Determination of aesculin in rat plasma by high performance liquid chromatography method and its application to pharmacokinetics studies

A sensitive and reproducible high performance liquid chromatography method with UV detection was described for the determination of aesculin in rat plasma. After deproteinization by methanol using metronidazole as internal standard (I.S.), solutes were evaporated to dryness at 40 °C under a gentle stream of nitrogen. The residue was reconstituted in 100 μl of mobile phase and a volume of 20 μl was injected into the HPLC for analysis. Solutes were separated on a Diamonsil C18 column (250 mm × 4.6 mm i.d., 5 μm particle size, Dikma) protected by a ODS guard column (10 mm × 4.0 mm i.d., 5 μm particle size), using acetonitrile–0.1% triethylamine solution (adjusted to pH 3.0 using phosphoric acid) (10:90, v/v) as mobile phase (flow-rate 1.0 ml/min), and wavelength of the UV detector was set at 338 nm. No interference from any endogenous substances was observed during the elution of aesculin and internal standard (I.S., metronidazole). The retention times for I.S and aesculin were 10.4 and 12.4 min, respectively. The limit of quantification was evaluated to be 57.4 ng/ml and the limit of detection was 24.0 ng/ml. The method was used in the study of pharmacokinetics of aesculin after intraperitoneal injection (i.p.) administration in rats.