LC-MS/MS method for quantitation of gemcitabine and its metabolite 2′,2′-difluoro-2′-deoxyuridine in mouse plasma and brain tissue: Application to a preclinical pharmacokinetic study

Abstract

A simple, sensitive, and relatively fast assay was developed and validated for the quantitation of gemcitabine (dFdC) and its major metabolite 2′,2′-difluoro-2′-deoxyuridine (dFdU) in mouse plasma and brain tissue. The assay used a small sample (25 μL plasma and 5 mg brain) for extraction by protein precipitation. After dilution of the supernatant extract, 1 μL was injected into HPLC system for reverse phase chromatographic separation with a total run time of 8 min. Chromatographic resolution of dFdC and dFdU was achieved on a Gemini C18 column (50 × 4.6 mm, 3 μm) utilizing gradient elution. Multiple reaction monitoring (MRM) with positive/negative ion switching was performed for detection of dFdC and its internal standard (dFdC-IS) in positive ion mode and dFdU and its IS (dFdU-IS) in negative ion mode. Two calibration curves ranging from 5−2000 ng/mL and 250−50,000 ng/mL were generated for dFdC and dFdU in mouse plasma, respectively. For measurement of dFdC and dFdU in mouse brain tissue, another two curves were used ranging from 0.02 to 40 ng/mg and 1–40 ng/mg, respectively. This assay demonstrated excellent precision and accuracy within day and between days for simultaneous measurement of dFdC and dFdU at all the concentration levels in both matrices. The other parameters such as selectivity, sensitivity, matrix effects, recovery, and storage stability were also assessed for both analytes in each matrix. Compared to the previously reported methods, the sample extraction in the current assay was simplified significantly, and the analysis time was greatly shortened. We successfully applied the validated method to the analysis of dFdC and dFdU in mouse plasma, brain, and brain tumor tissue in a preclinical pharmacokinetic study.