2.3. Verbascoside and isoverbascoside quantification from plant extracts and HPLC method validations

Under previously developed HPLC conditions [12], we quantitatively analyzed verbascoside and isoverbascoside by utilizing the Thermo Scientific UltiMate 3000 HPLC system coupled with a diode array detector. The mobile phase was an isocratic 20 % acetonitrile acidified with 2.5 % acetic acid flowing at 0.5 mL/min. The column used was a 4.6 × 150 Nacalai Tesque Cosmosil C18 reverse phase column. Standard curves were plotted using the area under the curve of both compounds at a 1.95–250 μg/mL concentration. The limit of detection and the limit of quantification were obtained by calculating the signal-to-noise ratio equal to 3:1 and 10:1, respectively. The accuracy was obtained by adding both standards of a known amount to the plant extract and expressed as the recovery percentage. The method precision was obtained by analyzing three concentrations of both standards in the same day (intra-day) and three different days (inter-day) and expressed as %RSD. Subsequently, 1 mg of the extracts was dissolved in the mobile phase and filtered prior injection. The injection volume was 10 μL. sample and standard analyses were performed in triplicate.