/100 mL H2O; 20 °C, 1 atm). CO2 is absorbed by alkaline
quantification in aqueous alkaline solutions and alkalin- solutions with the formation of carbonates. A very minor part
ized plasma and urine. Carbonate in acetonic solutions of CO2 dissolved in water forms H2CO3, which deprotonates
of these matrices (1:4 v/v) and added 13C-labeled car- to form HCO3- and CO32-. In many cells and tissues of animals,
bonate for use as the internal standard were heated in notably erythrocytes, renal cortex, gastric mucosa, and muscle,
the presence of the derivatization agent pentafluorobenzyl the enzyme carbonic anhydrase catalyzes the reversible hy-
(PFB) bromide for 60 min and 50 °C. Investigations with dratation of CO2 to form HCO3- and H+.1,2 In human blood,
12
CO32-, 13CO32-, (CH3)2CO, and (CD3)2CO in alkaline CO2/HCO3- is an important buffering system3 and is mainly
solutions and GC-MS and GC-MS/MS analyses under controlled by the lung and the kidney.
negative-ion chemical ionization (NICI) or electron ion- CO2 is the main greenhouse gas in the atmosphere. About
ization (EI) conditions of toluene extracts of the reactants 30% of the excess released into the atmosphere by human
revealed formation of two minor [i.e., PFB-OCOOH and activities since the industrialization has been absorbed by the
OdCO2-(PFB)2] and two major [i.e., CH3COCH2- oceans. CO2 has affected and will further affect the human
C(OH)(OPFB)2 and CH3COCHdC(OPFB)2] carbonate environment as well as the environmental envelope of oceanic
derivatives. The latter have different retention times (7.9 organisms, notably of coral reef ecosystems.4 The need to
and 7.5 min, respectively) but virtually identical reduce CO2 accumulation in the atmosphere has initiated much
EI and NICI mass spectra. It is assumed that work into developing new technologies for CO2 capture and
CH3COCH2-C(OH)(OPFB)2 is formed from the reaction utilization.5-8 Interestingly, because of its easy and abundant
of the carbonate dianion with two molecules of PFB availability, CO2 has attracted considerable attention as a
bromide to form the diPFB ester of carbonic acid, which renewable and environmentally friendly source of carbon. CO2
further reacts with one molecule of acetone. Subsequent is especially useful as a phosgene substitute and a versatile
loss of water finally generates the major derivative synthon for numerous synthetic targets.7
CH3COCHdC(OPFB)2. This derivatization reaction was Because of the general interest in CO2, many different
utilized to quantify total CO2/HCO3-/CO32- (tCO2) in analytical methods have been developed and are being used
human plasma and urine by GC-MS in the NICI mode to measure CO2, bicarbonate, and carbonate in various biologi-
by selected ion monitoring of the anions [M - H]- of cal systems. These analytical methods use detectors that utilize
CH3COCHdC(OPFB)2 at m/z 461 for the endogenous specific physicochemical properties of these species, such as
species and m/z 462 for the internal standard 13CO32-. absorption of light in the infrared region and thermal conduc-
Oral intake of the carboanhydrase inhibitor drug aceta-
(1) Geers, C.; Gros, G. Physiol. Rev. 2000, 80, 681–715.
zolamide by two healthy volunteers resulted in temporary
(2) Supuran, C. T. Curr. Pharm. Des. 2008, 14, 603–614.
(3) Jungas, R. L. Anal. Biochem. 2006, 349, 1–15.
†
Part of the special issue “Atmospheric Analysis as Related to Climate (4) Lough, J. M. J. Environ. Monit. 2008, 10, 21–29.
Change”. (5) Aresta, M.; Dibenedetto, A. Dalton Trans. 2007, 36, 2975–2992.
* To whom correspondence should be addressed. Address: Institute of Clinical (6) Yu, K. M. K.; Curcic, I.; Gabriel, J.; Tsang, S. C. E. ChemSusChem 2008,
Pharmacology, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 1, 893–899.
Hannover, Germany. Phone: +49 511 532 3959. Fax: +49 511 532 2750. E-mail: (7) Sakakura, T.; Kohno, K. Chem. Commun. 2009, 1312–1330.
[email protected]. (8) Riduan, S. N.; Zhang, Y. Dalton Trans. 2010, 39, 3347–3357.

10.1021/ac1007688  2010 American Chemical Society Analytical Chemistry, Vol. 82, No. 19, October 1, 2010 7897
Published on Web 06/16/2010
tivity (by CO2)9 and migration of carbonate in an electric field.10 (PFB-Br) were obtained from Aldrich (Steinheim, Germany).
In medicine, the CO2 partial pressure (pCO2) in blood is Acetone, hexadeutero-acetone (declared isotopic purity of 99
commonly measured potentiometrically, and HCO3- concentra- atom % at 2H), sodium carbonate, sodium bicarbonate, and
tion in plasma is calculated using the Henderson-Hesselbach sodium hydroxide were bought from Merck (Darmstadt,
equation. In healthy humans, pCO2 and plasma HCO3- con- Germany). For quantitative measurements stock solutions
centration amount to 36-42 mmHg and 20-27 mM, respec- (each 100 mM) of unlabeled and 13C-labeled carbonate in
tively. In clinical research and practice, gaseous CO2, in distilled water were freshly prepared.
particular the ratio of 13CO2/12CO2, is analyzed by the isotope Safety Considerations. PFB-Br is corrossive and an eye
ratio mass spectrometry methodology.11
irritant. Inhalation and contact with skin and eyes should be
To the best of our knowledge, quantification of bicarbonate
avoided. All work should be performed in a well-ventilated fume
or carbonate in inorganic and organic matter by means of the
hood.
GC-MS technology has not been reported so far. The reason for
Derivatization Procedure. Acetone, aqueous solutions, and
this is most likely the lack of suitable derivatization reactions to
biological fluids were stored in closed vials in an ice-bath. In
produce volatile and thermally stable derivatives of HCO3-/CO32-.
However, there are early reports that cyclic carbonate deriva- quantitative analyses, derivatization with PFB-Br was performed by
tives prepared from vicinal alkyl diols and β,β,β-trichloroeth- mixing a carbonate containing matrix (100 µL) with acetone (400
ylcarbonate12 or phosgene13 are susceptible to GC. In previous µL) and PFB-Br (10 µL) and by incubating the mixture at 50 °C for
work,14 we found that nitrate (NO3-), which is a structural 60 min. For quantitative measurements, biological fluids (100 µL)
analog of CO32-, can be converted in aqueous acetone to the were combined with a 100-µL aliquot of a solution of the internal
nitric acid pentafluorobenzyl (PFB) ester derivative (PFB- standard Na213CO3 in distilled water to reach final added concen-
ONO2) by means of the versatile derivatization agent alpha- trations of 5, 20, or 50 mM. PFB-Br derivatization was performed
bromo-2,3,4,5,6-pentafluorotoluene (pentafluorobenzyl bromide, by adding acetone (800 µL) and PFB-Br (20 µL) and by incubating
PFB-Br) and quantified in various biological fluids. We assumed the mixture at 50 °C for 60 min. After derivatization samples were
that CO32- would also form with this reagent a thermally stable, cooled to room temperature, acetone was evaporated under a
volatile and strongly electron-capturing PFB derivative. In the nitrogen stream and reaction products were extracted from the
present work, a unique derivatization reaction for CO32- with remaining aqueous phase by vortex-mixing with toluene (1 mL)
PFB-Br and acetone in aqueous solution and human plasma for 1 min without preceding pH correction. One 800-µL aliquot of
and urine is described. Using commercially available 13C-labeled the organic phase was transferred into a clean 1.8-mL autosampler
CO32- and 2H-labeled acetone, the major PFB/acetone deriva- glass vial.
tive of CO32- was found to undergo an unusual in-source
GC-MS and GC-MS-MS Conditions. In both instruments,
rearrangement/fragmentation under negative-ion chemical
toluene aliquots (1 µL) were injected in the splitless mode using
ionization (NICI) conditions. The specificity of the derivatization
the following temperature program: the column temperature was
reaction and the fragmentation allows for the accurate quan-
held at 70 °C for 1 min, then increased to 280 and 300 °C at a rate
tification of total CO2/HCO3-/CO32- (tCO2) in biological and
of 30 °C/min and 10 °C/min, respectively. The oven temperature
environmental samples by GC-MS using 13CO32- as internal
standard. was held at 300 °C for 1 min.
In GC-MS analyses on the MS engine 5890A, helium (50 kPa)
and methane (200 Pa) were used as the carrier and the reagent
EXPERIMENTAL SECTION
Instrumentation. A Hewlett-Packard MS engine 5890A con- gas for NICI, respectively. Electron energy and electron current
nected directly to a gas chromatograph 5890 series II equipped were set to 230 eV and 300 µA, respectively, for NICI. Electron
with an autosampler Hewlett-Packard model 7673 (Waldbronn, energy was 70 eV in the EI mode. Constant temperatures of 180,
Germany) was used for GC-MS analyses. GC-MS and GC-MS- 280, and 200 °C were kept at the ion source, interface, and injector,
MS analyses were also performed on a triple-stage quadrupole respectively. Quantification of carbonate was performed in the
mass spectrometer ThermoQuest TSQ 7000 (Finnigan MAT, San NICI mode by selected ion monitoring (SIM) of the ions with
Jose, CA) directly interfaced with a Trace 2000 series gas m/z 461 for carbonate and m/z 462 for the internal standard
chromatograph equipped with an autosampler AS 2000 (CE Na213CO3 with a dwell time of 50 ms for each ion. In quantitative
Instruments, Austin, TX). Fused-silica capillary columns Optima analyses the electron multiplier voltage was set to values
17 [15 m × 0.25 mm inside diameter, 0.25 µm film thickness] from between 1600 and 2000 V.
Macherey-Nagel (Düren, Germany) were used. GC-MS and GC-MS-MS spectra were also generated on the
Reagents. Sodium [13C]carbonate (declared isotopic purity TSQ 7000 instrument. Interface, injector, and ion-source were kept
of 99 atom % at 13C) and 2,3,4,5,6-pentafluorobenzyl bromide at 280, 280, and 180 °C, respectively. Electron energy and electron
(9) Beccaria, A. M.; Poggi, G.; Castello, G. J. Chromatogr. 1987, 395, 641–
current was set to 200 eV and 300 µA, respectively. Helium (50
647. kPa), methane (530 Pa), and argon (0.13 Pa collision pressure)
(10) Wildman, B. J.; Jackson, P. E.; Jones, W. R.; Alden, P. G. J. Chromatogr.
were used as carrier, reagent, and collision gases, respectively.
1991, 546, 459–466.
(11) Benson, S.; Lennard, C.; Maynard, P.; Roux, C. Forensic Sci. Int. 2006, Collision energy was set to 15 eV.
157, 1–22. Statistical Analysis. If not otherwise specified, quantitative
(12) Oehlenschläger, J.; Gercken, G. J. Chromatogr. 1979, 176, 126–128.
(13) Koppenhoefer, B.; Lin, B. J. Chromatogr. 1989, 481, 17–26.
analyses were performed in triplicate. Values are presented as
(14) Tsikas, D. Anal. Chem. 2000, 72, 4064–4072. mean ± standard deviation.
7898 Analytical Chemistry, Vol. 82, No. 19, October 1, 2010
Table 1. Ions in the Mass Spectra of the Major GC Peaka of [12C]carbonate and [13C]carbonate after Derivatization
with Pentafluorobenzyl Bromide in CH3COCH3 or CD3COCD3
12
carbonate/acetone CO32-/CH3COCH3 13
CO32-/CH3COCH3 12
CO32-/CD3COCD3 13
CO32-/CD3COCD3
negative-ion chemical ionization
[M-H/D]- 461 (31) 462 (35) 464 (23) 465 (17)
[M-H/D-H2O]- 443 (2) 444 (2) 446 (1) 447 (1)
[M-MeCO]- 419 (5) 420 (7) 419 (3) 420 (4)
[M-H/D-*CH(OH)3]- 397 (100) 397 (100) 400 (100) 400 (100)
[M-H/D-*CH(OH)3-H/DF]- 377 (10) 377 (10), 378 (11) 380 (9) 381 (10)
281 (5) 282 (5) not detected 285 (8)
217 (5) 217 (6) 221 (5) 221 (4)
electron ionization
[M-H/D]•+ 461 (2) 462 (3) 464 (5) 465 (7)
[PFB]•+ 181 (32) 181 (28) 181 (80) 181 (85)
[MeCO]•+ 43 (100) 43 (100) 46 (100) 46 (100)
a
Derivative 2; MS engine 5890A. Me, CH3 or CD3; *C indicates 12C or 13C; PFB, pentafluorobenzyl.

RESULTS AND DISCUSSION carbonate and PFB-Br, reacts with the diPFB ester of
Identity of Reaction Products. GC-MS analysis of toluene carbonate by the methylene carbanion of acetone to produce
extracts from the derivatization with PFB-Br of aqueous saturated two derivatives which have virtually identical NICI and EI
solutions of Na212CO3 and Na213CO3 in CH3COCH3 or mass spectra (Figure 1). Because of the clearly different GC
CD3COCD3 yielded several GC peaks. The mass spectra of four behavior, derivative 1 is more polar and less volatile (tR ) 7.9
peaks showed corresponding ions with a difference of 1 amu min) than derivative 2 (tR ) 7.5 min). Possible structures could
in the EI and in the NICI mode due to the mass difference of be CD3COCD2-C(OH)(OPFB)2 (MW 483) for derivative 1 and
the 13C and 12C isotopes. The retention times (tR) of the two CD3COCDdC(OPFB)2 (MW 464) for derivative 2 from
major GC peaks were 7.9 min (derivative 1) and 7.5 min Na212CO3 in CD3COCD3. The appearance of the ion at m/z 461
(derivative 2). The EI and NICI mass spectra of the larger GC in the EI and NICI mass spectra of Na212CO3 suggests that
peak (derivative 2) of the PFB derivatives of Na212CO3 and ionization of CH3COCHdC(OPFB)2 produces the radical cation
Na213CO3 produced in CH3COCH3 or CD3COCD3 are shown [CH3COC)C(OPFB)2]+• (i.e., [M-H]+•) and the anion
in Supporting Information, Figure 1. The most intense ions [CH3COCdC(OPFB)2]- (i.e., [M-H]-) by loss of one H• or
present in the EI and NICI mass spectra of derivative 2 of
H+, respectively (Table 1). That derivative 1 and derivative 2 have
Na212CO3 and Na213CO3 obtained from derivatization reactions
virtually identical NICI and EI mass spectra suggests that
performed in CH3COCH3 or CD3COCD3 are summarized in
ionization of CD3COCD2-C(OH)(OPFB)2 is accompanied by loss
Table 1. The most intense ions in the NICI mass spectra of the
of one water molecule (HOD; see Figure 1).
PFB derivative of Na213CO3 of derivative 1 were m/z 462
Formation of the diPFB/acetone derivatives of carbonate raises
(intensity, 100%) and m/z 317 (90%) in CH3COCH3 and m/z
the possibility of formation of monoPFB/acetone derivatives, as
465 (intensity, 80%) and m/z 317 (100%) in CD3COCD3.
well as formation of PFB derivatives without involvement of
The largest ions in the EI and NICI mass spectra of derivatized
acetone. Indeed, using Na212CO3 and Na213CO3 we observed very
Na213CO3 in CH3COCH3 had m/z values of 461 and 462,
minor GC peaks with relative retention times of 0.788 (deriva-
respectively (Supporting Information, Figure 1). The largest ions
tive 3) and 0.912 (derivative 4) with respect to the derivative
in the EI and NICI mass spectra of derivatized Na213CO3 in
2. These derivatives are likely to be PFBO-12COOH (MW 242)
CD3COCD3 had m/z values of 464 and 465, respectively (Table
1). These findings suggest that derivative 2 contains one 13C atom and PFBO-13COOH (MW 243) for derivative 3 and
from Na213CO3 and three D atoms from CD3COCD3. The (PFBO)2-12CO (MW 422) and (PFBO)2-13CO (MW 423) for
intense ions at m/z 43 ([CH3CO]+•) and m/z 46 ([CD3CO]+•) derivative 4 (Supporting Information, Figure 2).
in the EI mass spectra suggest that these derivatives contain Taken altogether, under alkaline conditions in aqueous acetone,
in their molecules one acetyl group from CH3COCH3 and carbonate reacts with PFB-Br to form at least four derivatives. The
CD3COCD3, respectively. The intense mass fragment at m/z most abundant derivatives are CH3COCHdC(OPFB)2 and its
181 was present in the EI mass spectra of the carbonate putative precursor CH3COCH2-C(OH)(OPFB)2, and they have
derivatives analyzed and corresponds to the PFB radical cation virtually identical MS properties under NICI and EI conditions.
([C6F5CH2]+•). To achieve molecular ions on the order of m/z Under the derivatization conditions used (i.e., basic pH, 50 °C,
461 to m/z 465 (Table 1), the PFB derivatives of Na212CO3/ 60 min) acetone not only serves as a solvent but also is a reactant
Na213CO3 must contain two PFB moieties (362 Da). and completes the reaction between carbonate and PFB-Br.
The data of Table 1 suggest that the carbonate dianion reacts Because derivative 2, the acetale CH3COCHdC(OPFB)2, is the
with two PFB-Br molecules in a nucleophilic substitution, most major reaction product, we focused on this derivative.
likely through a SN2 mechanism, to produce the diPFB ester In-Source Rearrangement/Fragmentation and Collision-
of carbonate. Because of the considerable nucleophilicity of Induced Dissociation in NICI Mode. The most intense ions in
acetone (pKa 20) and the alkaline conditions of the derivatiza- the NICI mass spectra of the derivatives of Na212CO3 and
tion reaction (pH about 12.5), it can be assumed that acetone, Na213CO3 in unlabeled acetone had the same m/z value of 397,
which is present at a very high molar excess over both suggesting that this ion does not contain the 12C/13C atom from
Analytical Chemistry, Vol. 82, No. 19, October 1, 2010 7899
Figure 1. Proposed mechanism for the reaction of Na212CO3 with PFB-Br and CD3COCD3 in aqueous alkaline solution to form two derivatives,
that is, derivative 1 and derivative 2. Derivative 2 is likely to be the acetale CD3COCDdC(OPFB)2, which is produced from derivative 1, that is,
CD3COCD2-C(OH)(OPFB)2, by loss of water (DOH). Derivatives 1 and 2 have different retention times in chromatography but produce virtually
identical mass spectra in NICI and EI conditions. Because of its hydroxyl group, CD3COCD2-C(OH)(OPFB)2 is less volatile emerges later from
the GC column than CD3COCDdC(OPFB)2.

Na212CO3/Na213CO3 (Table 1 and Supporting Information, mechanism, a 1,3-rearrangement, fragmentation starts with the
Figure 2A and B). Similarly, the most intense ions in the NICI intramolecular nucleophilic attack of the mesomeric alcoholate
mass spectra of the derivatives of Na212CO3 and Na213CO3 formed anion (from CD3COCD3) on the C-atom (from carbonate). After
in CD3COCD3 had the same m/z value of 400, suggesting that a sequence of several steps, this rearrangement is completed
this ion does not contain the 12C/13C atom from Na212CO3/ by the neutral loss of CH(OH)3 forming the substituted
Na213CO3, but it contains the CD3 group from CD3COCD3. The cyclobutadiene anion (Figure 3). Because of the highly conju-
loss of the carbonate-C atom from [CH3COCdC(OPFB)2]- gated system and the abundant strongly electron-capturing F
suggests that this ion rearranges within the ion-source under atoms, the anion [CD3C4(C6F5)2]- should be very stable and
NICI conditions, whereby a species with the molecular mass strongly electron-capturing. The driving forces for this rear-
of 64 Da leaves the anion. The corresponding neutral loss from
rangement and the subsequent fragmentation could be (1) the
[CH3COCd13C(OPFB)2]- is 13CH(OH)3 (65 Da).
inability of [M-H]- to directly lose CO2, (2) the possibility of
To investigate the origin of the common ion at m/z 397 in the
the formation of the neutral loss CH(OH)3, and (3) the
mass spectra of derivative 2 of Na212CO3 and Na213CO3 (Table 1
formation of a highly conjugated, stabilized anion. Indeed, the
and Supporting Information, Figure 2A and B), the precursor
anion [CD3C4(C6F5)2]- (m/z 400) undergoes only minor
anions at m/z 461 and m/z 462 were subjected to collision-induced
dissociation (CID). The tandem mass spectra shown in Figure 2 fragmentation to m/z 381 because of loss of a fluoride anion
indicate numerous product ions. Some product ions have same (Table 1). Subjection of the ions at m/z 397 ([CH3C4(C6F5)2]-)
m/z values, whereas other product ions differ each by 1 amu of derivative 2 from Na212CO3 and Na213CO3 (Table 1) to CID
because of 13C. Interestingly, no product ions at m/z 397 were (15 eV collision energy) resulted in virtually identical product ion
observed from CID of m/z 461 (Na212CO3) and m/z 462 mass spectra with three product ions at m/z 377, m/z 357, and
(Na213CO3). This finding strongly suggests that m/z 397 is m/z 337 because of the loss of 1, 2, and 3 HF molecules,
formed in the ion-source during NICI. respectively, with the 3 H most likely stemming from the original
In Figure 3, a mechanism for this unusual rearrangement for methyl group of [CH3C4(C6F5)2]- (Supporting Information,
[CD3COCDdC(OPFB)2]- is proposed. In accordance with this Figure 3A and B).
7900 Analytical Chemistry, Vol. 82, No. 19, October 1, 2010
Figure 2. GC-MS/MS spectra produced by collision-induced dissociation of the parent anions (P-) of the derivative 2 at m/z 461 of
CH3COCHd12C(OPFB)2 from Na212CO3 (A) and m/z 462 of CH3COCHd13C(OPFB)2 from Na213CO3 (B). Common product ions are highlighted
in bold. The instrument TSQ 7000 was used. Collision energy was 15 eV each.

It is worth mentioning that unusual fragmentation has been indicate that the gas-phase anion chemistry in the laboratory may
observed for R-nitro-pentafluorotoluene, although under CID be very complex and may lead to structurally unusual anions like
conditions in GC-MS/MS.15 These and previous observations16-18 those detected in remote galactic regions.19-24 Interestingly, in-

(15) Tsikas, D.; Schwedhelm, E.; Frölich, J. C. J. Chromatogr. A 2005, 1067, (17) Ingemann, S.; Nibbering, N. M. M.; Sullivan, S. A.; DeyPuy, C. H. J. Am.
337–345. Chem. Soc. 1982, 104, 6520–6527.
(16) Dawson, J. H. J.; Nibbering, N. M. M. Int. J. Mass Spectrom. Ion Phys. 1980, (18) Büker, H. H.; Nibbering, N. M. M.; Espinosa, D.; Mongin, F.; Schlosser,
33, 3–19. M. Tetrahedron Lett. 1997, 38, 8519–8522.

Analytical Chemistry, Vol. 82, No. 19, October 1, 2010 7901

Figure 3. Proposed mechanism for the rearrangement and fragmentation of the anion of CD3COCDdC(OPFB)2 at m/z 464 under NICI conditions
leading to the anions m/z 400 and m/z 381 and to the neutral loss of the alcohol CH(OH)3 (MW 64).

2
source fragmentation and decay are quite common phenomena H atoms (natural relative abundance, 0.015%), the abundance
in mass spectrometry and can be utilized, for instance, for protein ratio of m/z 461 to m/z 462, that is, PM+1/PM, was calculated
identification by MALDI mass spectrometry.25 according to Biemann26 as 0.204. The concentration of Na2CO3
Analysis of Carbonate in Aqueous Solution. Standard ([Na2CO3]) in aqueous solutions and biological fluids was
curves of Na2CO3 (range 0-20 mM) were prepared in aqueous calculated by means of the formula (F1) which considers the
solutions of Na213CO3 (5 mM, 20 mM or 50 mM). Expectedly, contribution of the 13C-isotope.
plotting of the peak area ratio (PAR) of m/z 461-462 versus
the concentration of Na2CO3 did not result in a straight line [Na2CO3] ) ([Na213CO3] × PAR)/(1 - 0.204 × PAR) (F1)
but yielded a hyperbolic curve when Na213CO3 was used at 5
mM. This observation is mainly attributed to the 13C-isotope whereas [Na213CO3] is the concentration of the internal standard
(natural relative abundance, 1.107%) of the anion Na213CO3 in the sample.
[CH3COCdC(OPFB)2]- (m/z 461), which originates from Linear regression analysis of the calculated Na2CO3 concen-
Na2CO3. The ion m/z 461 considerably contributes to the anion tration (y) and the concentration (x) of Na2CO3 added to an
[CH3COCd13C(OPFB)2]- (m/z 462), which originates from the alkaline aqueous solution of Na213CO3 (5 mM) in the range of
internal standard Na213CO3. Considering eighteen 13C atoms, 0-20 mM resulted in straight lines for both GC peaks with
three 17O atoms (natural relative abundance, 0.037%), and seven the regression equations y ) 2.89 + 1.06x (R ) 0.99686) for
the major GC peak and y ) 2.91 + 1.08x (R ) 0.99428) for the
(19) Langer, W. D.; Velusamy, T.; Kuiper, T. B.; Peng, R.; McCarthy, M. C.;
minor GC peak. The slope values of both regression equations
Travers, M. J.; Kovacs, A.; Gottlieb, C. A.; Thaddeus, P. Astrophys. J. 1997,
480, L63–L66. indicate mean accuracy values of 106% and 108%, respectively.
(20) Xu, L.; Huang, Y.; Giese, R. W. J. Mass Spectrom. 1998, 33, 615–620. The y-axis intercepts of 2.89 and 2.91 indicate presence of
(21) Blansky, J. S.; Bowie, J. H. J. Mass Spectrom. Rev. 1999, 18, 131–151.
Na2CO3 at a concentration of about 2.9 mM in the alkaline
(22) Dua, S.; Blansky, S. J.; Bowie, J. H. Rapid Commun. Mass Spectrom. 2000,
14, 118–121. solution used to prepare dilutions of Na2CO3.
(23) Sierra, M. A.; Gomez-Gallego, M.; Mancheno, M. J.; Martinez-Alvarez, R.; Imprecision (RSD) in these analyses ranged between 0.2% and
Raminez-Lopez, P.; Kayali, N.; Gonzalez, A. J. Mass Spectrom. 2003, 38,
5.6%. Instrumental precision was determined by 6-fold injection
151–156.
(24) Julian, R. R.; May, J. A.; Stoltz, B. M.; Beauchamp, J. L. J. Am. Chem. Soc. of 1-µL aliquots of the toluene extract from the derivatization of a
2003, 125, 4478–4486.
(25) Debois, D.; Bertrand, V.; Quinton, L.; De Pauw-Gillet, M. C.; De Pauw, E. (26) Biemann, K. Mass Spectrometry, Organic Chemical Application; McGraw-
Anal. Chem. 2010, 82, 4036–4045. Hill Book Company, Inc.: New York, 1962.

7902 Analytical Chemistry, Vol. 82, No. 19, October 1, 2010

5-mM solution of Na213CO3. On the assumption of complete
derivatization of carbonate and quantitative extraction of the
PFB derivative, the amount injected corresponds to 500 pmol
of Na213CO3. This amount was detected with an imprecision
(RSD) of 0.7% and a signal-to-noise (S/N) ratio of 2529:1 (RSD,
6.9%) for the major GC peak. A 6-fold injection of 1-µL aliquots
of a 1:1000-dilution (v/v) of the toluene extract mentioned
above resulted in an S/N ratio of 4.2 ± 0.67: 1 (RSD, 16%) for
the major GC peak (derivative 2). Thus, the amount of 500 fmol
of Na213CO3 is considered the lower limit of detection of the
method.
The shortcomings associated with the hyperbolic curves can
be overcome by using the internal standard Na213CO3 at concen-
trations higher than the highest expectable concentration of
Na2CO3 in the sample. Thus, using Na213CO3 at an added final
concentration of 50 mM linear regression analysis between the
PAR measured (y) and added Na212CO3 (x) resulted in straight
lines for both GC peaks: y ) 0.08 + 0.018x, R ) 0.99962 for
the major GC peak (derivative 2), and y ) 0.09 + 0.017x, R )
0.99962 for the minor GC peak (derivative 1). In such cases
Na2CO3 concentrations can be calculated by using Formula F2
which refuses the contribution of the 13C-isotope of Na212CO3
to the internal standard Na213CO3. Linear regression analysis
between the calculated Na2CO3 concentration (y) and the
concentration (x) of Na2CO3 added (0, 10, 20, 30, 40 mM) to
an alkaline aqueous solution of Na213CO3 (50 mM) resulted in
straight lines for both GC peaks with the regression equations
y ) 3.28 + 1.07x (R ) 0.99919) for the major peak and y ) Figure 4. Representative partial chromatograms from the GC-MS
4.07 + 1.02x (R ) 0.99986) for the minor peak. The slope values analysis of carbonate (SIM of m/z 461 and m/z 462) in the NICI mode
of both regression equations indicate mean accuracy values of in urine of a healthy volunteer before (A) and after (B) addition of 20
mM of Na2CO3 to the urine sample. The concentration of the internal
107% and 102%, respectively.
standard Na213CO3 was 50 mM with respect to the urine sample. The
PFB derivatives eluted at 7.33 (major GC peak, derivative 2) and 7.85
min (minor GC peak, derivative 1).
[Na2CO3] ) [Na213CO3] × PAR (F2)
up to 24 h after drug administration. Urine specimens were
Analysis of Carbonate in Human Urine. For quantitative stored at 4 °C in closed vials until measurement of carbonate,
analyses in urine samples the minor GC peak was considered creatinine and pH. Figure 5 shows that administration of the
because the first peak of the internal standard was not baseline- diuretics led to comparable pH increases in both volunteers. By
separated from an unknown compound with an m/z value of 462 contrast, the pharmacokinetics of carbonate excretion was quite
(Figure 4). The method was validated using a urine sample of a different in the volunteers. Creatinine-corrected tCO2 excretion
pH value of 6.5 and a Na213CO3 final concentration of 50 mM. reached a sharp maximum value of about 40 mmol/mmol
For Na2CO3 added to urine (0, 10, 20, 30, 40 mM) in duplicate, creatinine in volunteer A, whereas in volunteer B the maximum
recovery (91.6-106%) and imprecision (0.2-18%) were within was broader ranging between 23 and 25 mmol/mmol creati-
generally acceptable ranges. Linear regression analysis be- nine. Seven hours after drug administration the tCO2 excretion
tween measured (y) and added (x) Na2CO3 concentration rate decreased only slowly in the remaining 17 h in volunteer
resulted in a straight line with the regression equation y ) 9.87 B, but decreased more quickly in volunteer A. These differ-
+ 0.959x, R ) 0.99994. For NaHCO3 added to urine (0, 10, 20, ences are likely to be the result of the different pharmaceutical
30, 40 mM) in duplicate, recovery (93.2% to 103%) and preparations of the same drug, that is, regular release (Glau-
imprecision (3.3-5.2%) were also satisfactory. Linear regression pax) versus retarded release (Diamox).
analysis between measured (y) and added (x) NaHCO3 con- Analysis of Carbonate in Human Plasma. Unlike in urine,
centration resulted in a straight line with the regression in plasma samples both GC peaks (derivative 2 and derivative 1)
equation y ) 7.74 + 0.976x, R ) 0.99909. can be considered for carbonate quantification because the
The applicability of the method for urinary tCO2 was demon- unknown compound eluting in front of the derivative 2 did not
strated in a self-experiment on the diuretics acetazolamide, a interfere in urine samples with the internal standard (Figure 6).
carbonic anhydrase drug inhibitor.3 One author (volunteer A) The method was validated using Na213CO3 at a final concentration
received orally one and a half Glaupax 250-mg tablets (6.3 mg of 50 mM. Comparison of Na2CO3 concentrations obtained from
per kg bodyweight). The other author (volunteer B) received
(27) Tsikas, D.; Wolf, A.; Mitschke, A.; Gutzki, F. M.; Will, W.; Bader, M.
orally a 500-mg Diamox retard capsule (5 mg per kg body- J. Chromatogr. B [Online early access]. DOI: 10.1016/j.jchromb.2010.04.025.
weight). Urine samples were collected immediately before and Published Online April 24, 2010.

Analytical Chemistry, Vol. 82, No. 19, October 1, 2010 7903

Figure 5. Urine pH and creatinine-corrected carbonate excretion
before and after oral intake of the diuretics acetazolamide by the
authors. Volunteer A received one and a half 250-mg tablet Glaupax
(OmniVision, Puchheim, Germany), resulting in a dose of 6.3 mg/kg
bodyweight. Volunteer B received a 500-mg Diamox retard capsule
(Goldshield Pharmaceuticals Ltd., U.K.), resulting in a dose of 5 mg/
kg bodyweight. The time point of administration was few minutes after
collection of the first urine sample and is indicated by an arrow. The
concentration of the internal standard Na213CO3 was 50 mM in all
samples. The PFB derivatives eluted at 7.3 (major) and 7.8 min (minor
peak). The minor GC peak was used for quantification. Creatinine in
10-µL aliquots of urine samples was measured by GC-MS using
trideuteromethyl-creatinine as the internal standard (10 mM).27

both GC peaks yielded statistically insignificantly different

results (P ) 0.44, paired t test). The results were combined
for statistical analysis. For Na2CO3 added to pooled plasma (0,
12, 24, 36, 48 mM) in duplicate, recovery (99.4% to 112%), and
imprecision (0.1% to 7.3%) were within generally acceptable
ranges. Linear regression analysis between measured (y) and
added (x) Na2CO3 concentration resulted in a straight line with
the regression equation y ) 22.7 + 1.15x, R ) 0.99793, Figure 6. Representative partial chromatograms from the GC-MS
suggesting a mean basal Na2CO3 (actually tCO2) concentration analysis of carbonate (SIM of m/z 461 and m/z 462) in the NICI mode
in plasma of a healthy volunteer before (A, C) and after (B, D) addition
of 22.7 mM and a mean recovery of 115% for added Na2CO3. of 20 mM of Na2CO3 or NaHCO3, respectively The concentration of
the internal standard Na213CO3 was 50 mM with respect to the plasma
sample. The PFB derivatives eluted at 7.35 and 7.87 min. The
CONCLUSIONS
concentration of endogenous carbonate, that is, of the CO2/HCO3-/
The reaction of carbonate with PFB-Br and acetone in aqueous
CO32- system in this freshly obtained plasma sample was measured
phase yields two thermally stable and strongly electron-capturing to be 28 mM.
PFB derivatives. The major PFB derivative of carbonate was
identified as the acetale CH3COCHdC(OPFB)2. Under NICI out to the complex gas phase anion chemistry. This derivati-
conditions the anion of CH3COCHdC(OPFB)2 undergoes zation reaction offers the unique opportunity to derivatize and
unusual in-source rearrangement and fragmentation pointing quantify carbonate in environmental samples and in biological
7904 Analytical Chemistry, Vol. 82, No. 19, October 1, 2010
fluids by GC-MS with high accuracy, precision and sensitivity. SUPPORTING INFORMATION AVAILABLE
This method should be useful for routine quantitative analysis Electron ionization (EI) and negative-ion chemical ionization
of the CO2/HCO3-/CO32- system in human environmental and (NICI) mass spectra, and product ion mass spectra. This material
biological samples. is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENT
The expert laboratory assistance of A. Mitschke is gratefully Received for review March 25, 2010. Accepted June 1,
acknowledged. The authors thank F.M. Gutzki for performing GC- 2010.
MS/MS analyses. AC1007688

Analytical Chemistry, Vol. 82, No. 19, October 1, 2010 7905