Determination of phenolic compounds in plant extracts by HPLC-DAD

Abstract

Plants have the ability to synthesize secondary metabolites, biologically active substances involved in defense mechanisms against insects, pathogenic fungy and bacteria. Biochemical bases of their activity are related to the presence of specific molecules, among others, phenolic compounds. The aim of this study was to develop the validated method for the determination and quantification of phenolic acids, kaempferol and quercetin in ethanol leaf extracts of Morus alba L. and Halascya sendtneri (Boiss.) and leaf and bark extract of Ailanthus altissima (Mill.). The separation, quantification and validation of the individual phenols were performed by high performance liquid chromatography with diode-array detection (HPLC-DAD). The HPLC-DAD separation was achieved using a ZORBAX SB-Aq (5 μm particle size: 4.6 x 250 mm, Agilent). The mobile phase was acetonitrile with 2.0% acetic acid and Milli-Q water with 2.0% acetic acid in gradient mode, with the flow rate 1.0 ml/min. The obtained LOQs for all investigated phenolic acids were 0.03 μg/ml. The precision values, expressed as relative standard deviation (RSD, %), were lower than 10.19%. The developed HPLC-DAD chromatographic procedure exhibits linearity (R2>0.99) for the concentrations from 10.0 to 100.0 µg/ml with the repeatability RSD less than 12.00%. An efficient, sensitive and reliable method is developed which can be applied in the analysis of real plant material samples to ferulic, trans-cinnamic, 2-hydroxy cinamic, gallic, caffeic, p-coumaric and chlorogenic acid, kaempferol and quercetin.