Gradient HPLC-DAD for the Simultaneous Determination of Five Flavonoids in Plasma After Intravenously Administrated Ginkgo biloba Extract and its Application in the Study of Pharmacokinetics in Rats

A HPLC-DAD method was used and validated for the simultaneous determination of five flavonoids (rutin, quercitrin, quercetin, kaempferol, and isorahamnetin) in rat plasma. Chromatographic separation was performed using a Kromasil C18 column (250 × 4.6 mm, 5 µm) maintained at 35°C. The mobile phase was a mixture of methanol and 0.1% formic acid with a step linear gradient. At 1.0 mL/min flow rate, the eluent of five flavonoids were detected simultaneously at 350 nm with good separation. Under optimum conditions, good linear relationship between the peak area and the concentrations were obtained in the ranges of 0.2525 ∼ 20.2, 0.1208 ∼ 9.66, 0.1008 ∼ 20.16, 0.031 ∼ 2.46, and 0.098 ∼ 7.84 µg/mL for rutin, quercitrin, quercetin, kaempferol, and isorhamnetin, respectively. The correlation coefficient for each analyte was above 0.999. The intra-day and inter-day precisions were better than 7% and 10%. The detection limit (S/N = 3) for rutin, quercitrin, quercetin, kaempferol, and isorhamnetin were 0.01, 0.02, 0.006, 0.02 and 0.02 µg/mL, respectively. The method was validated for accuracy and precision, and it has been successfully applied to determine drug concentrations in rat plasma samples from rat that had been intravenously administrated Ginkgo biloba extract.