2.5. HPLC-PDA Analysis for Characterization of Caffeoylquinic Acids and GC–MS Analysis for Essential Oil Components

The phenolic profiles of the fractions were determined using HPLC (Waters e2695 Alliance system, Waters, Milford, MA, USA) coupled with a PDA detector. The separations were performed as described by Vilkickyte et al. [26], with minor modifications. Briefly, an ACE Super C18 (250 mm × 4.6 mm, 3 µm) column (ACT, Aberdeen, UK) was used, and the flow rate was 0.5 mL/min. The column temperature was set to 15 °C, with an injection volume of 10 µL. The mobile phase consisted of 0.1% trifluoroacetic acid (A) and acetonitrile (B), and the gradient solution pattern was 0 min, 85% A; 0–30 min, 70% A; 30–50 min, 40% A; 50–56 min, 10% A; 56–65 min, 15% A. To prepare the HPLC standard curves, each test compound was weighed out accurately, dissolved in 70% methanol (v/v) at a concentration of 1 mg/mL, and filtered through a 0.2 µm pore size PVDF syringe filter (Macherey-Nagel GmbH & Co. KG, Düren, Germany). Chromatographic peak identification was performed by comparing the analyte and reference compound (individual standard reference compounds of caffeoylquinic acids) retention time and the UV absorption spectra (Table A1). Quantification was performed at 320 nm for caffeoylquinic acids and at 350 nm for luteolin glycosides. For quantification, 5–7-point calibration curves were constructed by plotting the response of each analyte versus concentration (Table A1).

The gas chromatography–mass spectrometry (GC-MS) research was performed using a headspace methodology on a SHIMADZU GC-MS-QP2010 Ultra chromatography system with an Rxi-5ms (Restek Corporation, Benner Circle, Bellefonte, PA, USA) fused silica capillary column (30 m × 0.25 mm, 0.25 µm film coating). The oven temperature was programmed at 40 °C for 2 min, then increased by 3 °C /min to 150 °C, then 2 °C/min to 185 °C, then 10 °C/min to 250 °C, and then 20 °C/min to 310 °C for 5 min. The injector temperature was 260 °C, in split injection mode, with an injection volume of 1 µL and a split ratio of 1:5. The mass spectra scan range of m/z was 29–500 amu, with a mass scan time of 0.2 s, interface temperature of 280 °C, and IonSource temperature of 200 °C. Identification of the active compounds was done on the basis of retention index and library mass search database (NIST14, NIST14s, WR10, WR10R).