Considerations of Sample Preparation for Metabolomics Investigation

  • Teresa Whei-Mei FanEmail author
Part of the Methods in Pharmacology and Toxicology book series (MIPT)


Sample preparation is the gateway to metabolomic analysis, the importance of which cannot be overemphasized. There are general rules of thumb for sample preparation that help maximize sample integrity and metabolite recovery. The wide range of variations in metabolite functional groups, polarity, sizes, and stability precludes the use of a single extraction method in metabolomic studies. Common extraction methods for polar metabolites that utilize trichloroacetic acid or aqueous acetonitrile are suitable for both nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis while others that use chloroform/methanol/water partition or boiling water may not. Control of extract pH is crucial for consistent NMR assignments and chemical derivatization-linked MS analysis. Sequential polar and lipid extractions reduce sample size requirement and provide a better coverage for direct-infusion MS analysis of lipids, possibly by removing interfering salts. Cleanup of sample extracts, such as removal of fine particles or interfering cations, is often necessary but should be limited to reduce loss of metabolites.

Key words

NMR GC-MS FT-ICR-MS Trichloroacetic acid Perchloric acid Chloroform/methanol/water Acetonitrile Boiling water Mouse liver Human lung 



This work was supported in part by the National Cancer Institute grants # 1R01 CA101199-01 and 1R01CA118434-01A2, NIH Grant Number RR018733 from the National Center for Research Resources, National Science Foundation EPSCoR grant # EPS-0447479, Kentucky Challenge for Excellence, and the Brown Foundation. Dr. Zhengzhi Xie and Ms. Vennila Arumugum are acknowledged for respective assistance in sample processing and FT-ICR-MS analysis.



Butylated hydroxytoluene


Chloroform/methanol/water partition


Fourier transform-ion cyclotron resonance mass spectrometry


Gas chromatography-mass spectrometry


Reduced glutathione


Oxidized glutathione


Heteronuclear single quantum coherence spectroscopy


Compounds of identical chemical structure but differing in isotopic composition at individual atoms


Liquid chromatography-mass spectrometry




Perchloric acid


Trichloroacetic acid


Total correlation spectroscopy

Supplementary material


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Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  1. 1.Department of Chemistry, Center for Regulatory and Environmental Analytical Metabolomics (CREAM), and James Graham Brown Cancer CenterUniversity of LouisvilleLouisvilleUSA

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