Ultra-high performance supercritical fluid chromatography method for separation and quantitation of saikosaponins in herbal medicine

Abstract

Saikosaponins are the main active ingredients of Bupleuri Radix and have been shown to have hepatoprotective, immunomodulatory and anti-viral activities. Among the saikosaponins, saikosaponin a (SSa), saikosaponin b₁ (SSb₁) and saikosaponin b₂ (SSb₂) are a group of isomers, which are difficult to separate by HPLC. In this study, a new method for separation and quantitation of saikosaponins was established by using ultra-high performance supercritical fluid chromatography (UHPSFC). A Torus Diol column (100 mm × 3 mm, 1.7 μm) was applied in this study. The mobile phase CO₂ (A) was the main solvent with MeOH (B) as co-solvent. The results showed that the five saikosaponins were successfully separated within 22 min through optimization of chromatographic conditions. Besides, the UHPSFC method was applied for the quantitation of saikosaponins in a patent medicine Chaihu Dropping Pills, and demonstrated a good correlation coefficient (R²) ≥ 0.9990 in the range of 0.025 - 0.25 mg/mL. The recoveries of the five saikosaponins at three different concentrations were in the range of 90.23-99.84%. This study indicates that the proposed method has high separation efficiency in analyzing saikosaponins, which provides a new way for the separation and quantitation of saikosaponins in herbal medicines.

Keywords: Isomer; Saikosaponin; Separation; Ultra-high performance supercritical fluid chromatography (UHPSFC).