Carbohydrate profiling of bacteria by gas chromatography-mass spectrometry and their trace detection in complex matrices by gas chromatography-tandem mass spectrometry.

Bacterial cellular polysaccharides are composed of a variety of sugar monomers. These sugars serve as chemical markers to identify specific species or genera or to determine their physiological status. Some of these markers can also be used for trace detection of bacteria or their constituents in complex clinical or environmental matrices. Analyses are performed, in our hands, employing hydrolysis followed by the alditol acetate derivatization procedure. Substantial improvements have been made to sample preparation including simplification and computer-controlled automation. For characterization of whole cell bacterial hydrolysates, sugars are analyzed by gas chromatography-mass spectrometry (GC-MS). Simple chromatograms are generated using selected ion monitoring (SIM). Using total ion GC-MS, sugars can be readily identified. In more complex clinical and environmental samples, markers for bacteria are present at sufficiently low concentrations that more advanced instrumentation, gas chromatography-tandem mass spectrometry (GC-MS-MS), is preferred for optimal analysis. Using multiple reaction monitoring, MS-MS is used (replacing more conventional SIM) to ignore extraneous chromatographic peaks. Triple quadrupole and ion trap GC-MS-MS instruments have both been used successfully. Absolute chemical identification of sugar markers at trace levels is achieved, using MS-MS, by the product spectrum.     

Pulsed flow modulation two-dimensional comprehensive gas chromatography-tandem mass spectrometry with supersonic molecular beams

Pulsed flow modulation (PFM) two-dimensional comprehensive gas chromatography (GC x GC) was combined with quadrupole-based mass spectrometry (MS) via a supersonic molecular beam (SMB) interface using a triple-quadrupole system as the base platform, which enabled tandem mass spectrometry (MS-MS). PFM is a simple GC x GC modulator that does not consume cryogenic gases while providing tunable second GC x GC column injection time for enabling the use of quadrupole-based mass spectrometry regardless its limited scanning speed. The 20-ml/min second column flow rate involved with PFM is handled, splitless, by the SMB interface without affecting the sensitivity. The combinations of PFM GC x GC-MS with SMB and PFM GC x GC-MS-MS with SMB were explored with the analysis of diazinon and permethrin in coriander. PFM GC x GC-MS with SMB is characterized by enhanced molecular ion and tailing-free fast ion source response time. It enables universal pesticide analysis with full scan and data analysis with reconstructed single ion monitoring on the enhanced molecular ion and another prominent high mass fragment ion. The elimination of the third fragment ion used in standard three ions method results in significantly reduced matrix interference. GC x GC-MS with SMB improves the GC separation, and thereby our ability for sample identification using libraries. GC-MS-MS with SMB provides better reduction (elimination) of matrix interference than GC x GC-MS. However, it is a target method, which is not always applicable. GC x GC-MS-MS does not seem to further reduce matrix interferences over GC-MS-MS and unlike GC x GC-MS, it is incompatible with library identification, but it is beneficial to have both GC x GC and MS-MS capabilities in the same system.     

Solid-phase micro-extraction-gas chromatography-(tandem) mass spectrometry as a tool for pesticide residue analysis in water samples at high sensitivity and selectivity with confirmation capabilities

Gas chromatography-mass spectrometry (GC-MS) has been widely applied for pesticide monitoring because of its high sensitivity and specificity and for the potential of multi-residue and multi-class analysis. An analytical procedure was developed for the determination of pesticide multi-residues in water samples combining solid-phase micro-extraction (SPME) and gas chromatography-ion trap mass spectrometry. For SPME extraction a poly(dimethylsiloxane)-divinylbenzene coated fibre was selected whereas the mass spectrometer was operated under full scan, selected ion storage (SIS), microSIS (SIM) and MS-MS and the figures of merit compared. Quantitative and qualitative (confirmatory) capabilities of each operation mode are discussed. Using MS-MS, precision was typically below 10% and limits of detection (LODs) were improved by 1.3 to 20 times (to low- or sub-ppt levels) compared to microSIS, with the advantage of maintaining identification capabilities. The combination of selective extraction by SPME and highly selective determination by GC-MS-MS made possible ultra-selective and essentially error-free determination of pesticides in complex environmental samples. This aspect will be highlighted in the paper.     

Comparison of gas chromatography-pulsed flame photometric detection-mass spectrometry, automated mass spectral deconvolution and identification system and gas chromatography-tandem mass spectrometry as tools for trace level detection and identification

The complexity of a matrix is in many cases the major limiting factor in the detection and identification of trace level analytes. In this work, the ability to detect and identify trace level of pesticides in complex matrices was studied and compared in three, relatively new methods: (a) GC-PFPD-MS where simultaneous PFPD (pulsed flame photometric detection) and MS analysis is performed. The PFPD indicates the exact chromatographic time of suspected peaks for their MS identification and provides elemental information; (b) automatic GC-MS data analysis using the AMDIS ("Automated Mass Spectral Deconvolution and Identification System") software by the National Institute of Standards and Technology; (c) GC-MS-MS analysis. A pesticide mixture (MX-5), containing diazinon, methyl parathion, ethyl parathion, methyl trithion and ethion was spiked, in descending levels from 1 ppm to 10 ppb, into soil and sage (spice) extracts and the detection level and identification quality were evaluated in each experiment. PFPD-MS and AMDIS exhibited similar performance, both superior to standard GC-MS, revealing and identifying compounds that did not exhibit an observable GC peak (either buried under the chromatographic background baseline or co-eluting with other interfering GC peaks). GC-MS-MS featured improved detection limits (lower by a factor of 6-8) compared to AMDIS and PFPD-MS. The GC-PFPD-MS-MS combination was found useful in several cases, where no reconstructed ion chromatogram MS-MS peaks existed, but an MS-MS spectrum could still be extracted at the elution time indicated by PFPD. The level of identification and confirmation with MS-MS was inferior to that of the other two techniques. In comparison with the soil matrix, detection limits obtained with the loaded sage matrix were poorer by similar factors for all the techniques studied (factors of 5.8, >6.5 and 4.0 for AMDIS, PFPD-MS and MS-MS, respectively). Based on the above results, the paper discusses the trade-offs between detectivity and identification level with the compared three techniques as well as other more traditional techniques and approaches.     

Hyphenated techniques in anticancer drug monitoring. I. Capillary gas chromatography-mass spectrometry

Muramic acid (Mur) is present in the cell wall of Eubacteria and serves as a chemical marker for the trace detection of bacteria and bacterial cell wall debris in complex matrices. There have been numerous studies using a variety of derivatives of Mur, particularly in combination with gas chromatography-tandem mass spectrometry (GC-MS-MS) where the detection limit has been steadily lowered. A stable, halogenated derivative, the pentafluorobenzyl oxime (PFBO) acetate of Mur, has been developed by others and successfully used for GC with electron-capture detection. The current report is the first use of this derivative for GC-MS-MS analysis of Mur, or indeed any other carbohydrate, using negative ion chemical ionization (NICI) with GC-MS-MS. Mur was readily detected in settled surface dust (166 ng/mg), as well as dust collected from indoor air (1.4-5.9 ng/mg). Analyses of Mur as a PFBO acetate by GC-NICI-MS-MS or as alditol acetates by electron impact GC-electron impact ionization MS-MS serve as complementary approaches for trace detection in complex matrices.     

Simultaneous determination of phenolic acids and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one in wheat (Triticum aestivum L.) by gas chromatography-tandem mass spectrometry

A procedure using gas chromatography and tandem mass spectrometry (GC-MS-MS) has been developed for the identification and quantification of some allelochemicals in wheat (Triticum aestivum L.). The quantities of allelochemicals in wheat shoots ranged from 2.9 to 110 mg per kilogram of dry shoot residues. Compared with gas chromatography-mass spectrometry (GC-MS), the GC-MS-MS technique significantly increased instrument selectivity and sensitivity, thereby providing more reliable quantitation results in the determination of the phytotoxic compounds examined during this allelopathy research.     

Identification of isopropyl substituted β-blocking agents in human urine by gas chromatography and tandem mass spectrometry

Summary A method for the sensitive detection of isopropyl substituted β-blocking agents in human urine is presented. The sample preparation step involves enzymatic hydrolysis, solid phase extraction and derivatisation withN-methyl-N-trimethylsilyl-trifluoroacetamide. The instrumental analysis was performed using gas chromatography-mass spectrometry with an ion trap mass spectromeler. The mass spectrometer was operated in the scan as well as in the MS-MS mode.     

Determination of pesticide residues in olives and olive oil by matrix solid-phase dispersion followed by gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry

A novel analytical approach has been developed and evaluated for the quantitative analysis of a selected group of widely used pesticides (dimethoate, simazine, atrazine, diuron, terbuthylazine, methyl-parathion, methyl-pirimiphos, endosulfan I, endosulfan II, endosulfan sulphate, cypermethrin and deltamethrin), which can be found at trace levels in olive oil and olives. The proposed methodology is based on matrix solid-phase dispersion (MSPD), (with a preliminary liquid-liquid extraction in olive oil samples) using aminopropyl as sorbent material with a clean-up performed in the elution step with Florisil, followed by mass spectrometric identification and quantitation of the selected pesticides using both gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode and liquid chromatography tandem mass spectrometry (LC-MS-MS) in positive ionization mode. The recoveries obtained (with mean values between 85 and 115% (obtained at different fortification levels) with RSD values below 10% in most cases, confirm the usefulness of the proposed methodology for the analyses of these kind of complex samples with a high fat content. Moreover, the obtained detection limits, which were below 5 microg kg(-1) by LC-MS analyses and ranged from 10 to 60 microg kg(-1) by GC-MS meet the requirements established by the olive oil pesticide regulatory programs. The method was satisfactorily applied to different olives and olive oil samples.     

Application of liquid chromatography-electrospray ionization tandem mass spectrometry to the detection of 10 sulfonamides in honey

Liquid chromatography (LC) in combination with tandem mass spectrometry (MS-MS) has been applied to the separation and detection of 10 different sulfonamides in honey. The methodology encompasses a simple hydrolysis of the honey sample to liberate sugar-bound sulfonamides followed by liquid-liquid extraction of the 10 analytes, filtration, and analysis by LC-MS-MS. Conditions for reversed-phase LC and electrospray ionization (ESI) MS-MS in the positive ion mode were optimized for the 10 compounds under study, monitoring two characteristic mass transitions simultaneously for each analyte. The procedure is a qualitative confirmatory method for 10 sulfonamides at the low microg/kg level in honey. Typical recoveries of the analytes in honey ranged from 44 to 73% at a fortification level of 50 microg/kg. This study also addresses the issue of matrix-induced suppression of ionization, an effect often encountered in trace residue analysis of food matrices using LC-ESI-MS-MS based methods.     

Elucidation of urinary metabolites of fluoxymesterone by liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry

The suitability of liquid chromatography tandem mass spectrometry (LC-MS/MS) and gas chromatography mass spectrometry (GC-MS) for the elucidation of fluoxymesterone metabolism has been evaluated. Electrospray ionization (ESI) and collision induced dissociation (CID) fragmentation in LC-MS/MS and electron impact spectra (EI) in GC-MS have been studied for fluoxymesterone and two commercially available metabolites. MS(n) experiments and accurate mass measurements performed by an ion-trap analyser and a QTOF instrument respectively have been used for the elucidation of the fragmentation pathway. The neutral loss scan of 20 Da (loss of HF) in LC-MS/MS has been applied for the selective detection of fluoxymesterone metabolites. In a positive fluoxymesterone doping control sample, 9 different analytes have been detected including the parent compound. Seven of these metabolites were also confirmed by GC-MS including 5 previously unreported metabolites. On the basis of the ionization, the CID fragmentation, the accurate mass of the product ions and the EI spectra of these analytes, a tentative elucidation as well as a proposal for the metabolic pathway of fluoxymesterone has been suggested. The presence of these compounds has also been confirmed by the analysis of five other positive fluoxymesterone urine samples.     

Determination of hopane biomarkers in Arabian crude oils by gas chromatography-tandem mass spectrometry

Gas chromatography-mass spectrometry with the mass spectrometer operated in the tandem mode at a constant accelerating voltage was used to analyse for triterpanes in Arabian crude oils. Specific molecular parent-daughter ion pairs were selected for quantitative measurements owing to their metastable transitions in the first field-free region. More accurate relative distribution ratios of hydrocarbon biomarkers were determined by this sequential gating technique than by the conventional diagnostic daughter ion mass fragmentogram in common GC-MS. The data and spectra obtained from MS-MS operation may serve as unique “fingerprints” for these regional crudes     

Determination of trace food-derived hazardous compounds in Chinese cooked foods using solid-phase extraction and gas chromatography coupled to triple quadrupole mass spectrometry

Gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) with a triple quadrupole analyzer (QqQ) was applied for the first time to the determination of trace food-derived hazardous compounds in food samples. Thirteen heterocyclic amines were confirmed and quantified by the developed sensitive method. The method includes a solid-phase extraction procedure with a polystyrene copolymer cartridge (LiChrolut EN), followed by a derivatization reaction with N,N-dimethylformamide di-tert-butylacetal. Analyses were performed by gas chromatography with triple quadrupole mass spectrometry in the electron impact mode. The MS/MS fragmentation pathway of derived heterocyclic amines was studied and the differences of fragmentation characteristics were used successively to distinguish the isomers in absence of chemical standards. The excellent selectivity and sensitivity achieved in multiple reaction monitoring mode allowed us satisfactory quantitation and confirmation. The limits of quantitation of the method for these compounds ranged from 0.12 to 0.48ng/g of sample. The method developed was applied to the analysis of Chinese cooked foods, and the results demonstrated the potentiality of the GC-MS/MS method for the analysis of trace food-derived hazardous compounds in complex food matrices such as meat samples.     

Analysis of carcinogenic polycyclic aromatic hydrocarbons in complex environmental mixtures by LC-APPI-MS/MS

Here we developed a highly sensitive, fast and reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the detection and analysis of 16 different polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs that have been identified as carcinogens and classified according to their biological potency. Comparison to standard analysis procedures based on gas chromatography-mass spectrometry (GC-MS) instrumentation demonstrated an improved easiness of sample preparation and sensitivity of detection achieved with the new LC-MS/MS method employing an atmospheric pressure photoionization (APPI) source attached to an API 4000 mass spectrometer (LC-APPI-MS/MS). The favorable outcome could be confirmed by analyzing complex mixtures such as certain Standard Reference Materials (SRMs) obtained from the National Institute of Standards & Technology (NIST), i.e., SRM 1975 and SRM 2975, and several diesel exhaust soots provided by the German automobile industry. Certified concentrations of individual analytes provided by NIST not only could be confirmed, but additional extremely potent carcinogens such as several isomeric hexacyclic dibenzopyrenes (DBPs), 5-methylchrysene (5-MC), and others have been detected in these crude samples in a concentration range down to below 1 ng g(-1) raw material.     

Separation and identification of ceramides in the human stratum corneum by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry and electrospray multiple-stage mass spectrometry profiling

Summary A sensitive, selective, and rapid method is described for analysis of ceramides in the human stratum coracum by direct coupling of HPLC with an electrospray ion-trap mass spectrometry. Nonaqueous reversed-phase chromatography stabilizes the electrospray ionization, resulting in sensitivity that enables direct measurement of skin lipid extracts with no special sample preparation. Assignment of individual signals to the corresponding ceramide species is based on interpretation of the fragment spectra from MS-MS experiments. This enables much finer differentiation between ceramdies than that achievable by thin-layer chromatography. Summary A sensitive, selective, and rapid method is described for analysis of ceramides in the human stratum corneum by direct coupling of HPLC with an electrospray ion-trap mass spectrometry. Nonaqueous reversed-phase chromatography stabilizes the electrospray ionization, resulting in sensitivity that enables direct measurement of skin lipid extracts with no special sample preparation. Assignment of individual signals to the corresponding ceramide species is based on interpretation of the fragment spectra from MS-MS experiments. This enables much finer differentiation between ceramides than that achievable by thin-layer chromatography.     

顶空气相色谱-质谱联用技术的应用进展

顶空分析作为一种无有机溶剂萃取的样品处理技术,通常与气相色谱-质谱（GC-MS）技术结合用来分析复杂基质中的挥发性有机物。顶空气相色谱-质谱（HS-GC-MS）技术具有快速、高效、环保、灵敏度高等特点,在常规分析中发挥着重要作用。该文简要概述了静态顶空、动态顶空、顶空固相微萃取分析以及GC-MS联用技术,并介绍了整个顶空分析系统的影响因素和优化过程。根据基质类型的分类,综述了HS-GC-MS在食品和饮料、环境、生物等样品中的应用实例。HS-GC-MS的研究非常活跃,不断出现新应用,在分析挥发性有机物方面具有广阔前景。     

Comprehensive two-dimensional gas chromatography in combination with rapid scanning quadrupole mass spectrometry in perfume analysis

Single column gas chromatography (GC) in combination with a flame ionization detector (FID) and/or a mass spectrometer is routinely employed in the determination of perfume profiles. The latter are to be considered medium to highly complex matrices and, as such, can only be partially separated even on long capillaries. Inevitably, several monodimensional peaks are the result of two or more overlapping components, often hindering reliable identification and quantitation. The present investigation is based on the use of a comprehensive GC (GC x GC) method, in vacuum outlet conditions, for the near to complete resolution of a complex perfume sample. A rapid scanning quadrupole mass spectrometry (qMS) system, employed for the assignment of GC x GC peaks, supplied high quality mass spectra. The validity of the three-dimensional (3D) GC x GC-qMS application was measured and compared to that of GC-qMS analysis on the same matrix. Peak identification, in all applications, was achieved through MS spectra library matching and the interactive use of linear retention indices (LRI).     

Screening of nitrogen mustards and their degradation products in water and decontamination solution by liquid chromatography-mass spectrometry

Analysing nitrogen mustards and their degradation products in decontamination emulsions posed a significant challenge due to the different phases present in such matrices. Extensive sample preparation may be required to isolate target analytes. Furthermore, numerous reaction products are formed in the decontamination emulsion. A fast and effective qualitative screening procedure was developed for these compounds, using liquid chromatography-mass spectrometry (LC-MS). This eliminated the need for additional sample handling and derivatisation that are required for gas chromatographic-mass spectrometric (GC-MS) analysis. A liquid chromatograph with mixed mode column and isocratic elution gave good chromatography. The feasibility of applying this technique for detecting these compounds in spiked water and decontamination emulsion was demonstrated. Detailed characterisation of the degradation products in these two matrices was carried out. The results demonstrated that N-methyldiethanolamine (MDEA), N-ethyldiethanolamine (EDEA) and triethanolamine (TEA) are not the major degradation products of their respective nitrogen mustards. Degradation profiles of nitrogen mustards in water were also established. In verification analysis, it is important not only to develop methods for the identification of the actual chemical agents; the methods must also encompass degradation products of the chemical agents as well so as to exclude false negatives. This study demonstrated the increasingly pivotal role that LC-MS play in verification analysis.     

Quantitative gas chromatography/time-of-flight mass spectrometry: a review

Time-of-flight (TOF) instruments have recently gained popularity in quantitative analyses. Normally, TOF mass spectrometers are used for accurate mass measurements for empirical formula verification. However, over the past decade, they have been used quantitatively as well. Because of the fast separations and narrow peaks that result from gas chromatography separations, scanning mass spectrometers are not ideal detectors. TOF mass spectrometers, however, have the ability to collect spectra at a faster rate. Two-dimensional gas chromatography has also been introduced to further resolve peaks from complex matrices. Two-dimensional gas chromatography results in a faster separation as well as narrower peaks. This paper reviews the methods currently in the literature for the quantitation of compounds using one- and two-dimensional gas chromatography and TOF mass spectrometry detection.     

Quantification of glucuronidated and sulfated steroids in human urine by ultra-high pressure liquid chromatography quadrupole time-of-flight mass spectrometry

The urinary steroid profile is constituted by anabolic androgenic steroids, including testosterone and its relatives, that are extensively metabolized into phase II sulfated or glucuronidated steroids. The use of liquid chromatography coupled to mass spectrometry (LC-MS) is an issue for the direct analysis of conjugated steroids, which can be used as urinary markers of exogenous steroid administration in doping analysis, without hydrolysis of the conjugated moiety. In this study, a sensitive and selective ultra high-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS) method was developed to quantify major urinary metabolites simultaneously after testosterone intake. The sample preparation of the urine (1 mL) was performed by solid-phase extraction on Oasis HLB sorbent using a 96-well plate format. The conjugated steroids were analyzed by UHPLC-QTOF-MS^E with a single-gradient elution of 36 min (including re-equilibration time) in the negative electrospray ionization mode. MS^E analysis involved parallel alternating acquisitions of both low- and high-collision energy functions. The method was validated and applied to samples collected from a clinical study performed with a group of healthy human volunteers who had taken testosterone, which were compared with samples from a placebo group. Quantitative results were also compared to GC-MS and LC-MS/MS measurements, and the correlations between data were found appropriate. The acquisition of full mass spectra over the entire mass range with QTOF mass analyzers gives promise of the opportunity to extend the steroid profile to a higher number of conjugated steroids.     

Mass spectrometrically detected directly coupled high performance liquid chromatography/nuclear magnetic resonance spectroscopy/mass spectrometry for the identification of xenobiotic metabolites in maize plants

Reconstructed ion chromatograms have been used to identify relevant high performance liquid chromatography (HPLC) peaks in a directly coupled high performance liquid chromatography/nuclear magnetic resonance spectroscopy/mass spectrometry (HPLC/NMR/MS) experiment. This has been applied to a study of the metabolism of a model compound, 5-nitropyridone (2-hydroxy-5-nitropyridine), in maize plants grown hydroponically. By monitoring the on-flow reconstructed ion chromatogram corresponding to the 5-nitropyridone fragment at m/z 143, and additional molecular ions corresponding to metabolites identified as products from similar compounds, relevant peaks were identified rapidly for subsequent stopped-flow 1H NMR spectroscopic analysis. The combination of coupled HPLC/NMR/MS enabled the direct identification of three metabolites, namely the N-glucoside, N-malonylglucoside, and O-malonylglucoside. This work demonstrates the power of HPLC/NMR/MS for the structural elucidation of xenobiotic metabolites in complex biological matrices (such as plant material) with minimal sample preparation. In particular, using mass spectrometry for the initial identification of relevant HPLC peaks allows the analysis of complex samples without the necessity for other spectroscopic markers, such as 19F NMR signal for fluorinated compounds or UV spectroscopy for molecules with strong UV chromophores.