2.3.5. DPPH Assay

The determination of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity required the following sample preparation: 1 g of honey was dissolved in 5 mL of methanol 40% (*v*/*<sup>v</sup>*, with acidified water) and stirred for 15 min with a magnetic stirrer [31]. Then, 35 μL of honey solution was mixed with 250 μL of DPPH. The absorbance was measured at 515 nm using a QE65000 spectrometer (Ocean Optics, St. Petersburg, FL, USA). The results were expressed as % DPPH using the formula in Equation (1):

$$\% \text{DPPH} = \left(A\_0 - \frac{A\_1}{A\_0}\right) \times 100,\tag{1}$$

#### where *A*0 is the DPPH absorbance, *A*1 is the sample absorbance.

### 2.3.6. Determination of Sugars Composition

Sugars composition was determined according to the IHC (International Honey Commission) methods [8,29]. The samples were filtered through 0.45 μm PTFE membrane filters prior to the injection in the HPLC instrument (Schimadzu, Kyoto, Japan) equipped with a LC-20 AD liquid chromatograph, SIL-20A auto sampler, CTO-20AC column oven, and RID-10A refractive index detector. The separation was performed on a Phenomenex Luna® Omega 3 μm SUGAR 100 Å HPLC Column 150 × 4.6 mm. Peaks were identified based on their retention times and the determination of sugar content was made according to the external standard method on peak areas or peak heights. The mobile phase was acetonitrile:water (80:20, *v*/*v*), with a flow rate of 1.3 mL·min−1; column and detector temperature was 30 ◦C and the sample volume injection was 10 μL. Standard solutions of fructose, glucose, maltose, sucrose, turanose, trehalose, melesitose, and raffinose were individually injected to calculate the sugar content of each honey sample by using peak areas based on the retention time.

### 2.3.7. Determination of Polyphenols Composition

Honey solutions were prepared following the steps presented in Section 2.3.3 [30]. The samples were filtered through 0.45 μm PTFE membrane filters and then injected (with a volume of 10 μL) into the HPLC instrument (Schimadzu, Kyoto, Japan) for analysis using an SPD-M-20A diode array detector. The separation was carried out on a Phenomenex Kinetex 2.6 μm Biphenyl 100 Å HPLC Column 150 × 4.6 mm thermostated at 25 ◦C. Elution was carried out with a solvent system consisting of 0.1% acetic acid in water (solvent A) and acetonitrile (solvent B) as previously described by Palacios et al. [32] with modifications. The solvent flow rate was of 1 mL·min−1. The determined phenolic compounds were gallic acid, vanillic acid, protocatechuic acid and *p*-hydroxibenzoic acid at 280 nm, and chlorogenic acid, *p*-coumaric acid, caffeic acid, rosmarinic acid, myricetin, quercetin, luteolin and kaempherol at 320 nm. The obtained standard calibration curves showed high degrees of linearity (*R*<sup>2</sup> > 0.99). Data collection and subsequent processing were performed using the LC solution software version 1.21 (Shimadzu, Kyoto, Japan).

### 2.3.8. Determination of Organic Acids Composition

The method used to determine the organic acids involved a sample preparation of 0.5 g of honey mixed with 2.5 mL of 4% metaphosphoric acid (*w*/*v*), then the samples were vortexed. After, the samples were centrifuged for 5 min at 3500 rpm using a Z216-MK refrigerated centrifuge (Hermle Labortechnik, Wemingen, Germany) [33]. The sample was injected in the HPLC instrument (Schimadzu, Kyoto, Japan) with a diode array detector. The separation was carried out on a Phenomenex Kinetex® 5 μm C18 100 Å HPLC Column 250 × 4.6 mm. The mobile phase used was a mixture of 0.5% metaphosphoric acid and acetonitrile (50/50, *v*/*v*) at a flow rate of 0.8 mL·min−1. The volume of injection was 10 μL. The organic acids identification and quantification were carried out at 210 nm. The organic acids that were determined were acetic acid, lactic acid, propionic acid, butyric acid, and gluconic acid. The concentration of organic acids was expressed as mg/L.
