N E U R OM E T H O D S

Series EditorWolfgang Walz

University of SaskatchewanSaskatoon, SK, Canada

For further volumes:http://www.springer.com/series/7657

Neuromethods publishes cutting-edge methods and protocols in all areas of neuroscience aswell as translational neurological and mental research. Each volume in the series offers testedlaboratory protocols, step-by-step methods for reproducible lab experiments and addressesmethodological controversies and pitfalls in order to aid neuroscientists in experimentation.Neuromethods focuses on traditional and emerging topics with wide-ranging implications tobrain function, such as electrophysiology, neuroimaging, behavioral analysis, genomics,neurodegeneration, translational research and clinical trials. Neuromethods provides investi-gators and trainees with highly useful compendiums of key strategies and approaches forsuccessful research in animal and human brain function including translational “bench tobedside” approaches to mental and neurological diseases.

Metabolomics

Edited by

Paul L. Wood

Metabolomics Unit, College of Veterinary Medicine, Lincoln Memorial University,Harrogate, TN, USA

EditorPaul L. WoodMetabolomics UnitCollege of Veterinary MedicineLincoln Memorial UniversityHarrogate, TN, USA

ISSN 0893-2336 ISSN 1940-6045 (electronic)NeuromethodsISBN 978-1-0716-0863-0 ISBN 978-1-0716-0864-7 (eBook)https://doi.org/10.1007/978-1-0716-0864-7

© Springer Science+Business Media, LLC, part of Springer Nature 2021This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material isconcerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproductionon microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,computer software, or by similar or dissimilar methodology now known or hereafter developed.The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulationsand therefore free for general use.The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed tobe true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,expressed or implied, with respect to the material contained herein or for any errors or omissions that may have beenmade. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of SpringerNature.The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface to the Series

Experimental life sciences have two basic foundations: concepts and tools. The Neuro-methods series focuses on the tools and techniques unique to the investigation of the nervoussystem and excitable cells. It will not, however, shortchange the concept side of things ascare has been taken to integrate these tools within the context of the concepts and questionsunder investigation. In this way, the series is unique in that it not only collects protocols butalso includes theoretical background information and critiques which led to the methodsand their development. Thus it gives the reader a better understanding of the origin of thetechniques and their potential future development. The Neuromethods publishing programstrikes a balance between recent and exciting developments like those concerning newanimal models of disease, imaging, in vivo methods, and more established techniques,including, for example, immunocytochemistry and electrophysiological technologies. Newtrainees in neurosciences still need a sound footing in these older methods in order to applya critical approach to their results.

Under the guidance of its founders, Alan Boulton and Glen Baker, the Neuromethodsseries has been a success since its first volume published throughHumana Press in 1985. Theseries continues to flourish through many changes over the years. It is now published underthe umbrella of Springer Protocols. While methods involving brain research have changed alot since the series started, the publishing environment and technology have changed evenmore radically. Neuromethods has the distinct layout and style of the Springer Protocolsprogram, designed specifically for readability and ease of reference in a laboratory setting.

The careful application of methods is potentially the most important step in the processof scientific inquiry. In the past, new methodologies led the way in developing new dis-ciplines in the biological and medical sciences. For example, Physiology emerged out ofAnatomy in the nineteenth century by harnessing new methods based on the newly discov-ered phenomenon of electricity. Nowadays, the relationships between disciplines and meth-ods are more complex. Methods are now widely shared between disciplines and researchareas. New developments in electronic publishing make it possible for scientists thatencounter new methods to quickly find sources of information electronically. The designof individual volumes and chapters in this series takes this new access technology intoaccount. Springer Protocols makes it possible to download single protocols separately. Inaddition, Springer makes its print-on-demand technology available globally. A print copycan therefore be acquired quickly and for a competitive price anywhere in the world.

Saskatoon, SK, Canada Wolfgang Walz

v

Preface

Metabolomics was initially envisioned as an “omics” platform that would provide a snapshotof the entire metabolome. This vision has not been achieved since there is no single analyticalplatform that can monitor the wide diversity of chemical structures that compose themetabolome. As a result, metabolomics has evolved into a number of subfields that includelipidomics (see Volume 125 in this series), small molecule metabolomics, oxidative meta-bolomics, fluxomics, metallomics, and mass spectrometric imaging of small molecularweight metabolites and lipids.

In this volume, we will address the current status of analytical approaches utilized tosample defined molecular populations of metabolites via functional group derivatization,specialized chromatographic methods, and ionization techniques. All of these approachesrequire detailed experimental design to utilize the optimal methodology to address specificscientific questions. This is essential since many metabolites yield incredibly variable obser-vations as a result of their rapid ongoing dynamics. Examples include rapid postmortemdegradation (e.g., acetylcholine levels) and postmortem accumulation (e.g., GABA). There-fore, designing analytical platforms to monitor stable end products that are destined forexcretion (e.g., dipeptides as biomarkers of protein metabolism and uric acid as an endproduct of purine metabolism), stable oxidized metabolites (e.g., isoprostanes), non-rate-limiting precursor pools, non-reutilized modified nucleosides as cancer biomarkers (e.g.,7-methylguanine), metabolites indicative of abnormal intermediary metabolism (e.g., keto-glutaramic acid as a biomarker of hyperammonemia), biomarkers of immune activation(e.g., kyneurenine and cyclic phosphatidic acid 16:0), biomarkers of ECM collagen degra-dation (e.g., deoxypyridinoline), inflammatory biomarkers of nitrosative damage (e.g.,8-nitroguanine), biomarkers of polyamine metabolism and diamine transporter function(e.g., putrescine), sulfur amino acid status in redox reactions (e.g., cysteine), biomarkers ofuremia (e.g., guanidino acids), biomarkers of abnormal peroxisomal activity (e.g., dicarbox-ylic acid 16:0 and plasmalogens), and biomarkers of gut microbial metabolism (e.g., p-cresolsulfate).

To monitor this diversity of complex metabolites, a number of analytical approaches arerequired. Mass spectrometry is the most sensitive and specific methodology for metaboliteidentification and quantitation. In this volume, we review the key methods for sampleintroduction to the ion source, including direct flow, gas chromatography, liquid chroma-tography, and capillary electrophoresis. In addition, derivatization methods required forthese sample introduction techniques are presented in detail, including complex enantiose-lective assays. Both nontargeted and targeted analyses are included in our volume as well asthe emerging field of metallomics.

Finally, it is important to note that reporting metabolite levels from metabolomicsstudies is always reflective of relative and not absolute levels. Absolute levels cannot bereported until an isotopic variant of each metabolite of interest has been included in the assayto correct for extraction efficiency, ionization potential, and in the case of derivatization,reaction completion. With regard to stable isotope internal standards, it should be stressedthat 13C and/or 15N are the optimal isotopes since deuterated compounds have differentsolubility properties to endogenous metabolites (i.e., altered hydrogen bonding activity).

vii

Many investigators report their metabolomics data as absolute levels utilizing a singleinternal standard or as a mol%. These data are deceptive in that they are not corrected forthe different ionization capacities between metabolites. Development of absolute quantita-tion assays for critical metabolites is essential in translational research for the advancement ofbiomarkers to improve the delivery of healthcare in clinical and veterinary medicine. Thesewill include antecedent, screening, diagnostic, and prognostic assays as well as improvedanalytical approaches to improve the clinical stratification of patient subpopulations.

In summary, our Metabolomics volume is of great value to students, researchers,practicing physicians and veterinarians, as well as administrators involved in the funding ofresearch.

Harrogate, TN, USA Paul L. Wood

viii Preface

Contents

Preface to the Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vPreface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viiContributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

1 Flow Infusion ESI High Resolution Mass Spectrometry MetabolomicsAnalytical Platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Paul L. Wood

2 High-Throughput Metabolomics Using Flow InjectionAnalysis and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. . 9Estelle Rathahao-Paris, Sandra Alves, and Alain Paris

3 Two-Dimensional Liquid Chromatography in Metabolomicsand Lipidomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Miriam Perez-Cova, Roma Tauler, and Joaquim Jaumot

4 Metabolomics of Small Numbers of Cells Using Chemical IsotopeLabeling Combined with Nanoflow LC-MS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49Xian Luo and Liang Li

5 Untargeted Metabolomics to Interpret the Effects of Social Defeat:Integrating Chemistry and Behavioral Approaches in Neuroscience . . . . . . . . . . . 61Allen K. Bourdon and Brooke N. Dulka

6 Flow Infusion Electrospray Ionization High-ResolutionMass Spectrometry of the Chloride Adducts of Sphingolipids,Glycerophospholipids, Glycerols, Hydroxy Fatty Acids, Fatty AcidEsters of Hydroxy Fatty Acids (FAHFA), and Modified Nucleosides . . . . . . . . . . 69Paul L. Wood and Randall L. Woltjer

7 High Accurate Mass Gas Chromatography–Mass Spectrometryfor Performing Isotopic Ratio Outlier Analysis: Applicationsfor Nonannotated Metabolite Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77Yunping Qiu and Irwin J. Kurland

8 Enantioseparation and Detection of (R)-2-Hydroxyglutarateand (S)-2-Hydroxyglutarate by Chiral Gas Chromatography–TripleQuadrupole Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89Shinji K. Strain, Morris D. Groves, and Mark R. Emmett

9 Enantioselective Supercritical Fluid Chromatography (SFC)for Chiral Metabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101Robert Hofstetter, Andreas Link, and Georg M. Fassauer

10 Capillary Electrophoresis–Mass Spectrometry of HydrophilicMetabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113Masahiro Sugimoto

ix

11 Lipidomic Analysis of Oxygenated PolyunsaturatedFatty Acid–Derived Inflammatory Mediators in NeurodegenerativeDiseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121Mauricio Mastrogiovanni, Estefanıa Ifran,Andres Trostchansky, and Homero Rubbo

12 Characterization and Quantification of the Fatty Acid Amidome. . . . . . . . . . . . . . 143Kristen A. Jeffries, Emma K. Farrell, Ryan L. Anderson,Gabriela Suarez, Amanda J. Goyette Osborne, Mc Kenzi K. Heide,and David J. Merkler

13 Metabolomics Analysis of Complex Biological SpecimensUsing Nuclear Magnetic Resonance Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . 155Khushboo Gulati, Sharanya Sarkar, and Krishna Mohan Poluri

14 Untargeted Metabolomics Methods to Analyze Blood-Derived Samples . . . . . . . 173Danuta Dudzik and Antonia Garcıa

15 Multicompartmental High-Throughput Metabolomics Basedon Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189Raul Gonzalez-Domınguez, Alvaro Gonzalez-Domınguez,Ana Sayago, and Angeles Fernandez-Recamales

16 GC-MS Nontargeted Metabolomics of Neural Tissue . . . . . . . . . . . . . . . . . . . . . . . 199Carolina Gonzalez-Riano, Mª. Fernanda Rey-Stolle,Coral Barbas, and Antonia Garcıa

17 GC-MS of Pentafluourobenzyl Derivatives of Phenols, Amines,and Carboxylic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221Paul L. Wood

18 GC-MS of tert-Butyldimethylsilyl (tBDMS) Derivativesof Neurochemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229Paul L. Wood

19 UHPLC-MS/MS Method for Determination of BiologicallyImportant Thiols in Plasma Using New Derivatizing MaleimideReagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235Dominika Olesova, Andrej Kovac, and Jaroslav Galba

20 Untargeted Metabolomics Determination of PostmortemChanges in Brain Tissue Samples by UHPLC-ESI-QTOF-MSand GC-EI-Q-MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245Carolina Gonzalez-Riano, Antonia Garcıa, and Coral Barbas

21 Metallomics Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267Valderi Luiz Dressler, Graciela Marini Hiedrich,Vinicius Machado Neves, Eson Irineu Muller, and Dirce Pozebon

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

x Contents

Contributors

SANDRA ALVES • Sorbonne Universite, Faculte des Sciences et de l’Ingenierie, Institut Parisiende Chimie Moleculaire (IPCM), Paris, France

RYAN L. ANDERSON • Department of Chemistry, University of South Florida, Tampa, FL,USA; Department of Biochemistry and Molecular Genetics, University of Colorado—Denver Anschutz Medical Campus, Aurora, CO, USA

CORAL BARBAS • Center for Metabolomics and Bioanalysis (CEMBIO), Facultad deFarmacia, Universidad San Pablo CEU. CEU Universities, Madrid, Spain; Centre forMetabolomics and Bioanalysis (CEMBIO), University San Pablo CEU, Madrid, Spain

ALLEN K. BOURDON • Stepan Company, Northfield, IL, USAVALDERI LUIZ DRESSLER • Department of Chemistry, Federal University of Santa Maria,

Santa Maria, RS, BrazilDANUTA DUDZIK • Department of Biopharmaceutics and Pharmacodynamics, Faculty of

Pharmacy, Medical University of Gdansk, Gdansk, PolandBROOKE N. DULKA • University of Wisconsin-Milwaukee, Milwaukee, WI, USAMARK R. EMMETT • Department of Biochemistry and Molecular Biology, University of Texas

Medical Branch, Galveston, TX, USAEMMA K. FARRELL • Department of Chemistry, University of South Florida, Tampa, FL,

USA; The McCrone Group, Westmont, IL, USAGEORG M. FASSAUER • Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy,

University Greifswald—Germany, Greifswald, GermanyANGELES FERNANDEZ-RECAMALES • Department of Chemistry, Faculty of Experimental

Sciences, University of Huelva, Huelva, Spain; International Campus of Excellence CeiA3,University of Huelva, Huelva, Spain

JAROSLAV GALBA • Institute of Neuroimmunology, Slovak Academy of Sciences, Bratislava,Slovak Republic

ANTONIA GARCIA • Centre for Metabolomics and Bioanalysis (CEMBIO), Department ofChemistry and Biochemistry, Facultad de Farmacia, Universidad San Pablo-CEU, CEUUniversities, Madrid, Spain; Center for Metabolomics and Bioanalysis (CEMBIO),Facultad de Farmacia, Universidad San Pablo CEU. CEU Universities, Madrid, Spain;Centre for Metabolomics and Bioanalysis (CEMBIO), University San Pablo CEU, Madrid,Spain

ALVARO GONZALEZ-DOMINGUEZ • Department of Pediatrics, Hospital Universitario Puertadel Mar., Cadiz, Spain; Institute of Research and Innovation in Biomedical Sciences of theProvince of Cadiz (INiBICA), Cadiz, Spain

RAUL GONZALEZ-DOMINGUEZ • Department of Chemistry, Faculty of Experimental Sciences,University of Huelva, Huelva, Spain; International Campus of Excellence CeiA3,University of Huelva, Huelva, Spain

CAROLINA GONZALEZ-RIANO • Center for Metabolomics and Bioanalysis (CEMBIO),Facultad de Farmacia, Universidad San Pablo CEU. CEU Universities, Madrid, Spain;Centre for Metabolomics and Bioanalysis (CEMBIO), University San Pablo CEU, Madrid,Spain

AMANDA J. GOYETTE OSBORNE • Department of Chemistry, University of South Florida,Tampa, FL, USA

xi

MORRIS D. GROVES • Austin Brain Tumor Center, Texas Oncology/US Oncology Research,Austin, TX, USA

KHUSHBOO GULATI • Department of Biotechnology, Indian Institute of Technology Roorkee,Roorkee, Uttarakhand, India

MC KENZI K. HEIDE • Department of Chemistry, University of South Florida, Tampa, FL,USA

GRACIELA MARINI HIEDRICH • Department of Chemistry, Federal University of Santa Maria,Santa Maria, RS, Brazil

ROBERT HOFSTETTER • Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy,University Greifswald—Germany, Greifswald, Germany

ESTEFANIA IFRAN • Departamento de Bioquımica, Facultad de Medicina and Centro deInvestigaciones Biomedicas (CEINBIO), Universidad de la Republica, Montevideo,Uruguay

JOAQUIM JAUMOT • Department of Environmental Chemistry, IDAEA-CSIC, Barcelona,Spain

KRISTEN A. JEFFRIES • Department of Chemistry, University of South Florida, Tampa, FL,USA; Agricultural Marketing Service, USDA, Gastonia, NC, USA

ANDREJ KOVAC • Institute of Neuroimmunology, Slovak Academy of Sciences, Bratislava,Slovak Republic

IRWIN J. KURLAND • Stable Isotope and Metabolomics Core Facility, Diabetes Center,Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA

LIANG LI • Department of Chemistry, University of Alberta, Edmonton, AB, CanadaANDREAS LINK • Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy,

University Greifswald—Germany, Greifswald, GermanyXIAN LUO • Department of Chemistry, University of Alberta, Edmonton, AB, CanadaMAURICIO MASTROGIOVANNI • Departamento de Bioquımica, Facultad de Medicina and

Centro de Investigaciones Biomedicas (CEINBIO), Universidad de la Republica,Montevideo, Uruguay

DAVID J. MERKLER • Department of Chemistry, University of South Florida, Tampa, FL,USA

ESON IRINEU MULLER • Department of Chemistry, Federal University of Santa Maria, SantaMaria, RS, Brazil

VINICIUS MACHADO NEVES • Department of Chemistry, Federal University of Santa Maria,Santa Maria, RS, Brazil

DOMINIKA OLESOVA • Institute of Neuroimmunology, Slovak Academy of Sciences,Bratislava, Slovak Republic

ALAIN PARIS • Museum national d’Histoire naturelle, CEA, Unite Molecules deCommunication et Adaptation des Microorganismes (MCAM), Paris, France

MIRIAM PEREZ-COVA • Department of Environmental Chemistry, IDAEA-CSIC, Barcelona,Spain

KRISHNA MOHAN POLURI • Department of Biotechnology, Indian Institute of TechnologyRoorkee, Roorkee, Uttarakhand, India; Centre for Nanotechnology, Indian Institute ofTechnology Roorkee, Roorkee, Uttarakhand, India

DIRCE POZEBON • Department of Chemistry, Federal University of Santa Maria, SantaMaria, RS, Brazil; Chemistry Institute, Federal University of Rio Grande do Sul, PortoAlegre, RS, Brazil

YUNPING QIU • Stable Isotope and Metabolomics Core Facility, Diabetes Center, Departmentof Medicine, Albert Einstein College of Medicine, Bronx, NY, USA

xii Contributors

ESTELLE RATHAHAO-PARIS • Universite Paris Saclay, CEA, INRAE, DepartementMedicaments et Technologies pour la Sante (DMTS), SPI, Gif-sur-Yvette, France; SorbonneUniversite, Faculte des Sciences et de l’Ingenierie, Institut Parisien de Chimie Moleculaire(IPCM), Paris, France

Mª. FERNANDA REY-STOLLE • Center for Metabolomics and Bioanalysis (CEMBIO),Facultad de Farmacia, Universidad San Pablo CEU. CEU Universities, Madrid, Spain

HOMERO RUBBO • Departamento de Bioquımica, Facultad de Medicina and Centro deInvestigaciones Biomedicas (CEINBIO), Universidad de la Republica, Montevideo,Uruguay

SHARANYA SARKAR • Department of Biotechnology, Indian Institute of Technology Roorkee,Roorkee, Uttarakhand, India

ANA SAYAGO • Department of Chemistry, Faculty of Experimental Sciences, University ofHuelva, Huelva, Spain; International Campus of Excellence CeiA3, University of Huelva,Huelva, Spain

SHINJI K. STRAIN • Department of Neuroscience, Cell Biology and Anatomy, University ofTexas Medical Branch, Galveston, TX, USA

GABRIELA SUAREZ • Department of Chemistry, University of South Florida, Tampa, FL, USAMASAHIRO SUGIMOTO • Research and Development Center for Minimally Invasive Therapies

Health Promotion and Preemptive Medicine, Tokyo Medical University, Shinjuku, Tokyo,Japan; Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan

ROMA TAULER • Department of Environmental Chemistry, IDAEA-CSIC, Barcelona, SpainANDRES TROSTCHANSKY • Departamento de Bioquımica, Facultad de Medicina and Centro

de Investigaciones Biomedicas (CEINBIO), Universidad de la Republica, Montevideo,Uruguay

RANDALL L. WOLTJER • Department of Neurology, Oregon Health Science University andPortland VA Medical Center, Portland, OR, USA

PAUL L. WOOD • Metabolomics Unit, College of Veterinary Medicine, Lincoln MemorialUniversity, Harrogate, TN, USA

Contributors xiii