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Metabolic Flux Analysis in Eukaryotic Cells pp 33–50Cite as

Liquid Chromatography Methods for Separation of Polar and Charged Intracellular Metabolites for 13C Metabolic Flux Analysis

Part of the Methods in Molecular Biology book series (MIMB,volume 2088)

Abstract

Accurate quantification of mass isotopolog distribution (MID) of intracellular metabolites is a key requirement for 13C metabolic flux analysis (13C–MFA). Liquid chromatography coupled with mass spectrometry (LC/MS) has emerged as a frontrunner technique that combines two orthogonal separation strategies. While metabolomics requires separation of monoisotopic peaks, 13C-MFA imposes additional demands for chromatographic separation as isotopologs of metabolites significantly add to the number of analytes. In this protocol chapter, we discuss two liquid chromatography methods, namely, reverse phase ion-pairing and hydrophilic interaction chromatography (HILIC) that together can separate a wide variety of metabolites that are typically used for 13C metabolic flux analysis.

Key words

  • Sugar phosphates
  • Nucleotides
  • Reverse phase ion-pairing
  • HILIC
  • Metabolic flux analysis

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Acknowledgments

This work was supported by a grant from Department of Biotechnology (DBT), Government of India, awarded to PPW toward DBT-Pan IIT Center for Bioenergy (Grant No. BT/EB/PAN IIT/2012).

Author information

Authors and Affiliations

  1. Department of Chemical Engineering, Indian Institute of Technology Bombay, Mumbai, India

    Damini Jaiswal & Pramod P. Wangikar

  2. Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, USA

    Anjali Mittal & Deepak Nagrath

  3. Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA

    Deepak Nagrath

  4. DBT-PAN IIT Centre for Bioenergy, Indian Institute of Technology Bombay, Mumbai, India

    Pramod P. Wangikar

  5. Wadhwani Research Centre for Bioengineering, Indian Institute of Technology Bombay, Mumbai, India

    Pramod P. Wangikar

Authors
  1. Damini Jaiswal
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  2. Anjali Mittal
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  3. Deepak Nagrath
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  4. Pramod P. Wangikar
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Corresponding author

Correspondence to Pramod P. Wangikar .

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Editors and Affiliations

  1. Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA

    Deepak Nagrath

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Jaiswal, D., Mittal, A., Nagrath, D., Wangikar, P.P. (2020). Liquid Chromatography Methods for Separation of Polar and Charged Intracellular Metabolites for 13C Metabolic Flux Analysis. In: Nagrath, D. (eds) Metabolic Flux Analysis in Eukaryotic Cells. Methods in Molecular Biology, vol 2088. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0159-4_3

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  • DOI: https://doi.org/10.1007/978-1-0716-0159-4_3

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  • Publisher Name: Humana, New York, NY

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  • Online ISBN: 978-1-0716-0159-4

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