Objective: To investigate a new method for the enrichment of alkaloids

analyze the compositions of alkaloids

and look for new anti-asthmatic constituents in Ephedra sinica. Method: In order to reduce the interference by non-alkaloid components on qualitative analysis and to improve the efficiency of separation and preparation of alkaloids

alkaloids in E. sinica were enriched by using new solid phase extraction (SPE) packing in this study;and the fractions of the enriched alkaloids were prepared by applying high performance liquid chromatography (HPLC);HPLC-TOF-MS was used to identify the main components in each fraction;active constituents as β2-adrenergic receptor agonists were characterized by using label-free dynamic mass redistribution assays. Result: The results indicated that AC18 SPE packing had high selectivity for the enrichment of alkaloids in E. sinica. A mixed-mode chromatography method was developed to separate alkaloids based on a mixed-mode reversed phase/strong anion-exchange XCharge C18 column

and 14 fractions were prepared in total. In high performance liquid chromatography-mass spectrometry (HPLC-MS)

26 alkaloids were identified. Among the 14 fractions screened

11 fractions showed agonistic activity on β2-adrenergic receptor(β2-AR). Combined with HPLC-MS results for these 11 fractions

12 alkaloids displayed agonistic activity on β2-AR

including 5 groups of alkaloid and thier isomers:ephedrine/pseudoephedrine

methyl ephedrine/methyl pseudoephedrine

norephedrine/norpseudoephedrine (±) -1-phenyl-2-imido-1-propanol and (±) -tyramine betaine. Conclusion: The separation and characterization of alkaloid isomers in E. sinica provided guidance for discovering novel anti-asthmatic constituents.