An Experimental Design for the Analysis of 5-Caffeoylquinic Acid (5-CQA) in Ethanolic Extracts of Hibiscus (Hibiscus sabdariffa L.) Flower

This study optimized the extraction and analysis of 5-caffeoylquinic acid (5-CQA), the predominant chlorogenic acid in hibiscus flowers, using a sustainable ethanolic extraction method combined with high-performance liquid chromatography (HPLC) analysis. To identify the optimal solvent extraction conditions for 5-CQA from hibiscus, Response Surface Methodology (RSM) was employed, incorporating three factors: pH levels (2, 4.5, 7), extraction times (20, 40, 60 minutes) and solvent compositions (25v/v%, 50v/v%, 75v/v% ethanol). The temperature was maintained at the boiling point throughout the extraction period. RSM identified 25% ethanol, pH 7, and 25-minute heat treatment as the optimal extraction conditions for maximizing 5-CQA yield. Two HPLC columns; InertSustain Phenyl (A) and Synergi Polar (B), differing in length, diameter, and particle size were evaluated for chromatographic performance monitoring HPLC-UV signals at a wavelength of 325 nm. The mobile phase for both columns consisted of acetonitrile and acidified water (with orthophosphoric acid). Both HPLC columns achieved high correlation coefficients (0.9908 and 0.9965, A and B) between extracted 5-CQA and standard peaks. Under optimized conditions, the yields of 5-CQA were 1.43 mg/g and 1.48 mg/g dry hibiscus flower for columns A and B, respectively. Compared to literature-reported methods, the developed protocol offers high efficiency, reduced solvent usage, and minimal heat treatment, establishing it as a convenient, and sustainable alternative for 5-CQA extraction.