#171 HPTLC method development for simultaneous estimation of Andrographolide, Gallic acid and Quercetin from herbal extracts with implementation of Quality by Design (QbD)

Abstract

Development of Quality by Design (QbD) based sensitive, economic and robust high performance thin layer chromatographic (HPTLC) method for simultaneous estimation of Andrographolide (A), Gallic acid (G) and Quercetin (Q) in herbal extracts. The chromatographic separation was carried on Merck TLC aluminum sheets of silica gel 60 F254 (20 × 10 cm) with a mobile phase of toluene: ethyl acetate: methanol: formic acid (4:3:1:0.05) with densitometric scanning at 232 and 256 nm. QbD approach was implemented by investigating Critical Method Parameters (CMPs) through regular two-level factorial design by using a trial version of Design Expert-13 software and evaluated for their effect on selected critical analytical attributes that are retention factor (R_f) and peak area. The Pareto charts, 3D response surface plots, and contour plots (Figure 1) for the constructed model demonstrated a significant influence of the selected factors on responses of A, G, and Q. The results obtained using the regular two-level factorial design for screening of CMPs indicated that the composition of the mobile phase ratio had a significant effect on the retention factors and areas of A, G, and Q. The method parameter of saturation time and wavelength had a minor effect on the R_f values, while the wavelength had a significant effect on all areas. Under the optimized conditions, the biomarkers were suitably resolved with R_f values of 0.38±0.02, 0.24±0.02, and 0.50±0.02 for A, G, and Q, respectively, with the linearity in the concentration range of 400- 2400 ng/band for A, 200-1200 ng/band for G and Q, high accuracy of 98.9-99.8%, and intra- and interday precision of %RSD <2%. This validated HPTLC method developed through a QbD approach was successfully employed for quantification of A, G, and Q in selected herbal extracts. The optimized method was proved to be economical, robust, time-saving and thus, can be used for simultaneous estimation of A, G, and Q in other herbal extracts and herbal formulations.

Fig. Contour plots of Critical Analytical Attributes (CAA)