Measurement of daphnoretin in plasma of freely moving rat by liquid chromatography

Daphnoretin (7-hydroxyl-6-methoxy-3,7′-dicoumaryl ether), isolated from Wikstronemia indica C.A. Mey. (Thymelaceae), has been reported to induce rabbit platelet aggregation through protein kinase C activation and anticancer activity. In this study, we developed an automated blood sampling system coupled to a simple and sensitive HPLC system to determine plasma concentration of daphnoretin in rats. This method was applied to investigate the pharmacokinetics of daphnoretin in a freely moving rat. Separation of daphnoretin in the rat plasma was achieved using a reversed-phase C18 column (250 mm × 4.6 mm, 5 μm) with a mobile phase of methanol–10 mM NaH2PO4 (adjusted to pH 3.0 with H3PO4) (55:45, v/v), and the flow rate of 1.0 ml/min. The UV detector was set at 345 nm. The automated blood sampling system (DR-II has been applied for blood sampling in a conscious and freely moving rat. The blood samples were centrifuged at 3000 × g for 10 min and the plasma samples were then deproteinized by acetonitrile containing an internal standard (khellin 1 μg/ml). After centrifugation (8000 × g for 10 min), the aliquot of supernatant was injected into the HPLC system for analysis. The concentration–response relationship from the present method indicated linearity over a concentration range of 0.05–1.00 and 1.00–100 μg/ml. Intra- and inter-assay precision and accuracy of daphnoretin fell well within the predefined limits of acceptability (≤15%). After daphnoretin (500 mg/kg) was given orally, the maximum concentration was 0.17 μg/ml at the time of 5 min. The oral bioavailability was about 0.15%.