Application of Quality Risk Assessment and Design of Experiment in Optimizing Chromatographic Method for Estimation of Total Sarsasapogenin from the Roots of Asparagus racemosus Wild

The roots of the plant Asparagus racemosus (Wild) (family: Liliaceae) are used alone as a lactogenic and general tonic or incorporated as a major ingredient in many herbal and Ayurveda formulations. Shatavarin glycosides containing sarsasapogenin as aglycone were found bioactive, and present in predominantly higher amount. The experiments were planned to develop an analytical method for estimation of total sarsasapogenin from the extract using HPTLC, through a systematic approach after identifying the potential failure modes, termed as critical process parameters to achieve the analytical target profile. The associated risks were mitigated by studying the combined effect of the critical process parameters on area, Rf, and tailing factor corresponding to the sarsasapogenin peak, assigned as critical method attributes. The optimization studies were carried out through Design of Experiment approach and process parameters were finalized to achieve analytical target profile. The developed method could resolve the peak of sarsasapogenin from the hydrolyzed methanolic extract prepared from the root powder using silica gel-backed pre-coated aluminum plates. The optimized mobile phase was Hexane:Ethyl acetate:Formic acid (7.0:1.9:0.35, v/v/v); the plates were subjected to post-chromatographic derivatization using 5% v/v hydrochloric acid solution, and heating of the plats for 3 min at 110˚C. The chromatogram was recorded by scanning the plates in TLC scanner using fluorescence mode at 366 nm. The developed analytical method was found to yield linear response in the range of 40-280 ng/spot. The limit of quantification was determined to be 35.88 ng/spot. The method was found selective, accurate, and robust. The amount of sarsasapogenin was found to be determined using the developed method and found to be 2.31% w/w on dried weight basis from the dried root powder. This is the first report of a systematic approach in estimating sarsasapogenin from Asparagus sp.