Journal of Chromatography B (v.871, #2)

Hyphenated techniques for global metabolite profiling by Georgios Theodoridis; Ian D. Wilson (141-142).

Mass spectrometry for the identification of the discriminating signals from metabolomics: Current status and future trends by Erwan Werner; Jean-François Heilier; Céline Ducruix; Eric Ezan; Christophe Junot; Jean-Claude Tabet (143-163).
The metabolome is characterized by a large number of molecules exhibiting a high diversity of chemical structures and abundances, requiring complementary analytical platforms to reach its extensive coverage. Among them, atmospheric pressure ionization mass spectrometry (API-MS)-based technologies, and especially those using electrospray ionization are now very popular. In this context, this review deals with strengths, limitations and future trends in the identification of signals highlighted by API-MS-based metabolomics. It covers the identification process from the determination of the molecular mass and/or its elemental composition to the confirmation of structural hypotheses. Furthermore, some tools that were developed in order to address the MS signal redundancy and some approaches that could facilitate identification by improving the visualization and organization of complex data sets are also reported and discussed.
Keywords: Metabolomics; Metabonomics; Identification; Mass spectrometry;

The human cerebrospinal fluid metabolome by David S. Wishart; Michael J. Lewis; Joshua A. Morrissey; Mitchel D. Flegel; Kevin Jeroncic; Yeping Xiong; Dean Cheng; Roman Eisner; Bijaya Gautam; Dan Tzur; Summit Sawhney; Fiona Bamforth; Russ Greiner; Liang Li (164-173).
Keywords: Cerebrospinal fluid; CSF; Metabolome; Metabolomics; Nuclear magnetic resonance; Mass spectrometry;

A matter of fat: An introduction to lipidomic profiling methods by Lee D. Roberts; Gregor McCombie; Christopher M. Titman; Julian L. Griffin (174-181).
In recent years, lipidomics or lipid profiling, an extension of metabolomics where the lipid complement of a cell, tissue or organism is measured, has been the recipient of increasing attention as a research tool in a range of diverse disciplines including physiology, lipid biochemistry, clinical biomarker discovery and pathology. The advancement of the field has been driven by the development of analytical technologies, and in particular advances in liquid chromatography mass spectrometry and chemometric methods. In this review, we give an overview of the current methods with which lipid profiling is being performed. The benefits and shortcomings of mass spectrometry both in the presence and absence of chromatographic separation techniques such as liquid-, gas- and thin layer chromatography are explored. Alone these techniques have their limitations but through a combination many of the disadvantages may be overcome providing a valuable analytical tool for a variety of disease processes.
Keywords: Lipidomics; Mass spectrometry; Electrospray ionisation; Matrix assisted laser desorption/ionisation; Liquid chromatography; Gas chromatography; Thin layer chromatography; Lipid profiling; Metabolomics;

Retention index thresholds for compound matching in GC–MS metabolite profiling by Nadine Strehmel; Jan Hummel; Alexander Erban; Katrin Strassburg; Joachim Kopka (182-190).
The generation of retention index (RI) libraries is an expensive and time-consuming effort. Procedures for the transfer of RI properties between chromatography variants are, therefore, highly relevant for a shared use. The precision of RI determination and accuracy of RI transfer between 8 method variants employing 5%-phenyl-95%-dimethylpolysiloxane capillary columns was investigated using a series of 9 n-alkanes (C10–C36). The precision of the RI determination of 13 exemplary fatty acid methyl esters (C8 ME–C30 ME) was 0.22–0.33 standard deviation (S.D.) expressed in RI units in low complexity samples. In the presence of complex biological matrices this precision may deteriorate to 0.75–1.11. Application of the previously proposed Kováts, van den Dool or 3rd–5th order polynomial regression algorithms resulted in similar precision of RI calculation. For transfer of empirical van den Dool-RI properties between the chromatography variants 3rd order regression was found to represent the minimal necessary assumption. The range of typical regression coefficients was r 2  = 0.9988–0.9998 and accuracy of RI prediction between chromatography variants varied between 5.1 and 19.8 (0.29–0.69%) S.D. of residual RI error, RIpredicted  − RIdetermined (n  > 64). Accuracy of prediction was enhanced when subsets of chemically similar compound classes were used for regression, for example organic acids and sugars exhibited 0.78 (n  = 29) and 3.74 (n  = 37) S.D. of residual RI error, respectively. In conclusion, we suggest use of percent RI error rather than absolute RI units for the definition of matching thresholds. Thresholds of 0.5–1.0% may apply to most transfers between chromatography variants. These thresholds will not solve all matching ambiguities in complex samples. Therefore, we recommend co-analysis of reference substances with each GC–MS profiling experiment. Composition of these defined reference mixtures may best approximate or mimic the quantitative and qualitative composition of the biological matrix under investigation.
Keywords: Retention index matching; Gas chromatography; GC–MS; Metabolite profiling; Metabolomics;

Standardizing GC–MS metabolomics by Harin Kanani; Panagiotis K. Chrysanthopoulos; Maria I. Klapa (191-201).
Metabolomics being the most recently introduced “omic” analytical platform is currently at its development phase. For the metabolomics to be broadly deployed to biological and clinical research and practice, issues regarding data validation and reproducibility need to be resolved. Gas chromatography–mass spectrometry (GC–MS) will remain integral part of the metabolomics laboratory. In this paper, the sources of biases in GC–MS metabolomics are discussed and experimental evidence for their occurrence and impact on the final results is provided. When available, methods to correct or account for these biases are presented towards the standardization of a systematic methodology for quantitative GC–MS metabolomics.
Keywords: Quantitative systems biology; High-throughput “omic” techniques; Data correction and normalization; Data validation; Derivatization biases; TMS-derivatives; Methoximation; Metabolite extraction;

Gas chromatography/mass spectrometry in metabolic profiling of biological fluids by Kishore K. Pasikanti; P.C. Ho; E.C.Y. Chan (202-211).
One of the objectives of metabonomics is to identify subtle changes in metabolite profiles between biological systems of different physiological or pathological states. Gas chromatography mass spectrometry (GC/MS) is a widely used analytical tool for metabolic profiling in various biofluids, such as urine and blood due to its high sensitivity, peak resolution and reproducibility. The availability of the GC/MS electron impact (EI) spectral library further facilitates the identification of diagnostic biomarkers and aids the subsequent mechanistic elucidation of the biological or pathological variations. With the advent of new comprehensive two dimensional GC (GC × GC) coupled to time-of-flight mass spectrometry (TOFMS), it is possible to detect more than 1200 compounds in a single analytical run. In this review, we discuss the applications of GC/MS in the metabolic profiling of urine and blood, and discuss its advances in methodologies and technologies.
Keywords: Metabolic profiling; Gas chromatography; GC/MS; Metabonomics; Metabolomics; Urine; Blood;

GC–MS methods for metabolic profiling of microbial fermentation products of dietary polyphenols in human and in vitro intervention studies by Christian H. Grün; Ferdi A. van Dorsten; Doris M. Jacobs; Marie Le Belleguic; Ewoud J.J. van Velzen; Max O. Bingham; Hans-Gerd Janssen; John P.M. van Duynhoven (212-219).
Flavonoids, a subclass of polyphenols, are major constituents of many plant-based foods and beverages, including tea, wine and chocolate. Epidemiological studies have shown that a flavonoid-rich diet is associated with reduced risk of cardiovascular diseases. The majority of the flavonoids survive intact until they reach the colon where they are then extensively metabolized into smaller fragments. Here, we describe the development of GC–MS-based methods for the profiling of phenolic microbial fermentation products in urine, plasma, and fecal water. Furthermore, the methods are applicable for profiling products obtained from in vitro batch culture fermentation models. The methods incorporate enzymatic deconjugation, liquid–liquid extraction, derivatization, and subsequent analysis by GC–MS. At the level of individual compounds, the methods gave recoveries better than 80% with inter-day precision being better than 20%, depending on the matrix. Limits of detection were below 0.1 μg/ml for most phenolic acids. The newly developed methods were successfully applied to samples from human and in-vitro intervention trials, studying the metabolic impact of flavonoid intake. In conclusion, the methods presented are robust and generally applicable to diverse biological fluids. Its profiling character is useful to investigate on a large scale the gut microbiome-mediated bioavailability of flavonoids.
Keywords: Polyphenols; Flavonoids; Phenolic acids; Nutrition; Metabolism; Metabolomics; GC–MS; Profiling; Gut fermentation; Urine; Plasma; Feces;

Phenotype differentiation of three E. coli strains by GC-FID and GC–MS based metabolomics by Jing Tian; Chunyun Shi; Peng Gao; Kailong Yuan; Dawei Yang; Xin Lu; Guowang Xu (220-226).
Two mutants of E. coli with deletion of sdhAB and ackA-pta genes respectively and their wild-type strains were subjected to gas chromatography-flame-ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC–MS) metabolomics analysis. Intracellular metabolites of the three strains were profiled by GC-FID firstly. Methodological evaluation of the employed platform indicated that the limit of detection ranges were from 0.2 to 12.5 ng for some representative metabolites and the corresponding recoveries were varied from 68.7 to 122.7%. Secondly, multivariable data analysis was applied to the acquired data sets. As expected, the three phenotypes could be easily differentiated, and the perturbed metabolite pools in the genetically modified strains were screened. Lastly, the metabolites playing key roles in the differentiation were further identified by GC–MS. It was confirmed that succinic acid and aspartic acid were similarly affected in the modified strains. But proline content was altered contrarily. Additionally, deletion of sdhAB gene also affected the growth property of relevant mutant greatly. The potential mechanism was postulated accordingly.
Keywords: Metabolomics; E. coli; Phenotype; GC-FID; GC–MS;

Instrumental and experimental effects in LC–MS-based metabolomics by Lyle Burton; Gordana Ivosev; Stephen Tate; Gary Impey; Julie Wingate; Ron Bonner (227-235).
The experimental complexity of a metabolomics study can cause uncontrolled variance that is not related to the biological effect being studied and may distort or obscure the data analysis. While some sources can be controlled with good experimental techniques and careful sample handling, others are inherent in the analytical technique used and cannot easily be avoided. We discuss the sources and appearance of some of these artifacts and show ways in which they can be detected using visualization and statistical tools, allowing appropriate treatment prior to multivariate analysis (MVA).
Keywords: Metabolomics; LC–MS; Multivariate analysis;

Analytical strategies for LC–MS-based targeted metabolomics by Wenyun Lu; Bryson D. Bennett; Joshua D. Rabinowitz (236-242).
Recent advances in mass spectrometry are enabling improved analysis of endogenous metabolites. Here we discuss several issues relevant to developing liquid chromatography–electrospray ionization-mass spectrometry methods for targeted metabolomics (i.e., quantitative analysis of dozens to hundreds of specific metabolites). Sample preparation and liquid chromatography approaches are discussed, with an eye towards the challenge of dealing with a diversity of metabolite classes in parallel. Evidence is presented that heated electrospray ionization (ESI) generally gives improved signal compared to the more traditional unheated ESI. Applicability to targeted metabolomics of triple quadrupole mass spectrometry operating in multiple reaction monitoring (MRM) mode and high mass resolution full scan mass spectrometry (e.g., time-of-flight, Orbitrap) are described. We suggest that both are viable solutions, with MRM preferred when targeting a more limited number of analytes, and full scan preferred for its potential ability to bridge targeted and untargeted metabolomics.
Keywords: Targeted metabolomics; Liquid chromatography; Mass spectrometry; Hydrophilic interaction chromatography (HILIC); Ion-pairing chromatography; Heated electrospray ionization; Multiple reaction monitoring (MRM); Triple quadrupole; Full scan; Time-of-flight;

Application of the accurate mass and time tag approach in studies of the human blood lipidome by Jie Ding; Christina M. Sorensen; Navdeep Jaitly; Hongliang Jiang; Daniel J. Orton; Matthew E. Monroe; Ronald J. Moore; Richard D. Smith; Thomas O. Metz (243-252).
We report a preliminary demonstration of the accurate mass and time (AMT) tag approach for lipidomics. Initial data-dependent LC–MS/MS analyses of human plasma, erythrocyte, and lymphocyte lipids were performed in order to identify lipid molecular species in conjunction with complementary accurate mass and isotopic distribution information. Identified lipids were used to populate initial lipid AMT tag databases containing 250 and 45 entries for those species detected in positive and negative electrospray ionization (ESI) modes, respectively. The positive ESI database was then utilized to identify human plasma, erythrocyte, and lymphocyte lipids in high-throughput LC–MS analyses based on the AMT tag approach. We were able to define the lipid profiles of human plasma, erythrocytes, and lymphocytes based on qualitative and quantitative differences in lipid abundance.
Keywords: Lipidomics; AMT tag approach; Capillary liquid chromatography; Mass spectrometry;

Global metabolic profiling analysis on human urine by UPLC–TOFMS: Issues and method validation in nutritional metabolomics by Philippe A. Guy; Isabelle Tavazzi; Stephen J. Bruce; Ziad Ramadan; Sunil Kochhar (253-260).
Optimisation and method validation was assessed here for metabolic profiling analysis of urine samples using UPLC–TOFMS. A longer run time of 31 min revealed greater reproducibility, and the higher number of variables was identified as compared to shortened run times (10 and 26 min). We have also implemented two QC urine samples enabling the assessment of the quality and reproducibility of the data generated during the whole analytical workflow (retention time drift, mass precision and fluctuation of the ion responses over time). Based on the QC data, suitable standards for ensuring consistent analytical results for metabolomics applications using the UPLC–MS techniques are recommended.
Keywords: Metabolomics; Metabonomics; UPLC–TOFMS; Urine; Method validation;

UPLC–TOF-MS for plant metabolomics: A sequential approach for wound marker analysis in Arabidopsis thaliana by Elia Grata; Julien Boccard; Davy Guillarme; Gaetan Glauser; Pierre-Alain Carrupt; Edward E. Farmer; Jean-Luc Wolfender; Serge Rudaz (261-270).
The model plant Arabidopsis thaliana was studied for the search of new metabolites involved in wound signalling. Diverse LC approaches were considered in terms of efficiency and analysis time and a 7-min gradient on a UPLC–TOF-MS system with a short column was chosen for metabolite fingerprinting. This screening step was designed to allow the comparison of a high number of samples over a wide range of time points after stress induction in positive and negative ionisation modes. Thanks to data treatment, clear discrimination was obtained, providing lists of potential stress-induced ions. In a second step, the fingerprinting conditions were transferred to longer column, providing a higher peak capacity able to demonstrate the presence of isomers among the highlighted compounds.
Keywords: UPLC–TOF-MS; Arabidopsis thaliana; Metabolomics; Signalling; Oxylipins;

Automated SPE-RP-HPLC fractionation of biofluids combined to off-line NMR spectroscopy for biomarker identification in metabonomics by Serge Rezzi; Francia Arce Vera; François-Pierre J. Martin; Serena Wang; Dennis Lawler; Sunil Kochhar (271-278).
NMR-based metabonomics is a valuable and straightforward approach to measuring hundreds of metabolites in complex biofluids. However, metabolite identification is sometimes limited by overlapped signals in NMR spectra. We describe a new methodology using an automated hyphenation of solid phase extraction (SPE) with RP-HPLC combined to NMR spectroscopy, which allowed identification of 72 metabolites of various molecular classes in human urine. This methodology was also successfully applied to the fractionation of a cat urine sample to aid identification of aromatic compounds and felinine. The SPE-RP-HPLC method appears to be a reliable tool to support biomarker discovery in metabonomic studies.
Keywords: Solid phase extraction; High-pressure liquid chromatography; Metabonomics; Metabolomics; Nuclear magnetic resonance spectroscopy; Cat urine;

High temperature-ultra performance liquid chromatography–mass spectrometry for the metabonomic analysis of Zucker rat urine by Helen G. Gika; Georgios Theodoridis; Jon Extance; Anthony M. Edge; Ian D. Wilson (279-287).
The applicability and potential of using elevated temperatures and sub 2-μm porous particles in chromatography for metabonomics/metabolomics was investigated using, for the first time, solvent temperatures higher than the boiling point of water (up to 180 °C) and thermal gradients to reduce the use of organic solvents. Ultra performance liquid chromatography, combined with mass spectrometry, was investigated for the global metabolite profiling of the plasma and urine of normal and Zucker (fa/fa) obese rats (a well established disease animal model). “Isobaric” high temperature chromatography, where the temperature and flow rate follow a gradient program, was developed and evaluated against a conventional organic solvent gradient. LC–MS data were first examined by established chromatographic criteria in order to evaluate the chromatographic performance and next were treated by special peak picking algorithms to allow the application of multivariate statistics. These studies showed that, for urine (but not plasma), chromatography at elevated temperatures provided better results than conventional reversed-phase LC with higher peak capacity and better peak asymmetry. From a systems biology point of view, better group clustering and separation was obtained with a larger number of variables of high importance when using high temperature-ultra performance liquid chromatography (HT-UPLC) compared to conventional solvent gradients.
Keywords: High temperature liquid chromatography; Thermal gradient; Metabonomics; Metabolomics; UPLC; Metabolite profiling; Zucker rat;

Metabolic profiling of serum using Ultra Performance Liquid Chromatography and the LTQ-Orbitrap mass spectrometry system by Warwick B. Dunn; David Broadhurst; Marie Brown; Philip N. Baker; Christopher W.G. Redman; Louise C. Kenny; Douglas B. Kell (288-298).
Advances in analytical instrumentation can provide significant advantages to the volume and quality of biological knowledge acquired in metabolomic investigations. The interfacing of sub-2 μm liquid chromatography (UPLC ACQUITY®) and LTQ-Orbitrap mass spectrometry systems provides many theoretical advantages. The applicability of the interfaced systems was investigated using a simple 11-component metabolite mix and a complex mammalian biofluid, serum. Metabolites were detected in the metabolite mix with signals that were linear with their concentration over 2.5–3.5 orders of magnitude, with correlation coefficients greater than 0.993 and limits of detection less than 1 μmol L−1. Reproducibility of retention time (RSD < 3%) and chromatographic peak area (RSD < 15%) and a high mass accuracy (<2 ppm) were observed for 14 QC serum samples interdispersed with other serum samples, analysed over a period of 40 h. The evaluation of a single deconvolution software package (XCMS) was performed and showed that two parameters (snthresh and bw) provided significant changes to the number of peaks detected and the peak area reproducibility for the dataset used. The data were used to indicate possible biomarkers of pre-eclampsia and showed both the instruments and XCMS to be applicable to the reproducible and valid detection of disease biomarkers present in serum.
Keywords: UPLC; Orbitrap; XCMS; Metabolic profiling; UPLC–MS; Pre-eclampsia;

The application of reversed-phase ultra-performance liquid chromatography, based on the use of sub 2 μm particles, combined with time-of-flight mass spectrometry has been investigated for the production of global metabolite profiles from human urine. The stability and repeatability of the methodology, which employed gradient elution, was determined by the repeat analysis of a pooled quality control (QC) sample. As seen in previous studies conducted with conventional LC–MS an element of system conditioning was required to obtain reproducible data, as the initial injections were unrepresentative. However, once the system had equilibrated excellent repeatability in terms of retention time, signal intensity and mass accuracy was seen providing confidence that for this matrix, the within-day repeatability of UPLC–TOF-MS was sufficient to assure data quality in global metabolic profiling applications.
Keywords: Metabolite profiling; Ultra-performance liquid chromatography; Time-of-flight mass spectrometry; Metabonomics; Metabolomics;

Quantitative profiling of bile acids in biofluids and tissues based on accurate mass high resolution LC-FT-MS: Compound class targeting in a metabolomics workflow by Ivana Bobeldijk; Maarten Hekman; Jitske de Vries-van der Weij; Leon Coulier; Raymond Ramaker; Robert Kleemann; Teake Kooistra; Carina Rubingh; Andreas Freidig; Elwin Verheij (306-313).
We report a sensitive, generic method for quantitative profiling of bile acids and other endogenous metabolites in small quantities of various biological fluids and tissues. The method is based on a straightforward sample preparation, separation by reversed-phase high performance liquid-chromatography mass spectrometry (HPLC-MS) and electrospray ionisation in the negative ionisation mode (ESI−). Detection is performed in full scan using the linear ion trap Fourier transform mass spectrometer (LTQ-FTMS) generating data for many (endogenous) metabolites, not only bile acids. A validation of the method in urine, plasma and liver was performed for 17 bile acids including their taurine, sulfate and glycine conjugates. The method is linear in the 0.01–1 μM range. The accuracy in human plasma ranges from 74 to 113%, in human urine 77 to 104% and in mouse liver 79 to 140%. The precision ranges from 2 to 20% for pooled samples even in studies with large number of samples (n  > 250). The method was successfully applied to a multi-compartmental APOE*3-Leiden mouse study, the main goal of which was to analyze the effect of increasing dietary cholesterol concentrations on hepatic cholesterol homeostasis and bile acid synthesis. Serum and liver samples from different treatment groups were profiled with the new method. Statistically significant differences between the diet groups were observed regarding total as well as individual bile acid concentrations.
Keywords: Metabolomics; Targeted metabolite profiling; Accurate mass; High performance liquid chromatography; Bile acids; Body fluids; Tissues;

Reproducible and comprehensive sample extraction and detection of metabolites with a broad range of physico-chemical properties from biological matrices can be a highly challenging process. A single LC/MS separation method was developed for a 2.1 mm × 100 mm, 1.8 μm ZORBAX SB-Aq column that was used to separate human erythrocyte metabolites extracted under sample extraction solvent conditions where the pH was neutral or had been adjusted to either, pH 2, 6 or 9. Internal standards were included and evaluated for tracking sample extraction efficiency. Through the combination of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) techniques in both positive (+) and negative (−) ion modes, a total of 2370 features (compounds and associated compound related components: isotopes, adducts and dimers) were detected across all pHs. Broader coverage of the detected metabolome was achieved by observing that (1) performing extractions at pH 2 and 9, leads to a combined 92% increase in detected features over pH 7 alone; and (2) including APCI in the analysis results in a 34% increase in detected features, across all pHs, than the total number detected by ESI. A significant dependency of extraction solvent pH on the recovery of heme and other compounds was observed in erythrocytes and underscores the need for a comprehensive sample extraction strategy and LC/MS analysis in metabolomics profiling experiments.
Keywords: Metabolomics; Metabolome; Extraction; LC/MS; Ion source; ESI; APCI; Erythrocytes; pH;

Serum metabolic profiling of abnormal savda by liquid chromatography/mass spectrometry by Peiyuan Yin; Patamu Mohemaiti; Jing Chen; Xinjie Zhao; Xin Lu; Adilijiang Yimiti; Halmurat Upur; Guowang Xu (322-327).
Abnormal savda is a special symptom in Uigur medicine. The understanding of its metabolic origins is of great importance for the subsequent treatment. Here, a metabonomic study of this symptom was carried out using LC–MS based human serum metabolic profiling. Orthogonal signal correction partial least-squares discriminant analysis (OSC-PLS-DA) was used for the classification and prediction of abnormal savda. Potential biomarkers from metabonomics were also identified for a metabolic understanding of abnormal savda. As a result, our OSC-PLS-DA model had a satisfactory ability for separation and prediction of abnormal savda. The potential biomarkers including bilirubin, bile acids, tryptophan, phenylalanine and lyso-phosphatidylcholines indicated that abnormal savda could be related to some abnormal metabolisms within the body, including energy metabolism, absorption of nutrition, metabolism of lecithin on cell membrane, etc. To the best of our knowledge, this is the first study of abnormal savda based on serum metabolic profiling. The LC/MS-based metabonomic platform could be a powerful tool for the classification of symptoms and for the development of this traditional medicine into an evidence-based one.
Keywords: Metabonomics; Metabolic profiling; Abnormal savda; Uigur medicine;

Metabonomics evaluation of urine from rats given acute and chronic doses of acetaminophen using NMR and UPLC/MS by Jinchun Sun; Laura K. Schnackenberg; Ricky D. Holland; Thomas C. Schmitt; Glenn H. Cantor; Yvonne P. Dragan; Richard D. Beger (328-340).
Urinary metabolic perturbations associated with acute and chronic acetaminophen-induced hepatotoxicity were investigated using nuclear magnetic resonance (NMR) spectroscopy and ultra performance liquid chromatography/mass spectrometry (UPLC/MS) metabonomics approaches to determine biomarkers of hepatotoxicity. Acute and chronic doses of acetaminophen (APAP) were administered to male Sprague-Dawley rats. NMR and UPLC/MS were able to detect both drug metabolites and endogenous metabolites simultaneously. The principal component analysis (PCA) of NMR or UPLC/MS spectra showed that metabolic changes observed in both acute and chronic dosing of acetaminophen were similar. Histopathology and clinical chemistry studies were performed and correlated well with the PCA analysis and magnitude of metabolite changes. Depletion of antioxidants (e.g. ferulic acid), trigonelline, S-adenosyl-l-methionine, and energy-related metabolites indicated that oxidative stress was caused by acute and chronic acetaminophen administration. Similar patterns of metabolic changes in response to acute or chronic dosing suggest similar detoxification and recovery mechanisms following APAP administration.
Keywords: Acetaminophen; Metabonomics; Metabolomics;

An approach towards method development for untargeted urinary metabolite profiling in metabonomic research using UPLC/QToF MS by Max C.Y. Wong; Warren T.K. Lee; Jayme S.Y. Wong; Gary Frost; John Lodge (341-348).
The application of LC–MS for untargeted urinary metabolite profiling in metabonomic research has gained much interest in recent years. However, the effects of varying sample pre-treatments and LC conditions on generic metabolite profiling have not been studied. We aimed to evaluate the effects of varying experimental conditions on data acquisition in untargeted urinary metabolite profiling using UPLC/QToF MS. In-house QC sample clustering was used to monitor the performance of the analytical platform. In terms of sample pre-treatment, results showed that untreated filtered urine yielded the highest number of features but dilution with methanol provided a more homogenous urinary metabolic profile with less variation in number of features and feature intensities. An increased cycle time with a lower flow rate (400 μl/min vs 600 μl/min) also resulted in a higher number of features with less variability. The step elution gradient yielded the highest number of features and the best chromatographic resolution among three different elution gradients tested. The maximum retention time and mass shift were only 0.03 min and 0.0015 Da respectively over 600 injections. The analytical platform also showed excellent robustness as evident by tight QC sample clustering. To conclude, we have investigated LC conditions by studying variability and repeatability of LC–MS data for untargeted urinary metabolite profiling.
Keywords: Urine; Metabonomics; UPLC/QToF MS; Untargeted profiling; Quality control;

Analysis of human urine for specific compounds or metabolites is an established method for biomonitoring occupational or environmental exposures. Modern liquid chromatography–tandem mass spectrometry is not limited to single compounds but can simultaneously analyze whole classes of urine constituents with both high sensitivity and specificity. Individual differences in the composition of urine are very large in humans, which raises a number of problems that are not encountered in animal experimentation. In this report, we investigated whether analysis of glucuronides as a class could reflect differences between human individuals regarding the polymorphic activity of the cytochrome P450 enzyme CYP2D6. From a group of 152 students that had been classified for CYP2D6 activity, urine of 12 “poor metabolizers” and 35 “extensive metabolizers” was collected 90 min after ingestion of 10 mg of the antitussive drug dextromethorphan (DEX) and analyzed for glucuronides. Methods development included the following aspects: adjustment of urine samples to equal creatinine concentration to avoid differences between samples in retention times and ion suppression; on-line enrichment of low-level analytes by column switching; precursor ion scan vs. theoretical multiple reaction monitoring; use of quality control samples to check for reproducibility in large sample series; peak extraction and handling of null entries to build the data matrix; logarithmic data transformation and different scaling procedures; principal component analysis (PCA) vs. discriminant analysis. Our results show that an optimized procedure not only identified the known DEX metabolites as predictors of CYP2D6-specific metabolic pathways but also indicated the presence of additional, so far unknown path-specific glucuronide metabolites. We conclude that metabolite profiling of urine and other biofluids by modern mass spectrometric methodology may help characterize individual differences and become useful in drug development and personalized pharmacotherapy.
Keywords: Mass spectrometry; Liquid chromatography; Multivariate analysis; Drug metabolism; Metabolite profiling; Metabolomics; CYP2D6; Polymorphism; Humans; Urine; Phenotyping; Dextromethorphan;

An approach to enhancing coverage of the urinary metabonome using liquid chromatography–ion mobility–mass spectrometry by Emma L. Harry; Daniel J. Weston; Anthony W.T. Bristow; Ian D. Wilson; Colin S. Creaser (357-361).
The potential of drift tube ion mobility (IM) spectrometry in combination with high performance liquid chromatography (LC) and mass spectrometry (MS) for the metabonomic analysis of rat urine is reported. The combined LC–IM–MS approach using quadrupole/time-of-flight mass spectrometry with electrospray ionisation, uses gas-phase analyte characterisation based on both mass-to-charge (m/z) ratio and relative gas-phase mobility (drift time) following LC separation. The technique allowed the acquisition of nested data sets, with mass spectra acquired at regular intervals (65 μs) during each IMS separation (∼13 ms) and several IMS spectra acquired during the elution of a single LC peak, without increasing the overall analysis time compared to LC–MS. Preliminary results indicate that spectral quality is improved when using LC–IM–MS, compared to direct injection IM–MS, for which significant ion suppression effects were observed in the electrospray ion source. The use of reversed-phase LC employing fast gradient elution reduced sample preparation to a minimum, whilst maintaining the potential for high throughput analysis. Data mining allowed information on specific analytes to be extracted from the complex metabonomic data set. LC–IM–MS based approaches may have a useful role in metabonomic analyses by introducing an additional discriminatory dimension of ion mobility (drift time).
Keywords: Ion mobility–mass spectrometry; Reversed-phase HPLC; Metabonomics; Urinary metabolome;

Tannin-enriched extracts from raspberry, cloudberry and strawberry were analysed by liquid chromatography–mass spectrometric (LC–MS) techniques. The raspberry and cloudberry extracts contained a similar mixture of identifiable ellagitannin components and ellagic acid. However, the strawberry extract contained a complex mixture of ellagitannin and proanthocyanidin components that could not be adequately resolved to allow identification of individual peaks. Nevertheless, the negative ESI-MS spectra obtained by direct infusion mass spectrometric (DIMS) analysis described the diversity of these samples. For example, the predominance of signals associated with Lambertianin C in cloudberry and Sanguiin H6 in raspberry tannin extracts could be discerned and the diversity of signals from procyanidin and propelargonidin oligomers could be identified in the strawberry extract. The dose response for the main ellagitannin-derived signals in the raspberry tannin sample revealed a saturation effect probably due to ion suppression effects in the ion trap spectrometer. Nevertheless, DIMS spectra of whole berry extracts described qualitative differences in ellagitannin-derived peaks in raspberry, cloudberry and strawberry samples. In addition, positive mode DIMS spectra illustrated qualitative differences in the anthocyanin composition of berries of progeny from a raspberry breeding population that had been previously analysed by LC–MS. This suggests that DIMS could be applied to rapidly assess differences in polyphenol content, especially in large sample sets such as the progeny from breeding programmes.
Keywords: Anthocyanins; Berries; Breeding; Direct infusion mass spectrometry (DIMS); Ellagitannins; LC–MS; Polyphenols;

Metabolic analysis of body fluids by capillary electrophoresis using noncovalently coated capillaries by Rawi Ramautar; Oleg A. Mayboroda; André M. Deelder; Govert W. Somsen; Gerhardus J. de Jong (370-374).
The potential of capillaries noncovalently coated with charged polymers for the metabolic analysis of body fluids by CE is illustrated. Firstly, the usefulness of a coating consisting of a triple layer of polybrene–dextran sulfate–polybrene for the fast analysis of organic acids is described. The CE system allowed direct injections of CSF, plasma and urine samples, yielding good separation efficiencies. RSDs for migration times and peak areas of organic acids in plasma were <3% and <5%, respectively. The usefulness of the system is illustrated by the profiling of organic acids in plasma and urine samples. Secondly, a CE system comprising a bilayer coating of polybrene–poly(vinylsulfonate), which provides a considerable EOF at low pH is described. This system was combined with TOF-MS and used for the fast analysis of amino acids in cerebrospinal fluid (CSF) and urine with minimal sample pretreatment. RSDs for migration times and peak areas of amino acids in CSF and urine were <2% and <10%, respectively. The applicability of the system is demonstrated by the profiling of endogenous low-molecular weight metabolites in CSF from a healthy individual and a patient with complex regional pain syndrome.
Keywords: Metabolic analysis; Capillary electrophoresis; Noncovalently coated capillaries; Plasma; CSF; Urine;

The aim of this study was to develop a method for comprehensive profiling of metabolites involved in mammalian steroid metabolism. The study was performed using the partial filling micellar electrokinetic chromatography (PF-MEKC) technique for determination of endogenous low-hydrophilic steroids. The detection techniques in capillary electrophoresis were UV absorption and electrospray mass spectrometry (ESI-MS). Thirteen steroids were included in the method development, and the selected were metabolites involved in major pathways of steroid biosynthesis. Although only eight of them could be separated and detected with UV, they could be identified by ESI-MS using selected ion monitoring (SIM) technique. Tandem MS spectra were also collected. UV detection was more sensitive than MS due to better separation of compounds and the selective signal sensitivity. The lowest limits of detection were 10–100 ng/mL for cortisone, corticosterone, hydrocortisone and testosterone. The other steroids could be detected at 500–1000 ng/mL. The identification of cortisone, corticosterone, hydrocortisone, estrogen and testosterone were made in patient urine samples and their concentrations were 1–40 μg/L.
Keywords: Steroids; Partial filling; Micellar electrokinetic capillary electrophoresis; Mass spectrometry; Urine;