Simultaneous Determination of Two Types of Active Components with Different Physicochemical Properties in Kushen by HPLC-UV

摘要

A simple, reliable and accurate high performance liquid chromatography(HPLC) coupled with a UV detector at varied wavelength was developed for the quantitation of two types of anti-carcinogenic compounds, namely, Kushen alkaloids(KS-As) and Kushen flavonoids(KS-Fs), in Kushen. Their remarkable difference in physicochemical properties was circumvented by integrated optimization of column and mobile phase. The separation was achieved on an alkaline-resisting C18 column via gradient elution with acetonitrile(ACN) and 0.025%(volume ratio) diethylamine(DEA) in water as mobile phase. The flow rate was 0.7 mL/min, and the column temperature was maintained at 35 °C. UV detection wavelength was set at 225 nm to monitor KS-As, and then changed to 335 nm for KS-Fs at 26th min, which was kept for 30 min before returning to the original wavelength. All the calibration curves show good linearity(r2>0.999) within test ranges. The intra- and inter-day precision values of these components were less than 3.82%, and the average recovery rates obtained were in a range of 97.24%―102.64% for all the samples with RSD below 3.94%. This assay was successfully applied to determining the major active components in commercial herbal samples of Kushen. Moreover, the possible relationship between sample and the geographical region was visualized based on the contents of the 6 compounds in the light of principal component analysis(PCA). The results suggest that the proposed method facilitates the quality control of Kushen.