ABSTRACT
To establish a sensitive and specific LC-MS/MS method for determination of the binding conditions of ponicidin with bovine serum albumin (BSA) and analysis of its mechanism. The protein binding rates and related binding constants of ponicidin in BSA samples were determined by ultrafiltration and LC-MS/MS. Scatchard equation was used to calculate the binding constant (Ka) of ponicidin in BSA samples as well as the number of binding sites (n), and the mechanism on ponicidin binding with BSA was explored. The results showed that the average protein binding rate of ponicidin with BSA was 57.2%, mainly as grade Ⅰ intensive binding, and the relevant binding constant was 2.54×104 L•μg⁻¹, with a binding site number of n=0.75. The binding of ponicidin with BSA had no concentration dependence within the investigated concentrations. The established method in this study showed high sensitivity, specificity, simple operation and met the analysis requirements, and the calculation of binding constant laid foundation for the clinical drug interactions and pharmacokinetics research.
ABSTRACT
Objective: To establish HPLC-PDA fingerprint for study on Rabdosiae Rubescentis Herba (RRH) from different areas and to scientifically evaluate and effectively control the quality of RRH from different areas by HPLC fingerprint combined with principal component analysis (PCA). Methods: The fingerprint of RRH was established by HPLC-PDA and PCA and semblance analysis methods were applied to treating the experimental data in order to fine the similarity and difference among 18 batches of RRH from four different areas. The chromatographic procedure was carried out with DiamonsilTM C18 (250 mm × 4.6 mm, 5 μm) as an analytic column and a mixture consisting of acetonitrile-0.05% phosphoric acid in gradient as mobile phase, and the temperature of column was 30 ℃. The detection wavelength was set at 238 nm and the flow rate was 0.8 mL/min. Results: The common mode of HPLC characteristic chromatographic profile was set up with 19 common peaks, and four of them were identified as oridonin, ponicidin, lasiodonin, and rosmarinic acid. The similarities of 18 batches of RRH were between 0.421- 0.984. The result of PCA showed that the accumulative contribution rate of the first three factors was up to 87.3%, which were chosen to comprehensively evaluate the quality of RRH. Conclusion: The application of HPLC combined with PCA could quickly and objectively evaluate the quality difference of RRH in different batches.