Metabolite Measurement: Pitfalls to Avoid and Practices to Follow

Annu Rev Biochem. 2017 Jun 20;86:277-304. doi: 10.1146/annurev-biochem-061516-044952.


Metabolites are the small biological molecules involved in energy conversion and biosynthesis. Studying metabolism is inherently challenging due to metabolites' reactivity, structural diversity, and broad concentration range. Herein, we review the common pitfalls encountered in metabolomics and provide concrete guidelines for obtaining accurate metabolite measurements, focusing on water-soluble primary metabolites. We show how seemingly straightforward sample preparation methods can introduce systematic errors (e.g., owing to interconversion among metabolites) and how proper selection of quenching solvent (e.g., acidic acetonitrile:methanol:water) can mitigate such problems. We discuss the specific strengths, pitfalls, and best practices for each common analytical platform: liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance (NMR), and enzyme assays. Together this information provides a pragmatic knowledge base for carrying out biologically informative metabolite measurements.

Keywords: accuracy; mass spectroscopy; metabolite extraction; metabolomics; metabonomics; stability.

Publication types

  • Review
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / analysis
  • Animals
  • Chromatography, Liquid / standards*
  • Gas Chromatography-Mass Spectrometry / standards*
  • Glutathione / analysis
  • Guidelines as Topic
  • Humans
  • Liquid Phase Microextraction / methods
  • Magnetic Resonance Spectroscopy / standards*
  • Mass Spectrometry / standards*
  • Metabolomics / instrumentation
  • Metabolomics / methods
  • Metabolomics / standards*
  • Mice
  • NADP / analysis
  • Solvents


  • Solvents
  • NADP
  • Adenosine Triphosphate
  • Glutathione