Skip to main content
Log in

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

  • Original Article
  • Published:
Metabolomics Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Allwood, J. W., & Goodacre, R. (2010). An introduction to liquid chromatography-mass spectrometry instrumentation applied in plant metabolomic analyses. Phytochemical Analysis, 21(1), 33–47.

    Article  CAS  PubMed  Google Scholar 

  • Buscher, J. M., Czernik, D., Ewald, J. C., Sauer, U., & Zamboni, N. (2009). Cross-Platform comparison of methods for quantitative metabolomics of primary metabolism. Analytical Chemistry, 81(6), 2135–2143.

    Article  CAS  PubMed  Google Scholar 

  • Callahan, D. L., De Souza, D., Bacic, A., & Roessner, U. (2009). Profiling of polar metabolites in biological extracts using diamond hydride-based aqueous normal phase chromatography. Journal of Separation Science, 32(13), 2273–2280.

    Article  CAS  PubMed  Google Scholar 

  • Chalcraft, K., Kong, J., Waserman, S., Jordana, M., & McCarry, B. (2014). Comprehensive metabolomic analysis of peanut-induced anaphylaxis in a murine model. Metabolomics, 10(3), 452–460.

    Article  CAS  Google Scholar 

  • Chalcraft, K. R., & McCarry, B. E. (2013). Tandem LC columns for the simultaneous retention of polar and nonpolar molecules in comprehensive metabolomics analysis. Journal of Separation Science, 36(21–22), 3478–3485.

    Article  CAS  PubMed  Google Scholar 

  • Dunn, W. B., Broadhurst, D., Begley, P., et al. (2011). Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry. Nature Protocols, 6(7), 1060–1083.

    Article  CAS  PubMed  Google Scholar 

  • Fischer, S., Pesek, M., & Pesek, J. (2010). ANP chromatography for hydrophilic metabolites. Resource document. http://disruptechno2.free.fr/MicroSolv/HPLC columns/Presentations/ANP chromatography for hydrophilic metabolites.pdf. Accessed June 2010.

  • Greco, G., Grosse, S., & Letzel, T. (2013). Serial coupling of reversed-phase and zwitterionic hydrophilic interaction LC/MS for the analysis of polar and nonpolar phenols in wine. Journal of Separation Science, 36(8), 1379–1388.

    Article  CAS  PubMed  Google Scholar 

  • Horai, H., Arita, M., Kanaya, S., et al. (2010). MassBank: A public repository for sharing mass spectral data for life sciences. Journal of Mass Spectrometry, 45(7), 703–714.

    Article  CAS  PubMed  Google Scholar 

  • Jayamanne, M., Granelli, I., Tjernberg, A., & Edlund, P. O. (2010). Development of a two-dimensional liquid chromatography system for isolation of drug metabolites. Journal of Pharmaceutical and Biomedical Analysis, 51(3), 649–657.

    Article  CAS  PubMed  Google Scholar 

  • Kiffe, M., Graf, D., & Trunzer, M. (2007). Two-dimensional liquid chromatography/mass spectrometry set-up for structural elucidation of metabolites in complex biological matrices. Rapid Communications in Mass Spectrometry, 21(6), 961–970.

    Article  CAS  PubMed  Google Scholar 

  • Kind, T., Tolstikov, V., Fiehn, O., & Weiss, R. H. (2007). A comprehensive urinary metabolomic approach for identifying kidney cancerr. Analytical Biochemistry, 363(2), 185–195.

    Article  CAS  PubMed  Google Scholar 

  • Klavins, K., Drexler, H., Hann, S., & Koellensperger, G. (2014). Quantitative metabolite profiling utilizing parallel column analysis for simultaneous reversed-phase and hydrophilic interaction liquid chromatography separations combined with tandem mass spectrometry. Analytical Chemistry, 86(9), 4145–4150.

    Article  CAS  PubMed  Google Scholar 

  • Kuehnbaum, N. L., & Britz-McKibbin, P. (2013). New advances in separation science for metabolomics: Resolving chemical diversity in a post-genomic era. Chemical Reviews, 113(4), 2437–2468.

    Article  CAS  PubMed  Google Scholar 

  • Kulsing, C., Yang, Y., Munera, C., et al. (2014). Correlations between the zeta potentials of silica hydride-based stationary phases, analyte retention behaviour and their ionic interaction descriptors. Analytica Chimica Acta, 817, 48–60.

    Article  CAS  PubMed  Google Scholar 

  • Liu, Y., Xue, X., Guo, Z., Xu, Q., Zhang, F., & Liang, X. (2008). Novel two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography, an excellent orthogonal system for practical analysis. Journal of Chromatography A, 1208(1–2), 133–140.

    Article  CAS  PubMed  Google Scholar 

  • Nagele, E., Vollmer, M., & Horth, P. (2003). Two-dimensional nano-liquid chromatography-mass spectrometry system for applications in proteomics. Journal of Chromatography A, 1009(1–2), 197–205.

    Article  CAS  PubMed  Google Scholar 

  • Pesek, J. J., Matyska, M. T., Boysen, R. I., Yang, Y., & Hearn, M. T. W. (2013). Aqueous normal-phase chromatography using silica-hydride-based stationary phases. Trends in Analytical Chemistry, 42, 64–73.

    Article  CAS  Google Scholar 

  • Pesek, J. J., Matyska, M. T., Fischer, S. M., & Sana, T. R. (2008). Analysis of hydrophilic metabolites by high-performance liquid chromatography–mass spectrometry using a silica hydride-based stationary phase. Journal of Chromatography A, 1204(1), 48–55.

    Article  CAS  PubMed  Google Scholar 

  • Pesek, J. J., Matyska, M. T., Hearn, M. T. W., & Boysen, R. I. (2009). Aqueous normal-phase retention of nucleotides on silica hydride columns. Journal of Chromatography A, 1216(7), 1140–1146.

    Article  CAS  PubMed  Google Scholar 

  • Pluskal, T., Castillo, S., Villar-Briones, A., & Oresic, M. (2010). MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Bioinformatics, 11, 395. [Research Support, Non-U.S. Gov’t].

    Article  PubMed  PubMed Central  Google Scholar 

  • Rajab, M., Greco, G., Heim, C., Helmreich, B., & Letzel, T. (2013). Serial coupling of RP and zwitterionic hydrophilic interaction LC–MS: Suspects screening of diclofenac transformation products by oxidation with a boron-doped diamond electrode. Journal of Separation Science, 36(18), 3011–3018.

    CAS  PubMed  Google Scholar 

  • Smith, C. A., Want, E. J., O’Maille, G., Abagyan, R., & Siuzdak, G. (2006). XCMS: Processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Analytical Chemistry, 78(3), 779–787.

    Article  CAS  PubMed  Google Scholar 

  • van der Werf, M. J., Overkamp, K. M., Muilwijk, B., Coulier, L., & Hankemeier, T. (2007). Microbial metabolomics: Toward a platform with full metabolome coverage. Analytical Biochemistry, 370(1), 17–25.

    Article  PubMed  Google Scholar 

  • Wang, Y., Wang, J., Yao, M., et al. (2008). Metabonomics study on the effects of the ginsenoside Rg3 in a β-cyclodextrin-based formulation on tumor-bearing rats by a fully automatic hydrophilic interaction/reversed-phase column-switching HPLC − ESI-MS approach. Analytical Chemistry, 80(12), 4680–4688.

    Article  CAS  PubMed  Google Scholar 

  • Wishart, D. S., Jewison, T., Guo, A. C., et al. (2013). HMDB 3.0–The Human Metabolome Database in 2013. Nucleic Acids Research, 4, D801–D807. (Database issue).

    Article  Google Scholar 

  • Wu, Q., Yuan, H., Zhang, L., & Zhang, Y. (2012). Recent advances on multidimensional liquid chromatography–mass spectrometry for proteomics: From qualitative to quantitative analysis—A review. Analytica Chimica Acta, 731, 1–10.

    Article  CAS  PubMed  Google Scholar 

  • Yin, P., Wan, D., Zhao, C., et al. (2009). A metabonomic study of hepatitis B-induced liver cirrhosis and hepatocellular carcinoma by using RP-LC and HILIC coupled with mass spectrometry. Molecular BioSystems, 5(8), 868–876.

    Article  CAS  PubMed  Google Scholar 

  • Young, J. E., Matyska, M. T., & Pesek, J. J. (2011). Liquid chromatography/mass spectrometry compatible approaches for the quantitation of folic acid in fortified juices and cereals using aqueous normal phase conditions. Journal of Chromatography A, 1218(15), 2121–2126.

    Article  CAS  PubMed  Google Scholar 

  • Zhang, T., Creek, D. J., Barrett, M. P., Blackburn, G., & Watson, D. G. (2012). Evaluation of coupling reversed phase, aqueous normal phase, and hydrophilic interaction liquid chromatography with orbitrap mass spectrometry for metabolomic studies of human urine. Analytical Chemistry, 84(4), 1994–2001.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

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.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Damien L. Callahan or Malcolm J. McConville.

Additional information

James S. Pyke and Damien L. Callahan have contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 940 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pyke, J.S., Callahan, D.L., Kanojia, K. et al. A tandem liquid chromatography–mass spectrometry (LC–MS) method for profiling small molecules in complex samples. Metabolomics 11, 1552–1562 (2015). https://doi.org/10.1007/s11306-015-0806-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11306-015-0806-7

Keywords

Navigation