3.3. Solid-Phase Extraction and HPLC–ESI-MS/MS Analysis

Solid-phase extraction (SPE) method was used to extract phenolic compounds from honey according to a previous method with slight modifications [15]. Honey samples (10.0 g) were mixed with 30 mL acidulated water (pH = 2.0) and vortexed to completely blend. The solution was then centrifuged for 10 min at 10,000 rpm to remove impurities. Prior to extraction, the Oasis HLB cartridge column (Waters, Wexford, Ireland) was activated with methanol (10 mL) and acidulated water (pH 2.0, 10 mL). After the supernatant was loaded, the column was then washed with distilled water and the phenolic compounds were eluted with methanol into a 10 mL flask. The solvent was evaporated to dryness at 40 °C and the residue was dissolved in 1.0 mL of 0.1% formic acid:acetonitrile (70:30, v/v). The sample was then mixed on a vortexer for 2 min and filtered through a filter membrane with 0.25μm pore size. All measurements were performed in triplicate.

The extracts were analysed using an Agilent C18 column (100 mm × 2.1 mm, 2.7 μm; Agilent, Wilmington, DE, USA) within 50 min. The column temperature was maintained at 30°C. 0.1% formic acid in water (solvent A) and methanol (solvent B) were used as mobile phase, and the flow rate was 0.2 mL/min. The HPLC-MS conditions were as described in our previous study [15]. A typical TIC chromatogram from standard phenolic compounds and buckwheat honey sample is given in Figure 6A,B. The MS parameters used to measure phenolic acids and flavonoids and the transitions from precursor to product ions are shown in Supplementary Table S3. The identification and quantification of phenolic compounds was performed with MassHunter software (Agilent Technologies). The detailed description of the calibration curves is presented in Supplementary Table S4.

(A) TIC chromatogram of phenolic standard. (B) TIC chromatogram of buckwheat honey. Compound names and retention times (min) are as follows: (A): peak 1, gallic acid (2.74), peak 2, protocatechuic acid (5.05), peak 3, p-hydroxybenzoic acid (8.73), peak 4, caffeic acid (12.52), peak 5, p-coumaric acid (16.88), peak 6, ferulic acid (18.88), peak 6, benzoic acid (18.92), peak 7,rutin (23.42), peak 8, myricetin (24.41), peak 9, morin (25.47), peak 10, quercetin (26.70), peak 11, naringenin (26.82), peak 12, kaempferol (28.29), peak 13, apigenin (28.75), peak 14, pinocembrin (29.95), peak 15, CAPE (30.77), chrysin (30.95), galangin (31.11). (B): peak 2, protocatechuic acid (5.05), peak 3, p-hydroxybenzoic acid (8.73), peak 4, caffeic acid (12.52), peak 5, p-coumaric acid (16.88), peak 6, ferulic acid (18.88), peak 7, rutin (23.42), peak 11, naringenin (26.82), peak 14, pinocembrin (29.95), peak 15, CAPE (30.77), chrysin (30.95), galangin (31.11).