title: LC-MS/MS assay for the quantitation of FdCyd and its metabolites FdUrd and FU in human plasma.
abstract: 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 0.2mL 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 3000ng/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 120mg PO FdCyd 30min after 3000mg THU. Our LC-MS/MS assay will be an essential tool to further define the pharmacology of FdCyd in ongoing and future studies.
authors.affiliation: Cancer Therapeutics Drug Discovery Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA, United States.
authors.forename: Julianne L
authors.initials: JL
authors.lastname: Holleran
authors.affiliation: Cancer Therapeutics Drug Discovery Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA, United States; Department of Pharmacology and Chemical Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, United States.
authors.forename: Julie L
authors.initials: JL
authors.lastname: Eiseman
authors.affiliation: Cancer Therapeutics Drug Discovery Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA, United States; Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, United States.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Parise
authors.affiliation: Division of Cancer Treatment and Diagnosis, NCI, Bethesda, MD, United States.
authors.forename: Shivaani
authors.initials: S
authors.lastname: Kummar
authors.affiliation: Cancer Therapeutics Drug Discovery Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA, United States; Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, United States; Division of Hematology-Oncology, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States. Electronic address: beumerj@gmail.com.
authors.forename: Jan H
authors.initials: JH
authors.lastname: Beumer
mesh_terms: D001681:Biological Assay / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics; D005472:Fluorouracil / Q000097:blood* / Q000737:chemistry*; D006801:Humans; D010949:Plasma / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods; D013767:Tetrahydrouridine / Q000737:chemistry
chemical_list: C576827:5-fluoro-2'-deoxyuridine; D013767:Tetrahydrouridine; D005472:Fluorouracil; D003857:Deoxyuridine
keywords: Assay; FdCyd; Metabolites; Tandem mass spectrometry; Validation