UHPLC Analysis of Phenolic Compounds in Plant Extracts

UHPLC chromatographic system Acquity UPLC (Waters, Milford, MA, USA) comprising a binary pump, an autosampler, a column oven, and a diode array detector (DAD) was used for the determination of active compounds in the extracts. The system control, data acquisition, and data evaluation were executed using the Empower software (Waters, Milford, MA, USA). The reversed-phase Triart ExRS C18 (150 × 3 mm; 1.9 µm) column preceded by a guard column Ascentis Express C18 (5 × 4.6 mm) packed with 5 µm particles was used for the separations. The mobile phases consisted of aqueous acetic acid with pH 2.8 (A) and acetonitrile (B) with a flow rate of 0.35 mL/min. The gradient elution enabled the separation of the active compounds. The gradient started with 10% B in A as the initial conditions. Then, B was ramped to 22% in 8 min and 28% in the next 2.2 min. Then, a steep increase to 40% in 30 s followed by a further increase to 50% in 3 min and held for 0.2 min. Finally, the percentage of B was decreased to initial conditions in 0.2 min and held for 3.4 min to equilibrate the system. The temperature of the column was 30 °C. The sample was cooled at 6 °C in an autosampler and 2 µL were injected into the UHPLC system. The separation of all tested phenolic compounds lasted 15 min.
The analytes summarized in Table S1 were detected by DAD at 4 different wavelengths: (i) 254 nm selected for guaiaverin, hirsutrin, hyperoside, reynoutrin, quercitrin; (ii) 280 nm selected for gallic acid, epicatechin, catechin, phloridzin, phloretin; (iii) 320 nm applied for chlorogenic and caffeic acid; and (iv) 354 nm selected for rutin and quercetin. The 3D record was also recorded for wavelengths in the range of 210–400 nm to collect UV spectra for all detected peaks. Obtained UV spectra, together with retention times, were used for the identification of individual active compounds by comparing standard solutions and extracts.
Concentration levels for each analyte were calculated from the respective integrated peak areas compared to the standard (10 µg/mL) while within the framework of the previous measurements for the determination of phenolics in apples (unpublished results). Calibrations of all analytes proved to be linear in the whole tested range starting from 0.1 µg/mL.