Quantification of Gemcitabine Incorporation into Human DNA by LC/MS/MS as a Surrogate Measure for Target Engagement

Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
Analytical Chemistry (Impact Factor: 5.64). 08/2010; 82(15):6576-83. DOI: 10.1021/ac100984h
Source: PubMed


In this study, we report a method for direct determination of gemcitabine incorporation into human DNA. Gemcitabine (dFdC), a structural analog of the nucleoside deoxycytidine (dC), derives its primary antitumor activity through interruption of DNA synthesis. Unlike other surrogate measures, DNA incorporation provides a mechanistic end point useful for dose optimization. DNA samples (ca. 25 microg) were hydrolyzed using a two-step enzymatic procedure to release dFdC which was subsequently quantified by LC-ESI-MS/MS using stable isotope labeled internal standards and selected reaction monitoring (SRM). dFdC was quantitated and reported relative to deoxyguanosine (dG) since dG is the complementary base for both dFdC and dC. The SRM channel for dG was detuned using collision energy as the attenuating parameter in order to accommodate the difference in relative abundance for these two analytes (>104) and enable simultaneous quantification from the same injection. The assay was shown to be independent of the amount of DNA analyzed. The method was validated for clinical use using a 3 day procedure assessing precision, accuracy, stability, selectivity, and robustness. The validated ranges for dFdC and dG were 5-7500 pg/mL and 0.1-150 microg/mL, respectively. Results are presented which confirm that the ratio of dFdC to dG in DNA isolated from tumor cells incubated with dFdC increases with increased exposure to the drug and that dFdC can also be quantified from DNA extracted from blood.

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    • "In the current study, we observed heterogeneity in delivery between patients and within pancreatic tumors. Notably, the Advion measurement of gemcitabine DNA incorporation has high inter-assay precision and accuracy [15], making it unlikely that the variability in gemcitabine DNA incorporation was due to the measurement technique. "
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