Simultaneous determination of baicalin, rhein and berberine in rat plasma by column-switching high-performance liquid chromatography

A simple HPLC method using column-switching and ultraviolet detection was developed for the simultaneous determination of baicalin (BA), rhein (RH) and berberine (BE) in rat plasma. Plasma samples were injected directly onto a Capcell Pak MF C8 column (150 mm × 4.6 mm i.d.) to remove protein and to be pre-separated by an isocratic elution using 50 mmol/L phosphate sodium (pH 6.85)–acetonitrile (10:1, v/v). After the drug-containing fractions were transferred to a Kromasil C18 column (150 mm × 4.6 mm i.d.) by a valve switching step, the valve position was switched back and the main separation was performed by an isocratic elution using triethylamine adjusted 20 mmol/L phosphoric acid (pH 6.78)–acetonitrile (4:1, v/v). The flow rate was always 1.0 mL/min. The calibration curve showed excellent linear relationship (r ≥ 0.9997) over the concentration range of 0.4–7.9 μg/mL for baicalin, 0.2–7.8 μg/mL for rhein and 0.4–7.7 μg/mL for berberine in rat plasma. The intra- and inter-day assay precisions (R.S.D.) of three analytes were in the range of 0.34–4.3% and the accuracies were between 98.0% and 102.4%. Their recoveries were all greater than 95%. The method was successfully applied to the multi-constituents plasma concentration–time curve study after oral administration of a traditional Chinese medicine prescription Xiexin-Tang in rats.