, Volume 11, Issue 6, pp 1552–1562 | Cite as

A tandem liquid chromatography–mass spectrometry (LC–MS) method for profiling small molecules in complex samples

  • James S. Pyke
  • Damien L. Callahan
  • Komal Kanojia
  • Jairus Bowne
  • Sheena Sahani
  • Dedreia Tull
  • Antony Bacic
  • Malcolm J. McConville
  • Ute Roessner
Original Article


Liquid chromatography–mass spectrometry (LC–MS) methods using either aqueous normal phase (ANP) or reversed phase (RP) columns are routinely used in small molecule or metabolomic analyses. These stationary phases enable chromatographic fractionation of polar and non-polar compounds, respectively. The application of a single chromatographic stationary phase to a complex biological extract results in a significant proportion of compounds which elute in the non-retained fraction, where they are poorly detected because of a combination of ion suppression and the co-elution of isomeric compounds. Thus coverage of both polar and non-polar components of the metabolome generally involves multiple analyses of the same sample, increasing the analysis time and complexity. In this study we describe a novel tandem in-line LC–MS method, in which compounds from one injection are sequentially separated in a single run on both ANP and RP LC-columns. This method is simple, robust, and enables the use of independent gradients customized for both RP and ANP columns. The MS signal is acquired in a single chromatogram which reduces instrument time and operator and data analysis errors. This method has been used to analyze a range of biological extracts, from plant and animal tissues, human serum and urine, microbial cell and culture supernatants. Optimized sample preparation protocols are described for this method as well as a library containing the retention times and accurate masses of 127 compounds.


Reversed phase Aqueous normal phase Mass spectrometry Metabolomics Tandem liquid chromatography 



The authors thank Thomas Naderer for the supply of S. cerevisiae and E. coli cells and Liesbet Temmerman for C. elegans. J. Pyke would thanks Paul O’Donnell for and Richard EH Wettenhall. The authors thank Steve Fischer, Agilent Technologies, Santa Clara, U.S.A. for his suggestion of the 6-port configuration.

Conflict of interest

All authors declare they have no conflict of interest in the submission of this manuscript.

Compliance with ethical requirements

The authors declare there are no ethical implications and that this manuscript complies with the ethical requirements of authors outlined by the Committee on Publication Ethics (COPE).

Financial support

M McConville is an NHMRC Principal Research Fellow. U. Roessner is an ARC Future Fellow. The authors are grateful to the Victorian Node of Metabolomics Australia, which is funded through Bioplatforms Australia Pty Ltd, a National Collaborative Research Infrastructure Strategy (NCRIS), 5.1 biomolecular platforms and informatics investment, and co-investment from the Victorian State government and The University of Melbourne.

Supplementary material

11306_2015_806_MOESM1_ESM.docx (941 kb)
Supplementary material 1 (DOCX 940 kb)


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • James S. Pyke
    • 1
    • 2
  • Damien L. Callahan
    • 1
    • 3
  • Komal Kanojia
    • 1
  • Jairus Bowne
    • 1
  • Sheena Sahani
    • 1
  • Dedreia Tull
    • 1
  • Antony Bacic
    • 1
    • 4
  • Malcolm J. McConville
    • 1
    • 5
  • Ute Roessner
    • 1
  1. 1.Metabolomics Australia, School of Botany and Bio21 InstituteThe University of MelbourneParkvilleAustralia
  2. 2.Agilent TechnologiesMulgraveAustralia
  3. 3.Center for Chemistry and Biotechnology, School of Life and Environmental ScienceDeakin UniversityBurwoodAustralia
  4. 4.ARC Centre of Excellence in Plant Cell Walls, School of BotanyThe University of MelbourneParkvilleAustralia
  5. 5.Department of Biochemistry and Molecular BiologyUniversity of MelbourneParkvilleAustralia

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