ESTIMATION OF WITHAFERIN – A FROM ASHWAGANDHADI LEHYA USING HPLC

Ashwagandhadi lehya is an Ayurveda formulation, official in Ayurvedic Formulary of India and used as aphrodisiac, tonic, rejuvenating agents etc. It is one of the major components of Ashwagandhadi lehya is Withania somnifera which contain Withaferin –A. The study was undertaken to develop an analytical method using HPLC to estimate Withaferin – A, being a marker from, methanolic extract of Ashwagandhadi lehya as one of the quality parameters. Method development involved simple sample preparation without any purification, as well as isocratic separation of Withaferin-A. The method was subjected to validation involving establishing linearity, precision, accuracy, specificity, and robustness.Optimized mobile phase was acetonitrile: water (45: 55 v/v) contained 1% v/v glacial acetic acid. The column used was Phenomenex (250 × 4.6 mm, 5 µm) with flow rate set at 1 ml/min and chromatogram was recorded at 216 nm. The studies showed that peak corresponding to Withaferin – A within concentration range of 1-75 µg/ml was found to be correlated with respective concentration with correlation coefficient (r²) 0.99802 ± 0.0004. The developed analytical method was found precise, while accuracy was established using standard addition method. The average recovery was 95.59%, when determined at three different spiking levels (50%, 100% & 150%), in the pre-analyzed sample solution. LOD was found to be 0.3 µg/ml and LOQ was 0.1 µg/ml. Robustness study suggested that increase in acetonitrile proportion, increased R_T as well as increase in flow rate negatively impacted up on R_T. The content of Withaferin-A was estimated as 0.03 ± 0.001 mg % w/w. The results obtained from the studies showed that method was found to be accurate, precise and validated for estimation of Withaferin-A from Ashwagandhadi lehya.