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HPLC-DAD Method for Quantification of the Flavonoids with Antiradicalar Activity in the Hydroethanolic Extract from Tonina fluviatilis aubl. (Eriocaulaceae)
This work describes the isolation of flavonoids by preparative chromatography, and identification by NMR experiments, mass spectrometry (ESI-ITMS) and the development and validation for simultaneous quantification based on HPLC-DAD method for this flavonoids in the hydroethanolic extract of the aerial-parts from Tonina fluviatilis. The flavonoids identified and quantified in the extract were: 6,7-dimethoxyquercetin-3-O-β-D-glicopyranoside (1), 6-hidroxy-7-methoxyquercetin-3-O-β-D-glicopyranoside (2) and 6-methoxyquercetin-3-O-β-D-glicopyranoside (3). The HPLC-DAD analyses were performed using a C18 column and gradient programs with water and methanol, both acidified with 0.1% TFA, as mobile phase. The analysis of the samples spiked with known amounts of (1 – 3) demonstrated that the response was proportional to the concentrations of the samples with the determination coefficient being r2= 0.9989 (1) and r2= 0.9992 (2 and 3) for the linear range of the analytical calibration curves (10, 25, 50, 75, 100 and 250 µg.mL-1).The method is sensitive enough for the analysis of the three flavonoids quercetin derivatives, with (LOD within 8.90 and 10.64 µg.mL-1; LOQ within 26.99 and 32.22 µg.mL-1). Also, good results were obtained with respect to repeatability (coefficients of variation – CV < 2%) and recovery (higher than 90%) in three different concentrations (1, 20 and 40 µg.mL-1). The antiradicar activity (DPPH) for hydroethanolic extract was (29.1%) and substances 1 (39.5%) and 2-3 (87.7%) in the kidnapping concentration of 200 µg.mL-1 compared to positive controls (quercetin and gallic acid).
(Granted by FAPESP, CNPq).