Quantitative HPLC Analysis of Ascorbic Acid and Gallic Acid in Phyllanthus Emblica

Abstract

A reversed-phase high-performance liquid chromatographic method has been developed and validated for simultaneous estimation of ascorbic and gallic acid in Phyllanthus emblica L. (Euphorbiaceae). A C18 column was used with a gradient elution of methanol and 0.1% (v/v) acetic acid in HPLC-grade water as mobile phase at a flow rate of 0.9 mL min -1 . UV detection was performed at 278 nm. The method was validated for accuracy, precision, linearity, specificity and sensitivity in accordance with International Conference on Harmonisation guidelines. Amounts of ascorbic and gallic acid detected in freeze-dried extract of the plant were 4.58% and 0.59%. Total run time was 50 min. ascorbic and gallic acid was eluted with retention times of 3.60 and 10.77 min respectively. Validation revealed that the method is specific, accurate, precise, reliable and reproducible. Calibration plots were linear over the concentration ranges 30–90 μg mL -1 for ascorbic acid and 5–15 μg mL -1 for gallic acid, respectively. Limits of detection were 1.42 and 1.56 μg mL -1 and limits of quantification were 4.32 and 4.73 μg mL -1 for ascorbic and gallic acid, respectively. Recovery was 99.37 % and 98.68% for ascorbic and gallic acid, respectively and the coefficient of variance was <2.0% for both. In negative ESI mode, the spectra showed the signals at m/z of 174.98 for ascorbic acid and m/z of 168.98 for gallic acid .The high percentage recovery and low coefficient of variation confirm the suitability of the method for simultaneous analysis of ascorbic and gallic acid in Phyllanthus emblica .