title: Identification of metabolites from liquid chromatography-coulometric array detection profiling: gas chromatography-mass spectrometry and refractionation provide essential information orthogonal to LC-MS/microNMR.
abstract: Liquid chromatography-coulometric array detection (LC-EC) is a sensitive, quantitative, and robust metabolomics profiling tool that complements the commonly used mass spectrometry (MS) and nuclear magnetic resonance (NMR)-based approaches. However, LC-EC provides little structural information. We recently demonstrated a workflow for the structural characterization of metabolites detected by LC-EC profiling combined with LC-electrospray ionization (ESI)-MS and microNMR. This methodology is now extended to include (i) gas chromatography (GC)-electron ionization (EI)-MS analysis to fill structural gaps left by LC-ESI-MS and NMR and (ii) secondary fractionation of LC-collected fractions containing multiple coeluting analytes. GC-EI-MS spectra have more informative fragment ions that are reproducible for database searches. Secondary fractionation provides enhanced metabolite characterization by reducing spectral overlap in NMR and ion suppression in LC-ESI-MS. The need for these additional methods in the analysis of the broad chemical classes and concentration ranges found in plasma is illustrated with discussion of four specific examples: (i) characterization of compounds for which one or more of the detectors is insensitive (e.g., positional isomers in LC-MS, the direct detection of carboxylic groups and sulfonic groups in (1)H NMR, or nonvolatile species in GC-MS), (ii) detection of labile compounds, (iii) resolution of closely eluting and/or coeluting compounds, and (iv) the capability to harness structural similarities common in many biologically related, LC-EC-detectable compounds.
authors.affiliation: Department of Neurosurgery, Brigham and Women's Hospital, Department of Neurosurgery, Harvard Medical School, Boston, MA 02115, USA; Barnett Institute of Chemical and Biological Analysis, Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA.
authors.forename: Rose M
authors.initials: RM
authors.lastname: Gathungu
authors.affiliation: Department of Neurosurgery, Brigham and Women's Hospital, Department of Neurosurgery, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Susan S
authors.initials: SS
authors.lastname: Bird
authors.affiliation: Department of Neurosurgery, Brigham and Women's Hospital, Department of Neurosurgery, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Diane P
authors.initials: DP
authors.lastname: Sheldon
authors.affiliation: Barnett Institute of Chemical and Biological Analysis, Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA.
authors.forename: Roger
authors.initials: R
authors.lastname: Kautz
authors.affiliation: Barnett Institute of Chemical and Biological Analysis, Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Vouros
authors.affiliation: Bedford VA Medical Center, Bedford, MA 01730, USA.
authors.forename: Wayne R
authors.initials: WR
authors.lastname: Matson
authors.affiliation: Department of Neurosurgery, Brigham and Women's Hospital, Department of Neurosurgery, Harvard Medical School, Boston, MA 02115, USA. Electronic address: bkristal@partners.org.
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Kristal
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D007211:Indoles / Q000097:blood / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D055432:Metabolomics / Q000379:methods*; D055616:Microtechnology / Q000379:methods*
chemical_list: D007211:Indoles
keywords: GC–MS; LC–EC; LC–MS; Metabolite characterization; NMR; Secondary fractionation