A tandem liquid chromatography–mass spectrometry (LC–MS) method for profiling small molecules in complex samples
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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.
KeywordsReversed 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).
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.
- 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.
- 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.CrossRefPubMedGoogle 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.PubMedGoogle 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.CrossRefPubMedGoogle 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.CrossRefPubMedGoogle Scholar