Simultaneous determination of main phytoecdysones and triterpenoids in Radix Achyranthis Bidentatae by high-performance liquid chromatography with diode array-evaporative light scattering detectors and mass spectrometry

Abstract

A liquid chromatographic method was developed for simultaneous determination of two main types of bioactive compounds: four phytoecdysones and eight triterpenoids in Radix Achyranthis Bidentatae (RAB), i.e., polypodine B ( 1), ecdysterone ( 2), 25- R inokosterone ( 3), 25- S inokosterone ( 4), ginsenoside Ro ( 5), chikusetsusaponin IVa ( 6), zingibroside R 1 ( 7), chikusetsusaponin IVa ethyl ester ( 8), 28-deglucosyl-chikusetsusaponin IVa ( 9), PJS-1 ( 10), 28-deglucosyl-chikusetsusaponin IVa butyl ester ( 11), and oleanolic acid ( 12). Optimum separations were obtained with a Zorbax C18 column, using a gradient elution with 0.08% aqueous formic acid (containing 5% isopropyl alcohol) and acetonitrile as mobile phase. Phytoecdysones were detected by diode array detector (DAD) at 242 nm, whereas triterpenoids were monitored by evaporative light scattering detector (ELSD) connected in series with DAD, temperature for the drift tube was 110 °C and the nitrogen flow rate was 3.2 L min −1. The identity of the analytes was confirmed using retention times, ultraviolet absorbance and mass spectral data in comparison with reference compounds. The method was validated for acceptable precision (intra- and inter-day variation ≤ 4.87%), accuracy (recovery ≥ 88.9%) and sensitivity (LOD ≤ 0.43 μg mL −1 (DAD) and 26.0 μg mL −1 (ELSD), LOQ ≤ 0.97 μg mL −1 (DAD) and 46.5 μg mL −1 (ELSD), respectively). This rapid and reliable method was applied for the analysis of four cultivated and ten commercial samples. The results demonstrated that the method is suitable for routine analysis and quality control of RAB.