TY - JOUR
T1 - LC–MS/MS assay for the quantitation of FdCyd and its metabolites FdUrd and FU in human plasma
AU - Holleran, Julianne L.
AU - Eiseman, Julie L.
AU - Parise, Robert A.
AU - Kummar, Shivaani
AU - Beumer, Jan H.
N1 - Funding Information:
Support: This project was supported by NCI contract HHSN261201100015C, NCI/CTEP grant UM1-CA186690 (University of Pittsburgh), and used the UPCI Cancer Pharmacokinetics and Pharmacodynamics Facility (CPPF) and was supported in part by award P30-CA47904.
Publisher Copyright:
© 2016 Elsevier B.V.
PY - 2016/9/10
Y1 - 2016/9/10
N2 - 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.
AB - 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.
KW - Assay
KW - FdCyd
KW - Metabolites
KW - Tandem mass spectrometry
KW - Validation
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U2 - 10.1016/j.jpba.2016.07.027
DO - 10.1016/j.jpba.2016.07.027
M3 - Article
C2 - 27454087
AN - SCOPUS:84989883255
SN - 0731-7085
VL - 129
SP - 359
EP - 366
JO - Journal of Pharmaceutical and Biomedical Analysis
JF - Journal of Pharmaceutical and Biomedical Analysis
ER -