Comprehensive gas chromatography-time-of-flight mass spectrometry using soft and selective photoionization techniques

The hyphenation of gas chromatography and mass spectrometry (GC/MS) revolutionized organic analysis. In GC/MS coupling, usually electron impact ionization is applied, and molecules are identified by their fragment pattern. Although mass spectrometry in principle is a separation method, it is used predominantly as a spectrometric technique. However, if soft (i.e., fragmentation-free) ionization techniques are applied, the inherent separation character of MS is emphasized, which has similarities to a GC boiling point separation. By combining polar column GC separation and fast soft ionization time-of-flight mass spectrometry technology, a comprehensive separation of complex petrochemical samples can be obtained (GC x MS approach). Compounds of comparable physical-chemical properties are characteristically grouped together in a two-dimensional retention time-m/z representation. This resembles the separation characteristics of comprehensive two-dimensional gas chromatography (GC x GC) and, thus, represents a novel multidimensional separation approach. In this work, a gas chromatograph equipped with a polar separation column was coupled to a home-built laser ionization time-of-flight mass spectrometer. Laser-based, single-photon ionization was used for universal soft ionization and resonance-enhanced multiphoton ionization for selective ionization of aromatic compounds. A novel capillary-jet inlet system was used for the coupling. Multidimensional comprehensive analysis of complex petrochemical hydrocarbon samples using gas chromatography coupled to mass spectrometry with soft and selective photo ionization sources is first demonstrated.     

Simple coupling of gas chromatography to electrospray ionization mass spectrometry

A simple method for direct coupling of gas chromatography (GC) with electrospray ionization mass spectrometry (ESI/MS) has been developed. The outlet of the GC capillary column was placed between the ESI needle and the atmospheric pressure ionization (API) source of a mass spectrometer. The ionization occurs via dissolution of neutral compounds into the charged ESI droplet followed by ion evaporation or via a gas-phase proton transfer reaction between a protonated solvent molecule and an analyte. The mass spectra of organic volatile compounds showed abundant protonated molecules with little fragmentation, being very similar to those produced by normal liquid ESI. The quantitative performance of the system was evaluated by determining the limit of detection (LOD), linearity ( r (2)), and repeatability (RSD). The GC-ESI/MS method was shown to be stable, providing high sensitivity and good quantitative performance.     

Integration of continuous-flow accelerator mass spectrometry with chromatography and mass-selective detection

Physical combination of an accelerator mass spectrometry (AMS) instrument with a conventional gas chromatograph-mass spectrometer (GC/MS) is described. The resulting hybrid instrument (GC/MS/AMS) was used to monitor mass chromatograms and radiochromatograms simultaneously when (14)C-labeled compounds were injected into the gas chromatograph. Combination of the two instruments was achieved by splitting the column effluent and directing half to the mass spectrometer and half to a flow-through CuO reactor in line with the gas-accepting AMS ion source. The reactor converts compounds in the GC effluent to CO2 as required for function of the ion source. With cholesterol as test compound, the limits of quantitation were 175 pg and 0.00175 dpm injected. The accuracy achieved in analysis of five nonzero calibration standards and three quality control standards, using cholesterol-2,2,3,4,4,6-d6 as injection standard, was 100 +/- 11.8% with selected ion monitoring and 100 +/- 16% for radiochromatography. Respective values for interday precision were 1.0-3.2 and 22-32%. Application of GC/MS/AMS to a current topic of interest was demonstrated in a model metabolomic study in which cultured primary hepatocytes were given [(14)C]glucose and organic acids excreted into the culture medium were analyzed.     

Inlet ionization: a new highly sensitive approach for liquid chromatography/mass spectrometry of small and large molecules

Inlet ionization is a new approach for ionizing both small and large molecules in solids or liquid solvents with high sensitivity. The utility of solvent based inlet ionization mass spectrometry (MS) as a method for analysis of volatile and nonvolatile compounds eluting from a liquid chromatography (LC) column is demonstrated. This new LC/MS approach uses reverse phase solvent systems common to electrospray ionization MS. The first LC/MS analyses using this novel approach produced sharp chromatographic peaks and good quality full mass range mass spectra for over 25 peptides from injection of only 1 pmol of a tryptic digest of bovine serum albumin using an eluent flow rate of 55 μL min(-1). Similarly, full acquisition LC/MS/MS of the MH(+) ion of the drug clozapine, using the same solvent flow rate, produced a signal-to-noise ratio of 54 for the major fragment ion with injection of only 1 μL of a 2 ppb solution. LC/MS results were acquired on two different manufacturer's mass spectrometers using a Waters Corporation NanoAcquity liquid chromatograph.     

Analysis of amino acid isotopomers using FT-ICR MS

Fluxes through known metabolic pathways and the presence of novel metabolic reactions are often determined by feeding isotopically labeled substrate to an organism and then determining the isotopomer distribution in amino acids in proteins. However, commonly used techniques to measure the isotopomer distributions require derivatization prior to analysis (gas chromatography/mass spectrometry (GC/MS)) or large sample sizes (nuclear magnetic resonance (NMR) spectroscopy). Here, we demonstrate the use of Fourier transform-ion cyclotron resonance mass spectrometry with direct infusion via electrospray ionization to rapidly measure the amino acid isotopomer distribution in a biomass hydrolysate of the soil bacterium Desulfovibrio vulgaris Hildenborough. By applying high front-end resolution for the precursor ion selection followed by sustained off-resonance irradiation collision-induced dissociation, it was possible to determine exactly and unambiguously the specific locations of the labeled atoms in the amino acids, which usually requires a combination of 2-D 13C NMR spectroscopy and GC/MS. This method should be generally applicable to all biomass samples and will allow more accurate determination of metabolic fluxes with less work and less sample.     

Gas chromatography/electron ionization-mass spectrometry-selected ion monitoring screening method for a thorough investigation of polyhalogenated compounds in passive sampler extracts with quadrupole systems

Nontarget analysis and identification of unknown polyhalogenated compounds is important in acquiring a thorough picture of the present pollution status as well as for identifying emerging environmental problems. Such analyses usually require the application of electron ionization mass spectrometry because the resulting mass spectra frequently allow for compound identification. When quadrupoles are used as mass separators, the full scan technique often suffers from low sensitivity along with nonspecificity for polyhalogenated trace compounds which often result in interference by matrix compounds. We have developed a novel nontarget gas chromatography/electron ionization-mass spectrometry-selected ion monitoring (GC/EI-MS-SIM) method that overcomes these sensitivity and selectivity issues. Our method is based on the fact that the molecular ions and isotope patterns of polyhalogenated compounds involve the most relevant primary information with regard to the structure of polyhalogenated compounds. Additionally, the retention times of polyhalogenated compounds generally increase with increasing molecular weight. The retention time range of polyhalogenated compounds was divided in three partly overlapping segments of 112 u (segment A: m/z 300-412; segment B: m/z 350-462; segment C: m/z 450-562) that were screened in eight GC runs consisting of 15 consecutive SIM ions. This method was tested with a passive water sampler extract known to contain over 30 polyhalogenated compounds according to the sensitive analysis by GC/electron capture negative ion (ECNI)-MS. While none of these polyhalogenated compounds could be detected by GC/EI-MS in full scan mode, our nontarget GC/EI-MS-SIM method allowed for the detection of 38 polyhalogenated compounds. Only seven could be identified by means of reference standards while more than 15 of the unknowns could be traced back to at least the class of compounds based on the mass spectrometric data from the nontarget SIM runs. All compounds identified originated from halogenated natural products. The nontarget GC/EI-MS-SIM method combines the high sensitivity obtainable with quadrupole systems for trace analysis with the structural information essential for the identification of unknown pollutants.     

Gas chromatography connected to multiple channel electrospray ionization mass spectrometry for the detection of volatile organic compounds

This work successfully connected gas chromatography (GC) to seven-channel electrospray ionization (ESI) mass spectrometry to separate and detect a mixture of volatile organic compounds. Gaseous analyte was eluted separately from a GC column and directed into the central channel of the ESI source. The analyte was protonated by ion-molecule reactions between the analyte and the ions which were generated by electrospraying the acidic solution through the outside six channels surrounding the central channel. Real-time analysis of the organic reaction involving volatile and thermally unstable compounds (dimethylhydrazine ? azomethane + H(2)) was also achieved by continuously purging the air in the reaction vessel to the seven-channel ESI source.     

Performance evaluation of gas chromatography-atmospheric pressure chemical ionization-time-of-flight mass spectrometry for metabolic fingerprinting and profiling

Gas chromatography-atmospheric-pressure chemical ionization-time-of-flight mass spectrometry (GC-APCI-TOFMS) was compared to GC × GC-electron ionization (EI)-TOFMS, GC-EI-TOFMS, GC-chemical ionization (CI)-quadrupole mass spectrometry (qMS), and GC-EI-qMS in terms of reproducibility, dynamic range, limit of detection, and quantification using a mix of 43 metabolites and 12 stable isotope-labeled standards. Lower limits of quantification for GC-APCI-TOFMS ranged between 0.06 and 7.81 μM, and relative standard deviations for calibration replicates were between 0.4% and 8.7%. For all compounds and techniques, except in four cases, R(2) values were above 0.99. Regarding limits of quantification, GC-APCI-TOFMS was inferior to only GC × GC-EI-TOFMS, but outperformed all other techniques tested. GC-APCI-TOFMS was further applied to the metabolic fingerprinting of two Escherichia coli strains. Of 45 features that differed significantly (false discovery rate < 0.05) between the strains, 25 metabolites were identified through highly accurate and reproducible (Δm ± SD below 5 mDa over m/z 190-722) mass measurements. Starting from the quasimolecular ion, six additional metabolites were identified that had not been found in a previous study using GC × GC-EI-TOFMS and an EI mass spectral library for identification purposes. Silylation adducts formed in the APCI source assisted the identification of unknown compounds, as their formation is structure-dependent and is not observed for compounds lacking a carboxylic group.     

Ambient mass spectrometry with a handheld mass spectrometer at high pressure

The first coupling of atmospheric pressure ionization methods, electrospray ionization (ESI) and desorption electrospray ionization (DESI), to a miniature hand-held mass spectrometer is reported. The instrument employs a rectilinear ion trap (RIT) mass analyzer and is battery-operated, hand-portable, and rugged (total system: 10 kg, 0.014 m(3), 75 W power consumption). The mass spectrometer was fitted with an atmospheric inlet, consisting of a 10 cm x 127 microm inner diameter stainless steel capillary tube which was used to introduce gas into the vacuum chamber at 13 mL/min. The operating pressure was 15 mTorr. Ions, generated by the atmospheric pressure ion source, were directed by the inlet along the axis of the ion trap, entering through an aperture in the dc-biased end plate, which was also operated as an ion gate. ESI and DESI sources were used to generate ions; ESI-MS analysis of an aqueous mixture of drugs yielded detection limits in the low parts-per-billion range. Signal response was linear over more than 3 orders of magnitude. Tandem mass spectrometry experiments were used to identify components of this mixture. ESI was also applied to the analysis of peptides and in this case multiply charged species were observed for compounds of molecular weight up to 1200 Da. Cocaine samples deposited or already present on different surfaces, including currency, were rapidly analyzed in situ by DESI. A geometry-independent version of the DESI ion source was also coupled to the miniature mass spectrometer. These results demonstrate that atmospheric pressure ionization can be implemented on simple portable mass spectrometry systems.     

Ion-exchange chromatography/electrospray mass spectrometry for the identification of organic and inorganic species in topiramate tablets

An ion-exchange chromatograph/electrospray ionization mass spectrometer (IC/ESI-MS) was used successfully to identify organic and inorganic species present in topiramate tablets. An ion suppressor is placed between the column and detectors to replace sodium ions in the mobile phase with hydrogen ions supplied by the suppressor. The ensuing combination of the hydrogen ions with the mobile phase hydroxide ions produces water and thus allows simultaneous ion detection by an ion conductivity detector and a mass spectrometer. Analytes, including lactate, glycolate, chloride, formate, sulfate, and oxalate, were unambiguously identified by matching the mass spectra and retention times with those of the authentic compounds. Due to its capability of detecting positive and negative as well as neutral species, ESI-MS provides valuable information which is not available with ion conductivity detection alone. Though the coupling of ion-exchange chromatography to mass spectrometry has been reported previously, this is the first demonstration of IC/ESI-MS for the identification of unknown species in real samples. Finally, with the use of deuterium/carbon-13 labeling and MS/MS techniques, we have confirmed that oxalic acid (HOOC-COOH) is formed from formic acid (HCOOH) at the electrospray interface in the presence of the electric field. This observation not only confirms the identity of an unknown peak, but it also provides new insight into chemistry that can take place during electrospray ionization.     

Structure elucidation of an artifact discharging from rubber-based vial closures by means of gas chromatography/tandem mass spectrometry

The use of vial closures equipped with butyl rubber septa may lead to sample contamination by rubber additives discharging from the septum material. In this study, the structure elucidation of an artifact causing intense signals in gas chromatography/electron capture negative ion mass spectrometry (GC/ECNI-MS) and gas chromatographic analyses with electron capture detection is described. Tentative identification of the leached compound was achieved by employing tandem mass spectrometric techniques both in electron capture negative ion and in electron ionization modes. The artifact could thus be characterized as 2-benzothiazolyl-N,N-dimethyl dithiocarbamate, which is a known vulcanization accelerator for rubber. It is conceivable that the identified compound or related substances are also used in other applications. Therefore, two food-related matrixes were investigated for a possible migration of this compound into foods. During these analyses, the tentatively identified rubber additive was detected in an aqueous extract of a rubber seal ring for canning jars. GC/ECNI-MS provided better sensitivity and selectivity than GC/EI-MS for the determination of the rubber additive and other mercaptobenzothiazole-derived substances.     

Colloidal graphite-assisted laser desorption/ionization mass spectrometry and MSn of small molecules. 1. Imaging of cerebrosides directly from rat brain tissue

Graphite-assisted laser desorption/ionization (GALDI) mass spectrometry (MS) was investigated for analysis of cerebrosides in a complex total brain lipid extract. Conventional MALDI MS and GALDI MS were compared regarding lipid analysis by using high-vacuum (HV, <10-6 Torr) LDI time-of-flight mass spectrometry and intermediate-pressure (IP, 0.17 Torr) linear ion trap mass spectrometry. Cerebrosides were not detected or detected with low sensitivity in MALDI MS because of other dominant phospholipids. By using GALDI, cerebrosides were detected as intense mass peaks without prior separation from other lipid species while mass peaks corresponding to phosphatidylcholines (PCs) were weak. The signal increase for cerebrosides and the signal decrease for PCs in GALDI MS were more significant in HV than in IP. MSn experiments of precursor ions corresponding to cerebrosides and PCs in brain lipid extract were performed to identify the detected species and distinguish isobaric ions. Twenty-two cerebroside species were detected by GALDI whereas eight cerebroside species were detected by MALDI. Sulfatides in brain lipid extract were also easily detected by GALDI MS in the negative ion mode. By forming a colloidal graphite thin film on rat brain tissue, direct lipid profiling by imaging mass spectrometry (IMS) was performed. Chemically selective images for cerebrosides and sulfatides were successfully obtained. Imaging tandem mass spectrometry (IMS/MS) was performed to generate images of specific product ions from isobaric species.     

Wrong-way-round ionization of sulfonamides and tetracyclines enables simultaneous analysis with free and conjugated estrogens by liquid chromatography tandem mass spectrometry

The increasing demand to monitor multiple classes of analytes has been mirrored by increased analytical cost and decreased throughput. For instance, the analyses of estrogens and antibiotics by liquid chromatography with tandem mass spectrometry (LC-MS/MS) are typically performed in two separate methods because estrogen analysis requires electrospray with negative ionization, while sulfonamide and tetracycline antibiotics are analyzed under positive ionization. Therefore, we investigated the use of wrong-way-round (WWR) ionization to demonstrate that sulfonamides and tetracyclines can be analyzed at a high pH (10.4), allowing simultaneous analysis with free and conjugated estrogens. An LC-MS/MS method was developed for 28 compounds by polarity switching, based on WWR ionization brought about by the ability of ammonium ions to protonate basic compounds in the gas phase even at high pH. Mass spectral data suggest that gas-phase chemical ionization induced by ammonium ions to form adducts [M + NH(4)](+) occurred, with the subsequent dissociation to the molecular ion [M + H](+). Almost all compounds have an increased signal-to-noise (S/N) ratio of [M + H](+) for sulfonamides and tetracyclines when ionized in basic versus acidic mobile phases by direct injection (no column), indicating that detection limits were not compromised. This study demonstrates a successful application of WWR ionization for the simultaneous analysis of multiple classes of compounds in a single LC-MS/MS analysis.     

Determination of alpha- and beta-hydroxycarbonyls and dicarbonyls in snow and rain samples by GC/FID and GC/MS employing benzyl hydroxyl oxime derivatization

A flame ionization detector (FID) combined with capillary gas chromatography (GC/FID) and gas chromatography/ mass spectrometry (GC/MS) has been used to identify multifunctional carbonyls in wet precipitation samples. The carbonyl groups were first derivatized to O-benzylhydroxyloxime (BH oxime) by using O-benzylhydroxylamine. The BH oxime derivatives were then treated with N,O-bis(trimethylsilyl)acetamide for the hydroxyl group to derive their TMS ethers. The BH oxime/TMS derivatives were measured using GC/FID as well as GC/MS on positive EI and CI (isobutane was used as CI gas) modes. Three groups of carbonyl compounds (monoaldehydes, dicarbonyls, hydroxycarbonyls) were identified in the samples by using this method. We have identified, for the first time, a group of alpha- and beta-hydroxycarbonyls, glycolaldehyde, hydroxyacetone, and 4-hydroxy-2-butanone, in wet precipitation samples. Concentrations of hydroxycarbonyls ranged from 0.9 to 53.8 microg/L in the precipitation samples. Their concentration level is similar to that of low molecular weight dicarboxylic acids, which have been reported as major water-soluble organic compounds in rain.     

Protocol for liquid chromatography/mass spectrometry of glutathione conjugates using postcolumn solvent modification

A novel protocol for thermospray liquid chromatography/mass spectrometry (LC/MS) analysis of mixtures of glutathione conjugates is reported. Solvent conditions for optimal high-performance liquid chromatography are not always the same as for optimal thermospray ionization mass spectrometry. Labile glutathione conjugates that give poor spectra in aqueous ammonium acetate yield more intense molecular ion signals with increased percentages of acetonitrile. Direct injection thermospray ionization using 30-60% acetonitrile in aqueous ammonium acetate produced protonated molecular ions for glutathione conjugates of menadione, styrene oxide, pentachlorophenyl methyl sulfone, chlorodinitrobenzene, and chlorambucil. Since, the high percentages of organic modifier needed for good molecular ion intensity preclude chromatographic separation of these polar compounds, successful graphic separation of these polar compounds, successful LC/MS was facilitated by postcolumn addition of organic modifiers to the mobile phase. This new methodology allowed excellent chromatographic separations and thermospray ionization mass spectra to be obtained for a mixture of haloalkane glutathione conjugates. Moreover, cleavage of the gamma-glutamyl-cysteine amide bond of glutathione results in class-characteristic fragment ions. Changes in the fragmentation pathways in spectra acquired with and without organic modifiers shed light on the importance of the desolvation process in obtaining good molecular ion sensitivity in thermospray.     

A Laminar Flow Nebulizer for Aerosol MALDI

A laminar flow nebulizer was developed for use with aerosol matrix-assisted laser desorption/ionization mass spectrometry. The nebulizer consists of a glass pneumatic nebulizer combined with a laminar flow tube. Particles formed at the pneumatic nebulizer are entrained in an auxiliary gas stream that dries the particles in the laminar flow tube. The mass range of the reflectron time-of-flight mass spectrometer has been extended to greater than 5000 Da: a mass spectrum of bovine insulin is reported at a mass resolution of 100. Mass spectra of bovine insulin and the peptides bradykinin and angiotensin II contain features resulting from prompt analyte dissociation prior to ion acceleration.     

GC/MS with post-column switching for large volume injection of headspace samples: sensitive determination of volatile organic compounds in human whole blood and urine

When volatile or semivolatile compounds are measured by headspace (HS) gas chromatography (GC)/mass spectrometry (MS), the maximum gas volume to be injected is usually 0.5-1.0 mL; over the volume, the MS detector automatically shuts down due to impairment of the vacuum rate of the MS ionization chamber. To overcome the problem, we modified the gas flow routes of a new type of GC/MS instrument to create a postcolumn switching system, which can eliminate the large volume of gas before introduction of target compounds into the MS ionization chamber. Our HS-GC/MS system enabled injection of as large as 5 mL of HS gas without any disturbance. As the first example analysis, we tried to establish the analysis of naphthalene and p-dichlorobenzene in human whole blood and urine by this method with large volume injection. The limits of detection for both compounds in whole blood and urine were as low as about 10 and 5 pg/mL, respectively. The validation data and actual measurements were also demonstrated. The new GC/MS system has great potential to analyze any type of volatile or semivolatile organic compounds in biological matrixes with very high sensitivity and full automation.     

Quasi-simultaneous acquisition of hard electron ionization and soft single-photon ionization mass spectra during GC/MS analysis by rapid switching between both ionization methods: analytical concept, setup, and application on diesel fuel

This work describes the realization of rapid switching between hard electron ionization (EI) and soft single-photon ionization (SPI) integrated in a compact orthogonal acceleration time-of-flight mass spectrometer. Vacuum-ultraviolet (VUV) photons of 9.8 eV (126 nm) emitted from the innovative electron-beam-pumped rare-gas excimer light source (EBEL) filled with argon are focused into the ion chamber by an ellipsoidal mirror optic for accomplishing of SPI. This novel orthogonal acceleration time-of-flight mass spectrometer with switching capability was hyphenated to one-dimensional gas chromatography (GC) and comprehensive two-dimensional (2D) gas chromatography (GC × GC) for the first time. Within this demonstration study, a maximum switching frequency of 80 Hz was applied for investigation of a mineral-oil-type diesel sample. This approach allows the quasi-simultaneous acquisition of complementary information about the fragmentation pattern (EI) as well as the molecular mass (SPI) of compounds within a single analysis. Furthermore, by application of a polar GC column for separation, the SPI data can be displayed in a 2D contour plot, leading to a comprehensive 2D characterization (GC × MS), whereas the typical group-type assignment for diesel is also met.     

Calibration of a commercial solid-phase microextraction device for measuring headspace concentrations of organic volatiles

Solid-phase microextraction (SPME) is a versatile new technique for collecting headspace volatiles prior to GC analysis. The commercial availability of uniform SPME fibers makes routine, practical quantitation of headspace concentrations possible, but straightforward information for relating GC peak areas from SPME analyses to headspace concentrations has not been available. The calibration factors (amount absorbed by the fiber divided by headspace concentration) were determined for 71 compounds using SPME fibers with a 100 μm poly(dimethylsiloxane) coating. The compounds ranged from 1 to 16 carbons in size and included a variety of functional groups. Calibration factors varied widely, being 7000 times higher for tetradecane than for acetaldehyde. Most compounds with a Kovats retention index of <1300 on a nonpolar GC column (DB-1) equilibrated with the fiber in 30 min or less. A regression model is presented for predicting the calibration factor from GC retention index, temperature, and analyte functional class. The calibration factor increased with retention index but decreased with increasing sampling temperature. For a given retention index, polar compounds such as amines and alcohols were absorbed by the fibers in greater amounts than were hydrocarbons. Henry's law constants determined using SPME were in general agreement with literature values, which supported the accuracy of the measured calibration factors. An unexpected concentration dependence of calibration factors was noted, especially for nitrogen-containing and hydroxy compounds; calibration factors were relatively higher (the SPME fiber was more sensitive) at the lower analyte concentrations.