Determination of protein binding rates of cinobufagin and resibufogenin with different plasmas by HPLC

The aim of this paper is to report the development of a method for the determination of the binding rates of cinobufagin (CBG) and resibufogenin (RBG) with different plasma proteins.  Equilibrium dialysis method was used to imitate the binding process between cinobufagin, resibufogenin and plasma proteins.  HPLC was employed to determine the concentration of cinobufagin and resibufogenin inside and outside of the dialysis membrane, and then on the basis of which protein binding rates were calculated.  The calibration curves of both cinobufagin and resibufogenin with human, rats’ and Beagle dogs’ plasma were linear in the range of 0.50 − 40.00 μg·mL⁻¹, while the buffer solution was 0.25 − 4.00 μg·mL⁻¹.  The extract recovery of cinobufagin was in the range of (68.73 ± 2.16) % − (79.27 ± 1.62) %, while that of resibufogenin was (71.59 ± 4.31) % − (83.47 ± 2.63) %.  The average plasma protein binding rates with cinobufagin were 85.63%, 80.21% and 70.10% in human, rats’ and Beagle dogs’ plasma, whereas those binding rates with resibufogenin were 84.51%, 75.11% and 70.60%, respectively.  The results suggested that the protein binding rates of cinobufagin and resibufogenin were of middle strength, which in rats’, Beagle dogs’ and human plasma were decreased in the following order: human plasma > rats’ plasma > Beagle’ dogs plasma.