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Quantitative Profiling of Endogenous Metabolites Using Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry (HILIC-MS/MS)

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Book cover Microbial Metabolomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1859))

Abstract

Dynamic modeling of metabolic reaction networks requires absolute quantification of intracellular and extracellular metabolite concentrations with high precision and accuracy. This chapter presents a robust HILIC-ESI-MS/MS procedure for targeted quantitative profiling of more than 50 polar key metabolites in multicomponent endogenous extracts. Without using ion-pairing-agents or prior derivatization protocols, organic acids, amino acids, sugar phosphates, coenzymes, and nucleotides are measured on a triple quadrupole platform in positive and negative electrospray ionization modes with preoptimized MRM transitions. Robust polymer-based zwitterionic stationary phases (ZIC®-pHILIC) support alkaline mobile phase conditions (pH 9.2) for enhancing retention and chromatographic performance of polar analytes in bicratic elution mode without unfavourable column bleed. The quality of the method was extensively validated and demonstrated by absolute metabolite quantification in endogenous Escherichia coli extracts by comparative use of standard-based external calibration, isotope dilution, and standard addition as quantification strategies. In sum, alkaline ZIC®-pHILIC chromatography emerged as an efficient approach providing high selectivity and sensitivity for comprehensive metabolic studies.

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Acknowledgments

The work was funded by the Bundesministerium für Bildung und Forschung (grant number: 0315867, BMBF, Berlin, Germany) in cooperation with Evonik Industries AG.

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Authors and Affiliations

  1. Institute of Biochemical Engineering, University of Stuttgart, Stuttgart, Germany

    Attila Teleki & Ralf Takors

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  1. Attila Teleki
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  2. Ralf Takors
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Correspondence to Ralf Takors .

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Editors and Affiliations

  1. Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, USA

    Edward E.K. Baidoo

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Teleki, A., Takors, R. (2019). Quantitative Profiling of Endogenous Metabolites Using Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry (HILIC-MS/MS). In: Baidoo, E. (eds) Microbial Metabolomics. Methods in Molecular Biology, vol 1859. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8757-3_10

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  • DOI: https://doi.org/10.1007/978-1-4939-8757-3_10

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8756-6

  • Online ISBN: 978-1-4939-8757-3

  • eBook Packages: Springer Protocols

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