Development and Validation of HPLC Method for Quantification of Phytoconstituents in Phyllanthus emblica

A high performance liquid chromatography method coupled with diode array detection was developed to simultaneously determine eight different marker compounds in Phyllanthus emblica. These markers are ascorbic acid (1), gallic acid (2), methyl gallate (3), chlorogenic acid (4), ethyl gallate (5), ellagic acid (6), rutin (7) and chebulagic acid (8). HPLC analysis was carried out at wavelength 272nm. The developed method was able to determine the marker compounds with excellent resolution, precision and recovery. The chromatographic separation was performed on Thermo Scientific BDS HYPERSIL Phenyl reversed-phase column (250mm×4.6mm, 5μm). The mobile phase was consisted of 0.1% ortho-phosphoric acid: Methanol (95:05v/v) (A) and acetonitrile (B) at a flow rate of 1.5 ml/min gradient mode. Regression equations showed good linear relationships (R > 0.998) between the peak area of each marker and concentration. The assay was reproducible with overall intraand inter-day variation of less than 3.4%. The recoveries, measured at three concentration levels, varied from 97.8% to 101.1%. The method was applied to determine the amounts of the marker compounds in dried fruits of Phyllanthus emblica, and significant variations in phytoconstituents were observed.