Abstract
A rapid, sensitive, precise and robust high-performance thin layer chromatography (HPTLC) and reverse phase high- performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous determination of withaferin-A and withanolide-A in the methanolic extract of Withania somnifera. The HPTLC method for steroidal lactones was performed on F254TLC plates with toluene: ethyl acetate: methanol: formic acid (60:8:5:1) as mobile phase and densitometric determination was carried out at λ=220 nm in reflectance/absorbance mode. The calibration plots showed a good linear relationship in the concentration range of 400-3200 ng/band with r2= 0.999 for both withaferin-A and withanolide-A. The recovery for withaferin-A and withanolide-A was found to be 98.13% and & 99.16% respectively. Further, a reverse-phase high performance liquid chromatography (RP-HPLC) gradient method with 0.01M phosphate buffer (pH 2.5 with orthophosphoric acid, A) and acetonitrile (B), was used as mobile phase & λ=220 nm was used as detection wavelength. Calibration curves for both the phytoconstituents were found to be linear in the range from 10-320 μg/ml. The correlation coefficients of linear regression analysis (r2) were found to be 0.989 & 0.9999 for withaferin-A and withanolide-A respectively, with respect to peak area. Further, % w/w recovery were found to be 98.16% and 98% for withaferin-A and withanolide-A respectively. Thus, the proposed HPTLC & RP-HPLC method provides a good resolution of withaferin-A and withanolide-A from other phytoconstituents present in the methanolic extract of roots of Withania somnifera and can be useful for their selective quantification.