Protein Binding Rate Of In Vitro Stability Of The Rt-a And Its Metabolites

RT-A and its metabolite RT-B are highly active compounds of improve cardiovascular function, which were synthetized based on the structure of resveratrol and polydatin. They are two new candidate drugs in the stage of preclinical development. In accordance with the requirements of preclinical pharmacokinetic study, the stability of RT-A in vitro, the quantitative analysis method for determination of RT-B in rat plasma, and the determination of the rat plasma protein binding rate with RT-B were investigated. On the basis of experimental study, a scientific basis for the clinical development of the drug was provided.In this paper, HPLC was used to study the impact of temperature and pH on the stability of RT-A in buffer solution and its stability in rat plasma. The chromatography was performed on a Kromasil C₁₈ column with methanol-water-acetic acid (70:30:0.1, v/v/v) as the mobile phase and the absorbance was monitored at 318 nm. The column temperature was set at 40℃. The calibration curve was linear in the range of 1-80μg/mL with the correlation coefficient 0.998 3. The intra-day and inter-day precision were within 11 %, and accuracy were between 6.1 %. The chromatography of RT-A in plasma was the same to the above condition. The calibration curve was linear in the range of 1-40μg/mL with the correlation coefficient 0.997 5. The intra-day and inter-day precision were within 9.8 %, and accuracy were between 12.5 %. Experimental results show that the stability of RT-A was pH-and temperature-dependent. In neutral and acidic conditions, RT-A was found to be stable. When the pH value of the buffer solution was higher than 9.5, the decrease in the concentration level of RT-A was significant with an apparent first-order process after incubation at 37°C, the half-lives were 3.76 and 0.30 h at pH 9.5 and 11.0 respectively. A large amount of RT-B were detected simultaneously at pH 11.0. The stability of RT-A decrease with the increase of temperature, the half-lives were 50.2, 8.60, 5.37 and 1.54 h in phosphate buffers with pH 9.5 at 0, 25, 34 and 50℃. RT-A was unstable in plasma. Plasma enzyme inhibitor could improve the stability of RT-A in plasma.The HPLC method was developed to determine RT-B in rat plasma. The chromatography was performed on a Kromasil C₁₈ column with methanol-water- phosphoric acid (28:72:0.2, v/v/v) as the mobile phase and the absorbance was monitored at 318 nm. The column temperature was set at 40 ℃. The calibration curve was linear in the range of 0.04-10μg/mL with the correlation coefficient 0.999 4. The intra-day and inter-day precision were within 15 %, and accuracy were between 4.7 %. The assay was found to be simple and accurate to measure the concentrations of RT-B in plasma.The equilibrium dialysis to determine the plasma protein binding rate of RT-B was carried out to provide method for monitoring RT-B blood concentration. Drug-containing dialysis solution of low, middle and high concentration (1, 2 and 3μg/mL) were prepared(n =3), experiments were end after 4, 10, 24 and 48 h to inspect the balance time of plasma protein, finally 24 h was set for the final balance time. The plasma protein binding rate of RT-B at low, middle and high concentrations were (79.2±0.7) %, (80.1±0.9) % and (80.2±0.9) % respectively, and the mean plasma protein binding rate of RT-B was (79.8±0.8) %.