Simultaneous Determinations of Cinobufagin and Its Metabolites by Reverse-Phase High Performance Liquid Chromatography in Rat Serum and Urine

Abstract

A high-performance liquid chromatography was developed for simultaneous determinations of Cinobufagin (CB) and its metabolites deacetylcinobufagin, 3-epideacetyl-cinobufagin, 3-ketodeacetylcinobufagin, 3-epicinobufagin and 3-ketodeacetylcinobufagin in serum and urine of rat. The biological samples were extracted with diethylether-ethylacetate (4:1v/v) in the presence of bufalin as the internal standard. Recoveries for CB and all other compounds were in the range of 80.8%–100.2%. Excellent resolution was obtained by reversed-phase chromatography on a 4.6 mm I.D.×150 mm ODS column using acetonitrile:water (50:50 v/v) as the mobile phase at a flow-rate of 0.7 ml/min with UV detector at 300 nm. Standard curve data revealed linearity over a range of 10–500 ng per ml serum and urine. The detection limits of CB and its metabolites were less than 10 ng/ml. Coefficients of variation for the analysis were less than 10. CB and its some metabolites were observed in