Development and Validation of Stability Indicating Assay Methods for Betulinic Acid

Abstract

A simple, selective, precise stability indicating high performance thin layer chromatography and high performance liquid chromatography of isolated Betulinic acid was developed and validated. The stability indicating HPTLC method was developed and validated for quantification of Betulinic acid in different forced degradation conditions. Optimized mobile phase was used Chloroform: Toluene: Ethanol (4: 4: 1). The method is based on high performance thin layer chromatography using Precoated Silica Gel 60 F 254 plates with 250 μm thickness. Detection was carried out at 525nm wavelength. The Rf value of isolated Betulinic acid was 0.65 ± 0.02. The linear regression analysis data for the calibration plots showed good linear relationship with r= 0.998 with respect to peak area in the concentration range 200-900 ng per spot. The method was validated for precision. The limit of detection and quantification were 26.93 ng per spot and 81.61 ng per spot, respectively. The stability indicating HPLC method was developed and validated for quantification of Betulinic acid in different forced degradation conditions. Optimized mobile phase was used Acetonitrile: Water (93:7). The method is based on high performance liquid chromatography using Purosphere® STAR, Reversed Phase (C-18, 250mm × 4.6mm, 5μm) column. Detection was carried out at 207nm wavelength. The Rt value of isolated Betulinic acid was 7.55 ± 0.1. The linear regression analysis data for the calibration plots showed good linear relationship with r= 0.999 with respect to peak area in the concentration range 20-90 μg per ml. The method was validated for precision. The limit of detection and quantification were 0.3631 μg per ml and 1.101 μg per ml, respectively.Isolated Betulinic acid was subjected to acid and base hydrolysis, oxidation, photo degradation, and thermal degradation. The drug undergoes degradation under oxidation, acidic and basic conditions. This indicates that the drug is susceptible to acid hydrolysis, base hydrolysis and oxidation. As the methods could effectively quantify the isolated Betulinic acid in different forced degradation conditions, it can be employed as stability indicating method.