Validated HPTLC Fingerprint Analysis for Simultaneous Estimation of Quercetin, Kaempferol and Asiatic Acid in Leaves of Two Different Chemotypes of Centella asiatica

Quercetin, Kaempferol and Asiatic acid in the methanolic extract of leaves of Two Chemotypes of Centella asiatica was developed and validated. Methods: The HPTLC method was developed using a suitable mobile phase Toluene: Ethyl acetate: Chloroform: Formic acid (6:6:4:1). The plates were derivatized with anisaldehyde reagent and densitometric determination was performed at wavelength 530nm.Results: The Rf values for Asiatic acid, Quercetin, and Kaempferol was 0.33, 0.44 and 0.55 respectively. The linear regression analysis for the calibration showed a linear relationship with r2= 0.9764 and 0.9823 for Quercetin and Kaempferol respectively in the concentration range of 100-1000ng/band. In case of Asiatic acid, the linear regression analysis data for the calibration plots showed a linear relationship with r2=0.9996 in the concentration range of 100-500ng/band. Precision was confirmed by performing replicate analysis on a single day and different days. The accuracy of the method was validated by conducting recovery studies using thestandard addition method. The average recovery for Quercetin was 98.60% for Kaempferol was 90.08% and for Asiatic acid was 97.66%. System suitability parameter was evaluated by spotting five replicates of all three standards. The %RSD was found to be below 2% with respect to peak area as well as Rf value. Robustness ofmethod was evaluated by changing mobile phase concentration from Toluene: Ethyl Acetate: Chloroform: Formic Acid (6:6:4:1) to (5:5:5:1), for which %RSD of peak areas was found to be below 10%.Conclusion: The HPTLC method developed was found to be simple, precise, accurate, suitable and robust for routine quality control of the powder of leaves of Centella asiatica.