Simultaneous determination of gemcitabine prodrug, gemcitabine and its major metabolite 2′, 2′-difluorodeoxyuridine in rat plasma by UFLC-MS/MS

To improve bioavailability and provide resistance to deamination , an array of gemcitabine (dFdC) prodrugs carrying the acyl modifications has been successful in the optimization of pharmacokinetic properties of dFdC, but the reports about 4- N -carbobenzoxy-dFdC (Cbz-dFdC), a dFdC prodrug bearing alkyloxycarbonyl modification, are relatively rare. Notably, in vivo enzymatic hydrolysis was an absolutely essential factor for the activation of these prodrugs, which is correlated with the anti-tumor activity. Therefore, detailed metabolism studies of Cbz-dFdC should be carried out for a more authentic pharmacodynamic evaluation. In order to detect the pharmacokinetic characteristics of Cbz-dFdC, a selective, sensitive and accurate method for the simultaneous determination of Cbz-dFdC, along with dFdC and its major metabolite dFdU in rat plasma was developed and validated using UFLC–MS/MS techniques. Column was at 40 °C for separation using an eluent with acetonitrile and 0.1% formic acid , 1 mM ammonium formate at a flow rate of 0.2 mL/min. Detection was performed using ESI source in positive ion selected reaction monitoring mode by monitoring the following ion transitions m / z 398.1 → 202.2 (Cbz-dFdC), m / z 264.1 → 112.0 (dFdC), m / z 265.3 → 113.2 (dFdU) and m / z 246.1 → 112.0 (IS). Analytes were extracted by simple precipitation with acetonitrile containing internal standards followed by liquid-liquid extraction with ethyl acetate . The calibration curves of Cbz-dFdC, dFdC and dFdU were linear in the concentration range of 2 to 500 ng/mL, 2 to 500 ng/mL and 40 to 10,000 ng/mL, respectively. The assay ranges selected for the three analytes were appropriate and minimized the need for reanalysis. All the validation data, such as intra- and inter-day precision, accuracy, selectivity and stability, were within the required limits. In conclusion, the sensitive analytical assay was selective and accurate for the determination of rat plasma concentrations of Cbz-dFdC, dFdC and dFdU from a single LC–MS/MS analysis and well-suited to support pharmacokinetic studies.