Simultaneous Determination of Triterpenoid Saponins from Pulsatilla koreana using High Performance Liquid Chromatography Coupled with a Charged Aerosol Detector (HPLC-CAD)

Abstract

Several triterpenoid saponins from root of Pulsatilla koreana Nakai (Ranunculaceae) were studied and their biological activities were reported. It is difficult to analyze triterpenoid saponins using HPLC-UV due to the lack of chromophores. So, evaporative light scattering detection (ELSD) is used as a valuable alternative to UV detection. More recently, a charged aerosol detection (CAD) has been developed to improve the sensitivity and reproducibility of ELSD. In this study, we developed and validated a novel method of high performance liquid chromatography coupled with a charged aerosol detector for the simultaneous determination of four triterpenoid saponins: pulsatilloside E. pulsatilla saponin H, anemoside B-4 and cussosaponin C. Analytes were separated by the Supelco Ascentis (R) Express C18 column (4.6 mm x 150 mm, 2.7 mu m) with gradient elution of methanol and water at a How rate of 0.8 mL/min at 30 degrees C. We examined various factors that could affect the sensitivity of the detectors, including various concentrations of additives, the pH oldie mobile phase, and the CAD range. Linear calibration curves were obtained within the concentration ranges of 2 - 200 mu g/mL for pulsatilloside E, anemoside B-4 and cussosaponin C, and 5 - 500 mu g/mL for pulsatilla saponin H with correlation coefficient (R-2) greater than 0.995. The limit of detection (LOD) and quantification (LOQ) were 0.04 - 0.2 and 2 - 5 mu g/mL, respectively. The validity of the developed HPLC-CAD method was confirmed by satisfactory values of linearity, intra- and inter-day accuracy and precision. This method could be successfully applied to quality evaluation, quality control and monitoring of Pulsatilla koreana.