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

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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

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gemcitabine
Tissue culture
Culture Media
Plasmas
Liquids
Plasma (human)
Cytidine Deaminase
Bioreactors
Cell culture
Tetrahydrouridine
Cell Culture Techniques
Antimetabolites
Deoxycytidine
Phosphorylation
Pharmacokinetics
Sulfonic Acids
Metabolism
Liver

Keywords

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

Cite this

@article{ac19d06900184cda8f7edb0c5e101610,
title = "High-performance liquid chromatographic method for the determination of gemcitabine and 2′,2′-difluorodeoxyuridine in plasma and tissue culture media",
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.",
keywords = "Gemcitabine, HPLC, In vitro bioreactor cell culture, UV, dFdU",
author = "Kirstein, {Mark N} and Iman Hassan and Guire, {Dan E.} and Weller, {Dennis R.} and Dagit, {Jason W.} and Fisher, {James E.} and Remmel, {Rory P}",
year = "2006",
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journal = "Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences",
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TY - JOUR

T1 - High-performance liquid chromatographic method for the determination of gemcitabine and 2′,2′-difluorodeoxyuridine in plasma and tissue culture media

AU - Kirstein, Mark N

AU - Hassan, Iman

AU - Guire, Dan E.

AU - Weller, Dennis R.

AU - Dagit, Jason W.

AU - Fisher, James E.

AU - Remmel, Rory P

PY - 2006/5/1

Y1 - 2006/5/1

N2 - 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.

AB - 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.

KW - Gemcitabine

KW - HPLC

KW - In vitro bioreactor cell culture

KW - UV

KW - dFdU

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