Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Anal. Chem. 2010, 82, 6862–6869 Gas Chromatography-Combustion-Mass Spectrometry with Postcolumn Isotope Dilution for Compound-Independent Quantification: Its Potential to Assess HS-SPME Procedures Sergio Cueto Díaz, Jorge Ruiz Encinar,* Alfredo Sanz-Medel, and J. Ignacio García Alonso* Department of Physical and Analytical Chemistry, University of Oviedo, Julián Clavería 8, 33006 Oviedo, Spain A quadrupole GC-MS instrument with an electron ionization (EI) source has been modified to enable application of postcolumn isotope dilution analysis for the standardless quantification of organic compounds injected in the gas chromatograph. Instrumental modifications included the quantitative conversion of the separated compounds into CO 2, using a postcolumn combustion furnace, and the subsequent mixing of the gas with a constant flow of 13 CO2 diluted in helium. The online measurement of the 12 CO2/ 13 CO2 (44/45) ratio in the EI-MS allowed us to obtain quantitative data without resorting to compound-specific standards. Validation of the procedure involved the analysis of standard solutions containing different families of organic compounds (C 9-C20 linear hydrocarbons, BTEX and esters) obtaining satisfactory results in all cases in terms of absolute errors (
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
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Anal. Chem. 2010, 82, 6745-6750 Let
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purchased from Invitrogen-Molecular
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Figure 3. Electropherograms of TPP-
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Anal. Chem. 2010, 82, 6751-6755 Res
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(pH 8.0) with cysteine and cystamin
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Figure 4. Ion mobility mass spectra
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GOx glucose + O298 gluconic acid +
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coater at 2000 rpm for 20 s, and th
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Figure 5. Comparison of cyclic volt
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in channels with either no grooves
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indicators of atmospheric processin
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Figure 1. GC/MS total ion chromatog
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Table 2. Concentrations and Stable
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on a substrate are preferred. 20-24
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Figure 4. SERS analysis of NAADP co
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Anal. Chem. 2010, 82, 6775-6781 Hig
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tion, 2 µL of proprionaldehyde wer
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Figure 4. Analysis of 2a by HPLC-MS
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eaction of 1a with PBH can be condu
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Numerous references had demonstrate
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Figure 1. TEM images of the prepare
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Figure 4. Schematic representation
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esult in a big SPR signal change wi
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also reduces chemical noise, which
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Table 1. Extraction Yields, Liquid
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scanning of AMPP amides of the anal
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Anal. Chem. 2010, 82, 6797-6806 δ
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Figure 1. Schematic view of the pre
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from the specimen and enclosed in a
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Figure 4. (A) IRMS mass-44 chromato
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Table 3. Ambient Measurement Result
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Anal. Chem. 2010, 82, 6807-6813 Dir
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Polymerase (1 U per sample). Reacti
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Page 75 and 76: Table 2. Concentrations and Ratios
Page 77 and 78: Anal. Chem. 2010, 82, 6821-6829 Mac
Page 79 and 80: mg/mL protein, followed by separati
Page 81 and 82: Figure 2. Productivity of SEQUST an
Page 83 and 84: Figure 4. High-resolution MS/MS spe
Page 85 and 86: Figure 6. Characterization of PSMs
Page 87 and 88: containing T-T mismatches. 23 Based
Page 89 and 90: Figure 1. Extinction spectra of sol
Page 91 and 92: Figure 3. (A) The value of Ex650 nm
Page 93 and 94: Figure 5. Extinction spectra and co
Page 95 and 96: wished to explore the dehydration o
Page 97 and 98: constant medium for separation; we
Page 99 and 100: Figure 2. Standards of (Pi)n, n ) 1
Page 101 and 102: Figure 5. Quantitative calibration
Page 103 and 104: Anal. Chem. 2010, 82, 6847-6853 Met
Page 105 and 106: Figure 1. 226 Ra spectrum by liquid
Page 107 and 108: Table 1. Counting Properties and De
Page 109 and 110: Table 3. Analysis of 226 Ra in Sedi
Page 111 and 112: mercial microarray scanner and fabr
Page 113 and 114: Figure 2. Optical transmission meas
Page 115 and 116: Figure 5. Volcano plots detailing t
Page 117: data as well. The 41 genes in Table
Page 121 and 122: Figure 1. Schematic illustration of
Page 123 and 124: Table 1. Absolute Quantification Re
Page 125 and 126: ment. Therefore, the long-time drea
Page 127 and 128: Figure 1. Chemically actuated micro
Page 129 and 130: Figure 2. Influence of a surfactant
Page 131 and 132: Figure 5. Device to eject and mix s
Page 133 and 134: Anal. Chem. 2010, 82, 6877-6886 Imm
Page 135 and 136: NaCl, phosphate buffer saline (PBS)
Page 137 and 138: Figure 2. Product ion mass spectra
Page 139 and 140: -70 °C resolved the problem, givin
Page 141 and 142: Table 1. Intraday Precision and Acc
Page 143 and 144: Anal. Chem. 2010, 82, 6887-6894 Fer
Page 145 and 146: Figure 1. Infrared spectra of (A) u
Page 147 and 148: Figure 3. Cyclic voltammograms obta
Page 149 and 150: Figure 6. Calculated charge from ch
Page 151 and 152: Anal. Chem. 2010, 82, 6895-6903 Ele
Page 153 and 154: Table 1. Chemical Structure, pKa Va
Page 155 and 156: pH with a tilted baseline (Figure 1
Page 157 and 158: Table 2. Linearity and Detection Li
Page 159 and 160: ascorbic acid (AA), uric acid (UA),
Page 161 and 162: the multielement capabilities, the
Page 163 and 164: RESULTS AND DISCUSSION Sulfur Detec
Page 165 and 166: Table 2. Molecular Properties and C
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dilution and hybridization buffer.
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solution under appropriate incubati
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Figure 4. Standardization curve for
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Anal. Chem. 2010, 82, 6919-6925 Ele
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the ×10 objective, to have a large
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Figure 2. With a suitable removal o
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the NB signal in a much better foot
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Scheme 1. Reactions of Selenium Rea
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Figure 2. (a) ESI-MS spectrum showi
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Figure 4. (a) ESI-MS spectrum showi
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Anal. Chem. 2010, 82, 6933-6939 Dif
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trode 28 by a finite element using
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Figure 4. Comparison between simula
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Figure 6. Comparison between simula
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educed in the vicinity of double bo
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Figure 2. Normalized product ion ab
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Figure 4. EID (a) and IRMPD (b) of
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Anal. Chem. 2010, 82, 6947-6957 Ide
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Figure 1. Schematic flowchart showi
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difference, ppm compound Table 1. I
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isoforms, its successful use, in th
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Figure 5. Extracted ion current ESI
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m/z 1172.935 was observed for Ser14
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linked products via affinity tags.
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Scheme 2. Fragmentation Mechanism o
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Figure 1. (A) ESI-LTQ-CID-MS 2 prod
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Figure 3. (A) ESI-LTQ-CID-MS 2 prod
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Figure 5. (A) MALDI-TOF/TOF product
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Anal. Chem. 2010, 82, 6969-6975 Ana
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Figure 3. Equilibrium response as a
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Figure 6. The average measured resp
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for the fill time, and we find that
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nucleotide tails. 3-5 Thus, the amo
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allow the use of higher aptamer con
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quent ligation of the aptamers afte
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Anal. Chem. 2010, 82, 6983-6990 Imp
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Figure 2. Configuration editor wind
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Figure 4. Dependencies between volu
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Since the time for liquid expulsion
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Anal. Chem. 2010, 82, 6991-6999 Int
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Figure 1. Representation of the Car
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Table 2. Capillary Electrophoresis
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Figure 4. (A) Typical raw electroph
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field. DNA profiles were delivered
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Another approach to handle this lim
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(principal components, PCs) in whic
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Figure 5. Relevant score plots and
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CONCLUSIONS In this paper we have i
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electroactive-species loaded liposo
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Figure 2. Qdot-based FLFTS response
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Figure 6. Fluorescence imaging of Q
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Anal. Chem. 2010, 82, 7015-7020 Sel
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Table 1. Effect of 1 D-LC Condition
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Figure 3. Peak-production rate vers
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Anal. Chem. 2010, 82, 7021-7026 Cel
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luciferase (T7 control vector) was
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Figure 3. Inhibitory effects of luc
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Anal. Chem. 2010, 82, 7027-7034 Pat
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Electrochemistry. Electrochemical m
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Figure 2. SEM images of Au substrat
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Table 3. Film Thickness Measurement
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Anal. Chem. 2010, 82, 7035-7043 Qua
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Figure 1. (A) FL spectra of BSPOTPE
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Figure 4. (A) Variation in the FL i
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Figure 6. (A) FL spectra of BSPOTPE
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Scheme 1. Proposed Mechanism for Fl
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one or more drawbacks including poo
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Figure 2. Free fluorophores do not
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Anal. Chem. 2010, 82, 7049-7052 Dev
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Figure 2. Comparative analysis of d
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