Measurement of the anticancer agent gemcitabine and its deaminated metabolite at low concentrations in human plasma by liquid chromatography-mass spectrometry

摘要

A liquid chromatography/mass spectrometry (LC-MS) method has been developed and validated for the determination of the anticancer agent gemcitabine (dFdC) and its metabolite 2',2'-difluoro-2'-deoxyuridine (dFdU) in human plasma. An Oasis HLB solid phase extraction cartridge was used for plasma sample preparation. Separation of the analytes was achieved with a YMC ODS-AQ (5 μm, 120 A, 2.0 mm * 150 mm) column. The initial composition of the mobile phase was 2% methanol/98% 5 mM ammonium acetate at pH 6.8 (v/v), and the flow rate was 0.2 ml/min. An isocratic gradient was used for 3 min, followed by a linear gradient over 4 min to 30% methanol/70% 5 mM ammonium acetate at pH 6.8. The gradient returned to the initial conditions over 2 min and remained there for 6 min. The retention times of dFdC, dFdU, and the internal standard 5'-deoxy-5-fluorouridine (5'-DFUR) were 11.46, 12.63, and 13.58 min. The mass spectrometer was operated under negative electrospray ionization conditions. Single-ion-monitoring (SIM) mode was used for analyte quantitation at m/z 262 for [dFdC–H]~-, m/z 263 for [dFdU–H]~-, and m/z 245 for [5'-DFUR–H]~-. The average recoveries for dFdC, dFdU, and 5'-DFUR were 88.4, 84.6, and 99.3%, respectively. The linear calibration ranges were 5–1000 ng/ml for dFdC, and 5–5000 ng/ml for dFdU. The intra- and inter-assay precisions (%CV) were ≤3 and ≤7% at three concentration levels (50.0, 500, and 5000 ng/ml). The limits of quantitation (defined as 10 times of signal-to-noise ratio) were 3.16 ng/ml for dFdC, and 1.35 ng/ml for dFdU with 50-l sample injections. This method has been used for measuring plasma concentrations of dFdC and dFdU in samples from adult cancer patients in a Phase I trial of weekly dFdC given as 150 (or lower) mg/(m~2 24-h) infusion. The average plasma dFdC concentrations at 22- and 23-h into the infusion were 18.3 and 16.8 ng/ml at 150 and 100 mg/m2, respectively; the values for dFdU averaged 2950 and 1372 ng/ml.