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


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 



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).


  1. 1.
    Mathew AK, Padmanaban VC (2013) Metabolomics: the apogee of the omics trilogy. Int J Pharm Pharm Sci 5:45–48. Scholar
  2. 2.
    Fiehn O, Kopka J, Dörmann P et al (2000) Metabolite profiling for plant functional genomics. Nat Biotechnol 18:1157–1161. Scholar
  3. 3.
    McAtee AG, Jazmin LJ, Young JD (2015) Application of isotope labeling experiments and 13C flux analysis to enable rational pathway engineering. Curr Opin Biotechnol 36:50–56. Scholar
  4. 4.
    Orth JD, Thiele I, Palsson BØ (2010) What is flux balance analysis? Nat Biotechnol 28:245–248. Scholar
  5. 5.
    Lee SY, Park JM, Kim TY (2011) Application of metabolic flux analysis in metabolic engineering, 1st edn. Elsevier Inc, AmsterdamGoogle Scholar
  6. 6.
    Ohta E, Dempo Y, Fukusaki E et al (2014) Molar-based targeted metabolic profiling of cyanobacterial strains with potential for biological production. Metabolites 4:499–516. Scholar
  7. 7.
    Luo B, Groenke K, Takors R et al (2007) Simultaneous determination of multiple intracellular metabolites in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle by liquid chromatography-mass spectrometry. J Chromatogr A 1147:153–164. Scholar
  8. 8.
    Lu W, Bennett BD, Rabinowitz JD (2008) Analytical strategies for LC-MS-based targeted metabolomics. J Chromatogr B Analyt Technol Biomed Life Sci 871:236–242. Scholar
  9. 9.
    Young JD, Shastri AA, Stephanopoulos G, Morgan JA (2011) Mapping photoautotrophic metabolism with isotopically nonstationary 13C flux analysis. Metab Eng 13:656–665. Scholar
  10. 10.
    Alagesan S, Gaudana SB, Sinha A, Wangikar PP (2013) Metabolic flux analysis of Cyanothece sp. ATCC 51142 under mixotrophic conditions. Photosynth Res 118:191–198. Scholar
  11. 11.
    Buszewski B, Noga S (2012) Hydrophilic interaction liquid chromatography (HILIC)-a powerful separation technique. Anal Bioanal Chem 402:231–247. Scholar
  12. 12.
    Keasling JD, Adams PD, Benites VT et al (2018) Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli. Metab Eng 47:60–72. Scholar
  13. 13.
    Prasannan CB, Jaiswal D, Davis R, Wangikar PP (2018) An improved method for extraction of polar and charged metabolites from cyanobacteria. PLoS One 13(10):1–16. Scholar
  14. 14.
    Jaiswal D, Prasannan CB, Hendry JI, Wangikar PP (2018) SWATH tandem mass spectrometry workflow for quantification of mass isotopologue distribution of intracellular metabolites and fragments labeled with isotopic 13C carbon. Anal Chem 90:6486–6493. Scholar
  15. 15.
    Hendry JI, Prasannan C, Ma F et al (2017) Rerouting of carbon flux in a glycogen mutant of cyanobacteria assessed via isotopically non-stationary 13C metabolic flux analysis. Biotechnol Bioeng 114:2298–2308. Scholar
  16. 16.
    McCloskey D, Utrilla J, Naviaux RK et al (2014) Fast Swinnex filtration (FSF): a fast and robust sampling and extraction method suitable for metabolomics analysis of cultures grown in complex media. Metabolomics 11:198–209. Scholar
  17. 17.
    Ikeda TP, Shauger AE, Kustu S (1996) Salmonella typhimurium apparently perceives external nitrogen limitation as internal glutamine limitation. J Mol Biol 259:589–607. Scholar
  18. 18.
    Schaub J, Schiesling C, Reuss M, Dauner M (2006) Integrated sampling procedure for metabolome analysis. Biotechnol Prog 22:1434–1442. Scholar
  19. 19.
    Lu W, Kimball E, Rabinowitz JD (2006) A high-performance liquid chromatography-tandem mass spectrometry method for quantitation of nitrogen-containing intracellular metabolites. J Am Soc Mass Spectrom 17:37–50. Scholar
  20. 20.
    Ståhlberg J (1999) Retention models for ions in chromatography. J Chromatogr A 855:3–55. Scholar
  21. 21.
    Qian T, Cai Z, Yang MS (2004) Determination of adenosine nucleotides in cultured cells by ion-pairing liquid chromatography-electrospray ionization mass spectrometry. Anal Biochem 325:77–84. Scholar
  22. 22.
    Lu W, Clasquin MF, Melamud E et al (2010) Metabolomic analysis via reversed-phase ion-pairing liquid chromatography coupled to a stand alone Orbitrap mass spectrometer. Anal Chem 82:3212–3221CrossRefGoogle Scholar
  23. 23.
    Mccloskey D, Gangoiti JA (2015) A pH and solvent optimized reverse-phase ion-paring-LC-MS/MS method that leverages multiple scan-types for targeted absolute quantification of intracellular metabolites. Metabolomics 11:1338–1350. Scholar
  24. 24.
    Jaiswal D, Sengupta A, Sohoni S, Sengupta S (2018) Genome features and biochemical characteristics of a robust , fast growing and naturally transformable Cyanobacterium Synechococcus elongatus PCC 11801 isolated from India. Sci Rep 8:1–13. Scholar
  25. 25.
    Alpert AJ (1990) Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds. J Chromatogr A 499:177–196. Scholar
  26. 26.
    Navarro-Reig M, Ortiz-Villanueva E, Tauler R, Jaumot J (2017) Modelling of hydrophilic interaction liquid chromatography stationary phases using chemometric approaches. Meta 7:6–9. Scholar
  27. 27.
    Kawachi Y, Ikegami T, Takubo H et al (2011) Chromatographic characterization of hydrophilic interaction liquid chromatography stationary phases: Hydrophilicity, charge effects, structural selectivity, and separation efficiency. J Chromatogr A 1218:5903–5919. Scholar
  28. 28.
    Creek DJ, Chokkathukalam A, Jankevics A et al (2012) Stable isotope-assisted metabolomics for network-wide metabolic pathway elucidation. Anal Chem 84:8442–8447. Scholar
  29. 29.
    Trammell SA, Brenner C (2013) Targeted, LCMS-based metabolomics for quantitative measurement of NAD+ metabolites. Comput Struct Biotechnol J 4:e201301012. Scholar
  30. 30.
    Bustamante S, Jayasena T, Richani D et al (2018) Quantifying the cellular NAD+ metabolome using a tandem liquid chromatography mass spectrometry approach. Metabolomics 14:15. Scholar
  31. 31.
    Tautenhahn R, Cho K, Uritboonthai W et al (2012) An accelerated workflow for untargeted metabolomics using the METLIN database. Nat Biotechnol 30:826–828. Scholar
  32. 32.
    Jewison T, Knox C, Neveu V et al (2012) YMDB: the yeast Metabolome database. Nucleic Acids Res 40:D815–D820. Scholar
  33. 33.
    Wishart DS, Tzur D, Knox C et al (2007) HMDB: the human metabolome database. Nucleic Acids Res 35:521–526. Scholar
  34. 34.
    Pence HE, Williams A (2010) ChemSpider: an online chemical information resource. J Chem Educ 87:1123–1124. Scholar
  35. 35.
    Tsugawa H, Cajka T, Kind T et al (2015) MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis. Nat Methods 12:523–526. Scholar
  36. 36.
    Li H, Cai Y, Guo Y et al (2016) MetDIA: targeted metabolite extraction of multiplexed MS/MS spectra generated by data-independent acquisition. Anal Chem 88:8757–8764. Scholar

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

Personalised recommendations