A High Performance Thin Layer Chromatographic Method Using a Design of Experiment Approach for Estimation of Phytochemicals in Extracts of Leaves.

OBJECTIVES

A systematic design of experiment (DoE) based sensitive, robust high performance thin layer chromatographic (HPTLC) method was established for simultaneous estimation of gallic acid (GA), quercetin (QT), and rutin (RT) from ethanolic and aqueous leaf extracts of .

MATERIALS AND METHODS

The chromatographic separation was carried on Merck TLC aluminum sheets of silica gel 60 F254 (10×10 cm) with mobile phase of toluene: ethyl acetate: methanol: formic acid (4.9:4.1:2:0.5, v/v/v/v) with densitometric scanning at 300 nm. The critical method parameters were initially identified by regular two level factorial design and further systematically optimized using a central composite design, evaluating the effect on selected critical analytical attributes, retention factor (RF), and peak area.

RESULTS

The Pareto charts, 3D response surface plots, and polynomial equations for the generated models suggested significant influence of the selected factors on responses of QT, GA, and RT. The desirability and overlay plots employed provided appropriate solutions that were experimentally validated. Under the optimized conditions, the biomarkers were suitably resolved with RF values of 0.64±0.02, 0.80±0.03, and 0.22±0.02 for GA, QT, and RT, respectively, with wide linear dynamic range (200-1200 ng/band each), high accuracy (98.1-99.4%), and intra- and interday precision (%RSD <2%). When employed for quantification of these biomarkers in extracts, the ethanolic and aqueous extracts exhibited higher content of QT (993.5 μg/g and 832 μg/g, respectively). The ethanolic extract showed a larger amount of RT (701 μg/g). In contrast, aqueous extract exhibited a higher proportion of GA (591.1 μg/g) compared to ethanolic extract (150 μg/g).

CONCLUSION

This validated HPTLC method developed through a DoE approach was successfully employed for quantification of GA, QT, and RT from extracts and may also be extended for their simultaneous estimation in other herbal extracts, thereby reducing time, and may serve as a cost effective tool for analysis.