Determination of Relative Contents of Isoflavones in Radix Puerariae Extract by Mass Spectrometry Guided High Performance Liquid Chromatography Using Quantitative Analysis of Multi-component with Single Marker

This study developed a method for the qualitative and quantitative analysis of isoflavones in Radix Puerariae extract using mass spectrometry (MS) and high performance liquid chromatography (HPLC). The relative contents of various kinds of isoflavones in the extract were determined using the quantitative analysis of multi-component with single marker (QAMS) method with puerarin as a single external standard. Chromatography was accomplished using a EcoPak C18 Plus column (4.6 mm × 150 mm, 5 μm). Using Radix Puerariae slices as control, the optimized chromatographic conditions were determined as follows: elution with a mobile phase consisting of 0.1% ammonia solution (pH 7.5), acetonitrile and methanol at a flow rate of 0.8 mL/min, an injection volume of 10 μL, column temperature of (35 ± 0.2) ℃, and detection wavelength of 250 nm. The MS conditions were set as follows: electrospray ionization source (ESI), full scan mode, negative ion mode detection, scan range of m/z 200–1 000, electrospray voltage of 12 000 psi, ion source temperature of 600 ℃, and nitrogen as carrier gas. The results showed that the separation of isoflavone compounds from Radix Puerariae slices was good, and 21 peaks were obtained with good reproducibility by HPLC with an ultra violet (HPLC-UV) (relative standard deviations (RSD) < 0.7%). The retention time reproducibility of 21 ion current peaks in MS was good (RSD < 1.0%), and the retention times of the chromatographic and ion current peaks were matched with each other. Totally 18 isoflavones were identified based on relevant literature. Good linearity was observed within the puerarin range of 0.052–500 μg/mL (R2 = 0.999 2), and the detection limit was 0.015 µg/mL (RSN ≥ 3). Furthermore, using puerarin as a reference standard, the contents of the 18 isoflavone compounds were relatively quantified. MS is useful for qualitative identification of plant extracts, which has complex composition, with the advantages of low consumption of materials, simple operation, rapidity and accurate results. The method proposed in this study can comprehensively evaluate the quality of extracts at a lower cost and conveniently, providing a reference for the development and application of Radix Puerariae as both an edible and medicinal material in health foods.