Purification and Identification of Genistein in Ginkgo biloba Leaf Extract

The Ginkgo biloba leaf extract was hydrolyzed under acidic conditions and neutralized with 5% aqueous NaOH. The hydrolysate was extracted with a mixture of ethyl acetate and tetrahydrofuran (7: 3, v/v), and was concentrated by the vacuum evaporation of organic solvent. Genistein was isolated from the hydrolysate using silica gel column chromatography with a gradient elution of dichloromethane and acetic ether and further recrystallized in dichloromethane. The whole process was monitored using high performance liquid chromatograph, equipped with photodiode array detector and electrospray ionization mass spectrometer/mass spectrometer (HPLC-PDA-ESI-MS/MS). The HPLC chromatogram with UV at 260 nm showed that the purity of genistein was higher than 98%. The structure of genistein was identified using UV absorption curves, nuclear magnetic resonance (H-1 NMR, C-13 NMR) spectra, ESI-MS in negative mode and tandem MS spectra. The fragmentation mechanism for genistein was also discussed in detail: collision induced dissociation (CID) of the [M - H](-) I-ion (m/z 269) showed that the quasi-molecular ions [M - H](-) were prone to process rearrangement in the skeleton and lose neutrals, such as C3O2, CO2, CO. The Retro-Diels-Alder fragmentation was another characteristic decomposition of the ions.