A Validated HPLC Method for Simultaneous Determination of Caffeoyl Phenylethanoid Glucosides and Flavone 8- C -glycosides in Haberlea rhodopensis

Abstract

A HPLC-UV method for analysis of the main compounds: caffeoyl phenylethanoid glucosides myconoside (1) and paucifloside (2) and flavone 8-C-glycosides: hispidulin 8-C-β-galactopyranoside (3), hispidulin 8-C-(2″-O-syringoyl-β-glucopyranoside) (4), hispidulin 8-C-(6-O-acetyl-β-glucopyranoside) (5) and hispidulin 8-C-(6-0-acetyl-2″-0-syringoyl-β-glucopyranoside) (6) in Haberlea rhodopensis leaves was developed and validated. Compound 3 was isolated for the first time from the title species. Ultrasound extraction with 80% methanol at room temperature allowed a good recovery of analytes (from 87.2 % for 1 to 109.8 % for 3) and the precision of the entire procedure was between 1.6% and 6.9%. The subsequent HPLC separation and quantification was achieved using a Hypersil ODS C18 column and UV detection at 280 nm. The mobile phase comprised methanol and 0.1 % o-phosphoric acid, and gradient elution mode was applied. The detection limits ranged from 0.042 μg/mL (6) to 0.18 μg/mL (5). The total amount in leaves of the assayed phenolic compounds was 374.2 mg/g. Myconoside was found to be the dominant compound in H. rhodopensis extract (332.2 ±0.7 mg/g dw) and reached up to 88.8% of the analyzed mixture in leaves, while the total content of flavone C-glycosides was 17.1 mg/g dw.