Simultaneous analysis of six components in "Chai-Hu-Kuei-Chih-Tang" by high performance liquid chromatography

An HPLC method was applied for the simultaneous determination of six marker substances in the Chinese medicinal preparation "Chai-Hu-Kuei-Chih-Tang". These substances were paeoniflorin in Paeoniae Radix, cinnamic acid in Cinnamomi Ramulus, baicalin, baicalein, wogonin in Scutellariae Radix, and glycyrrhizin in Glycyrrhizae Radix. Extracted samples were run through the HPLC column (Cosmosil 5 C18-AR, 4.6 mm Φ × 250 mm) at 30°C and the column was developed with a mixture of 15% acetonitrile - 20 mM sodium 1-hexane sulfonate (pH 3.5) and 45% acetonitrile - 20 mM sodium 1-hexane sulfonate (pH 3.5) aqueous solution employing linear gradient elution method at a flow-rate of 1.0-1.3 ml/min. An UV detector was used for the detection of the marker substances. The detection wavelength varied with time. It was 230 nm during 0-15 min, 275 nm during 15-38 min, 270 nm during 38-60 min, 250 nm during 60-68 min, and lastly 275 nm during 68-75 min. Relative standard deviations of intra- and inter-day analysis were less than 5%. This separation method could be successfully applied for the simultaneous determination of six marker substances in "Chai-Hu-Kuei-Chih-Tang".