Simultaneous determination of glycyrrhizin, glycyrrhetic acid and glycyrrhetic acid mono-glucuronide in Shakuyaku-kanzo-to incubated with rat feces by semi-micro high-performance liquid chromatography

A method for semi-micro high-performance liquid chromatography (HPLC) has been established for the simultaneous determination of glycyrrhizin (GL), glycyrrhetic acid (GA) and glycyrrhetic acid mono-glucuronide (GAMG) in incubation mixtures of rat feces with Shakuyaku-kanzo-to decoction (combination of licorice root and peony root). The analysis could be accomplished within 20 min with a TSKgel ODS-80TsQA (150 x 2.0 mm i.d.) column by linear gradient elution using a mobile phase containing aqueous phosphoric acid and acetonitrile at a flow rate of 0.2 ml x min(-1), a thermostatic oven at 25 degrees C, and detection at 254 nm. The detection limits of these compounds were 0.1-0.85 pmol per injection (5 microl). The concentrations of GL and its metabolites in the incubation mixture after continuous consumption of Shakuyaku-kanzo-to were significantly different compared with those of untreated control. GL-hydrolysis of rat feces was enhanced by pre-consumption of Shakuyaku-kanzo-to.