Analysis of Cupressuflavone and Amentoflavone from Cupressus sempervirens L. and its Tissue Cultured Callus using HPLC-DAD Method

Abstract

An HPLC-DAD method has been optimized and validated to obtain qualitative and quantitative profiles of cupressuflavone, and amentoflavone from Cupressus sempervirens L. compared to the content of micropropagated plants. The plants analyzed were collected from two geographical sources in Egypt; Cupressus from the north coast and Cupressus from Future University (Cairo, Egypt). Isocratic reversed phase HPLC separation was achieved using a C18 column (150mm×4.6mm I.D., 5μm). Good resolution between cupressuflavone and amentoflavone was achieved using a mixture of water: acetonitrile: formic acid (60: 40: 0.1%, v/v) as a mobile phase at a flow rate of 1mLmin-1. Quantitation was achieved with photodiode array detector at the wavelength of maximum absorbance (330nm) based on peak area. The method was effective in the determination of the analyses of interest without any interference of other compounds or the matrix. The method was linear over a concentration range of 0.5-40μgml-1 (r2 >0.999) for biflavonoids. The limits of detection were 0.15 and 0.14μgml-1 and the limits of quantitation were 0.47 and 0.43μgml-1 for cupressuflavone and amentoflavone, respectively. Intraday and Interday precision (RSD%) was less than ±3% and intraday and Interday accuracy (RE%) of the method was found to be less than ±15% at all concentrations tested. The procedure was relatively simple, precise, rapid, reproducible, and could be routinely used for analysis of the cupressuflavone and amentoflavone flavonoids.