Gas chromatography/flame ionisation detection mass spectrometry for the detection of endogenous urine metabolites for metabonomic studies and its use as a complementary tool to nuclear magnetic resonance spectroscopy

Metabonomics is a relatively new field of research in which the total pool of metabolites in body fluids or tissues from different patient groups is subjected to comparative analysis. Nuclear magnetic resonance (NMR) spectroscopy is the technology that is currently most widely used for the analysis of these highly complex metabolite mixtures, and hundreds of metabolites can be detected without any upfront separation. We have investigated in this study whether gas chromatography (GC) separation in combination with flame ionisation detection (FID) and mass spectrometry (MS) detection can be used for metabolite profiling from urine. We show that although GC sample preparation is much more involved than for NMR, hundreds of metabolites can reproducibly be detected and analysed by GC. We show that the data quality is sufficiently high--particularly if appropriate baseline correction and time-warping methods are applied--to allow for data comparison by chemometrics methods. A sample set of urines from eleven healthy human volunteers was analysed independently by GC and NMR, and subsequent chemometrics analysis of the two datasets showed some similar features. As judged by NIST database searches of the GC/MS data some of the major metabolites that are detected by NMR are also visible by GC/MS. Since in contrast to NMR every peak in GC corresponds to a single metabolite, the electron ionisation spectra can be used to quickly identify metabolites of interest if their reference spectra are present in a searchable database. In summary, we show that GC is a method that can be used as a complementary tool to NMR for metabolite profiling of urine samples.     

Human urinary metabolite profile of tea polyphenols analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry with data-dependent acquisition

Tea is rich in polyphenols and has a variety of biological activities. In order to better understand the biological effects of tea constituents on human health, markers for their exposure and their metabolic fates are needed. Previously, we have characterized several catechin metabolites in the blood and urine, but more information on the metabolite profile of tea polyphenols is needed. In the present study, the human urinary metabolite profile of tea polyphenols was investigated using liquid chromatography/electrospray ionization tandem mass spectrometry with data-dependent acquisition. With data-dependent MS/MS analysis by collecting the MS2 and MS3 spectra of the most intense ions in the sample, we identified more than twenty metabolites of tea polyphenols from human urine samples. (-)-Epigallocatechin (EGC) glucuronide, methylated EGC glucuronide, methylated EGC sulfate, (-)-epicatechin (EC) glucruronide, EC sulfate, methylated EC sulfate, as well as the glucuronide and sulfate metabolites of the ring-fission metabolites of tea catechins, 5-(3',4',5'-trihydroxyphenyl)-gamma-valerolactone (M4), 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone (M6) and 5-(3',5'-dihydroxyphenyl)-gamma-valerolactone (M6'), were the major human urinary metabolites of tea polyphenols. To our knowledge, this is the first report of the direct simultaneous analysis of the human urinary metabolite profile of tea polyphenols using single sample analysis. This method can also be used for thorough investigations of the metabolite profiles of many other dietary constituents.     

Analysis of gin essential oil mixtures by multidimensional and one-dimensional gas chromatography/mass spectrometry with spectral deconvolution

The composition of essential oils and their mixtures used to formulate gin is usually too complex to separate all sample components by standard capillary gas chromatography (GC). In particular, minor constituents that possess important organoleptic properties can be masked by co-elution with major sample components. A solution is provided that combines gas chromatography/mass spectrometry (GC/MS) with "interactive" spectral deconvolution software. Sequential two-dimensional (2D) GC/MS is used to produce a target compound library, with orthogonal GC-GC providing the separation power required to obtain peak retention times and the corresponding mass spectra needed for the deconvolution database. The combination of these two techniques, mass spectral deconvolution and automated sequential 2D-GC/MS, offers a very effective synergy for both identifying key constituents that determine the perception of flavor and aroma and the quality control needed to analyze mixtures of complex essential oils.     

Development and validation of a gas chromatography/mass spectrometry metabonomic platform for the global profiling of urinary metabolites

This paper presents a simple and reliable gas chromatography/mass spectrometry (GC/MS) method for the metabonomic analysis of human urine samples. The sample preparation involved the depletion of excess urea via treatment with urease and subsequent protein precipitation using ice-cold ethanol. An aliquot of the mixture was separated, dried, trimethylsilyl (TMS)-derivatized and 1.0 microL of the derivatized extract was injected into the GC/MS system via split injection (1:10). Approximately 150 putative metabolites belonging to different chemical classes were identified from the pooled human urine samples. All the identified metabolites were selected to evaluate precision and stability of the GC/MS assay. More than 95% of the metabolites demonstrated good reproducibility, with intra-day and inter-day precision values below 15%. Metabolic profiling of 53 healthy male and female urine samples in combination with pattern recognition techniques was performed to further validate the GC/MS metabolite profiling assay. Principal component analysis (PCA) followed by orthogonal partial least squares analysis (OPLS) revealed differences between urinary metabolite profiles of healthy male and female subjects. This validated GC/MS metabolic profiling method may be further applied to the metabonomic screening of urinary biomarkers in clinical studies. Copyright (c) 2008 John Wiley & Sons, Ltd.     

单叶蔓荆子挥发油成分的GC/MS分析

采用气相色谱／质谱（ＧＣ／ＭＳ）联用技术对山东泰山产单叶蔓荆子挥发油的化学成分进行了分析,分离出３０多个峰,确认了其中２８种成分,占总离子流的９５％以上,并对挥发油的主要化学成分Δ３－蒈烯,用气相色谱／傅里叶红外光谱（ＧＣ／ＦＴＩＲ）法进行了验证。     

Toward Depth-Resolved Analysis of Plant Metabolites by Nanospray Desorption Electrospray Ionization Mass Spectrometry

Nanospray desorption electrospray ionization (nano-DESI) is one of the ambient desorption ionization methods for mass spectrometry (MS), and it utilizes a steady-state liquid junction formed between two microcapillaries to directly extract analytes from sample surfaces with minimal sample damage. In this study, we employed nano-DESI MS to perform a metabolite fingerprinting analysis directly from a Hypericum leaf surface. Moreover, we investigated whether changes in metabolite fingerprints with time can be related to metabolite distribution according to depth. From a raw Hypericum leaf, the mass spectral fingerprints of key metabolites, including flavonoids and prenylated phloroglucinols, were successfully obtained using ethanol as a nano-DESI solvent, and the changes in their intensities were observed with time via full mass scan experiments. In addition, the differential extraction patterns of the obtained mass spectral fingerprints were clearly visualized over time through selected ion monitoring and pseudo-selected reaction monitoring experiments. To examine the correlation between the time-dependent changes in the metabolite fingerprints and depth-wise metabolite distribution, we performed a nano-DESI MS analysis against leaves whose surface layers were removed multiple times by forming polymeric gum Arabic films on their surfaces, followed by detaching. The preliminary results showed that the changes in the metabolite fingerprints according to the number of peelings showed a similar pattern with those obtained from the raw leaves over time.     

Rapid screening and characterization of drug metabolites using multiple ion monitoring dependent product ion scan and postacquisition data mining on a hybrid triple quadrupole‐linear ion trap mass spectrometer

Multiple ion monitoring (MIM)‐dependent acquisition with a triple quadrupole‐linear ion trap mass spectrometer (Q‐trap) was previously developed for drug metabolite profiling. In the analysis, multiple predicted metabolite ions are monitored in both Q1 and Q3 regardless of their fragmentations. The collision energy in Q2 is set to a low value to minimize fragmentation. Once an expected metabolite is detected by MIM, enhanced product ion (EPI) spectral acquisition of the metabolite is triggered. To analyze in vitro metabolites, MIM‐EPI retains the sensitivity and selectivity similar to that of multiple reaction monitoring (MRM)‐EPI in the analysis of in vitro metabolites. Here we present an improved approach utilizing MIM‐EPI for data acquisition and multiple data mining techniques for detection of metabolite ions and recovery of their MS/MS spectra. The postacquisition data processing tools included extracted ion chromatographic analysis, product ion filtering and neutral loss filtering. The effectiveness of this approach was evaluated by analyzing oxidative metabolites of indinavir and glutathione (GSH) conjugates of clozapine and 4‐ethylphenol in liver microsome incubations. Results showed that the MIM‐EPI‐based data mining approach allowed for comprehensive detection of metabolites based on predicted protonated molecules, product ions or neutral losses without predetermination of the parent drug MS/MS spectra. Additionally, it enabled metabolite detection and MS/MS acquisition in a single injection. This approach is potentially useful in high‐throughout screening of metabolic soft spots and reactive metabolites at the drug discovery stage. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination and excretion study of gestrinone in human urine by high performance liquid chromatography and gas chromatography/mass spectrometry

Gestrinone was studied by HPLC for screening and by GC/MS for confirmation. Three unknown peaks were found by HPLC which are probably the metabolites of gestrinone, and conjugated gestrinone in dosed human urine. The metabolites and gestrinone were excreted as the conjugated forms. The total amounts of metabolite 1 and conjugated gestrinone, recovered after 48 h, were 0.20 and 0.32 mg, respectively. When metabolite 1 was tested by LC/MS and LC/MS/MS, the parent ion was m/z 327, [MH](+), and fragment ions were seen at m/z 309 [MH - H(2)O](+), 291 [MH - 2H(2)O](+), 283, 263 and 239. The TMS-enol-TMS ether derivative of gestrinone has three peaks in the GC/MS chromatogram formed by tautomerism. The reproducibility of the derivatization method was stable and recoveries were over 87% when spiked into blank urine.     

Comprehensive analysis of yeast metabolite GC x GC-TOFMS data: combining discovery-mode and deconvolution chemometric software

The first extensive study of yeast metabolite GC x GC-TOFMS data from cells grown under fermenting, R, and respiring, DR, conditions is reported. In this study, recently developed chemometric software for use with three-dimensional instrumentation data was implemented, using a statistically-based Fisher ratio method. The Fisher ratio method is fully automated and will rapidly reduce the data to pinpoint two-dimensional chromatographic peaks differentiating sample types while utilizing all the mass channels. The effect of lowering the Fisher ratio threshold on peak identification was studied. At the lowest threshold (just above the noise level), 73 metabolite peaks were identified, nearly three-fold greater than the number of previously reported metabolite peaks identified (26). In addition to the 73 identified metabolites, 81 unknown metabolites were also located. A Parallel Factor Analysis graphical user interface (PARAFAC GUI) was applied to selected mass channels to obtain a concentration ratio, for each metabolite under the two growth conditions. Of the 73 known metabolites identified by the Fisher ratio method, 54 were statistically changing to the 95% confidence limit between the DR and R conditions according to the rigorous Student's t-test. PARAFAC determined the concentration ratio and provided a fully-deconvoluted (i.e. mathematically resolved) mass spectrum for each of the metabolites. The combination of the Fisher ratio method with the PARAFAC GUI provides high-throughput software for discovery-based metabolomics research, and is novel for GC x GC-TOFMS data due to the use of the entire data set in the analysis (640 MB x 70 runs, double precision floating point).     

气相色谱-质谱联用分析白兰叶油成分

采用气相色谱－质谱联用技术对苏州产白兰叶油的化学成分进行了分析,分离出３３个峰。鉴定了其中２７种成分,占总峰面积的９７％,并对油中的主要化学成分芳樟醇用气相色谱－傅立叶红外光谱法进行了验证。     

Metabolomics analysis reveals variation in Schisandra chinensis metabolites from different origins

Wu Wei Zi (Schisandra chinensis), an important herbal medicine, is mainly distributed in the northeast of China. Its phytochemical compositions, which depend on geographical origin, climatic conditions and cultural practices, may vary largely among Wu Wei Zi from different areas. In this study, we applied a comprehensive metabolite profiling approach using GC–TOF‐MS, ultra‐performance LC (UPLC) quadrupole TOF (QTOF) MS and inductively coupled plasma MS to systematically investigate the metabolite variations of S. chinensis from four different areas including Heilongjiang, Liaoning, Jilin, and Shanxi of China. A total of 65 primary metabolites, 35 secondary metabolites and 64 inorganic elements were identified. Several primary metabolites, including shikimic acid and tricarboxylic acid cycle intermediates, were abundant in those located in Heilongjiang, Jilin, and Liaoning. Besides, bioactive lignans are also highly abundant in those from northeastern China than those from northwestern China. Inorganic elements varied significantly among the different locations. Our results suggested that the metabolite profiling approach using GC–TOF‐MS, ultra‐performance LC quadrupole TOF MS, and inductively coupled plasma MS is a robust and reliable method that can be effectively used to explore subtle variations among plants from different geographical locations.     

Joint MS‐based platforms for comprehensive comparison of rat plasma and serum metabolic profiling

Metabolomics is a rapidly growing field in the comprehensive understanding of cellular and organism‐specific responses associated with perturbations induced by medicines, chemicals and environment. Blood matrices are frequently used in clinical and biological studies. In this study, we compared metabolic profiling between rat plasma and serum using complementary platforms of gas chromatography–mass spectrometry (GC‐MS) and liquid chromatography–quadruple time‐of‐flight–mass spectrometry (LC‐QTOF‐MS). The sample types that were tested included plasma prepared with K₂EDTA and serum collected using venous blood collection protocols. The results of peak area variation for each detected metabolite/feature in the quality control samples showed a good reproducibility in LC‐QTOF‐MS and better reproducibility in GC‐MS. In GC‐MS analysis: (a) 25.8% of the defined metabolites differed serum from plasma profiling (t‐test, p < 0.05); and (b) serum possessed higher sensitivity than plasma for its generally higher peak intensity in the metabolic profiling. In LC‐QTOF‐MS analysis, 13 (in positive ion mode) and seven (in negative ion mode) important metabolites were identified as mainly contributing to the separation between serum and plasma. Copyright © 2014 John Wiley & Sons, Ltd.     

Development of a mass spectrometric hydroxyl‐position determination method for the hydroxyindole metabolites of JWH‐018 by GC‐MS/MS

One of the many issues of designer drugs of abuse like synthetic cannabinoids (SCs) such as JWH‐018 is that details on their metabolism has yet to be fully elucidated and that multiple metabolites exist. The presence of isomeric compounds poses further challenges in their identification. Our group has previously shown the effectiveness of gas chromatography‐electron ionization‐tandem mass spectrometry (GC‐EI‐MS/MS) in the mass spectrometric differentiation of the positional isomers of the naphthoylindole‐type SC JWH‐081, and speculated that the same approach could be used for the metabolite isomers. Using JWH‐018 as a model SC, the aim of this study was to differentiate the positional isomers of its hydroxyindole metabolites by GC‐MS/MS. Standard compounds of JWH‐018 and its hydroxyindole metabolite positional isomers were first analyzed by GC‐EI‐MS in full scan mode, which was only able to differentiate the 4‐hydroxyindole isomer. Further GC‐MS/MS analysis was performed by selecting m/z 302 as the precursor ion. All four isomers produced characteristic product ions that enabled the differentiation between them. Using these ions, MRM analysis was performed on the urine of JWH‐018 administered mice and determined the hydroxyl positions to be at the 6‐position on the indole ring. GC‐EI‐MS/MS allowed for the regioisomeric differentiation of the hydroxyindole metabolite isomers of JWH‐018. Furthermore, analysis of the fragmentation patterns suggests that the present method has high potential to be extended to hydroxyindole metabolites of other naphthoylindole type SCs in identifying the position of the hydroxyl group on the indole ring. Copyright © 2016 John Wiley & Sons, Ltd.     

Liquid-phase microextraction for sample preparation in analysis of unconjugated anabolic steroids in urine

The applicability of in-vial two-phase liquid-phase microextraction (LPME) in porous hollow polypropylene fiber was studied for the sample preparation of unconjugated anabolic steroids in urine. Four different anabolic steroids - metabolites of fluoxymesterone, 4-chlorodehydromethyltestosterone, stanozolol and danazol - were used as test compounds and methyltestosterone as an internal standard. A standard two-phase LPME method for use with liquid chromatography/mass spectrometry (LC/MS) was set up and the influence of different parameters, including the nature of organic solvent, extraction time, salting-out and temperature, on the LPME process was investigated. Taking advantage of the preliminary studies, a novel two-phase LPME method utilizing simultaneous in-fiber silylation was developed and validated for gas chromatographic/mass spectrometric (GC/MS) analysis of a danazol metabolite in urine. In all, LPME allowed a very straightforward, simple and selective way to prepare urine samples for steroid analysis, being most suitable for hydrophobic steroids. The LPME method with in-fiber derivatization for GC/MS analysis exhibited high sensitivity, repeatability and linearity and enabled simultaneous filtration, extraction, enrichment and derivatization of the analyte from urine matrix without any other steps in sample pretreatment.     

Strategy of using microsome-based metabolite production to facilitate the identification of endogenous metabolites by liquid chromatography mass spectrometry

One of the challenges in metabolomic profiling of complex biological samples is to identify new and unknown compounds. Typically, standards are used to help identify metabolites, yet standards cannot be purchased or readily synthesized for many unknowns. In this work we present a strategy of using human liver microsomes (HLM) to metabolize known endogenous human metabolites (substrates), producing potentially new metabolites that have yet to be documented. The metabolites produced by HLM can be tentatively identified based on the associated substrate structure, known metabolic processes, tandem mass spectrometry (MS/MS) fragmentation patterns and, if necessary, accurate mass measurements. Once identified, these metabolites can be used as references for identification of the same compounds in complex biological samples. As a proof of principle, a total of 9 metabolites have been identified from individual HLM incubations using 5 different substrates. Each metabolite was used as a standard. In the analysis of human urine sample by liquid chromatography MS/MS, four spectral matches were found from the 9 microsome-produced metabolite standards. Two of them have previously been documented as endogenous human metabolites, the third is an isomer of a microsome-metabolite and the fourth compound has not been previously reported and is also an isomer of a microsome-metabolite. This work illustrates the feasibility of using microsome-based metabolism to produce metabolites of endogenous human metabolites that can be used to facilitate the identification of unknowns in biological samples. Future work on improving the performance of this strategy is also discussed.     

Chemical composition database establishment and metabolite profiling analysis of Yangyin qingfei decoction

The chemical fingerprinting and metabolite profile in a rat plasma sample after intragastric administration of Yangyin qingfei decoction (YYQFD, 14 g/kg) were investigated. First, YYQFD was analyzed by UPLC/Q‐TOF MS to establish the chemical composition database by comparing their retention behavior, accurate molecular mass and MS² data with those of references or known compounds in the literature. In this database, 100 chemical constituents with information on retention time, molecular mass, molecular formula, MS² data and compound name were identified, which can provide compound information for further metabolite profiling studies. Furthermore, 64 compounds including 37 prototypes and 27 metabolites were detected in the dosed rat plasma sample, and the metabolic pathways of YYQFD were hydrolyzation, hydroxylation, dehydrogenation, glucuronidation, glucosylation, sulfation and mixed modes. Among the five component herbs in the YYQFD, Glycyrrhizae Radix et Rhizome and Fritillariae Thunbergii bulbs were actively metabolized, contributing 16 and 7 metabolites, respectively. It is suggested that chemical characterization and metabolite profiling studies are valuable to elucidate the material basis of herbal preparations.     

LC–MS Analysis of the Essential Oils of Achillea millefolium and Achillea crithmifolia

The chemical composition of the hydro-distilled essential oils of Achillea millefolium and Achillea crithmifolia was analyzed by GC, GC–MS, ¹³C NMR and high resolution LC–MS. For the first time, the use of the combination of different chromatographic and spectral methods, primarily the advantage of LC-Orbitrap over standard methods, enabled the detection of azulenes and their progenitors, in minute quantities, in previously believed proazulene free Achillea taxon (A. crithmifolia). Furthermore, the LC-Orbitrap hyphenated method provided the means for detection of these and related non-volatile (GC-injector compared to water steam distillation) metabolites up to know unreported as essential oil constituents.     

气相色谱/质谱在不同种类硫酸盐还原菌的鉴定中应用探讨

采用气相色谱／质谱联用技术分析和鉴定吉林、长庆、胜利、新疆宝浪和江汉油田的硫酸盐还原菌（ｓｕｌｆａｔｅ－ｒｅｄｕｃｉｎｇ ｂａｃ６ｅｉａ,ＳＲＢ）代谢产物,利用透射电镜分析和观察五油田ＳＲＢ的形貌。结果表明,吉林、长庆、胜利、新疆宝浪ＳＲＢ呈杆状,培养基中的代谢产物主要以丙酸为主,江汉油田的ＳＲＢ呈弧状,培养基中的代谢产物主要以苯甲酸为主。该技术为油田提供了一种鉴定ＳＲＢ代谢产物判别其种类的快捷准     

Trace mycotoxin analysis in complex biological and food matrices by liquid chromatography-atmospheric pressure ionisation mass spectrometry

Mycotoxins are toxic secondary metabolites produced by filamentous fungi that are growing on agricultural commodities. Their frequent presence in food and their severe toxic, carcinogenic and estrogenic properties have been recognised as potential threat to human health. A reliable risk assessment of mycotoxin contamination for humans and animals relies basically on their unambiguous identification and accurate quantification in food and feedstuff. While most screening methods for mycotoxins are based on immunoassays, unambiguous analyte confirmation can be easily achieved with mass spectrometric methods, like gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/mass spectrometry (LC/MS). Due to the introduction of atmospheric pressure ionisation (API) techniques in the late 80s, LC/MS has become a routine technique also in food analysis, overcoming the traditional drawbacks of GC/MS regarding volatility and thermal stability. During the last few years, this technical and instrumental progress had also an increasing impact on the expanding field of mycotoxin analysis. The aim of the present review is to give an overview on the application of LC-(API)MS in the analysis of frequently occurring and highly toxic mycotoxins, such as trichothecenes, ochratoxins, zearalenone, fumonisins, aflatoxins, enniatins, moniliformin and several other mycotoxins. This includes also the investigation of some of their metabolites and degradation products. Suitable sample pre-treatment procedures, their applicability for high sample through-put and their influence on matrix effects will be discussed. The review covers literature published until July 2006.     

卷烟主流烟气粒相物中中性化学成分的全二维气相色谱飞行时间质谱(GC×GC-TOF MS)谱图特征识别

建立了全二维气相色谱-飞行时间质谱(GC×GC-TOF MS)分析卷烟主流烟气中中性化学成分的方法。以较长的弱极性柱HP-5MS(50 m×0.2 mm i.d.×0.33μm)作为第一维柱,较短的薄液膜中等极性柱DB-17MS(1.7 m×0.1 mm i.d.×0.1μm)作为第二维柱,对优质烟叶单料卷烟烟气的中性成分进行定性分析,经过人工纠错等分析初步鉴定出匹配度大于700的1 464种成分,重点讨论了中性香味羰基化合物全二维点阵的谱图特征,为烟气和复杂体系的深入研究提供了方法学基础。