Simultaneous determination of seven flavonoids in Nervilia fordii with HPLC

The study is to develop an HPLC method for simultaneous determination of rhamnazin (1), rhamnocitrin (2), rhamnetin (3), rhamnazin-3-O-β-D-glucopyranoside (4), rhamnazin-3-O-β-D-xylopyranosyl-(1→4)- β-D-glucopyranoside (5), rhamnazin-3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranoside (6), and rhamnocitrin- 3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranoside (7) in Nervilia fordii.  The separation was performed on a Kromasil C₁₈ column (250 mm × 4.6 mm, 5 μm) with 0.4% phosphoric acid − acetonitrile as the mobile phase in a gradient elution at a flow rate of 1.0 mL·min⁻¹.  The detect wavelength was set at 256 nm, and the column temperature was set at 40 ℃.  There were good linear relationships between the logarithm values of concentrations and those of the peak areas of seven flavonoids (1−7) in the range of 0.55−70.00 μg·mL⁻¹ (r = 0.999 7), 0.86−110.00 μg·mL⁻¹ (r = 0.999 7), 0.39−50.00 μg·mL⁻¹ (r = 0.999 7), 0.55−70.00 μg·mL⁻¹ (r = 0.999 5), 1.33−170.00 μg·mL⁻¹ (r = 0.999 8), 1.33−170.00 μg·mL⁻¹ (r = 0.999 8), 0.16−20.00 μg·mL⁻¹ (r = 0.999 5), respectively.  The recoveries of the seven flavonoids were between 97.19%−99.45%, the relative standard deviations (RSDs) were between 0.91%−2.69%.  The established method is rapid, accurate with high repeatability, which could provide scientific evidence for the quality control of Nervilia fordii.