QbD-Driven RP-HPLC Method Development and Validation for Trigonelline Quantification in Fenugreek Seeds and Phytosomal Formulation

Abstract

Background: Trigonelline is alkaloid known for its exceptional biological activities. The aim of the present work was to develop and validate Quality by Design based RP-HPLC method for estimation of trigonelline. Materials and Methods: The optimization process involves the design of experiments, utilizing response surface plots, contour plots, and overlay plots. The effective implementation of this method leads to a reduced retention time of 3 min, utilizing a reversed-phase C-18 column (5 mm, 4.6 mm, 250 mm, ZORBAX column). The mobile phase consists of methanol and water (40:60 v/v), with a flow rate of 1 mL/min at 265 nm. Results: The analytical technique has shown a linear response at 5-25 µg/mL, with R2=0.9994. The limits of detection and quantification were determined to be 0.425 µg/mL and 1.288 µg/mL, respectively, highlighting the sensitivity. Both precision assays, robustness and ruggedness demonstrated %RSD less than 2, indicating a reliable method. The accuracy was found to be 98.56% -99.76%, indicating the broad applicability. The established method was successfully employed to estimate the trigonelline content in fenugreek seeds and phytosomes with results showing 0.48% and 86.74%, respectively. Conclusion: This technique can be used to estimate trigonelline, for ensuring quality in phytopharmaceuticals.