Cosmosil c18

Under previously developed HPLC conditions [12 (link)], 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.

The LC/MS/MS experiment was performed according to our previously reported methodology [15] (link) with the exception of the column and solvent system. The column was a Cosmosil C₁₈ (4.6 mm × 250 mm, 3.5 μm, Nacalai, Inc., San Diego, CA, USA), and the constituents of the solvent system were 0.1% aqueous formic acid (A) and methanol:water (6:4, v/v, B). The gradient conditions of the mobile phase were: from 0% to 10% B over 10 min, from 10% to 100% B over 50 min, and isocratic elution for 10 min. MS/MS experiments were performed using a 3200 Q TRAP LC/MS/MS system (Applied Biosystems, Forster, CA, USA) with a Turbo VTM source and a Turbo Ion Spray probe (500 °C). The mass spectrometer was operated in positive and negative ion mode. Nitrogen was used as a nebulizing as well as a drying gas. The flow rates in both cases were 45 psi. The capillary voltage was set at 5.5 kV and the source temperature was set at 500 °C. The resolutions of the first and third quadrupole were between 0.6 and 0.8 (unit resolution). Mass spectra were recorded between m/z 100 and 1000 with a step size of 0.1 amu.