The first method for the simultaneous determination of polyphenolic antioxidants in extracts from leaves of Cirsium palustre based on high performance liquid chromatography combined with flow injection chemiluminescence detection (HPLC-FI-CL) has been developed. The extracts were prepared by using methanol as extraction medium and two types of extraction methods (reflux and ultrasound assisted extraction). The post-column CL determination of polyphenols was based on their enhancing effect on the chemiluminescence intensity generated in manganese(IV)-hexametaphosphate-formaldehyde system in a phosphoric acid medium. Main antioxidants determined in C. palustre leaves were eriodictyol-7-O-glucoside, luteolin-7-O-glucoside and 6-hydroxyluteolin-7-O-glucoside belonging to flavonoids, and chlorogenic acid belonging to phenolic acids. Chromatographic separation was carried out on a C18 column with gradient elution by using a mobile phase containing 0.25% (v/v) phosphoric acid in water (solvent A) and 100% methanol (solvent B). Under the optimized conditions of chromatographic separation and CL detection the validation of the method was performed. The calibration curves showed good linearity in the concentration range from 0.5 to 40 µg mL(-1). The HPLC-FI-CL method was successfully applied to the determination of four polyphenolic compounds in methanolic extracts from leaves of C. palustre. The accuracy of the developed method was confirmed by the comparison of the results with those obtained by an HPLC-PDA method. The relative error of determination does not exceed 6.1%. However, the HPLC-FI-CL method is characterized by 40-65 times higher sensitivity compared to the HPLC-PDA method.
Keywords: Antioxidant activity; Chemiluminescence; Cirsium palustre (L.); High performance liquid chromatography; Manganese(IV); Polyphenols.
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