Five isoflavones, puerarin, daidzin, daidzein, 3′-hydroxy-puerarin and 3′-methoxy-puerarin were isolated and purified simultaneously from Pueraria lobata for the first time by high performance counter-current chromatography (HPCCC) using a system consisting of hexane–ethyl acetate–n-butanol–ethanol–water (0.5 : 2 : 1 : 0.5 : 3.5, v/v/v/v/v). In total, 155 mg of puerarin, 41 mg of daidzin, 12 mg of 3′-hydroxy-puerarin, 6 mg of 3′-methoxy-puerarin and 106 mg of daidzein were purified from 5.1 g of the ethyl acetate extract of P. lobata; the purities of the five isoflavones as determined by high performance liquid chromatography (HPLC) were 98.77%, 96.53%, 97.59%, 90.21% and 98.36%, respectively. The structures of the five compounds were identified by their retention time and the electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn) in the positive ion mode, and confirmed by 1H-NMR experiments. The characteristic ESI-MS fragmentation patterns of the five compounds are discussed.
