HPTLC METHOD VALIDATION FOR DETECTION AND QUANTIFICATION OF BETULINIC ACID IN ANCISTROCLADUS HEYNEANUS WALL EX. J. GRAHAM
Keywords:
HPTLC, Betulinic Acid, Ancistrocladus heyneanus, Rf value, Method Validation, International Conference on Harmonization (ICH)Abstract
Objective: The present study aims at standardization of Thin Layer Chromatography (TLC) parameters and Validation of High Performance Thin Layer Chromatography (HPTLC) method for detection and quantification of Betulinic Acid (BA) in Ancistrocladus heyneanus Wall ex. J. Graham.
Methods: HPTLC was performed on 20x10 cm HPTLC Plates coated with Silica gel 60 F254 using mobile phase Toluene: Ethyl Acetate: Methanol (16:2:2 v/v) and Anisaldehyde Sulphuric acid as derivatizing agent. The double developed plate was scanned at 540 nm for determination of Rf values and absorbance spectra corresponding to each band. The Method Validation was carried out according to ICH (International Conference on Harmonization) guidelines. The standard of Betulinic acid assayed along with sample for determination of concentration of Betulinic acid in stem extract.
Results and Discussion: The TLC parameters were standardized and the Rf of BA was determined to be 0.67. The validation data was scrutinized. The values of Linearity (r2> 0.99), Method precision (% RSD=1.41), Intermediate precision (% RSD- 2.55-3.29), Accuracy (% recovery 93.6) were determined. The concentration of Betulinic Acid in the Stem extract of Ancistrocladus heyneanus was found to be 0.05 %.
Conclusion: The results of Method Validation obtained found satisfactory and indicate the successful validation of HPTLC method for Quantitative determination of Betulinic Acid (BA) from stem extract of Ancistrocladus heyneanus Wall ex. J. Graham.
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