An on-line HPLC system for detection of antioxidant compounds in some plant extracts by comparing three different methods

•A new gradient flow program was developed for the simultaneous identification of fifteen phenolic standards in HPLC.•Three on-line HPLC antioxidant methods (ABTS/DPPH/FRAP) were compared by using Echinacea, green tea and yaylacayi extracts.•By using DAD detector, peak identification was facilitated.•By the developed HPLC system, characterization and antioxidant activity determination of each peaks made simultaneously.•FRAP method was found to be superior over ABTS and DPPH methods in on-line HPLC-post column applications. Echinacea (Echinacea purpurea), green tea (Camellia sinensis) and yaylacayi (Thymus praecox OPIZ subsp. grossheimii (Ronniger) Jalas) are herbs used in traditional medicine because of their beneficial health effects. A high performance liquid chromatography (HPLC) post-column method was developed by coupling HPLC with on-line antioxidant assays using diode array (DAD) and ultraviolet (UV) detectors. Thus antioxidant compounds and fifteen phenolic standards were detected in echinacea, green tea and yaylacayi methanol extracts simultaneously. Later on, the developed on-line HPLC coupled system was used to compare three different antioxidant methods, ABTS [2,2′-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)], DPPH (2,2-diphenyl-1-picrylhydrazyl and FRAP (Ferric reducing antioxidant power). Eight compounds with antioxidant activity were identified as gallic acid, protocatechuic aldehyde, p-OH benzoic acid, chlorogenic acid, caffeic acid, vanillin, ferulic acid, and rosmarinic acid. For validation purposes, LOD (limit of detection) (0.36–14.68μg/mL) and LOQ (limit of quantification) (1.35–48.92μg/mL) values were calculated for six standards. The results showed that while the post-column ABTS and FRAP assays were repeatable, sensitive and supportive of each other, the post-column DPPH assay did not provide reliable results. Also due to the base-line noise and drifts in post-column DPPH assay, identification and quantitation of analytes could not be done accurately.