Fast separation of flavonoids by supercritical fluid chromatography using a column packed with a sub-2 μm particle stationary phase

The purpose of this study was to compare the effects of different chromatographic columns for the separation of seven flavonoids. Four different stationary phases are available, including bridged ethyl hybrid, BEH and the same hybrid phase modified with 2-ethylpyridine, CSH fluorophenyl, and HSS C-18 SB. The analytes included calycosin, genistein, medicarpin, calycosin-7-O-beta-D-glucoside, formononetin, formononetin-7-O-beta-D-glucoside, and liquiritigenin. The CSH fluorophenyl column was determined to be the most suitable and provided the fastest separation within 17 min using gradient elution with carbon dioxide as the mobile phase and methanol as the co-solvent. Good peak shapes were obtained, and the values of the peak asymmetry were close to 1.0 for all of the flavonoids. The resolution was more than 1.41 for all of the separated peaks. Baseline separation on the optimal columns was achieved by changing the co-solvent type and adjusting the temperature and pressure. Quantitative performance was evaluated under optimized conditions, and method validation was accomplished. The validation parameters, such as linearity, sensitivity, precision, and accuracy, were satisfactory. Good repeatability of both peak area (relative standard deviation < 1.02%) and retention time (relative standard deviation < 0.88%) was observed. The optimized chromatographic methods were successfully used for the determination of seven flavonoids in Radix astragali. The sensitivity was sufficient for the analysis of real samples.