High sensitive assay employing column switching chromatography to enable simultaneous quantification of an amide prodrug of gemcitabine (LY2334737), gemcitabine, and its metabolite dFdU in human plasma by LC-MS/MS

J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Aug 1:932:117-22. doi: 10.1016/j.jchromb.2013.06.008. Epub 2013 Jun 13.

Abstract

In this study we report a high sensitive method for the simultaneous analysis of LY2334737 (2'-deoxy-2',2'-difluoro-N-(1-oxo-2-propylpentyl)-cytidine), an amide prodrug of gemcitabine (2', 2'-difluoro-deoxycytidine), along with its active drug gemcitabine and its major metabolite dFdU (2',2'-difluoro-deoxyuridine) by LC-MS/MS. Quantification of all three analytes within a single analysis was challenging because the physio-chemical properties of LY2334737 were significantly different from gemcitabine and dFdU and was accomplished by incorporating column-switching. The assay was fully validated to quantify LY2334737 from 0.1 to 100ng/mL, gemcitabine from 0.25 to 100ng/mL and dFdU from 1 to 1000ng/mL in order to cover the diverse concentration ranges expected in clinical samples. A 25-fold dilution was also validated to accommodate any samples outside this range. Overall, the assay had good accuracy (ranging from -7.0 to 1.2% relative error) and precision (ranging from 2.1 to 8.4% relative standard deviation). Extraction efficiency was greater than 80% for all three analytes and there were no matrix effects. Plasma samples were stable for 24h at room temperature, 660 days in frozen storage, and at least 4 freeze-thaw cycles, at both -20 and -70°C. Data from clinical trials showed that plasma concentrations for LY2334737, gemcitabine, and dFdU were successfully quantified from a single LC-MS/MS analysis and that the assay ranges selected for the three analytes were appropriate and minimized the need for reanalysis.

Keywords: Column switching; Gemcitabine; LC–MS/MS; Prodrug.

Publication types

  • Evaluation Study

MeSH terms

  • Antimetabolites, Antineoplastic / blood*
  • Antimetabolites, Antineoplastic / metabolism
  • Chromatography, Liquid / methods
  • Deoxycytidine / analogs & derivatives*
  • Deoxycytidine / blood
  • Deoxycytidine / metabolism
  • Deoxyuridine / analogs & derivatives*
  • Deoxyuridine / blood
  • Deoxyuridine / metabolism
  • Floxuridine / analogs & derivatives*
  • Floxuridine / blood
  • Floxuridine / metabolism
  • Gemcitabine
  • Humans
  • Prodrugs / metabolism
  • Prodrugs / pharmacokinetics*
  • Sensitivity and Specificity
  • Tandem Mass Spectrometry / methods*

Substances

  • Antimetabolites, Antineoplastic
  • LY2334737
  • Prodrugs
  • Floxuridine
  • Deoxycytidine
  • Deoxyuridine
  • 2',2'-difluoro-2'-deoxyuridine
  • Gemcitabine