A Protocol for Untargeted Metabolomic Analysis: From Sample Preparation to Data Processing

Amanda L. Souza, Gary J. Patti

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

5 Scopus citations


Untargeted metabolomics has rapidly become a profiling method of choice in many areas of research, including mitochondrial biology. Most commonly, untargeted metabolomics is performed with liquid chromatography/mass spectrometry because it enables measurement of a relatively wide range of physiochemically diverse molecules. Specifically, to assess energy pathways that are associated with mitochondrial metabolism, hydrophilic interaction liquid chromatography (HILIC) is often applied before analysis with a high-resolution accurate mass instrument. The workflow produces large, complex data files that are impractical to analyze manually. Here, we present a protocol to perform untargeted metabolomics on biofluids such as plasma, urine, and cerebral spinal fluid with a HILIC separation and an Orbitrap mass spectrometer. Our protocol describes each step of the analysis in detail, from preparation of solvents for chromatography to selecting parameters during data processing.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages26
StatePublished - 2021

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • Accurate mass
  • Data-dependent acquisition
  • High-resolution
  • Liquid chromatography
  • Mass spectrometry
  • Metabolites
  • Metabolomics
  • Profiling
  • Quality assurance
  • Quality control


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