ESTIMATION OF SCOPOLETIN FROM A POLY-HERBAL FORMULATION COMPOSITION USING HPLC COUPLED WITH FLUORESCENCE DETECTOR THROUGH THE CONCEPT OF DESIGN OF EXPERIMENT.

HPLC coupled with a fluorescence detector was used to analyze bioactive phytoconstituent scopoletin from a poly-herbal composition derived from the extract prepared from roots of Argyreia nervosa, roots of Withania somnifera, and fruits of Tribulus terrestris. This analytical method was developed as a quality control tool for standardization of the composition to be formulated to enhance spermatogenesis. Chromatographic separation was achieved using Luna® (250 mm ×4.6 mm,100 Å,5 μm) C 18 column as a stationary phase, and water (0.01 M glacial acetic acid): methanol: acetonitrile (60:20:20, %v/v/v) as the mobile phase; passed through the column at a set flow rate of 1.0 ml min -1 . The elute in the flow cell was excited at 345 nm and the chromatogram was recorded at 444 nm as the emission wavelength. As a part of the analytical Quality by Design approach, systemic studied were conducted to identify potential risks affecting the critical attributes (area, resolution, retention time) of the analytical method, and mitigating the potential risks after optimizing the chromatographic parameters with the help of the Design of Experiment approach. The developed analytical method was subjected to the validation studies, which showed a linear relationship (r 2 = 0.9982) between the concentration and the area corresponding to scopoletin peak in the concentration range of 10-130 ng ml -1 . The method was found selective, sensitive, and precise. The recovery of the scopoletin was found in a range of 99.53 - 102.13 %; confirmed the accuracy of the analytical method. The amount of scopoletin was estimated to be 0.146 %w/w from the polyherbal composition.