Development and Validation of an HPLC-DAD Method for the Simultaneous Analysis of Phenolic Compounds

Phenolic compounds are natural substances that exhibit different functional bioactivities and provide health-protective actions against chronic illnesses. The vast potential of these compounds in health and other sectors demands the establishment of analytical procedures for their immediate and simultaneous analysis. In this study, a high-performance liquid chromatography with diode-array detection (HPLC-DAD) method was developed and validated for the simultaneous analysis of gallic acid, catechin, epicatechin, rutin hydrate, caffeic acid, syringic acid, ellagic acid, p-coumaric acid, trans-ferulic acid, myricetin, resveratrol, and quercetin. The chromatographic separation of the selected polyphenols was carried out in a reversed-phase Inertsil ODS-3 column (250mm x 4.5mm x 5µm) at a flow rate of 0.8 mL/min, injection volume of 20 µL, and column temperature of 30°C. The detection and quantification of phenolic compounds were done at specific wavelengths (254, 275, 305, and 325 nm) using gradient elution for 40 minutes, with acidified water and acetonitrile solution as mobile phase. Validation of the established analytical procedure showed that the coefficient of determination (R2 > 0.99), limit of detection (0.01 to 0.35 µg/mL), limit of quantitation (0.03 to 1.07 µg/mL), recovery values (98.33 to 101.12%), and repeatability (RSD < 5%) respectively indicated a linear, sensitive, accurate, and precise analytical method for the simultaneous chromatographic analysis of the 12 phenolic compounds. Overall, the developed HPLC-DAD procedure can offer adequate confidence for the identification and quantification of specific polyphenols and can be modified or updated for future analysis of phenolic compounds in different plant extracts.