Validated Method for the Analysis of Frangulins A and B and Glucofrangulins A and B Using HPLC and UHPLC

In the present study, robust and validated HPLC and UHPLC methods for the quantitative determination of frangulins A and B (3 and 4) and glucofrangulins A and B (1 and 2) in the bark of Frangula alnus have been developed. The HPLC method allowed the separation of the analytes in 25 min and the UHPLC method in just 13 min. The HPLC method used an MN Nucleodur C₁₈ 125 × 4 mm column with 3 μm particles, while the UHPLC method used a Waters Acquity UPLC BEH C₁₈, 100 × 2.1 mm column with 1.7 μm particles. Mobile phase A consisted of water and 1.25 mL/L phosphoric acid (85%), while mobile phase B consisted of CH₃CN/MeOH (20:80). The flow rates were set to 1 mL/min for the HPLC method and 0.4 mL/min for the UHPLC method, with the column temperature held at 50 °C and the detection wavelength being 435 nm for either method. A fractional factorial design was used to check the robustness of the methods. The resolution of the analytes was never less than 1.5 when the factors were varied in the tested range. The conditions for ultrasonic extraction were optimized by response surface methodology and found to be 68% CH₃CN in the extraction solvent, 35 °C extraction temperature, and a duration of 25 min. The extraction procedure was determined to be very robust against small deviations of these factors.