HPLC determination of quercetine-type flavonoids, vitexin and clorogenic acid in hawthorn raw material and pharmaceutical preparations

A simple HPLC method with photodiode-array (PDA) ultraviolet detection was developed for the simultaneous determination of five active polyphenol components (chlorogenic acid, vitexin, hyperoside, isoquercitrin and quercetine) of hawthorn (Crataegus monogyna Jacq. (Lindm) or Crataegus laevigata (Poir.) D.C.) in dry herbal material (fruits and flower-bearing branches), dry extracts and Forticor® hard capsules. Following extraction from cited materials, these five compounds were successfully separated, using a Lichrospher® 100 RP 18e column with a gradient elution of mobile phase A (500 mL of water + 9.8 mL of 85% H₃PO₄ (w/w)) and mobile phase B (acetonitrile), during 70 minutes. The flow-rate was set at 1ml/min and the eluent was detected at 360nm.

The method is linear over the studied range of 1.05–210, 6.25–250, 25–250, 7.5–150, 1.04–10.4 µg/mL for chlorogenic acid, vitexin, hyperoside, isoquercitrin and quercetine, respectively. The correlation coefficient for each analyte was greater than 0.999.

The intra-day and inter-day precision of the analysis was better than 2 and 3%,

respectively. The accuracy of the analysis is verified by the standard addition method, using three different concentrations of each component in the tested materials, with recovery values obtained in the range of 98.04–102.47% (RSD ≤1.85%).

The detection limits were experimentally found to be 0.6, 0.5, 0.5, 0.8 and 0.3 µg/ml chlorogenic acid, vitexin, hyperoside, isoquercitrin and quercetine, respectively.

The developed method was used in the routine control of hawthorn raw materials and pharmaceutical dosage form made from hawthorn berries or flower-bearing branches.

Acknowledgements: The authors wish to thank Serbian Ministry of Science for financial support project number TR 20137.