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 x 4.6 mm, 5 microm) 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 x g for 10 min and the plasma samples were then deproteinized by acetonitrile containing an internal standard (khellin 1 microg/ml). After centrifugation (8000 x 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 microg/ml. Intra- and inter-assay precision and accuracy of daphnoretin fell well within the predefined limits of acceptability (< or = 15%). After daphnoretin (500 mg/kg) was given orally, the maximum concentration was 0.17 microg/ml at the time of 5 min. The oral bioavailability was about 0.15%.
Lin
Lie-Chwen
LC
National Research Institute of Chinese Medicine, 155-1, Li-Nong Street Section 2, Taipei 112, Taiwan.
Yang
Kuo-Yi
KY
Chen
Yen-Fei
YF
Wang
Shau-Chun
SC
Tsai
Tung-Hu
TH
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003374:Coumarins / Q000097:blood* / Q000493:pharmacokinetics; D008297:Male; D013058:Mass Spectrometry; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
D003374:Coumarins; C035316:daphnoretin