Box—Behnken Design-Assisted Optimization for Simultaneous Estimation of Quercetin, Kaempferol, and Keto-β-Boswellic Acid by High-Performance Thin-Layer Chromatography Method

Summary A simple, specific, and quantitative high-performance thin-layer chromatography (HPTLC) method has been developed for the simultaneous determination of quercetin, kaempferol, and keto-β-boswellic acid in the herbal extracts of Spinacia oleracea and Boswellia serrata . Chromatographic development was performed using n -hexane—ethyl acetate—glacial acetic acid (6.4:3.6:0.2, % v/v ) as optimized mobile phase on TLC aluminum plate precoated with silica gel 60 F 254 and optimized resolution between quercetin and kaempferol (R 12 ) and between kaempferol and KBBA (R 23 ) were 2.11 and 2, respectively. The detection of quercetin, kaempferol, and keto-β-boswellic acid was carried out at 272 nm. Box—Behnken design was applied for optimization of the chromatographic conditions and combination of factors such as the volume of n -hexane (A), solvent front (B), and chamber saturation time (C) which were likely to affect R F , and resolution of quercetin, kaempferol, and keto-β-boswellic acid were identified from preliminary trials and further optimized using response surface design. The drugs were satisfactorily resolved with R F values of 0.22 ± 0.02, 0.40 ± 0.02, and 0.54 ± 0.02 for quercetin, kaempferol, and keto-β-boswellic acid, respectively. The linear ranges were found to be 1500–3500, 200–600, and 1040–3120 ng band −1 for quercetin, kaempferol, and keto-β-boswellic acid. The accuracy and precision measured were less than 2% relative standard deviation for all markers. The sensitivity of the method in terms of limit of detection (LOD) and limit of quantitation (LOQ) were measured. Among three factors, the significant factor found was the volume of hexane that resulted in a change in the R F of all three markers. The proposed method was found to be accurate, precise, reproducible, robust, and specific and can be applicable for the simultaneous determination of quercetin, kaempferol, and keto-β-boswellic acid for the quality control testing of extracts and medicinal plants.