Development and validation of RP-HPLC method for the determination of stigmasterol in the botanical extract of .

A simple, rapid, accurate and precise RP-HPLC method was developed for the determination of stigmasterol in botanical extract of . Separation was achieved with acetonitrile and acetic acid in water (75:25% v/v) in isocratic mode at 210 nm. Single sharp peak of standard stigmasterol was detected at retention time 3.17 min which overlay with the peak of plant extract at 3.14 min. The calibration curve was found to be linear in a concentration range of 2-10 μg/ml with correlation coefficient of 0.998. The LOD and LOQ were found to be 1.50 μg/ml and 4.55 μg/ml respectively. Accuracy and precision was determined with overall recovery of 99.6-100.1% for stigmasterol and RSD values in both intra-day and inter-day repeatability assay lesser than 0.340%, respectively. The robustness study also indicated that there is no influence of minor changes in detecting wavelength and flow rate of mobile phase on the response.