Identification of isoflavonoids in Radix Puerariae for quality control using on-line high performance liquid chromatography-diode array detector-electrospray ionization-mass spectrometry coupled with post-column derivatization

Radix Puerariae (RP) is a botanical supplement widely used as a nutraceutical in functional foods. The developed method by coupling HPLC-DAD-IT-TOF-MSn with post-column derivatization (PCD) has been used for separation and identification of 22 isoflavones in RP. In addition, a total of 15 isoflavones were simultaneously determined in one single HPLC run with photodiode array detection (DAD), 8 of which were quantified in Radix Puerariae for the first time. Content determination results were used to comprehensively evaluate qualities of Radix Puerariae samples from different natural habitats in China. A YMC™ C18 column (150mm×4.6mm i.d.; 5μm particle size) and gradient elution (acetonitrile and water with 0.02% (v/v) of formic acid) were used during the analysis. All calibration curves showed good linearity (r>0.9996) within the test ranges. The intra-day and inter-day stability for 15 analytes were less than 1.32% and 1.87%, respectively. The recoveries were between 95.1% and 103.5%. The established method was simple, feasible and practical, and could be applied to routine analysis of Radix Puerariae and its preparation. The research set a good example for the comprehensive quality control of Radix Puerariae. ► LC/MS coupled with post-column derivatization is used for structure identification. ► 22 isoflavonoids were separated and accurately identified in Radix Puerariae. ► 15 isoflavonoids were simultaneous determinated for quality control. ► 8 characteristic isoflavonoids were quantified firstly. ► 20 samples of Puerariae Radix were clustered analyzed based on content determination.