Standardization of an Extract

The presence of ursolic acid was identified by performing High-performance thin layer chromatography (HPTLC). The solvent system used for the analysis was toluene: ethyl acetate (8:2 v/v) as a binary mobile phase system. For spot detection, anisaldehyde sulfuric acid was used as the spraying reagent. Standard analytical grade of ursolic acid was used as a reference by dissolving in HPLC grade methanol. The obtained concentration was 1,070 µg/ml, which was further used for preparing the subsequent working standards. Camag Linomat V HPTLC system (Switzerland) equipped with 100 µl Camag syringe and scanner III was used for the current qualitative estimation. Sample and standard as narrow bands of a width of 3 mm were applied to precoated silica gel aluminum plate 60F-254.

An isocratic HPLC method was developed and validated for the identification of ursolic acid. A prepacked column, C18 (25 cm × 4.6 mm) 5 µm, with UV detector (210 nm) was used for the current study. The injection volume was 20 µl and its flow rate was maintained at 0.6 ml/min. Run time for standard and sample were 45 min and the data acquisition was done. For HPLC analytical development studies, methanol and acetonitrile (30:70, v/v) were selected as the binary mobile phase system. Before using, it was filtered and degassed. Thereafter, the standard solution was prepared by adding 10 mg of ursolic acid in methanol (10 ml) and subjected for sonication. The solution was settled to room temperature and diluted further with the help of diluent up to the mark.

Then the sample solution was prepared by taking 1.5 g of the test sample in iodine flask and adding 25 ml of water followed by 20 min of sonication. Reflux was done for about 30 min in reflux assembly and filtered. The process was repeated twice and the filtrate was evaporated to dryness. The obtained residue was dissolved in methanol and the volume was made up of 10 ml methanol. A further 0.5 ml of this solution was taken into a 10 ml volumetric flask and diluted with methanol. Injection of equal volumes of the standard solution was done and chromatograms were recorded along with measurement of peak area. The percentage of ursolic acid was calculated by using the formula:

At= Peak area of a test sample, As = Peak area of reference standard, Cs = Concentration of reference standard, Cu = Concentration of test sample, P = Potency of ursolic acid working standard

The developed method was validated according to ICH guidelines (Baranwal et al., 2012a). The parameters adopted for its validation are Linearity, Specificity, Accuracy, Range, Precision, Repeatability, Intermediate precision, Robustness, Limit of Detection, and Limit of Quantification (Manukumar and Umesha, 2015).