Rapid determination of gemcitabine and its metabolite in human plasma by LC-MSMS through micro protein precipitation with minimum matrix effect

Abstract

A novel micro protein precipitation was proposed and employed effectively for bio-sample preparation of only 5 μl of human plasma for rapid liquid chromatography- tandem mass spectrometric (LC-MSMS) determination of gemcitabine (dFdC) and its deaminated metabolite, 2′, 2′-difluorodeoxyuridine (dFdU).). Gemcitabine-13C, 15N2 Hydrochloride was the internal standard. Baseline chromatographic separation was achieved with an Alltima C 18 column (2.1×100 mm, 5μm) using isocratic mobile phase [10 mM ammonium acetate (pH = 6.8): methanol, 90:10]. The run time was only 5 min. The mass spectrometer was operated under a positive electrospray ionization condition and a multiple reaction monitoring mode. The linear calibration ranges were 2-2,000 ng/ml for gemcitabine and 20-20,000 ng/ml for dFdU. The recoveries for three analytes were in the same magnitude range from 87.7 to 89.7% with small variation. The intra and inter-day precisions for dFdC and dFdU were ≤ 5 and ≤ 7 and their accuracy ranged from 98 to 105.3% for dFdC and 93.8 to 104.9% for dFdU, respectively. Ion suppression effect was near negligible. This well validated assay has been applied for quantification of gemcitabine and dFdU in our gemcitabine phase II clinical trial study on Asian non-small cell lung cancer patients treated with different infusional rates of gemcitabine.