Development and Validation of a Multidetector HPLC Method for the Determination of Antioxidant Flavonoids of some Hypericum L. Species

A multidetector approach for the HPLC analysis of methanolic extracts of three Hypericum species is performed under isocratic conditions through a C18 (ODS) analytical column. Variable wavelength UV-VIS detector (λmax: 230 nm), fluorescence detector (ex: 250 nm em: 450 nm), and a programmable electrochemical detector (+1.30 V vs. Ag/AgCl) were employed simultaneously for detection. The mobile phase used was a combination of methanol and 0.01 M orthophosphoric acid (pH 7) (50:50, v/v). Retention time values were assigned relatively to those of the standards, and the flavonoid contents were quantified by the standard addition method. Rutin, quercetin-3′-glucoside (isoquercitrin), luteolin-4′-glucoside, quercetin-4′-glucoside, quercetin, naringenin, luteolin, and apigenin were chosen as the model compounds to undergo the validation studies with the three different detector systems. For the comparison of the detector performances, analytical method validation parameters were studied and displayed. Defined methods yielded pretty good results regarding linearity, precision (reproducibility), accuracy, limit of detection, and quantification.