Fast gas chromatography combustion isotope ratio mass spectrometry

We report here the first coupling of fast GC to IRMS, in a system capable of 250 ms peak widths (fwhm) at 1 mL/min flow rates, one-fifth as narrow as any previously reported GCC-IRMS system. We developed an optimized postcolumn interface that results in minimal peak broadening, using a programmable temperature vaporization injector in place of a rotary valve or backflush system to divert solvent, a narrow capillary combustion reactor followed by a cryogenic water trap with narrow-bore (<0.20 mm i.d.) transfer lines, and a narrow i.d. open split to the IRMS directly inserted into the column effluent. Quantitative combustion was demonstrated with CH4 injections. A comparison of CO2 injections with different fwhm peak widths (250, 2500, and 7500 ms) showed similar precisions, SD(delta13C)=0.2-0.3 per thousand, for injections of >600 pmol C on column; precision for the narrow peaks (250 ms) was considerably better for injections<150 pmol C on column. SD(delta13C)<1 per thousand was achievable for injections of 5-15 pmol on column for 250 ms wide peaks, 10-fold better precision than 2500 ms wide peaks, and within a factor of 3 of the counting statistics limit. For a mixture of 15 fatty acid methyl esters (FAME), 1.5 nmol C of each on column yielded typical SD(delta13Cpdb)=0.4 per thousand for fast GC and 0.5 per thousand for conventional GC. For 14 of the 15 FAME, delta13C values between the two systems were within+/-1.5 per thousand and not significantly different. Fast GCC-IRMS required one-third the run time (450 s vs 1400 s) to achieve comparable resolution. Mean peak widths for fast GCC-IRMS of the FAME were 720 ms, compared to 650 ms by fast GC with flame ionization detection. At a 15-fold dilution (100 pmol C on column for each FAME), fast GCC-IRMS achieved approximately 2-fold better precision and accuracy than similar injections on conventional GCC-IRMS. Finally, a mixture of 10 steroids (approximately 7 nmol C (100 ng) each on column) was analyzed with mean precision of SD(delta13C)=0.2 per thousand in 620 s by fast GCC-IRMS, while conventional GCC-IRMS required 1200 s and achieved poorer resolution. delta13C values for the two system were similar (Deltadelta13C1 nmol C) and achieves modest precision (approximately 1 per thousand) near the counting statistics limit on low level components.     

Comprehensive two-dimensional gas chromatography combustion isotope ratio mass spectrometry

We report the first coupling of comprehensive two-dimensional gas chromatography (GC x GC) to online combustion isotope ratio mass spectrometry (C-IRMS). A GC x GC system, equipped with a longitudinally modulated cryogenic system (LMCS), was interfaced to an optimized low dead volume combustion interface to preserve <300 ms full width at half-maximum (fwhm) fast GC peaks generated on the second GC column (GC2). The IRMS detector amplifiers were modified by configuration of resistors and capacitors to enable fast response, and a home-built system acquired data at 25 Hz. Software was home-written to handle isotopic time shifts of less than one bin (40 ms) and to integrate peak slices to recover isotope ratios from cryogenically sliced peaks. The performance of the GC x GCC-IRMS system was evaluated by isotopic analysis of urinary steroid standards. Steroids were separated by a nonpolar GC1 column (30 m x 0.25 mm, 5% phenyl), modulated into multiple 4- or 8-s cryogenic slices by the LMCS, and then separated on a polar GC2 column (1 or 2 m x 0.1 mm, 50% phenyl). GC2 peak widths from a 1-m column averaged 276 ms fwhm. Steroid standard sliced peaks were successfully reconstructed to yield delta(13)C VPDB values with average precisions of SD(delta(13)C) = 0.30 per thousand and average accuracies within 0.34 per thousand, at 8 ng on column. Two steroids, coeluting in GC1, were baseline separated in GC2 and resulted in delta(13)C VPDB values with average precisions of SD(delta(13)C) = 0.86 per thousand and average accuracies within 0.26 per thousand, at 3 ng on column. Results from this prototype system demonstrate that the enhanced peak capacity and signal available in GC x GC is compatible with high-precision carbon isotope analysis.     

Stable nitrogen isotope analysis of amino Acid enantiomers by gas chromatography/combustion/isotope ratio mass spectrometry

The analysis of the stable nitrogen isotope compositions of individual amino acid stereoisomers through the use of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is presented. Nitrogen isotopic compositions of single amino acids or of their enantiomers is possible without the labor-intensive and time-consuming preparative-scale chromatographic procedures required for conventional stable isotope analysis. Following hydrolysis and derivatization, single-component isotope analysis is accomplished on nanomole quantities of each of the stereoisomers of an amino acid, utilizing the effluent stream of gas chromatographic separation. Nitrogen isotope fractionation is minimal during acylation of the amino acid, with no additional nitrogen being added stoichiometrically to the derivative. Thus, the isotopic composition of the nitrogen in the derivative is that of the original compound. Replicate stable nitrogen isotope analyses of 11 amino acids, and their trifluoroacetyl (TFA)/isopropyl (IP) ester derivatives, determined by both conventional isotope ratio mass spectrometry (IRMS) and GC/C/IRMS, indicate that the GC procedure is highly reproducible (standard deviations typically 0.3-0.4‰) and that isotopic differences between the amino acid and its TFA/IP derivative are, in general, less than 0.5‰.     

Compound-specific chlorine isotope analysis: a comparison of gas chromatography/isotope ratio mass spectrometry and gas chromatography/quadrupole mass spectrometry methods in an interlaboratory study

Chlorine isotope analysis of chlorinated hydrocarbons like trichloroethylene (TCE) is of emerging demand because these species are important environmental pollutants. Continuous flow analysis of noncombusted TCE molecules, either by gas chromatography/isotope ratio mass spectrometry (GC/IRMS) or by GC/quadrupole mass spectrometry (GC/qMS), was recently brought forward as innovative analytical solution. Despite early implementations, a benchmark for routine applications has been missing. This study systematically compared the performance of GC/qMS versus GC/IRMS in six laboratories involving eight different instruments (GC/IRMS, Isoprime and Thermo MAT-253; GC/qMS, Agilent 5973N, two Agilent 5975C, two Thermo DSQII, and one Thermo DSQI). Calibrations of (37)Cl/(35)Cl instrument data against the international SMOC scale (Standard Mean Ocean Chloride) deviated between instruments and over time. Therefore, at least two calibration standards are required to obtain true differences between samples. Amount dependency of δ(37)Cl was pronounced for some instruments, but could be eliminated by corrections, or by adjusting amplitudes of standards and samples. Precision decreased in the order GC/IRMS (1σ ≈ 0.1‰), to GC/qMS (1σ ≈ 0.2-0.5‰ for Agilent GC/qMS and 1σ ≈ 0.2-0.9‰ for Thermo GC/qMS). Nonetheless, δ(37)Cl values between laboratories showed good agreement when the same external standards were used. These results lend confidence to the methods and may serve as a benchmark for future applications.     

A thermogravimetry-capillary gas chromatography/mass spectrometry interface

An interface and gas chromatograph oven are described that couple a thermogravimetric analyzer with a mass spectrometer and permit multiple capillary gas chromatographic separations of volatile thermal decomposition products generated during a single thermogravimetric analysis. Examples of the use of this apparatus for identifying the volatile products generated during poly(vinyl butyral) thermal decomposition in the presence of γ-alumina and catalytic cracking of poly(styrene) and poly(ethylene) are described. TG-GC/MS analyses employing isothermal, temperature programmed, and subambient temperature ramp gas chromatography separations are described. The apparatus permits repetitive temperature-programmed capillary gas chromatographic analyses of thermogravimetric effluent containing more than 25 constituents in 3-min intervals.     

Isotopic analysis of atmospheric formaldehyde by gas chromatography isotope ratio mass spectrometry

Little is known about the isotopic composition of formaldehyde in the atmosphere, a chemical intermediate in hydrocarbon oxidation. Here, we present a promising new method to analyze the carbon (delta 13C) and hydrogen (delta D) isotopic composition of atmospheric formaldehyde. The direct isotopic analytical technique described uses continuous-flow gas chromatography-isotope ratio mass spectrometry, which provides flexibility for either isotopic analysis without correction for derivative functional groups. Current levels of precision of measurement are +/-1.1 and +/-50 per thousand (1 sigma) for delta 13C and delta D analyses, respectively. Concentration of formaldehyde in ambient air is also determined, coincident with isotopic measurement, to a precision of +/-15%. The method has the required sensitivity for analyses of formaldehyde in urban air on relatively small volume grab samples of whole air (10-70L STP), potentially providing high temporal resolution. This is particularly advantageous for studying formaldehyde given its short lifetime and large variability in the atmosphere.     

Improved performance and maintenance in gas chromatography/isotope ratio mass spectrometry by precolumn solvent removal

The crucial step in current concepts to interface isotope ratio mass spectrometry (IRMS) to gas chromatography (GC) is efficient solvent removal. This is due to the essential postcolumn conversion of the analytes into simple gases, which is performed by either combustion or pyrolysis. The capacity of this step merely suffices to convert the analytes. Already small amounts of solvent present in the respective furnace can cause severe damage. In conventional GC/IRMS interfaces, the solvent is removed after passage of the GC column. Either back-flushing or flow diversion is employed for this purpose. Both techniques necessitate the use of numerous components such as unions, tee pieces, valves, and capillary connections. Often this results in significant deterioration of the chromatographic resolution. In contrast, accurate GC/IRMS measurements require baseline separation of adjacent peaks. Moreover, maintenance of conventional interfaces may be tedious and time consuming, mostly because the numerous connections are prone to leakage. In order to avoid these drawbacks, we propose a concept to efficiently remove the solvent before passage of the GC column. It is based on the use of a cooled injection system operated in solvent vent mode, where the solvent elimination is supported by an auxiliary pump. Most unions and tee pieces thus can be removed. The chromatographic resolution is considerably enhanced. In particular, analysis of high-boiling and polar compounds can be improved. At the same time, the maintenance of the system is significantly facilitated. Under the chosen conditions, partial losses of low-boiling analytes during solvent elimination were not associated with significant isotope fractionation.     

Temperature-programmed high-performance liquid chromatography coupled to isotope ratio mass spectrometry

The utility of liquid chromatography coupled to the isotope ratio mass spectrometry technique (LC-IRMS) has already been established through a variety of successful applications. However, the analytical constraint related to the use of aqueous mobile phases limits the LC separation mechanism. We report here a new strategy for high-precision (13)C isotopic analyses based on temperature-programmed LC-IRMS using aqueous mobile phases. Under these conditions, the isotopic precision and accuracy were studied. On one hand, experiments were carried out with phenolic acids using isothermal LC conditions at high temperature (170 degrees C); on the other hand, several experiments were performed by ramping the temperature, as conventionally used in a gas chromatography-based method with hydrosoluble fatty acids and pulses of CO 2 reference gas. In isothermal conditions at 170 degrees C, despite the increase of the CO 2 background, p-coumaric acid and its glucuronide conjugate gave reliable isotopic ratios compared to flow injection analysis-isotopic ratio mass spectrometry (FIA-IRMS) analyses (isotopic precision and accuracy are lower than 0.3 per thousand). On the opposite, for its sulfate conjugate, the isotopic accuracy is affected by its coelution with p-coumaric acid. Not surprisingly, this study also demonstrates that at high temperature (170 degrees C), a compound eluting with long residence time (i.e., ferulic acid) is degraded, affecting thus the delta (13)C (drift of 3 per thousand) and the peak area (compared to FIA-IRMS analysis at room temperature). Quantitation is also reported in isothermal conditions for p-coumaric acid in the range of 10-400 ng/mL and with benzoic acid as an internal standard. For temperature gradient LC-IRMS, in the area of the LC gradient (set up at 20 degrees C/min), the drift of the background observed produces a nonlinearity of SD (delta (13)C) approximately 0.01 per thousand/mV. To circumvent this drift, which impacts severely the precision and accuracy, an alternative approach, i.e., eluting the compound on the plateau of temperature studied was reported here. Other experiments with temperature-programmed LC-IRMS experiments are also reported with the presence of methanol in the injected solution to mimic residual solvent originating from the sample preparation or to slightly increase the solubility of the targeted compound for high-precision measurement.     

Applications of Hadamard transform to gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry

Successful application of the Hadamard transform (HT) technique to gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) is described. Novel sample injection devices were developed to achieve multiple sample injections in both GC and LC instruments. Air pressure was controlled by an electromagnetic valve in GC, while a syringe pump and Tee connector were employed for the injection device in LC. Two well-known, abused drugs, 3,4-methylenedioxy-N-methylamphetamine (MDMA) and N, N-dimethyltryptamine (DMT), were employed as model samples. Both of the injection devices permitted precise successive injections, resulting in clearly modulated chromatograms encoded by Hadamard matrices. After inverse Hadamard transformation of the encoded chromatogram, the signal-to-noise (S/N) ratios of the signals were substantially improved compared with those expected from theoretical values. The S/N ratios were enhanced approximately 10-fold in HT-GC/MS and 6.8 in HT-LC/MS, using the matrices of 1023 and 511, respectively. The HT-GC/MS was successfully applied to the determination of MDMA in the urine sample of a suspect.     

Closed-loop, multiobjective optimization of two-dimensional gas chromatography/mass spectrometry for serum metabolomics

Metabolomics seeks to measure potentially all the metabolites in a biological sample, and consequently, we need to develop and optimize methods to increase significantly the number of metabolites we can detect. We extended the closed-loop (iterative, automated) optimization system that we had previously developed for one-dimensional GC-TOF-MS (O'Hagan, S.; Dunn, W. B.; Brown, M.; Knowles, J. D.; Kell, D. B. Anal. Chem. 2005, 77, 290-303) to comprehensive two-dimensional (GCxGC) chromatography. The heuristic approach used was a multiobjective version of the efficient global optimization algorithm. In just 300 automated runs, we improved the number of metabolites observable relative to those in 1D GC by some 3-fold. The optimized conditions allowed for the detection of over 4000 raw peaks, of which some 1800 were considered to be real metabolite peaks and not impurities or peaks with a signal/noise ratio of less than 5. A variety of computational methods served to explain the basis for the improvement. This closed-loop optimization strategy is a generic and powerful approach for the optimization of any analytical instrumentation.     

Mass profile monitoring in trace analysis by gas chromatography/mass spectrometry.

The advantages of gas chromatography/mass spectrometry (GC/MS) selected-ion monitoring (SIM) in the mass profile (MP) mode at medium mass resolving power were investigated for analyses requiring detection of low-picogram quantities of analytes in complex mixtures. The mass profile monitoring provides a certainty at least 10 times greater than that achieved by conventional GC/MS-SIM in the peak-top monitoring mode, and it can be operated at lower mass resolving power to compensate for the loss of sensitivity in the MP mode. The examination of mass profile peak shape, central mass shift, and sequential changes during GC elution not only reveals the presence of interfering compounds but also results in accurate mass measurement for those interferences. The latter feature takes the MP mode beyond the target mass analysis that GC/MS-SIM was originally designed for. This additional dimension of information is particularly useful for those complex and incompletely characterized matrices that are frequently encountered in environmental and biological sample analyses.     

Correction of H3+ contributions in hydrogen isotope ratio monitoring mass spectrometry

Two fundamentally different approaches, termed "pointwise" and "peakwise," are currently used to correct hydrogen isotope ratio monitoring data for the presence of H3+ ion contributions. Consideration of the underlying assumptions shows that the peakwise approach is valid only for peaks with the same functional shape and only when background signals do not vary. The pointwise correction is much more versatile and can be used even when peak shapes and sizes, as well as background signals, vary significantly. It is not exact and is limited in accuracy by (1) the signal-broadening effects of electronic time constants, (2) the analog-to-digital conversion frequency, and (3) the highest frequency of the sample signal. To minimize errors for typical gas chromatographic signals, time constants of <500 ms and analog-to-digital sampling intervals of < or =250 ms are needed. Errors are further minimized by matching sample and standard peaks in both amplitude and D/H ratio. Using the pointwise algorithm, we demonstrate that a series of 14 homologous n-alkanes varying in concentration over a 5-fold range can be analyzed with a mean precision of 2.3 per thousand and no systematic errors.     

Microbial metabolomics with gas chromatography/mass spectrometry

An analytical method was set up suitable for the analysis of microbial metabolomes, consisting of an oximation and silylation derivatization reaction and subsequent analysis by gas chromatography coupled to mass spectrometry. Microbial matrixes contain many compounds that potentially interfere with either the derivatization procedure or analysis, such as high concentrations of salts, complex media or buffer components, or extremely high substrate and product concentrations. The developed method was extensively validated using different microorganisms, i.e., Bacillus subtilis, Propionibacterium freudenreichii, and Escherichia coli. Many metabolite classes could be analyzed with the method: alcohols, aldehydes, amino acids, amines, fatty acids, (phospho-) organic acids, sugars, sugar acids, (acyl-) sugar amines, sugar phosphate, purines, pyrimidines, and aromatic compounds. The derivatization reaction proved to be efficient (>50% transferred to derivatized form) and repeatable (relative standard deviations <10%). Linearity for most metabolites was satisfactory with regression coefficients better than 0.996. Quantification limits were 40-500 pg on-column or 0.1-0.7 mmol/g of microbial cells (dry weight). Generally, intrabatch precision (repeatability) and interbatch precision (reproducibility) for the analysis of metabolites in cell extracts was better than 10 and 15%, respectively. Notwithstanding the nontargeted character of the method and complex microbial matrix, analytical performance for most metabolites fit the requirements for target analysis in bioanalysis. The suitability of the method was demonstrated by analysis of E. coli samples harvested at different growth phases.     

Determination of the the H3 factor in hydrogen isotope ratio monitoring mass spectrometry

The H3 factor, K, is a parameter required in high-precision, mass spectrometric analyses of hydrogen isotopic abundances. When H2 is used as the sample gas, R* = R - Ki2, where R* is the true HD/H2 ratio, R is the observed (mass 3)/(mass 2) ion-current ratio, and i2 is the ion current at mass 2. Four different methods for the determination of K were defined and tested under conditions characteristic of isotope ratio monitoring systems. Three of these were peak-based. The fourth employed steady flows of H2 from a conventional inlet system. Results obtained using the latter method were more precise (standard deviation of K = 0.1 versus approximately 0.6 ppm mV(-1) for the peak-based methods). However, use of the resulting values of K for correction of isotope ratio monitoring GC/MS results led to systematic errors as large as 9 per thousand, whereas use of the peak-based values led to no systematic errors. Values of K were only weakly dependent on the pressure of He, declining approximately 5% for each 10-fold increase in P(He). Small variations in partial pressures of H2O and CH4, potential contaminants under isotope ratio monitoring conditions, had no significant effect on values of K.     

Differential metabolomics using stable isotope labeling and two-dimensional gas chromatography with time-of-flight mass spectrometry

This work describes an approach to differential metabolomics that involves stable isotope labeling for relative quantification as part of sample analysis by two-dimensional gas chromatography/mass spectrometry (GCxGC/MS). The polar metabolome in control and experimental samples was extracted and differentially derivatized using isotopically light and heavy (D6) forms of the silylation reagent N-methyl-N-tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA). MTBSTFA derivatives are of much greater hydrolytic stability than the more common trimethylsilyl derivatives, thus diminishing the possibility of isotopomer scrambling during GC analysis. Subsequent to derivatization with MTBSTFA, differentially labeled samples were mixed and analyzed by GCxGC/MS. Metabolites were identified, and the isotope ratio of isotopomers was quantified. The method was tested using three classes of metabolites; amino acids, fatty acids, and organic acids. The relative concentration of isotopically labeled metabolites was determined by isotope ratio analysis. The accuracy and precision, respectively, in quantification of standard mixtures was 9.5 and 4.77% for the 16 amino acids, 9.7 and 2.83% for the mixture of 19 fatty acids, and 14 and 4.53% for the 20 organic acids. Suitability of the method for the examination of complex samples was demonstrated in analyses of the spiked blood serum samples. This differential isotope coding method proved to be an effective means to compare the concentration of metabolites between two samples simultaneously.     

Data analysis tool for comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry

Data processing and identification of unknown compounds in comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GC×GC/TOFMS) analysis is a major challenge, particularly when large sample sets are analyzed. Herein, we present a method for efficient treatment of large data sets produced by GC×GC/TOFMS implemented as a freely available open source software package, Guineu. To handle large data sets and to efficiently utilize all the features available in the vendor software (baseline correction, mass spectral deconvolution, peak picking, integration, library search, and signal-to-noise filtering), data preprocessed by instrument software are used as a starting point for further processing. Our software affords alignment of the data, normalization, data filtering, and utilization of retention indexes in the verification of identification as well as a novel tool for automated group-type identification of the compounds. Herein, different features of the software are studied in detail and the performance of the system is verified by the analysis of a large set of standard samples as well as of a large set of authentic biological samples, including the control samples. The quantitative features of our GC×GC/TOFMS methodology are also studied to further demonstrate the method performance and the experimental results confirm the reliability of the developed procedure. The methodology has already been successfully used for the analysis of several thousand samples in the field of metabolomics.     

Long-term performance of a gas chromatography/tandem mass spectrometry assay for tebufelone in plasma

An automated gas chromatography/tandem mass spectrometry (GC/MS/MS) based method for the rapid determination of tebufelone (TE) in animal and human plasma has been routinely applied in our laboratory to more than 3000 samples over a 2-year period. The selectivity of MS/MS conducted on a triple quadrupole instrument, combined with the use of a stable-isotope-labeled internal standard, results in excellent analytical figures of merit, as well as minimal sample preparation, rapid analysis, and high assay reliability. The work described here goes beyond initial method development and validation studies by evaluating the long-term performance of quantitative GC/MS/MS. Electron ionization produces M.+ ions for TE and the [13C, 18O]TE internal standard, which are selected in Q1 and undergo collisionally activated dissociation in Q2. Quantitation is based on monitoring daughter ions at m/z 248 and 251, respectively, in Q3. A linear range of 1-3000 ng of TE/sample (20 pg to 60 ng injected) provides access to an effective concentration range of 0.5-30,000 ppb TE in plasma (0.1-2-g samples). The assay shows no bias and less than 10% relative standard deviation over this range. In the automated mode, less than 7 min elapse from injection to report printout and more than 70 plasma samples are routinely prepared and analyzed in a day. Such performance is consistently maintained throughout long-term application.