LC–MS/MS assay for the quantitation of FdCyd and its metabolites FdUrd and FU in human plasma

Julianne L. Holleran, Julie L. Eiseman, Robert A. Parise, Shivaani Kummar, Jan H. Beumer

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


The hypomethylating agent 5-fluoro-2′-deoxycytidine (FdCyd, NSC 48006) is being evaluated clinically both via the intravenous route and via the oral route in combination with 3,4,5,6-tetrahydrouridine (THU), a potent inhibitor of FdCyd catabolism. To determine the pharmacokinetics of FdCyd and downstream metabolites, we developed and validated an LC–MS/MS assay for the quantitation of FdCyd, 5-fluoro-2′-deoxyuridine (FdUrd), and 5-fluorouracil (FU) in 0.2 mL human plasma. After acetonitrile protein precipitation, the sample was split and separate chromatography was achieved for FdCyd with a Synergi Polar-RP column and for FdUrd and FU with a Shodex Asahipak NH2P-50 2D column. Gradients of 0.1% acetic acid in acetonitrile and water were used. Detection with a Quattromicro quadrupole mass spectrometer with electrospray ionization in positive-ion (FdCyd) or negative-ion (FdUrd and FU) multiple reaction monitoring (MRM) mode. The assay was linear from 5 to 3000 ng/mL for all three analytes and proved to be accurate (96.7-105.5%) and precise (<8.1%CV), and fulfilled FDA criteria for bioanalytical method validation. We demonstrated the suitability of this assay for measuring FdCyd and metabolites FdUrd and FU in plasma from a patient who was administered 120 mg PO FdCyd 30 min after 3000 mg THU. Our LC–MS/MS assay will be an essential tool to further define the pharmacology of FdCyd in ongoing and future studies.

Original languageEnglish (US)
Pages (from-to)359-366
Number of pages8
JournalJournal of Pharmaceutical and Biomedical Analysis
StatePublished - Sep 10 2016
Externally publishedYes


  • Assay
  • FdCyd
  • Metabolites
  • Tandem mass spectrometry
  • Validation

ASJC Scopus subject areas

  • Analytical Chemistry
  • Pharmaceutical Science
  • Drug Discovery
  • Spectroscopy
  • Clinical Biochemistry


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