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Anal Biochem. 2014 Jun 1;454:23-32. doi: 10.1016/j.ab.2014.01.020. Epub 2014 Mar 18.

Identification of metabolites from liquid chromatography-coulometric array detection profiling: gas chromatography-mass spectrometry and refractionation provide essential information orthogonal to LC-MS/microNMR.

Author information

1
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.
2
Department of Neurosurgery, Brigham and Women's Hospital, Department of Neurosurgery, Harvard Medical School, Boston, MA 02115, USA.
3
Barnett Institute of Chemical and Biological Analysis, Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA.
4
Bedford VA Medical Center, Bedford, MA 01730, USA.
5
Department of Neurosurgery, Brigham and Women's Hospital, Department of Neurosurgery, Harvard Medical School, Boston, MA 02115, USA. Electronic address: bkristal@partners.org.

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.

KEYWORDS:

GC–MS; LC–EC; LC–MS; Metabolite characterization; NMR; Secondary fractionation

PMID:
24657819
PMCID:
PMC4034759
DOI:
10.1016/j.ab.2014.01.020
[Indexed for MEDLINE]
Free PMC Article

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