Analytical and Bioanalytical Chemistry

, Volume 409, Issue 25, pp 5955–5964 | Cite as

An LC-MS chemical derivatization method for the measurement of five different one-carbon states of cellular tetrahydrofolate

  • Li Chen
  • Gregory S. Ducker
  • Wenyun Lu
  • Xin Teng
  • Joshua D. RabinowitzEmail author
Research Paper


The cofactor tetrahydrofolate (THF) is used to reduce, oxidize, and transfer one-carbon (1C) units required for the synthesis of nucleotides, glycine, and methionine. Measurement of intracellular THF species is complicated by their chemical instability, signal dilution caused by variable polyglutamation, and the potential for interconversion among these species. Here, we describe a method using negative mode liquid chromatography-mass spectrometry (LC-MS) to measure intracellular folate species from mammalian cells. Application of this method with isotope-labeled substrates revealed abiotic interconversion of THF and methylene-THF, which renders their separate quantitation particularly challenging. Chemical reduction of methylene-THF using deuterated sodium cyanoborohydride traps methylene-THF, which is unstable, as deuterated 5-methyl-THF, which is stable. Together with proper sample handling and LC-MS, this enables effective measurements of five active folate pools (THF, 5-methyl-THF, methylene-THF, methenyl-THF/10-formyl-THF, and 5-formyl-THF) representing the biologically important 1C oxidation states of THF in mammalian cells.

Graphical abstract

Chemical derivatization with deuterated cyanoborohydride traps unstable methylene-THF as isotope-labeled 5-methyl-THF, enabling accurate quantification by LC-MS.


Folate Chemical derivatization Methylene-THF LC-MS Interconversion One-carbon metabolism 



This research was supported in part by funding to J.D.R. from the U.S. National Institutes of Health (NIH) (R01CA16359-01A1) and Stand Up to Cancer (SU2C-AACR-DT0509). G.S.D. was supported by a postdoctoral fellowship (PF-15-190-01-TBE) from the American Cancer Society. W.L. was supported by NIH (CA211437).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2017_514_MOESM1_ESM.pdf (589 kb)
ESM 1 (PDF 588 kb)


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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Li Chen
    • 1
  • Gregory S. Ducker
    • 1
  • Wenyun Lu
    • 1
  • Xin Teng
    • 1
  • Joshua D. Rabinowitz
    • 1
    Email author
  1. 1.Lewis-Sigler Institute for Integrative Genomics and Department of ChemistryPrinceton UniversityPrincetonUSA

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