Validation and application by HPLC for simultaneous determination of vitexin-2″-O-glucoside, vitexin-2″-O-rhamnoside, rutin, vitexin, and hyperoside

Abstract

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 quantitation (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. Intra- 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.

Keywords: Flavonoids; Hawthorn; High-performance liquid chromatography; Leaves and fruits; Preparations.

Figure 1

Chemical structures and UV_max of five flavonoids in Chinese hawthorn leaves and fruits.

Figure 2

Typical chromatograms of five flavonoids in hawthorn leaves, hawthorn fruits and the preparations containing hawthorn leaves or fruits determined by HPLC-UV. (A) mixed standard solution of vitexin-2"-O-glucoside(I), vitexin-2"-O-rhamnoside(II), rutin(III), vitexin(IV), and hyperoside(V); (B) an unknown sample of hawthorn leaves; (C) an unknown sample of Yixintong tablets; (D) an unknown sample of hawthorn fruits; (E) an unknown sample of Jiangzhining tablets; (F) an unknown sample of Baohe pills; and (G) an unknown sample of Dashanzha pills.