An expedient assay for determination of gemcitabine and its metabolite in human plasma using isocratic ion-pair reversed-phase high-performance liquid chromatography

Abstract

An expedient method is presented for determination in human plasma of gemcitabine and its metabolite 2′,2′-difluorodeoxyuridine (dFdU) by ion-pair reversed-phase HPLC. Samples were simply prepared by protein precipitation. Separation was processed on a Thermo Hypersil column (250 × 4.6 mm, 5 μm Hypersil® BDS C18) with UV detection at 272 nm. The mobile phase consisted of 17% methanol and 83% phosphate buffer (20 mM, pH 3.1) containing 10 mM sodium 1-heptanesulfonate with a flow rate of 0.8 mL/min. The lower limit of quantification (LLOQ) of gemcitabine was 0.08 μg/mL with linear response over the range 0.08-20.0 μg/mL, and LLOQ of dFdU was 0.1 μg/mL with linear response over the range 0.1-50.0 μg/mL. Assay accuracy for both compounds was within ± 4%. The coefficient of variation (CV %) for intra- and interday precision for both compounds was