Sensitive analysis and pharmacokinetic study of a novel gemcitabine carbamate prodrug and its active metabolite gemcitabine in rats using LC-ESI-MS/MS

FY363 is a new chemical entity of gemcitabine analog, which has been shown to have a significant inhibitory effect on cell proliferation in a variety of tumor cell lines in vitro. As a carbamate derivative, FY363 would be converted to the active metabolite gemcitabine through enzyme action in vivo. In order to clarify the exposure of FY363 prototype and its metabolite gemcitabine in vivo after administration of FY363, a sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed and validated to simultaneously determine FY363 and gemcitabine in rat plasma after liquid-liquid extraction with ethyl acetate. Chromatographic separation was achieved on a highly stable polar column of Synergi 4u Polar-RP 80A (4 μm, 4.6 × 250 mm) which has a unique ether - phenyl bonded phase. Gradient elution was accomplished with mobile phase system consisting of 5 mM ammonium formate buffer containing 0.1% formic acid and mixed organic solvents containing methanol-acetonitrile (3:2, v/v). Multiple reaction monitoring transitions were performed on triple quadrupole mass spectrometric detection in positive-ion mode with an electrospray ionization source. The calibration curves showed good linearity (r > 0.99) over the established concentration range of 1.0-1000 ng/mL both for FY363 and gemcitabine. The assay was validated to be selective, robust and reproducible. This well validated method was successfully applied to demonstrate the pharmacokinetic behavior and the metabolic transformation of FY363 in rats. Results revealed that about 20% of FY363 were converted into its active metabolite gemcitabine in rats by comparing the exposure of gemcitabine after the FY363 administration with that after direct gemcitabine administration at equimolar dose.