DoE-assisted RP-HPLC Method for Simultaneous Estimation of Caffeine, Eugenol, and Zingerone in Ashwagandha Green Tea

Background: The objective of this research was to develop a reliable RP-HPLC analytical technique for the concurrent determination of Caffeine, Eugenol, and Zingerone in a marketed formulation. Aim: This research aimed to develop a novel High-Performance Liquid Chromatography (HPLC) method that is simple and accurate for standardising the three Phytoconstituents, Caffeine, Eugenol, and Zingerone, in TATA TE-A-ME Ashwagandha Green Tea. Green tea is used because it has anti–oxidant, anti-inflammatory, and anti-bacterial properties. It includes dried variants of the Tulsi, Ginger, Turmeric, and Liquorice plant species. Method: A C-18 column was used as the stationary phase, while Methanol and water (35: 65 v/v) were used as the mobile phase in the method development and validation. For Caffeine, Eugenol, and Zingerone, detection and quantification were carried out at 254 nm. Result: The Retention time was 4.76, 8.64 and 10.48 for Caffeine, Eugenol and Zingerone, respectively. In terms of linearity, precision, specificity, Limit of Detection (LOD), Limit of Quantification (LOQ), accuracy, and robustness, the method was validated in accordance with ICH guidelines. The calibration plots for Caffeine, Eugenol and Zingerone in the range showed a satisfactory linear relationship according to the regression analysis data. Conclusion: The study integrates the Experiment Planning (DOE) methodologies with High-Performance Liquid Chromatography in the Reverse Phase (RP-HPLC) to develop an analytical method for estimating phytoconstituents. Design of Experiments is applied to Robustness. Major Findings: The developed and validated HPLC method is simple, precise, specific and robust, making it suitable for the simultaneous estimation of eugenol, zingerone, and caffeine in commercial formulations.