Abstract

The objective of the study was to develop and validate a simple, robust and accurate method for detection and simultaneous estimation of lupeol and beta-sitosterol as dual bioactive markers in ethanol and n-hexane extracts of Cissus quadrangularis Linn. and its marketed formulation using reverse phase highperformance liquid chromatography for standardization by integrated quality by design approach. A reverse phase high-performance liquid chromatography method with photodiode array detection was developed for simultaneous estimation and standardization of two major phytosterols compounds, betasitosterol and lupeol, in marketed formulation and extracts of Cissus quadrangularis Linn. by integrated quality by design approach. The design of experiments was generated by Box-Behnken three level 3 factorial design to optimize the pH of the mobile phase, percentage of the buffer and organic modifier for better separation between lupeol and beta-sitosterol. The optimized chromatographic separation was performed on a Merck Hibar LiChrospher^® reverse phase C₁₈ (250 mm×4.6 mm, 5 μm) column as a stationary phase with isocratic elution program using a mixture of mobile phase A: Methanol and acetonitrile in the ratio of 35:15 v/v and mobile phase B: 20 mM ammonium acetate 40 % (pH 4.5) with the flow rate of 1.0 ml/ min at 210 nm. The experimental results of the predicted method by the design of experiment were similar to the suggested responses and all fall in acceptance criteria with the desirability of 0.961. The developed method was validated as per International Conference on Harmonisation guidelines Q2(R1). The betasitosterol and lupeol showed good regression (R²>0.9995) within test ranges and the percent recovery was found to be 1.77 % w/w and 3.007 % w/w in marketed formulation. The recovery in ethanol extract was found to be 0.926 % and 3.63 %, and 0.065 % and 0.19 %, respectively, in n-hexane extract. The method was found to be highly specific without the interference of impurities. Thus, the method could be extended for the marker-based standardization and routine quality control of Cissus quadrangularis Linn. extracts and their formulation.