RP-HPLC-DAD detrmination of flavonoids: separation of quercetin, luteolin and apigenin in Marchantia Convoluta

High performance liquid chromatography coupled with photodiode array detector (HPLC-DAD) has been reported to quantify isolated flavonoids or these compounds in complex biological matrices, such as Chinese herbal drugs and products from factories. This work was designed, therefore, to develop an HPLC-DAD system to separate quercetin, luteolin and apigenin and to quantify them in extractive solutions from Marchantia convoluta. Flavonoids were analyzed on a Kromasil RP-C₁₈ column; using a mobile phase, consisted of methanol- acetonitrile-acetic acid-phosphoric acid-H₂O (200:100:10:10:200, V/V); under the following conditions: detecting wavelength, 352 nm; flow rate, 0.60 ml/min; the sensitivity, 0.05 AUFS and the volume of injecting sample, 6.0 µl. The HPLC system was operated at ambient temperature (28±1°C). The method showed linearity for quercetin, luteolin and apigenin in the range 2.0-20.8, 2.2-24.0 and 1.6-20.0 µg/ml respectively, and the R.S.D. of the slope of the three lines was, respectively, 0.33%, 1.21% and 2.49% for quercetin, luteolin and apigenin. The aqueous and ethanol 80% extractive solutions showed linear response 1.5-15 µl/ml and ethanol 50% extractive solution in range 1.0-10 µl/ml. Precision and accuracy were determined for ethanol 80% extractive solution, in concentration of 10 µl/ml. The recoveries were 95.92-98.10%, 92.18-95.13% and 98.72-103.19% for quercetin, luteolin and apigenin respectively. RSD of results was 2.83-3.62%. The HPLC method showed an excellent performance in separating the flavonoids quercetin, luteolin and apigenin in Marchantia convoluta extracts, since the presence of interference has been previously evaluated and the mobile phase was chose carefully.