Rapid and simultaneous quantification of seven bioactive components in Radix Astragali based on pressurized liquid extraction combined with HPLC-ESI-MS/MS analysis

Abstract

A simple, rapid and sensitive pressurized liquid extraction (PLE) and high-performance liquid chromatography tandem mass spectrometric (HPLC-MS/MS) method has been developed for the simultaneous quantification of seven main bioactive components (calycosin, calycosin-7-O-β-D-glycoside, formononetin, formononetin-7-O-glycoside, astragaloside IV, astragaloside II and astragaloside III) in Radix Astragali. A gradient elution program was developed using a mobile phase consisting of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. Chromatographic separation was successfully achieved on an Agilent Zorbax XDB C₁₈ column (2.1 mm × 50 mm, 3.5 μm) with a flow rate of 0.50 mL min⁻¹. Detection was achieved on a triple quadrupole mass spectrometer using a multiple reaction monitoring (MRM) mode with an electrospray ionization source (ESI). The assay was fully validated to demonstrate its specificity, linearity, recovery, matrix effect, accuracy, precision and stability. The limits of detection and limits of quantification of the seven bioactive components were in the ranges of 0.03–0.60 and 0.10–2.00 ng mL⁻¹, respectively. The intra- and inter-day precisions were all less than 15%. Most importantly, the running time for analyzing one sample is only 4.5 min. The established PLE and HPLC-ESI-MS/MS method could serve as a simple, rapid and sensitive method for the quality evaluation of Radix Astragali.