IMPROVED AND RAPID HPLC-PDA METHOD FOR IDENTIFICATION AND QUANTIFICATION OF SWERTIAMARIN IN THE AERIAL PARTS OF ENICOSTEMMA AXILLARE

A simple, improved, rapid and precise, reverse phase high performance liquid chromatography-photo diode array (HPLC-PDA) method has been developed and validated for the separation, identification and quantification of swertiamarin in aerial parts of Enicostemma axillare. The highest (38.12±1.74%) amount of swertiamarin was found in cold water and the lowest amount (20.13±0.84%) in hot water extracts obtained by using two different solvents. The separation of swertiamarin was achieved on a C18 column using the solvent system consisting of a mixture of methanol-water (1:1) as a mobile phase in a gradient flow elution mode followed by UV detection at 238 nm. The highest amount of swertiamarin was detected at 238 nm instead of detection at 227 nm and 254 nm. The developed method was validated using International Conference on Harmonisation (ICH) guidelines. Calibration curves offered good linear regression (r2 0.998) within the test ranges. In intraday assay, the maximum relative standard deviation (RSD; %) values were found to be 0.12, 0.1 and 1.59 for low (6 μg/mL), medium (20 μg/ mL and high (60 μg/mL) concentrations of swertiamarin. In interday assay, the maximum RSD (%) values were found to be 2.68, 2.71 and 2.88 for low, medium and high concentrations, respectively. Limit of detection (LOD) and limit of quantification (LOQ) were calculated to be 4 μg/mL and 6 μg/mL whereas analytical recovery ranged from 95.80%–101.76%.