ABSTRACT
Saponins and sterones are two main characteristic components in Achyranthis Bidentatae Radix. In order to control the quality of Achyranthis Bidentatae Radix more effectively, a high-performance liquid chromatography (HPLC) method was established by using double external standards calibration method (DESCM) for simultaneous determination of the contents of achyranthoside C, achyranthoside D, β-ecdysterone, 25R-inokosterone and 25S-inokosterone in Achyranthis Bidentatae Radix. Chromatographic separation was achieved on an Agilent Poroshell 120 EC-C18 (150 mm × 4.6 mm, 2.7 µm) using 0.1% phosphoric acid in water and 0.1% phosphoric acid in acetonitrile as mobile phase. The flow rate was 0.8 mL·min-1 and the column temperature was set as 35 ℃, the injection volume was 5 μL and the total analytical time was 30 min. β-Ecdysterone was used as the reference to calculate the relative correction factors (RCF) and relative retention time (RRT) of 25R-inokosterone and 25S-inokosterone, achyranthoside D was used for achyranthoside C. The RCFs of 25R-inokosterone, 25S-inokosterone, and achyranthoside C were 1.116, 1.056, and 0.888 1, respectively. The double external standards calibration method (DESCM) and external standard method (ESM) were used to calculate the contents of five ingredients in Achyranthis Bidentatae Radix samples from different sources and the variation between the results was within acceptable limits (RE ≤ 5%). The results showed that the contents of two saponins and three sterones of Achyranthis Bidentatae Radix were 0.597%-1.916% and 0.044%-0.150% respectively. The total content of saponins was about 10 times that of sterones. In conclusion, the established DESCM allowed simultaneous determination of five ingredients (achyranthoside C, achyranthoside D, β-ecdysterone, 25R-inokosterone, and 25S-inokosterone) in Achyranthis Bidentatae Radix, providing a scientific and feasible overall quality evaluation method for Achyranthis Bidentatae Radix.