Determination of rutin in human plasma by high-performance liquid chromatography utilizing solid-phase extraction and ultraviolet detection

Abstract

An HPLC method for determining a flavonol glycoside, rutin, in human plasma is presented for application to the pharmacokinetic study. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using kaempferol-3-rutinoside as an internal standard. Solid-phase extraction was performed on an Oasis MAX cartridge possessing reversed-phase and anion-exchange functions (recovery, approximately 80%). The HPLC assay was carried out using a Luna ODS-2 column (150×2.1 mm I.D., 5 μm particle size). The mobile phase was acetonitrile–10 m M ammonium acetate solution containing 0.3 m M EDTA–glacial acetic acid (16.5:82.5:1, v/v, pH 3.8). The flow-rate was 0.3 ml/min. The detection wavelength was set at 370 nm. Calibration of the overall analytical procedure gave a linear signal ( r>0.9999) over a concentration range of 3–1000 ng/ml of rutin in plasma. The lower limit of quantification was ca. 5 ng/ml of rutin in plasma. The detection limit (defined as signal-to-noise ratio of about 3) was approximately 0.75 ng/ml. A preliminary experiment to investigate the plasma concentration of rutin after oral administration of 500 mg of rutin to a healthy volunteer demonstrated that the present method was suitable for determining rutin in human plasma.