LC–DAD Analysis of Six Bioactive Constituents in Rat Urine and Bile after Oral Administration of Luan-Pao-Prescription

Abstract

A simple and sensitive high-performance liquid chromatographic method with diode-array detection has been developed and validated for determination of six bioactive constituents, baicalin (BG), wogonoside (WG), baicalein (BA), wogonin (WO), oroxylin A (OA), and rhein (RHE), in rat urine and bile after oral administration of Luan-Pao-Prescription. The separation was performed on a C18 column at 30 °C with a mobile phase gradient prepared from 0.5% aqueous glacial acetic acid and acetonitrile. Detection was at 320 nm for BG, WG, BA, WO, and OA, and 254 nm for RHE, for both biological fluids. Because of the properties of the compounds of interest in urine and bile, two methods of sample preparation were used—solid phase extraction for monitoring BG and WG and liquid–liquid extraction for BA, WO, OA, and RHE. The HPLC method was validated for linearity, LOD, LLOQ, precision, accuracy, and recovery from the biological fluids in accordance with ICH guidelines. The method was successfully used to investigate urinary and biliary excretion of the six constituents in rats.