We developed an approach for screening bioactive compounds from botanical drug 
using multiple target-immobilized magnetic beads coupled with high performance 
liquid chromatography-mass spectrometry. This novel approach was called magnetic 
beads based multi-target affinity selection-mass spectrometry (MT-ASMS). It can 
enrich and identify different types of ligands from mixture extracts. Multiple 
targets (maltase, invertase, lipase) were immobilized on the magnetic beads by 
covalent linkage using 1-(3-dimethyl-aminopropyl)-3-ethyl-carbodiimide (EDC) and 
N-hydroxysuccinimide (NHS) as reaction reagents, respectively. The properties of 
enzyme conjugated magnetic beads were characterized using transmission electron 
microscopy, X-ray diffractometer and vibration sample magnetometer. Several 
factors including pH, ion strength, incubation time and temperature were 
optimized using three known ligands (caffeic acid, ferulic acid, and 
hesperidin). The established MT-ASMS approach was applied to screening for 
ligands from a Chinese medicine "Tang-Zhi-Qing", which was used to treat type II 
diabetes in China. Seven bound compounds were identified via liquid 
chromatography-mass spectrometry (LC/MS). Five active compounds including 
2,3,4,6-tetra-O-galloyl-D-glucose, 1,2,3,4-tetra-O-galloyl-D-glucose, 
1,2,3,4,6-penta-O-galloyl-d-glucose, quercetin-3-O-β-D-glucuronide and 
quercetin-3-O-β-D-glucoside were identified and their activities were validated 
by conventional inhibitory assay. Our findings suggested that the proposed 
approach is efficient in screening compounds with multiple activities from 
extracts of botanical drugs.