Development of a HPLC Method for Quantification of Amentoflavone in Leaf Extracts of Three Calophyllum Species
This study describes the development and validation of a method that can quantify amentoflavone (AF) in methanol extracts of leaves of three Malaysian Calophyllum species via a high performance liquid chromatography (HPLC) technique. Chromatographic analysis was conducted using a reverse phase C18 column with water/acetonitrile (55:45) as the mobile phase with a flow rate of 1.0 mL/min and detection by UV at 270 nm. The method was validated in terms of linearity, precision and accuracy in accordance with International Conference on Harmonization (ICH) guidelines. The calibration curve showed good linearity (r2 > 0.9998) in a concentration range of 2.5 – 100 μg/mL with low detection and quantification limits of 1.33 and 4.02 μg/mL, respectively. The coefficient of variation for intra-day and inter-day precision was less than 2%, while recovery was more than 80%, indicating this method has good precision and accuracy. The developed HPLC method was found to be suitable and reliable for its intended application. Using this method, the amount of AF quantified from C. incrassatum, C. canum and C. rubiginosum leaf extracts was 9.42, 30.39 and 24.23 μg/mL, respectively.