High-performance liquid chromatographic method for the determination of gemcitabine and 2′,2′-difluorodeoxyuridine in plasma and tissue culture media

Mark N. Kirstein, Iman Hassan, Dan E. Guire, Dennis R. Weller, Jason W. Dagit, James E. Fisher, Rory P. Remmel

Research output: Contribution to journalArticlepeer-review

48 Scopus citations

Abstract

Gemcitabine, a pyrimidine antimetabolite undergoes metabolism by plasma and liver cytidine deaminase to form the inactive compound, 2′,2′-difluorodeoxyuridine (dFdU). The parent molecule is activated by intracellular phosphorylation. To evaluate the population pharmacokinetics in patients receiving gemcitabine, and to test the relation between gemcitabine infusion rate and antitumor activity in an in vitro bioreactor cell culture system, we developed and validated a sensitive and specific HPLC-UV method for gemcitabine and dFdU. Deproteinized plasma is vortexed, centrifuged, and 25 μL of the acidified extract sample is injected onto a Waters Spherisorb 4.6 mm × 250 mm, 5 μm C18 column at 40 °C. The mobile phase (flow rate, 1.0 mL/min) consists of 10:90 (v/v) acetonitrile-aqueous buffer (50 mM sodium phosphate and 3.0 mM octyl sulfonic acid, pH 2.9). Gemcitabine, dFdU, and the internal standard, 2′-deoxycytidine (2′dC) were detected with UV wavelength set at 267 nm. The standard curves for gemcitabine in both matrices ranged from 2 to 200 μM, and for dFdU in plasma, from 2 to 100 μM. Within-run and between-run component precision (CV%) was ≤6.1 and 5.7%, respectively for both human plasma and tissue culture media, and for dFdU, 2.3 and 2.7%. Total accuracy ranged from 98.7 to 106.2% for human plasma and from 96.9 to 99.2% for tissue culture media, respectively, and for dFdU, from 96.5 to 99.6%. Tetrahydrouridine (THU), an inhibitor of cytidine deaminase is used to prevent breakdown in human plasma. With one method we can measure gemcitabine in both plasma and tissue culture media. Utility is demonstrated by evaluation of the disposition of gemcitabine in an in vitro bioreactor cell culture system.

Original languageEnglish (US)
Pages (from-to)136-142
Number of pages7
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume835
Issue number1-2
DOIs
StatePublished - May 1 2006

Keywords

  • Gemcitabine
  • HPLC
  • In vitro bioreactor cell culture
  • UV
  • dFdU

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