**1. Introduction**

Honey is a natural food product well known not only for its nutritional value, but also for its antimicrobial, antiviral, antifungal, anticancer, and antidiabetic properties, as several in vitro and in vivo studies have demonstrated [1]. From a compositional point of view, honey is a highly concentrated solution of complex mixture of sugars: fructose (38%), glucose (31%), water (17%), maltose (7%), as well as trisaccharides, other higher carbohydrates, sucrose, minerals, vitamins, and enzymes. Its composition depends strongly on the plant species from which the nectar or the honeydew was collected, and other factors, such as postharvest treatments, geographical, environmental or climate conditions [2,3]. Honey is among the top ten foods with the highest adulteration rate in the European Union, that implies a detrimental to its quality and consumers safety [4]. To protect this valuable food, a Codex standard for honey was adopted by the Codex Alimentarius Commission in 1981, being further revised in 1987, 2001 and 2019, to regulate its production and storage, establishing parameters to guarantee its quality [5]. In 2001, the European Council, following the Codex recommendations, established the Directive

**Citation:** Vazquez, L.; Armada, D.; Celeiro, M.; Dagnac, T.; Llompart, M. Evaluating the Presence and Contents of Phytochemicals in Honey Samples: Phenolic Compounds as Indicators to Identify Their Botanical Origin. *Foods* **2021**, *10*, 2616. https://doi.org/ 10.3390/foods10112616

Academic Editors: Olga Escuredo and M. Carmen Seijo

Received: 4 October 2021 Accepted: 25 October 2021 Published: 28 October 2021

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2001/110/EC [6], amended 2014/63/EU [7] that laid down the production and trading parameters of honey within the member states of the EU. However, several countries issue national provisions, decisions, and guidelines defining their own physicochemical, organoleptic and microscopic characteristics, enhancing the difficulties of the applicability of harmonized regulations [8].

The identification of honey botanical origin is a valuable information to assure honey quality. In this way, the analysis of its phenolic composition has been employed as a tool for its classification and authentication [1,9,10]. Phenolic compounds are secondary metabolites of plants generally involved in their defense against ultraviolet radiation or pathogens and have been recognized as the main responsible for the antioxidant activity of honey [11–13]. The most abundant phenol- types in honey are flavonoids, especially flavones and flavanols, as well as phenolic acids derived from benzoic and cinnamic acids [2,14].

Several analytical procedures have been reported to determine honey physicochemical properties including colour, viscosity, pH, moisture, free acidity, electrical conductivity, sugars, HMF (hydroxymethylfurfural) content, formol index and insoluble solids [15–19], but due to the high number of existing honey varieties, more specific techniques are needed. The use of chromatography coupled to mass detectors (MS) to obtain a deep chemical characterization of this product is a very valuable option. However, the major drawback for honey analysis is sample preparation since it is a very complex matrix. To establish the honey aromatic profile, the combination of solid-phase microextraction (SPME) with gas chromatography-mass spectrometry (GC-MS) has been the main employed technique [20–22]. On the other hand, for the determination of more polar analytes, including phenolic compounds, traditional sample preparation involves the use of solid-liquid or liquid-liquid (SLE, LLE) before LC-MS or HPLC-UV analysis. However, these techniques are long time consuming, requiring large amounts of organic solvents and further clean-up steps before analysis. Microwave-assisted extraction (MAE) and ultrasound assisted extraction (UAE) have been also proposed as extraction techniques to determine phenolic compounds in honey, but their use was not satisfactory in the presence of thermosensitive flavonoids such as quercetin, kaempferol or myricetin, that are almost degraded as consequence of radiation. On the other hand, both extraction techniques seemed to be suitable for the extraction of phenolic acids [20].

Therefore, the goal of this work is the development of a miniaturized analytical methodology to obtain the phenolic profile of Galician honeys (Northwest Spain) from different varieties and nectar sources. A green, fast and low-cost sample preparation strategy based on vortex extraction (VE) followed by ultrasound assisted extraction (UAE) employing aqueous- based solvents was assessed. Individual quantification of 41 target phenolic compounds was carried out by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Other indexes such as the total phenolic content (TPC) and antioxidant activity (AA) were also evaluated. Finally, advanced statistical tools such as analysis of variance (ANOVA) and principal component analysis (PCA) were employed to obtain models that allow classifying the different honeys according to their origins.

### **2. Materials and Methods**

### *2.1. Chemicals, Reagents and Materials*

The target phenolic compounds, their CAS numbers, molecular mass, log Kow, retention time and MS/MS transitions are summarized in Table 1. Methanol and ultrapure water, both MS grade, were supplied by Scharlab (Barcelona, Spain). Hydrochloric acid, formic acid, Folin–Ciocalteu's phenol reagen<sup>t</sup> (2M), 2,2-diphenyil-1-picrylhydrazyl (DPPH), and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox®) were purchased from Sigma–Aldrich (Darmstadt, Germany). Sodium carbonate was supplied by Panreac (Barcelona, Spain).

**Table 1.** Target phenolic compounds: CAS number, molecular mass (Mm), log Kow, retention time, ionization mode and MS/MS transitions.



**Table 1.** *Cont.*

1 "−"and "+" indicate negative and positive ionization modes, respectively. 2 Underlined MS/MS transition used for quantification purpose.<sup>3</sup> Isomers: 2,4/3,4-dihydroxybenzoic acid.

Phenolic individual standard stock solutions (500–1000 μg mL−1) were prepared in methanol. Further dilutions and mixtures were prepared in acidified water (0.1% formic acid)/methanol (80:20, *v*/*v*) (AW/MeOH). All solutions were stored at −20 ◦C and protected from light. All chemicals and reagents were of analytical grade.

A vortex stirrer by Velp Scientifica (Usmate, Italy) and an ultrasound bath (50 kHz) from JP Selecta (Barcelona, Spain) were employed to perform the extractions.
