Water 996 photodiode array detector

HPLC analysis was performed with an HPLC equipment (Water 996 Photodiode Array Detector, Water 717 Plus AutoSampler, Water 600s Controller, Water 626 Pump, Millennium system). A C18 column (4.6 × 250 mm, 5 μm) was employed with the mobile phase of 0.2% phosphoric acid: acetonitrile (15:85) and a flow velocity at 1.0 mL/min at room temperature. Chromatograms were detected at 240 nm using a DAD detector. A standard solution of geniposide (2.1 mg) dissolved in 2 mL methanol was prepared and serially diluted to form different concentrations (0.5, 0.25, 0.125, and 0.0625 mg/mL). The sample solution was diluted 1:10 in methanol.

DKT concentrations in the samples were measured by HPLC-UV (Hewlett Packard series 1100 HPLC Pump combined with Agilent Technologies 1200 Series Autosampler). A volume of 75 µL was injected into the HPLC system (Waters 515 HPLC Pump with Waters 717 Autosampler). DKT was detected with an UV lamp at 262 nm (Water 996 Photodiode Array Detector). The mobile phase consisted of 60:40 mixture of acetonitrile and purified water A (both containing 0.1% TFA). Stationary phase was a C-18 Agilent Eclipse XDB (4.6 × 150 mm; 3.5 µm). Elution flow was 1 mL/min and retention time for DKT was 3.95 min. Calibration curves were made in mobile phase based on a stock solution of DKT in methanol. Linearity was observed between 1.5 µg/mL and 300 µg/mL covering all the experimental sample values. The observed peaks were integrated using Millenium software (Agilent Technologies, County of Santa Clara, CA, USA). The developed analytical method met the standards for precision and accuracy.

Extracts were analyzed using HPLC (Waters 2695 Alliance HPLC; Waters Inc., Milford, MA, USA) with the octadecylsilane column (Prontosil 120–5-C18-SH-EPS 5.0 μm (200 × 4.6 mm; Bischoff, Leonberg, Germany). According to the previously published method [59 (link)], the solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) were used as mobile phase with the flow rate of 0.8 mL/min. The mobile phase B gradient was as follows: 16–25%, 0 to 35 min; 25–50%, 35 to 40 min; 50–65%, 40 to 47 min; 65–16%, 47 to 50 min. The injection volume was 5 μL. The peaks of 12 standard isoflavones were detected at 254 nm (Water 996 photodiode array detector (Waters Inc.)).