Simultaneous determination of 1-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl) uracil (FAU) and 1-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl) 5-methyluracil (FMAU) in human plasma by liquid chromatography/tandem mass spectrometry.

Abstract

A liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) assay was developed and validated for simultaneous determination of 1-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl) uracil (FAU) and its active metabolite 1-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl) 5-methyluracil (FMAU) in human plasma. FAU and FMAU were extracted from plasma samples using solid-phase extraction with Waters Sep-Pak® Vac C₁₈ cartridge. Chromatographic separation was achieved on a Waters Atlantis T3 C₁₈ column with a gradient mobile phase consisting of methanol and water with 0.45% formic acid (v/v) running at a flow rate of 0.2 ml/min. The analytes were monitored by triple quadrupole mass spectrometer under positive ionization mode. The lower limit of quantitation (LLOQ) was 10 and 2 ng/ml for FAU and FMAU in plasma, respectively. Calibration curves were linear over FAU and FMAU plasma concentration range of 10-2000 and 2-1000 ng/ml, respectively. The intra-day and inter-day accuracy and precision were within the generally accepted criteria for bioanalytical method (<15%). The method has been successfully employed to characterize the plasma pharmacokinetics of FAU and FMAU in cancer patients receiving 1-h intravenous infusion of FAU 50 mg/m².