Quantification of Lupeol in Polyherbal Formulation by High-Performance Thin-Layer Chromatography Method Using Design of Experiments as a Tool to Assess Optimization of Method Development and Extraction Efficiency

A simple, specific, and quantitative high-performance thin-layer chromatographic (HPTLC) method has been developed for the quantitative determination of lupeol in 2 marketed formulations, namely, Manasamitra vatakam and Amree plus capsule. Chromatographic development was performed by using a pre-coated silica gel 60 F-254 aluminum-backed plate, and the development was carried out using toluene-ethyl acetate (9.48:0.52, V/V) as the optimized mobile phase. The developed TLC plates were derivatized by using anisaldehyde-sulfuric acid reagent. The detection of lupeol was carried out at 600 nm. Box-Behnken design was applied for optimization of the chromatographic conditions, and combinations of factors, such as mobile phase composition (volume of ethyl acetate) (A), chamber saturation time (B), and migration distance (C) likely to affect R-F, were identified from preliminary trials and further optimized using a response surface design. Among 3 factors, the significant factor found was the volume of ethyl acetate that resulted in higher change in the R-F value and can be considered as a critical method parameter. Full factorial design was applied for optimization of extraction efficiency. The factors selected for the optimization process were volume of methanol (A) and duration of extraction (B) with percentage yield of extract as response. The linear ranges were found to be 500-3000 ng per band. The accuracy and precision measured were less than 2% relative standard deviation for lupeol. The sensitivity of the method in terms of the limit of detection (LOD) and the limit of quantification (LOQ) was measured. The proposed method was found to be accurate, precise, reproducible, robust, and specific and can be applicable for the determination of lupeol in the quality-control testing of extract and polyherbal formulations.