*3.3. VE-UAE-LC-MS/MS Performance*

Under the optimal experimental conditions that involve the use of only 0.1 g of honey sample diluted in 1 mL of AW/MeOH, the whole VE-UAE-LC-MS/MS method was validated in terms of linearity, accuracy and precision. Limits of detection (LODs) were also calculated. Results are summarized in Table 2. Calibration curves were prepared in AW/MeOH containing the 41 target phenolic compounds, covering a concentration range for most compounds from 5 to 10,000 ng <sup>L</sup>−1, with 11 concentration levels (5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000 and 10,000 ng <sup>L</sup>−1) and three replicates per level. The method showed a good linearity, with coefficients of determination (R2) higher than 0.99. Instrumental precision was evaluated within a day (*n* = 4) and amongs<sup>t</sup> days (*n* = 5) for all the calibration concentration levels. Relative standard deviation (RSD) values for 200 μg L −1 are shown in Table 2, with mean values about 10%. To assess the accuracy of the proposed method, recovery studies were carried out employing a multi-floral honey sample (MF28). It is worth noting that only a few methods demonstrated accuracy for such a high number of phenolic compounds in honey samples and most of them imply the use of artificial matrices [20] or further experimental steps, mainly based on solid-phase extraction (SPE) to remove matrix components such as sugars [27]. The study was performed spiking

the honey sample with the 41 target phenolic compounds at 2 μg g<sup>−</sup>1. Results depicted in Table 2 show that recovery values ranged between 70 and 100% for most compounds with RSD values lower than 8%. Recovery percentages obtained in other studies that apply UAE were higher for some phenolic acids (gallic acid, p-coumaric acid) while higher values were obtained in the present study for other compounds, such as myricetin and kaempferol. The degradation of some flavonoids during the extraction procedure assisted with irradiation was demonstrated [20]. Those undesirable effects were not observed in the present study since UAE was only applied for 1 min.


**Table 2.** VE-UAE\_LC-MS/MS performance: Linearity, precision, recoveries and LODs.


**Table 2.** *Cont.*

1 Sum of both isomers: 2,4/3,4- dihydroxybenzoic acid; n.c. Not calculated since the concentration in the sample was higher than the spike level (see sample MF28 in Table S2c).

> Limits of detection (LODs) were calculated as the compound concentration giving a signal-to-noise ratio of three (S/N = 3) employing the honey sample spiked with the target compounds. Results depicted in Table 2 show that they were at the ng g<sup>−</sup><sup>1</sup> level for all target phenolic compounds.

### *3.4. Analysis of Real Honey Samples*

### 3.4.1. TPC and AA

TPC and AA results for the 91 analyzed samples are summarized in Tables S1 and S2 for samples collected in 2018 and 2019, respectively. The ranges, mean and median concentrations for TPC, AA and individual phenolic compounds are shown in Table 3.

Results for TPC were similar in the two evaluated seasons. As shown in Table 3, TPC values ranged between 48–203 mg GAE 100g−1. As expected, a relationship seems to exist between the total concentration of target phenolic compounds and the TPC values, since the highest TPC was found in the heather sample HE1 that shows the highest sum of phenolic compounds, 252 μg g<sup>−</sup>1. On the other hand, most of EU and BL honey samples achieved low TPC.

The AA index ranged between 15–1017 μmol TRE 100 g<sup>−</sup><sup>1</sup> for the two seasons. Samples of honeydew achieved the highest antioxidant activity, reaching 1006 and 1017 μmol TRE 100 g<sup>−</sup><sup>1</sup> in sample HD4 and HD11, respectively. Results are in concordance, since most honey samples with high TPC values achieved high AA, as well. In the same way, the honey sample (EU11) with the lowest TPC (48 mg GAE 100 g<sup>−</sup>1) also reached the minimum AA concentration (15 μmol TRE 100 g<sup>−</sup>1).

Results of TPC and AA obtained in the Galician honeys were in consonance with those reported in other honeys from the same and different origin [19,28]. Thus, both indexes do not allow differentiating Galician honeys from other honeys, although they might allow to distinguish between the different honey varieties (see also Section 3.5).



### 3.4.2. Individual Phenolic Content

Individual target phenolic compounds concentrations, as well as the sum of them for the 91 analyzed samples are summarized in Tables S1 and S2, and concentration ranges for the analyzed varieties are shown in Table 3.

Among the 41 target phenolic compounds, 22 were found in the samples of the 2018 season whereas 25 were detected in samples of 2019. The highest concentration of individual phenolic compounds was found in the heather variety (HE), with total phenolic compounds concentrations reaching 252 μg g<sup>−</sup>1, especially owing to the high content of 3-hydroxyphenylacetic acid (242 μg g<sup>−</sup><sup>1</sup> in sample HE1). The sum of phenolic compounds was highly influenced by the concentration of this compound since it was found in 33 of the 91 analyzed samples at a mean value of 35 μg g<sup>−</sup><sup>1</sup> and in the range 0.41–242 μg g<sup>−</sup>1. It is worth noting that these high 3-phenoxyphenylacetic acid contents do not confer high antioxidant activities to the HE honey, compared with those containing honeydew.

Regarding those samples that were not highly affected by 3-phenoxyphenylacetic acid, honeydew honeys (HD) contained high concentration of the sum of phenolic compounds, with 35 and 25 μg g<sup>−</sup><sup>1</sup> in 2018 and 2019 seasons, respectively. In the same way, the mixture chestnut/honeydew variety (CN/HD) reached concentrations up to 30 μg g<sup>−</sup><sup>1</sup> for the sum of the target phenolic compounds. On the other hand, the lowest concentration for the sum of phenolic compound was detected in a eucalyptus honey (EU11) with 3.4 μg g<sup>−</sup>1. In general, results were similar in both seasons and the values were in concordance with the TPC and AA.

The most abundant phenolic compound, detected in all the analyzed honey samples, was p-hydroxybenzoic acid, in a concentration range from 0.68 to 5.1 μg g<sup>−</sup>1. Other benzoicand hydroxycinnamic- derivates acids, such as gallic or protocatechuicacid, were found at high concentrations, up to 10 μg g<sup>−</sup><sup>1</sup> in honeydew (HD), 8.0 μg g<sup>−</sup><sup>1</sup> in chestnut/honeydew (CN/HD) and 4.6 μg g<sup>−</sup><sup>1</sup> in blackberry (BL) varieties. It is important to note that 7 of the 14 chestnut samples collected during the 2019 campaign contained honeydew, which could contribute to the concentration increase. In HD samples, gentisic acid (present in 86 honeys) reached the highest mean concentration of 2.7 μg g<sup>−</sup>1. Additionally, p-coumaric acid, which was found in all samples, achieved concentrations up to 12 μg g<sup>−</sup><sup>1</sup> in multi-floral (MF) honeys. P-coumaric acid content fluctuations could be observed within the same variety; however, the highest concentrations and fluctuations occurred in multi-floral (MF) honeys, with values ranging from 0.1 to 12 μg g-1.

Veratric acid was detected only in six honey samples of several types (BL, CN, HD and MF) at concentrations around 1.5 μg g<sup>−</sup>1.

As regards aldehydes, 3-hydroxybenzaldehyde and 4-hydroxybenzaldehyde appeared in several samples of different honey varieties (EU, BL HE, CN and MF) at concentrations of up to 1.2 and 2.0 μg g<sup>−</sup>1, respectively. It is important to note the absence of these two aldehydes in the honeydew samples as well as in the chestnut honeys with honeydew (CN/HD), except in sample HD10 in which 3-hydroxybenzaldehyde appeared at a concentration of 0.10 μg g<sup>−</sup>1.

Concerning other families, 2 of the 3 targeted flavonols were found in the samples: quercetin in 69 samples and kaempferol in 40 honey samples, whereas myricetin was not detected in any sample. Additionally, the flavones chrysin and apigenin were found in 91 and 84 honey samples, respectively. They were present at concentrations up to 5.9 μg g<sup>−</sup>1, for chrysin in BL8, and 0.43 μg g<sup>−</sup>1, for apigenin in MF5. In contrast, flavanol compounds (catechin, epicatechin, gallocatechin gallate) were not detected in the analyzed samples.
