Measurement of the anticancer agent gemcitabine and its deaminated metabolite at low concentrations in human plasma by liquid chromatography-mass spectrometry

Yan Xu, Bruce Keith, Jean L. Grem

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Abstract

A liquid chromatography/mass spectrometry (LC-MS) method has been developed and validated for the determination of the anticancer agent gemcitabine (dFdC) and its metabolite 2′,2′-difluoro-2′- deoxyuridine (dFdU) in human plasma. An Oasis® HLB solid phase extraction cartridge was used for plasma sample preparation. Separation of the analytes was achieved with a YMC ODS-AQ (5μm, 120Å, 2.0mm×150mm) column. The initial composition of the mobile phase was 2% methanol/98% 5mM ammonium acetate at pH 6.8 (v/v), and the flow rate was 0.2ml/min. An isocratic gradient was used for 3min, followed by a linear gradient over 4min to 30% methanol/70% 5mM ammonium acetate at pH 6.8. The gradient returned to the initial conditions over 2min and remained there for 6min. The retention times of dFdC, dFdU, and the internal standard 5′-deoxy-5-fluorouridine (5′-DFUR) were 11.46, 12.63, and 13.58min. The mass spectrometer was operated under negative electrospray ionization conditions. Single-ion- monitoring (SIM) mode was used for analyte quantitation at m/z 262 for [dFdC-H]-, m/z 263 for [dFdU-H]-, and m/z 245 for [5′-DFUR-H]-. The average recoveries for dFdC, dFdU, and 5′-DFUR were 88.4, 84.6, and 99.3%, respectively. The linear calibration ranges were 5-1000ng/ml for dFdC, and 5-5000ng/ml for dFdU. The intra- and inter-assay precisions (%CV) were ≤3 and ≤7% at three concentration levels (50.0, 500, and 5000ng/ml). The limits of quantitation (defined as 10 times of signal-to-noise ratio) were 3.16ng/ml for dFdC, and 1.35ng/ml for dFdU with 50-μl sample injections. This method has been used for measuring plasma concentrations of dFdC and dFdU in samples from adult cancer patients in a Phase I trial of weekly dFdC given as 150 (or lower) mg/(m2 24-h) infusion. The average plasma dFdC concentrations at 22- and 23-h into the infusion were 18.3 and 16.8ng/ml at 150 and 100mg/m2, respectively; the values for dFdU averaged 2950 and 1372ng/ml.

Original languageEnglish (US)
Pages (from-to)263-270
Number of pages8
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume802
Issue number2
DOIs
Publication statusPublished - Apr 5 2004

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Keywords

  • 2′,2′-Difluoro-2′-deoxyuridine
  • Gemcitabine

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology

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