Ion Pair Chromatography for Endogenous Metabolites LC-MS Analysis in Tissue Samples Following Targeted Acquisition

  • Filippos Michopoulos
Part of the Methods in Molecular Biology book series (MIMB, volume 1738)


A protocol for the preparation of tissue extracts for the targeted analysis of ca. 150 polar metabolites, including those involved in central carbon metabolism is described, using a reversed-phase ion pair U(H)PLC-MS method. Data collection enabled by multiple-reaction monitoring provides highly specific, sensitive acquisition of metabolic intermediates with a wide range of physicochemical properties and pathway coverage. Technical aspects are discussed for method transfer along with the basic principles of sample sequence setup, data analysis, and validation. General comments are given to help the assessment of data quality and system performance.

Key words

Metabonomics Targeted analysis Mass spectrometry Metabolites Ion pair chromatography 


  1. 1.
    Bales JR et al (1998) Metabolic profiling of body fluids by proton NMR: self-poisoning episodes with paracetamol (acetaminophen). Magn Reson Med 6(3):300–306CrossRefGoogle Scholar
  2. 2.
    Nicholson JK, Wilson ID (1989) High resolution proton magnetic resonance spectroscopy of biological fluids. Prog Nucl Magn Reson Spectrosc 21(4–5):449–501CrossRefGoogle Scholar
  3. 3.
    Barzilai A et al (1991) Phosphate metabolites and steroid hormone receptors of benign and malignant breast tumors. A nuclear magnetic resonance study. Cancer 67(11):2919–2925CrossRefGoogle Scholar
  4. 4.
    Gavaghan CL et al (2000) An NMR-based metabonomic approach to investigate the biochemical consequences of genetic strain differences: application to the C57BL10J and Alpk:ApfCD mouse. FEBS Lett 484(3):169–174CrossRefGoogle Scholar
  5. 5.
    Kurhanewicz J et al (1995) Citrate as an in vivo marker to discriminate prostate cancer from benign prostatic hyperplasia and normal prostate peripheral zone: detection via localized proton spectroscopy. Urology 45(3):459–466CrossRefGoogle Scholar
  6. 6.
    Lynch MJ, Nicholson JK (1997) Proton MRS of human prostatic fluid: correlations between citrate, spermine, and myo-inositol levels and changes with disease. The Prostat 30(4):248–255CrossRefGoogle Scholar
  7. 7.
    Marx A et al (1996) Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing. Biotechnol Bioeng 49(2):111–129CrossRefGoogle Scholar
  8. 8.
    Gika HG et al (2010) Does the mass spectrometer define the marker? A comparison of global metabolite profiling data generated simultaneously via UPLC-MS on two different mass spectrometers. Anal Chem 82(19):8226–8234CrossRefGoogle Scholar
  9. 9.
    Dunn WB, Ellis DI (2005) Metabolomics: current analytical platforms and methodologies. TrAC Trends Anal Chem 24(4):285–294CrossRefGoogle Scholar
  10. 10.
    Lenz EM, Wilson ID (2007) Analytical strategies in Metabonomics. J Proteome Res 6(2):443–458CrossRefGoogle Scholar
  11. 11.
    Lindon JC, Nicholson JK (2008) Analytical technologies for metabonomics and metabolomics, and multi-omic information recovery. TrAC Trends Anal Chem 27(3):194–204CrossRefGoogle Scholar
  12. 12.
    Theodoridis G, Gika HG, Wilson ID (2008) LC-MS-based methodology for global metabolite profiling in metabonomics/metabolomics. TrAC Trends Anal Chem 27(3):251–260CrossRefGoogle Scholar
  13. 13.
    Shulaev V (2006) Metabolomics technology and bioinformatics. Brief Bioinform 7(2):128–139CrossRefGoogle Scholar
  14. 14.
    Smith CA et al (2006) XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal Chem 78(3):779–787CrossRefGoogle Scholar
  15. 15.
    Xia J et al (2012) MetaboAnalyst 2.0—a comprehensive server for metabolomic data analysis. Nucleic Acids Res 40(W1):W127–W133CrossRefGoogle Scholar
  16. 16.
    Lu W, Bennett BD, Rabinowitz JD (2008) Analytical strategies for LC–MS-based targeted metabolomics. J Chromatogr B 871(2):236–242CrossRefGoogle Scholar
  17. 17.
    Buescher JM et al (2010) Ultrahigh performance liquid chromatography− tandem mass spectrometry method for fast and robust quantification of anionic and aromatic metabolites. Anal Chem 82(11):4403–4412CrossRefGoogle Scholar
  18. 18.
    Michopoulos F et al (2014) Targeted profiling of polar intracellular metabolites using ion-pair-high performance liquid chromatography and-ultra high performance liquid chromatography coupled to tandem mass spectrometry: applications to serum, urine and tissue extracts. J Chromatogr A 1349:60–68CrossRefGoogle Scholar
  19. 19.
    Gika HG et al (2012) Quantitative profiling of polar primary metabolites using hydrophilic interaction ultrahigh performance liquid chromatography–tandem mass spectrometry. J Chromatogr A 1259:121–127CrossRefGoogle Scholar
  20. 20.
    Schiesel S, Lämmerhofer M, Lindner W (2010) Multitarget quantitative metabolic profiling of hydrophilic metabolites in fermentation broths of β-lactam antibiotics production by HILIC–ESI–MS/MS. Anal and Bioanal Chem 396(5):1655–1679CrossRefGoogle Scholar
  21. 21.
    Yuan M et al (2012) A positive/negative ion-switching, targeted mass spectrometry-based metabolomics platform for bodily fluids, cells, and fresh and fixed tissue. Nat Protoc 7(5):872–881CrossRefGoogle Scholar
  22. 22.
    Kloos D et al (2014) Analysis of biologically-active, endogenous carboxylic acids based on chromatography-mass spectrometry. TrAC Trends Anal Chem 61:17–28CrossRefGoogle Scholar
  23. 23.
    Kloos D et al (2012) Derivatization of the tricarboxylic acid cycle intermediates and analysis by online solid-phase extraction-liquid chromatography–mass spectrometry with positive-ion electrospray ionization. J Chromatogr A 1232:19–26CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.IMED OncologyAstraZenecaMacclesfieldUK

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