HPLC DETERMINATION OF PHENOLIC ACIDS IN ARNICAE FLOS

The quantitative determination of protocatechuic, chlorogenic and caffeic acids in Arnica montana by reverse phase high-performance liquid chromatography (RP-HPLC) was carried out. Samples of Arnicae flos from eight different origins were selected for the assay as follows: one Bulgarian and one Polish cultivated collection, two cultivars, three collections grown in the Botanical Garden in Finland and one is purchased from a Pharmacy Drugstore. The effect of extracting solvent was investigated and optimal extraction of assayed phenolic acids was achieved by 80% methanol. The subsequent HPLC separation of the analytes was performed on Hypersil ODS C-18 column using linear gradient elution with a mobile phase composed of 20 mM phosphate buffer (pH 3.22) and methanol, and with UV detection at 280 and 310 nm. The detection limits were 0.19 mu g/ml, 0.51 mu g/ml and 0.33 mu g/ml for protocatechuic, chlorogenic and caffeic acids, respectively and the quantification limits were 0.57 mu g/ml, 1.53 mu g/ml and 0.99 mu g/ml, respectively. Chlorogenic acid was the dominant phenolic acid in the studied samples being present in amounts between 0.68 +/- 0.04 mg/g dry weight in the sample from Poland and 2.06 +/- 0.17 mg/g in Arnicae flos collected from the Botanical Garden at Joensuu University, Finland. Caffeic and protocatechuic acids were detected in lower quantities. The caffeic acid was accounted for up to 0.16 +/- 0.08 mg/g dry weight in cultivar "ARBO" and it was lowest in the sample "Pharmacy Drugstore" - 0.02 +/- 0.002 mg/g. The protocatechuic acid ranged from 0.01 +/- 0.001 mg/g to 0.14 +/- 0.01 mg/g. The results revealed that the sample from the Botanical Garden at Joensuu University, Finland, tended to have the highest amounts of phenolic acids compared to the other samples, especially with cultivar "ARBO". The Bulgarian collection is a promising source of valuable phenolic compounds.