Analytical Chemistry Chemical Cytometry Quantitates Superoxide

corresponding compound if its chemical formula is known. Until
now such methodology has been exclusively applied to the
quantitative determination of compounds containing ICPMS
detectable elements for trace-elemental speciation of metals 9 or
semimetals. 10,11 Unfortunately, the use of the ICPMS for the
detection of carbon, hydrogen, nitrogen, or oxygen is seriously
hampered by the low ionization yields of these elements in the
ICP and the high carbon background under normal ICP operating
conditions (atmospheric pressure). In fact, there are only two
reports so far describing the use of HPLC and the postcolumn
addition of 13 C-labeled species for the quantification of organic
compounds by ICP-MS. 12,13 In both cases, the observed detection
limits were not satisfactory.
For the MS detection of organic compounds, previously
separated by GC, electron ionization (EI) is the most common
ionization source employed. It provides both structural and
quantitative information of any volatile organic compound injected
in the gas chromatograph. Unfortunately, as pointed out before,
it requires specific analytical standards as its response is structurespecific
for each single molecule subjected to analysis. 14 Recently,
we have introduced a new quantitative detection concept in gas
chromatography, based on the postcolumn addition of 13 CO2 for
carbon isotope dilution analysis using EI. 15 This concept
constitutes a patented procedure in which organic compounds
separated by liquid or gas chromatography are converted
quantitatively into carbon dioxide, by an oxidation or combustion
reaction, and then are mixed with a postcolumn flow of
enriched 13 CO2. 16 This procedure should provide quantitative
information of every single compound previously separated in
the chromatograph without the need for individual standards.
We selected electron ionization because it operates under high
vacuum conditions providing much better sensitivity and lower
background for carbon detection than the above-mentioned ICP
atmospheric source. For the correct application of isotope
dilution analysis conversion of all carbon containing compounds
to a unique chemical species is required (isotope equilibration).
The isotopic equilibrium between the 13 C-containing species
continuously added postcolumn ( 13 CO2) and the separated
organic compounds is reached by their quantitative conversion
into CO2, after the chromatographic separation, in a combustion
furnace. 17 As the only species to be finally measured by EI is
CO2, compound independent response and isotopic equilibration
is finally obtained. This approach can become a universal
quantitative detector in GC-MS analysis, without the classical
need for specific standards and, what is more, it is compatible
with the exceptional structural identification capabilities provided
by current GC-EI-MS instruments. Thus, in this work
(9) Sariego-Muñiz, C.; Marchante-Gayón, J. M.; García-Alonso, J. I.; Sanz-Medel,
A. J. Anal. At. Spectrom. 2001, 16, 587–592.
(10) Giusti, P.; Schaumlöffel, D.; Ruiz-Encinar, J.; Szpunar, J. J. Anal. At. Spectrom.
2005, 20, 1101–1107.
(11) Heilmann, J.; Heumann, K. G. Anal. Chem. 2008, 80, 1952–1961.
(12) Vogl, J.; Heumann, K. G. Anal. Chem. 1998, 70, 2038–2043.
(13) Smith, C.; Jensen, B. P.; Wilson, I. D.; Abou-Shakra, F.; Crowther, D. Rapid
Commun. Mass Spectrom. 2004, 18, 1487–1492.
(14) Mark, T. D. Int. J. Mass Spectrom. Ion Phys. 1982, 45, 125–145.
(15) Cueto-Díaz, S.; Ruiz-Encinar, J.; Sanz-Medel, A.; García-Alonso, J. I. Angew.
Chem., Int. Ed. 2009, 48, 2561–2564.
(16) Ruiz-Encinar, J.; García-Alonso, J. I World Intellectual Property Organization,
Spain; International Patent WO 2007/042597 A1, 2007.
(17) Merritt, D. A.; Freeman, K. H.; Ricci, M. P.; Studley, S. A.; Hayes, J. M.
Anal. Chem. 1995, 67, 2461–2473.
we describe the instrumental developments and its analytical
features for integral characterization and determination of
organic compounds in detail.
Furthermore, the compound-independent quantification provided
by the developed approach can be an inestimable tool in
the optimization and quantitative assessment of sample extraction
and preconcentration procedures applied prior to GC-MS analysis.
For instance, solid-phase microextraction (SPME) is today a
widely used preconcentration technique in Gas Chromatography. 18
Fiber absorption and recovery are common parameters used when
validating a given SPME procedure. However, those two parameters
are almost impossible to compute using conventional
approaches. 19 In fact, it is necessary to inject specific liquid
standards and to assume that the transfer efficiency in the GC
injector for every compound under analysis is the same when
using conventional injection and the thermal desorption. Thus,
the important topic of absolute absorption yields will be evaluated
in this work, as exemplified for the HS-SPME analysis of BTEX
(Benzene, Toluene, Ethylbenzene, and o,m,p-Xylenes) in different
water samples, using the proposed IDA-EI-MS quantification
procedure.
EXPERIMENTAL SECTION
Reagents and Materials. Solid enriched Na2CO3 (99% 13 C
enrichment) was obtained as a highly pure chemical reagent
(purity >98%) from Cambridge Isotopes Laboratories (Andover,
Massachusetts, USA). A standard mixture of n-alkanes (40 µg/
mL each in n-hexane) was purchased from Fluka (Seelze,
Germany). Phosphoric acid (99% puriss.) was purchased from
Sigma-Aldrich (St. Louis, USA). Individual compounds (undecane,
dodecane, tridecane, pentadecane, butyl butyrate, and
hexyl butanoate) with certified purities ranging from 99 to 99,7%
were used as analytical standards (Fluka, Seelze, Germany or
Sigma-Aldrich, St. Louis, USA). A solution of 1,2,4-trimethylbenzene
in methanol (5000 µg/mL) and a BTEX standard
mixture (2000 µg/mL each in methanol) were both from
Supelco (Bellefonte, USA). Ultrapure water (18.2 MΩcm) was
obtained with a Milli-Q system (Millipore, Bedford, MA).
n-Hexane for organic trace analysis grade was purchased from
Merck (Darmstadt, Germany). SPME holder and fibers were
purchased from Supelco (Bellefonte, USA).
Instrumentation. GC-MS. A Konik-Tech (Sant Cugat del
Vallés, Spain) 4000-B Gas Chromatograph coupled to a Konik-
Tech MS-Q12 quadrupole mass spectrometer with an electron
ionization source was used. This instrument uses a specially
designed injector which keeps the septum at relatively low
temperature, does not require a septum purge flow, and minimizes
losses of organic compounds in the injector port. The analytical
column was a 30 m long (0.32 mm internal diameter, 0.25 µm
stationary phase) DB-XLB column (Agilent J&W Scientific, Santa
Clara, USA). The sample volume injected in the GC was always 1
µL using manual injection. Sample injection was performed in the
splitless (1 min)/split mode. Carrier gas flow was set at 1 mL/
min for all samples and conditions.
Six-Way Valve. A manually actuated 0.25 mm bore stainlesssteel
six-way two-position valve (VICI AG International, Schenkon,
(18) Vas, G.; Vekey, K. J. Mass Spectrom. 2004, 39, 233–254.
(19) Langelfeld, J. J.; Hawthorne, S. B.; Miller, D. J. Anal. Chem. 1996, 68,
144–155.
Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
6863