Quality evaluation of golden saxifrage (Chrysosplenium alternifolium L.) through simultaneous determination of four bioactive flavonoids by high-performance liquid chromatography with PDA detection.

To control the quality of Chrysosplenium alternifolium L., a simple, fast and reliable method of high-performance liquid chromatography coupled with a photodiode array detector (HPLC-PDA) was developed and validated for simultaneous quantitative determination of four bioactive polymethoxylated flavonoids, namely chrysosplenosides B and D, and chrysosplenols B and D. Separation of the four analytes was accomplished on a C18 Hypersil ODS column (5 microm, 125 mm x 4 mm, i.d.) with an acetonitrile 10-100% (v/v) elution gradient, recorded at 345 nm. The equilibration of the methanol extracts and standard solution to 30% (v/v) of water was found to be necessary when minimizing viscosity differences between injections and the mobile phase, and thereby when minimizing distortions of analyte peaks and maximizing the resolution of critical bands of chrysosplenosides B and D. The correlation coefficients of all the calibration curves showed excellent linearity (r=0.9999) over the wide test range. The relative standard deviation of the method was less than 3.53 and 4.41% for intra- and inter-day assays, and the average recoveries were between 95.3 and 103.5%. High sensitivity was demonstrated with detection limits between 0.012 and 0.029 microg/ml (0.24-0.58 ng). C. alternifolium was found to be a valuable source of the flavonoids with the total content ranging from 2.456 to 4.314% of dry weight, depending on harvest time and cultivation area. The total flavonoids were also determined using the pharmacopeial UV-spectrophotometric method and a notable underestimation was found in comparison to the developed HPLC method.