Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Figure 2. Intensity chromatograms for the n-alkanes mixture
obtained in the SIM mode: (a) at m/z ) 71 operating the setup in the
qualitative configuration; (b) at m/z 44 and 45 operating the setup in
the quantitative configuration.
and the filling of the reservoir, traces of this compound possibly
present in the Helium gas used to dilute de 13 CO2 and traces
of air still present in the EI source. The high enrichment
obtained provides a very wide range of optimum analyte/spike
ratios and then a sample containing compounds in a large range
of concentrations could be quantified in the same analysis
without affecting the accuracy of the results. 3
The short-term (30 min) and long-term (9 h) stability of the
12 C/ 13 C isotope ratio is given in the Supporting Information
(Figures SI-3 and SI-4). In both cases the measured isotope ratios,
0.0339 ± 0.0002 and 0.034 ± 0.001 (n ) 8), respectively, were
remarkably stable. Similar results were obtained in different days
indicating that the setup was robust enough for routine analysis.
Evaluation of the GC-Combustion-IDMS System. The
overall performance of the developed instrumentation was evaluated
here by analyzing a standard solution of a mixture of
n-alkanes (C9-C20). To study peak broadening, 1 µL of a
solution containing approximately 5 µg/g of these compounds
in n-hexane was injected in the chromatograph both in the
qualitative (combustion oven bypassed) and quantitative (through
the oven) modes. The Selected Ion Monitoring chromatogram
obtained at m/z ) 71 (fragment characteristic of n-alkanes) is
shown in Figure 2a, whereas the chromatogram obtained at
masses 44 and 45 after combustion and postcolumn isotope
dilution analysis is shown in Figure 2b. As can be observed, no
significant peak broadening due to the combustion unit or to the
different connections was observed, being the peak width measured
at the half height equal to that found when operating the
6866 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
Figure 3. Mass flow chromatograms obtained for the n-akanes
mixture.
GC-MS in the conventional way (0.02 min, Figure 2a). Only a small
increase in retention times was observed (∼20 s) because of the
combustion furnace. The mass flow chromatogram obtained after
the application of the online isotope dilution equation (eq 1) is
shown in Figure 3. As can be observed, the sensitivity is roughly
constant for all compounds which is in clear contrast with the
results shown in Figure 2a for mass 71 without combustion (SIM
detection). The peak areas in the mass flow chromatogram were
linear with the amount of carbon injected over 2 orders of
magnitude (the maximum range assayed). The detection limit
obtained for tetradecane was 9 ppb (ng/g) based on three times
the standard deviation of the baseline. Taking into account that
the injection volume was 0.5 µL, this value corresponds to an
absolute limit of detection of 3 pg of tetradecane injected. If we
take into account the peak width at the baseline (4s approximately
in this particular case), then we can make our detection limit
independent of the column employed and the carrier gas flowrate.
The value found was 0.8 pg C s -1 , being in the same order
that those provided by a mass spectrometer in full scan mode
(1 pg C s -1 ) and clearly better than those of a flame ionization
detector (10 pg C s -1 ). This detection limit is mainly limited
by the background level observed at m/z ) 44, and explains
the difference in comparison with the detection limit achievable
using the mass spectrometer in SIM mode bypassing the oven
(typically 0.1 pg C s -1 ) in spite of the fact that both sensitivities
were very similar. In contrast, it should be mentioned that the
EI detection limit observed is 3 orders of magnitude lower than
that obtained for carbon using 13 C postcolumn isotope dilution
and ICP-MS detection (0.7 ng C s -1 ). 12
For the quantitative analysis of the standard mixture of
n-alkanes (C9-C20) we needed first to quantify the mass flow
of postcolumn 13 CO2. Out of the two possibilities of using
internal standard (IS) or external standard, we selected an IS
because it provided the advantage of compensation for small
variations in the sample volume injected (we were using manual
injection). Moreover, as recently pointed out by Heilmann and
Heumann, 11 an additional advantage of using an internal
standard is that the mass flow of enriched carbon dioxide does
not need to be determined. The peak areas of both the analyte
and the internal standard in the mass flow chromatogram are
related to the unknown mass flow of spike (eq 1). However,
the ratio of peak areas analyte/IS will be equal to the actual ratio
of concentrations in the injected sample and independent of the
mass flow of spike used in the calculations. Thus, quantification