Development and Validation of HPTLC Method for Quantitative Estimation of Oleanolic acid as Marker in Total Methanolic extract of Fruits of Randia dumetorum Lamk.

The objective of the present investigation was to develop a validated HPTLC method for the determination of oleanolic acid as marker in the Methanolic extract of fruits of Randia dumetorum Lamk. Analysis of oleanolic acid was performed on TLC aluminium plates pre-coated with silica gel 60F-254 as the stationary phase. The mobile phase consists of Toluene: Ethyl acetate: Glacial acetic acid (7:3:0.1 v/v/v). Linear ascending development was carried out in twin trough glass chamber. The plate was sprayed with 10% sulphuric acid, heated at 110°C and immediately scanned at 540nm using Camag TLC scanner III. The system was found to give compact spots for oleanolic acid (Rf value of 0.58 ± 0.01). The linear regression analysis data for the calibration plots showed good linear relationship with r2 = 0.9922 ± 0.0002 in the concentration range 50-500ng per spot. The mean value (± S.D) of slope and intercept were 5.989 ± 0.0491 and 211.547 ± 4.5092 respectively. According to ICH guidelines the method was validated for precision, recovery, robustness and ruggedness. The limits of detection and quantification were 10 ng/spot and 30 ng/spot respectively. The oleanolic acid content of methanolic extracts was 3.45%. Recovery values from 99.38 – 100.79 % showed excellent reliability and reproducibility of the method. Statistical analysis of the data showed that the method is reproducible and selective. Since the proposed mobile phase effectively resolves oleanolic acid, the developed HPTLC method can be applied for identification and quantification of oleanolic acid in herbal extracts and formulations.