SIMULTANEOUS ESTIMATION OF CAFFEIC AND CHLOROGENIC ACID CONTENT IN AMMI MAJUS SEED BY TLC AND HPLC

Objective: The present investigation formulated in order to identify and quantify of caffeic and chlorogenic acid content in Ammi majus seed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Methods: In the present study identification and quantification of caffeic and chlorogenic acid in Ammi majus seeds were investigated. Identification of these phenolic acids were done by using thin layer chromatography performed on a sheet of aluminium plates of silica gel as a stationary phase, suitable solvent systems which is Toluene: Ethyl acetate: Formic acid: Water (15:90:5:5), also use UV light and 5 % Fecl 3 for detection. This identification was further augmented by using high performance liquid chromatography and then quantified by using C18 column, isocratic mobile phase (acetonitrile: water (0.5% orthophosphoric acid) 15:85) and UV/Visible 320 nm detector. Results: Thin layer chromatography analysis of seed extracts revealed the presence of caffeic and chlorogenic acid in Ammi majus seed . The HPLC method was validated for linearity, accuracy, precision and limit of detection and quantification. The percentage of caffeic acid 0.031% in higher quantity than chlorogenic acid. Calibration curve was linear over concentration 5-200 µg/ml with correlation coefficients 0.998 and 0.999 for caffeic and chlorogenic acid. The relative standard deviation of the method was less than 2% for intra and inter-day precision assay and the average recoveries were between 97.47-101.92%. The proposed method was found to be linear, accurate, and precise. Conclusion: This study confirms the presence of caffeic and chlorogenic acid in Ammi majus seed extracts and the percentage of caffeic acid higher than chlorogenic acid.