A simple, precise, and rapid high-performance liquid chromatographic method was developed and validated for the simultaneous determination of vitexin-2"-O-glucoside, vitexin-2"-O-rhamnoside, rutin, vitexin, and hyperoside. The HPLC separation was performed using a Shim-pack VP-ODS C18 column (250 mm × 4.6 mm i.d. , 5 μm) with the isocratic mobile phase consisting of tetrahydrofuran/acetonitrile/0.05% phosphoric acid solution (20:3:77, v/v/v), and the flow rate was set at 1.0 mL/min. UV detection was carried out at a wavelength of 360 nm and the whole analysis took 25 min. The method was linear in the range of 4.12-206.00 μg/mL for vitexin-2"-O-glucoside, 4.05-202.50 μg/mL for vitexin-2"-O-rhamnoside, 1.64-82.00 μg/mL for rutin, 1.74-87.00 μg/mL for vitexin, and 1.41-70.60 μg/mL for hyperoside with the correlation coefficient for each analyte more than 0.998.The limit of detection (LOD) and limit of qnantitation (LOQ) were 0.6 and 2 ng for vitexin-2"-O-glucoside, 0.6 and 2 ng for vitexin-2"-O-rhamnoside, 0.3 and 1 ng for rutin, 1 and 3 ng for vitexin, and 0.5 and 2 ng for hyperoside, respectively. Lntra- and inter-day precision and accuracy (RSD) were less than 3%. The developed HPLC method was successfully applied to the analysis of five flavonoids in hawthorn leaves, hawthorn fruits, and the preparations containing hawthorn leaves or fruits.
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