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

  • Damini Jaiswal
  • Anjali Mittal
  • Deepak Nagrath
  • Pramod P. WangikarEmail author
Protocol
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 

Notes

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).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Damini Jaiswal
    • 1
  • Anjali Mittal
    • 2
  • Deepak Nagrath
    • 2
    • 3
  • Pramod P. Wangikar
    • 1
    • 4
    • 5
    Email author
  1. 1.Department of Chemical EngineeringIndian Institute of Technology BombayMumbaiIndia
  2. 2.Department of Chemical EngineeringUniversity of MichiganAnn ArborUSA
  3. 3.Department of Biomedical EngineeringUniversity of MichiganAnn ArborUSA
  4. 4.DBT-PAN IIT Centre for BioenergyIndian Institute of Technology BombayMumbaiIndia
  5. 5.Wadhwani Research Centre for BioengineeringIndian Institute of Technology BombayMumbaiIndia

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