Separation and determination of resibufogenin and cinobufagin in Chansu using reversed-phase liquid chromatography with γ-cyclodextrin as mobile-phase modifier

A simple, accurate, sensitive, and robust reversed-phase high-performance liquid chromatography (HPLC) method employing cyclodextrins as mobile phase additives has been developed in order to separate and determine resibufogenin and cinobufagin. Various factors affecting the separation for them, such as the nature of cyclodextrins, organic solvent, the concentration of γ-cyclodextrin, and temperature, were systematically studied. γ-cyclodextrin, as an effective mobile phase additive, can markedly improve the separation for resibufogenin and cinobufagin. The role of γ-cyclodextrin in the developed HPLC method is attributed to the formation of the inclusion complex between resibufogenin (or cinobufagin) and γ-cyclodextrin. So, the apparent formation constant (K(f) ) of the resibufogenin (or cinobufagin)/γ-cyclodextrin inclusion complex and the thermodynamic parameters of the inclusion process also were investigated. Resibufogenin (or cinobufagin) forms the 1:1 inclusion complexes with γ-cyclodextrin, and the resibufogenin/γ-cyclodextrin complex is more stable than the cinobufagin/γ-cyclodextrin complex. The K(f) values of resibufogenin and cinobufagin decrease with the increase of the temperature. The thermodynamic parameters of the inclusion reveal that the inclusion process between resibufogenin (or cinobufagin) and γ-cyclodextrin is spontaneous, exothermic, and enthalpically driven. Finally, the optimized method was successfully applied to separate and determine of resibufogenin and cinobufagin in the different Chansu (Bufonis venenum) samples.