Simultaneous Determination of Quercetin,Luteolin and Isorhamnetin in Pollen of Paeonia Rockii by RP-HPLC

OBJECTIVE To develope a RP-HPLC method for simultaneous determination of three flavonoids (quercetin, luteolin, isorhamnetin) in pollen from Paeonia rockii.METHODS The samples were pretreated, including reflux extraction with 75% ethanol for 2 h(with the ratio of liquor to material of 25:1), and then the extracting solution was hydrolyzed with 15% hydrochloric acid for another 2.5 h. Agilent Eclipse plus C₁₈ column (250 mm×4.6 mm, 5 μm) was adopted. The mobile phase was methanol-acetonitrile-0.1% phosphoric acid (25:13:62) at the flow rate of 1.0 mL·min^-1. The detection wavelength was 360 nm and the column temperature was 35℃.RESULTS Calibration curves were found to be linear in the range of 1.3-26.0 μg·mL^-1 for luteolin, 0.7-14.0 μg·mL^-1 for quercetin and 8.4-168.0 μg·mL^-1 for isorhamnetin. The correlation coefficients were greater or equal to 0.999 9. The average recoveries(n=9) of three flavonoids were 99.8%, 100.3% and 100.8%, respectively.CONCLUSION The developeded method is simple and efficient, which can be used for quality control of pollen of Paeonia rockii.