Development and validation of a fast and simple RP-HPLC method for the determination of diosmin and hesperidin in combined tablet dosage form

Marjan Piponski, Tanja Bakovska Stoimenova, Marina Topkoska, Stefan Stefov, Magdalena Piponska, Gordana Trendovska Serafimovska


A fast, simple, accurate and robust reversed phase HPLC method for the simultaneous determination of two flavonoids, hesperidin and diosmin, in combined tablets was developed and validated. This method uses a short C8 column with dimensions of 75 mm × 4 mm with 5 µm particles thermostated at 30 °C, and a mobile phase composed of formic acid (pH 4.1 and 0.05%, V/V) and methanol (58:42, V/V), delivered at a flow rate of 1.2 ml/min, with UV detector signal monitoring at 280 nm and an injection volume of 5 µl. These chromatographic conditions yielded chromatograms with symmetric peaks of hesperidin, eluting at a ~2 min retention time, and diosmin, at a ~4.5 min retention time, with a total run time cycle of 6 min. The method validation parameters confirmed excellent values for accuracy, linearity and reproducibility. This method is suitable for routine analysis in pharmaceutical and food quality control laboratories.


Diosmin, Hesperidin, HPLC, UV, Method

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