Separation of quercetin, sexangularetin, kaempferol and isorhamnetin for simultaneous HPLC determination of flavonoid aglycones in inflorescences, leaves and fruits of three Sorbus species.

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the simultaneous determination of four flavonol aglycones (quercetin, QU; sexangularetin, SX; kaempferol, KA; isorhamnetin, IS) in hydrolyzed extracts from different plant parts of Sorbus aucuparia L., Sorbus aria (L.) Crantz. and Sorbus intermedia (Ehrh.) Pers. Separation of the four compounds was accomplished on a C18 Lichrosphere 100 column (5 microm, 250 mm x 4.6mm, i.d.) with a methanol gradient elution and recorded at 370 nm. The high resolution of critical bands - SX, KA and IS - was achieved with retention of the last peak (IS) in 19.5 min. The equilibration of the standard mixture by addition of HCl to an acid concentration equal that of hydrolyzed extracts injected was found to be necessary when minimizing calibration error. The correlation coefficients of all the calibration curves showed good linearity (r>0.9991) over the test range. The relative standard deviation of the method was less than 2.8% for intra- and inter-day assays, and the average recoveries were between 95.5 and 102.5%. High sensitivity was demonstrated with detection limits between 0.050 and 0.085 microg/ml. The level of total aglycones was found to be in the range of 687-1,515 mg/100g of dry weight in the inflorescences, 424-1,078 mg/100g in the leaves and 20-60 mg/100g in the fruits depending on the Sorbus species.