4.1.4. Analysis of Phenolic Compounds Using Ultra-Performance Liquid Chromatography–Quadruple–Time of Flight Mass Spectrometry (UPLC–QTOF–MS)

Phenolic compounds were identified using the Acquity ultraperformance liquid chromatography (UPLC) system coupled with a quadruple-time of flight mass spectrometry (Q-TOF-MS) instrument (Waters Corp., Milford, MA, USA) equipped with an electrospray ionization (ESI) source. Separation of individual phenolics was carried out using a Acquity UPLC^R HSS T3 C18 column (150 × 2.1 mm², 1.8 µm; Corp., Milford, MA, USA) at 30 °C according to the method presented by Zakłos-Szyda et al. [44] with some modifications. The mobile phase was a mixture of 0.1% formic acid (A) and acetonitrile (B). The gradient program was as follows: initial conditions—99% (A), 12 min 65% (A), 12.5 min 0% (A), 13.5 min 99% (A). The flow rate was 0.45 mL/min, and the injection volume was 5 µL. The mass spectrometer was operated in negative mode for a mass range of 150–1500 Da, the fixed source temperature was 100 °C, the desolvation temperature was 250 °C, the desolvation gas flow was 600 L/h, the cone voltage was 45 V, the capillary voltage was 2.0 kV, and the collision energy was 50 V. Leucine enkephalin was used as a lock mass. The instrument was controlled by Mass-Lynx^TM V 4.1 software. Phenolic compounds were identified using their UV-Vis characteristics. MS and MS² properties were determined using data gathered in house and from the literature. Based on the qualitative analysis of phenolic compounds, the total contents of flavanols, flavonols, flavalignans, hydroxycinnamic acids and iridoids were calculated. The contents are expressed in mg/g of extract for equivalents of (+)- catechin, quercetin 3-glucoside, cinchonine, chlorogenic acid, and quercetin 3-rutinoside, respectively.