*2.2. Honey Samples*

Ninety-one honey samples from Galicia (Northwest Spain) were kindly supplied by the protected geographical indication (P.G.I.) Mel de Galicia. Samples were received in glass jars sealed with aluminum caps. They were stored in the original containers at controlled temperature (15 ◦C) and kept away from light until their analysis.

The methodology used for the study of the botanical characteristics was based on the determination of the pollen contained in the honey by centrifugation. In total, 52% of the honey samples contained between 2000 and 10,000 grains of pollen per gram of honey, according to the classes of Maurizio; 39% of these samples contained between 10,000 and 50,000 grains of pollen per gram of honey [19]. The pollen spectrum of the samples consisted of 82 different pollen types, with 45% of them likely to be labelled as monofloral, while the remaining 55% were considered multi-floral, in which was included 16% whose majority origin was honeydew (HD). As regards monofloral honeys, the chestnut (CN, 34%), the blackberry (BL, 27.3%), the eucalyptus (EU, 25%) and, to a lesser extent, the heather (HE, 13.7%) stand out.

It should be noted that, as for the main proportion of the honey produced in Galicia, the main types were *Castanea, Eucalyptus, Erica, Rubus and Cytisus*, all of them in the dominant category or as companion in the pollen spectrum of honey.

### *2.3. VE-UAE Procedure*

Under the optimal experimental conditions (see Section 3.2), 0.1 g of honey were weighted in a 1.8 mL glass vial and 1 mL of acidified water (0.1% formic acid)/methanol (80:20, *v*/*v*) (AW/MeOH) was added. The vial was sealed with an aluminum cap furnished with PTFE-faced septa and the solution was stirred by vortex for 1 min. Afterwards, the vial was immersed in an ultrasound bath for 1 min (20 ◦C, 50 KHz). The obtained extract was filtered through 0.22 μm polytetrafluoroethylene (PTFE) filters and directly injected in the LC-MS/MS system for phenols analysis (see Section 2.6). The experimental procedure is summarized in Figure 1.

**Figure 1.** Schematic representation of the VE-UAE experimental procedure.

### *2.4. Determination of TPC*

The total phenolic content (TPC) of honey samples was determined according to the Folin–Ciocalteu (FC) colorimetric method described by Singleton and Rosssi [23]. Honey sample preparation was performed employing a modified method of Pauliuc et al. [16]. Briefly, 0.5 g of honey sample were diluted in 5 mL of methanol/water (40:60, *<sup>v</sup>*/*<sup>v</sup>*, pH = 2, HCl) and magnetically stirred for 15 min. Afterwards, 1.3 mL of this solution was diluted (1:10, *v*/*v*) in water up to a final volume of 13 mL. Then, an aliquot of 5 mL was placed on a Falcon tube and 100 μL of Folin–Ciocalteu's phenol reagen<sup>t</sup> and 1 mL of Na2CO3 solution

(20%, w/v) were added. The Falcon tubes were kept away from light for 30 min. Afterwards, the absorbance was measured at 760 nm in a UV-Vis spectrophotometer Shimadzu UVmini-1240 (Kyoto, Japan). The TPC was quantified employing a calibration curve prepared with gallic acid standards solutions ranging between 1–20 mg L−<sup>1</sup> (R<sup>2</sup> = 0.9990) and expressed as mg of gallic acid equivalent (GAE) per 100 g of honey (mg GAE 100 g<sup>−</sup>1).

### *2.5. Determination of AA*

The antioxidant activity (AA) was determined by a modified method of Brand– Williams et al. [24]. Briefly, 200 μL of the honey solution (0.5 g of honey diluted in 5 mL of methanol/water, 40:60, *<sup>v</sup>*/*<sup>v</sup>*, pH = 2, HCl) were introduced in a Falcon tube and 3.9 mL of the DPPH reagen<sup>t</sup> solution (0.1 mM in methanol) were added. After 30 min in the absence of light, the absorbance was measured at 515 nm. The AA was quantified employing a calibration curve prepared with Trolox® (0.1–0.9 mmol TRE g<sup>−</sup>1, R<sup>2</sup> = 0.9970). The AA were expressed as micromoles of Trolox® equivalents (TRE) per 100 g of honey (μmol TRE 100 g<sup>−</sup>1).
