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

摘要

In this study, we report a method for direct determinationnof gemcitabine incorporation into human DNA. Gemcitabinen(dFdC), a structural analog of the nucleosidendeoxycytidine (dC), derives its primary antitumor activitynthrough interruption of DNA synthesis. Unlike othernsurrogate measures, DNA incorporation provides a mechanisticnend point useful for dose optimization. DNA samplesn(ca. 25 μg) were hydrolyzed using a two-step enzymaticnprocedure to release dFdC which was subsequentlynquantified by LC-ESI-MS/MS using stable isotope labeledninternal standards and selected reaction monitoring (SRM).ndFdC was quantitated and reported relative to deoxyguanosinen(dG) since dG is the complementary base for bothndFdC and dC. The SRM channel for dG was detuned usingncollision energy as the attenuating parameter in order tonaccommodate the difference in relative abundance fornthese two analytes (>104) and enable simultaneousnquantification from the same injection. The assay wasnshown to be independent of the amount of DNAnanalyzed. The method was validated for clinical usenusing a 3 day procedure assessing precision, accuracy,nstability, selectivity, and robustness. The validatednranges for dFdC and dG were 5-7500 pg/mL andn0.1-150 μg/mL, respectively. Results are presentednwhich confirm that the ratio of dFdC to dG in DNAnisolated from tumor cells incubated with dFdC increasesnwith increased exposure to the drug and thatndFdC can also be quantified from DNA extracted fromnblood.