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

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2003-02, Vol.785 (1), p.65-72
• Main Authors: Keith, Bruce, Xu, Yan, Grem, Jean L.
• Format: Article
• Language: English
• Subjects: Antimetabolites, Antineoplastic - blood; Chromatography, High Pressure Liquid - methods; Deoxycytidine - analogs & derivatives; Deoxycytidine - blood; Gemcitabine; Humans; Reference Standards; Reproducibility of Results; Sensitivity and Specificity
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/S1570-0232(02)00859-0

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 μ M (0.13–39.44 μg/ml), and 2′,2′-difluorodeoxyuridine (dFdU), the deaminated, inactive metabolite, over the range of 1.0–227 μ M (0.26–60 μg/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™ C 18 column, 5 μm, 150×4.6 mm, and a Waters C 18, 4 μm, 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 m M 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 analytes 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.