Measurement of the anti-cancer agent gemcitabine in human plasma by high-performance liquid chromatography

A reversed-phase HPLC assay has been developed to determine the concentration of the anti-metabolite 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) in human plasma over the concentration range of 0.5-150 muM (0.13-39.44 mug/ml), and 2',2'-difluorodeoxyuridine (dFdU), the deaminated, inactive metabolite, over the range of 1.0-227 muM (0.26-60 mug/ml). After the addition of 20 nmol 2'-fluorodeoxycytidine (FdC) as an internal standard, 0.5-ml samples of plasma were subjected to acetonitrile precipitation, followed by analysis using a gradient reversed-phase HPLC assay with UV detection. A Phenomenex Columbus(TM) C-18 column, 5 mum, 150x4.6 mm, and a Waters C-18,C- 4mum, Nova-Pak Sentry guard column were used to achieve separation. FdC, dFdC and dFdU were monitored at 282, 269 and 258 nm, respectively, on a Waters 996 photodiode array detector. The mobile phase, run at a total flow-rate of 1.5 ml/min, was composed of two solvents: 50 mM ammonium acetate pH 5.0 in either 2% (solvent A) or 10% methanol (solvent B, v/v); 100% solvent A was run for 17 min, followed by a linear gradient to 100% solvent B over 14 min. FdC, dFdC and dFdU were resolved from endogenous compounds and had retention times of 13.6 +/- 0.5, 18.1 +/- 1.1 and 29.0 +/- 0.6 min, respectively. The assay was useful in measuring the plasma levels of both atialytes in samples obtained from adult cancer patients participating in a Phase I trial of gemcitabine given as either a 1- or 2-h infusion weekly for 3 of 4 weeks. (C) 2002 Elsevier Science B.V. All rights reserved.