The cytotoxic agent Gemcitabine (2',2'-difluoro-2'-deoxycytidine) has been proved to be effective in the treatment of malignant gliomas. A rapid, sensitive and specific ultra performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) assay using microdialysis sampling was developed and validated to quantify gemcitabine and its major metabolite 2',2'-difluoro-2'-deoxyuridine (dFdU) in Sprague-Dawley rat bearing 9L glioma. Microdialysis probes were surgically implanted into the area of rat brain tumor in the striatal hemisphere, and artificial cerebrospinal fluid was used as a perfusion medium. The samples were analyzed directly by UPLC-MS/MS after the addition of 5-bromouracil as an internal standard (IS). Separation was achieved on Agilent SB-C(18) (50 mm × 2.1mm I.D., 1.8 μm) column at 40 °C using an isocratic elution method with acetonitrile and 0.1% formic acid (4:96, v/v) at a flow rate of 0.2 mL/min. Detection was performed using electrospray ionization in positive ion selected reaction monitoring mode by monitoring the following ion transitions m/z 264.0→112.0 (gemcitabine), m/z 265.1→113.0 (dFdU) and m/z 190.9→173.8 (IS). The calibration curves of gemcitabine and dFdU were linear in the concentration range of 0.66-677.08 ng/mL and 0.31-312.00 ng/mL, respectively. The lower limit of quantification of gemcitabine and dFdU were 0.66 ng/mL and 0.31 ng/mL, respectively. The lower limit of detection of gemcitabine and dFdU were calculated to be 0.2 ng/mL and 0.1 ng/mL, respectively. All the validation data, such as intra- and inter-day precision, accuracy, selectivity and stability, were within the required limits. The validated method was simple, precise and accurate, which was successfully employed to determinate the concentrations of gemcitabine and dFdU in the extracellular fluid of rat brain tumor.
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