**6. Evaluation of the phenolic content**

Analytical procedures used to determine polyphenols in a honey sample include their extrac‐ tion from the matrix as well as their separation and quantification. The determination begins with an extraction step by means of solvents, which are mostly mixtures of water‐alcohol in different proportions. Aqueous ethanol solutions (25–70 % v/v) are used in some work for 12–24 hours under stirring [42, 43]. While the methanolic extraction is used in different pro‐ portions with water [1, 44], there is still work using combined techniques of aqueous extrac‐ tion, with heating or acidification, and subsequent ethanol extraction [40, 45]. Few studies conduct extraction with other solvents such as ethyl acetate [46].

The filtered or centrifuged extracts and different profiling techniques can be used for the determination of phenolic compounds. Liquid chromatography is considered to be the most useful separation technique for the analysis of polyphenols in different samples. Coupled with various detection techniques, such as a diode array detector (DAD) [1, 21, 40, 47] and/or mass spectrometry, it enables both identification and quantification of poly‐ phenols [42, 45, 46]. Since phenolic components can vary greatly, the suitable technique is liquid chromatography coupled with various types of mass detection, LC–MS enables high selectivity, sensitivity and universality when analyzing various polyphenolic components in their complex matrices.

Honey is a supersaturated solution of sugars (70–75%), of which fructose (38% w/w) and glucose (31% w/w) are the main contributors, 20–25% of water and about 3–5% for various substances [22, 38]. Hundreds of bioactive substances have already been found in honeys from different regions. This wide variation occurs when honeybees collect nectar from plants, incorporating secondary metabolites product of vegetables. This metabolism is rather vari‐ able and primarily depends on the botanical and geographical origin of the floral source, although certain external factors also play a role, such as seasonal and environmental factors

Honey antioxidant activity appeared to be a result of the combined effect of a range of compounds. Phenolic compounds (flavonoids and phenolic acids), as well as non‐phenolic (ascorbic acid, carotenoid‐like substances, organic and amino acids, and proteins including certain enzymes such as glucose oxidase and catalase) can contribute to honey antioxidant

The honey phenolic compounds are the main antioxidant compounds of honey. They are the phenolic acids and flavonoids, which are considered potential markers of the honey botanical origin. The phenolic acids are divided in two subclasses: the substituted benzoic acids and cinnamic acids. The flavonoids present in honey are divided into three classes with similar structure: flavonols, flavones and flavanones. These are important due to their contribution to honey color, taste and flavor and also due to their beneficial effects

Large amount of research in honey also reports strong correlation between the total pheno‐ lic content and the antioxidant activity of honey extracts. Because of that, several literature reports have sought to identify and isolate them. Despite the relevant importance of poly‐ phenolic compounds, which are recognized as the major constituents and responsible for the health‐promoting properties of honey, their identification and quantification are of great

Analytical procedures used to determine polyphenols in a honey sample include their extrac‐ tion from the matrix as well as their separation and quantification. The determination begins with an extraction step by means of solvents, which are mostly mixtures of water‐alcohol in different proportions. Aqueous ethanol solutions (25–70 % v/v) are used in some work for 12–24 hours under stirring [42, 43]. While the methanolic extraction is used in different pro‐ portions with water [1, 44], there is still work using combined techniques of aqueous extrac‐ tion, with heating or acidification, and subsequent ethanol extraction [40, 45]. Few studies

The filtered or centrifuged extracts and different profiling techniques can be used for the determination of phenolic compounds. Liquid chromatography is considered to be the most useful separation technique for the analysis of polyphenols in different samples.

interest for understanding their contributions to the overall bioactivity of honey [40].

**6. Evaluation of the phenolic content**

conduct extraction with other solvents such as ethyl acetate [46].

and its processing [22, 40].

activity [40, 41].

294 Honey Analysis

on health [21].

Determination of a polyphenolic profile of honey is a complex task, so it is essential to develop separation and detection techniques, which would enable an unambiguous determination of as many components as possible. Tandem mass spectrometry is the detection method of choice when a comprehensive analysis of nontarget analyte is needed [46].

A wide variety of compounds isolated from honey and propolis come from flora, region and climate differences, where the nectar or sap was collected [12, 48, 49]. The phenolic com‐ pounds extracted, isolated and characterized can be classified into two major groups: pheno‐ lic acids and flavonoids.

The group of phenolic acids is divided into two main groups: derivatives of hydroxyben‐ zoic acid (**Figure 3A**) and the hydroxycinnamic acid derivatives (**Figure 3B**). The benzoic acid derivatives include salicylic acid, gentistic, p‐hydroxybenzoic, protocatechuic, vanil‐ lin, gallic, syringic and others. These are the most simple phenolic compounds found in foods [49, 50].

**Figure 3.** (A) General structure of derivatives of benzoic acid (benzoic acid, R1 = R2 = H). (B) General structure of the derivatives of hydroxycinnamic acid (hydroxycinnamic acid R1 = H) [12].

Hydroxycinnamic acid derivatives include p‐coumaric, caffeic, ferulic, among others. They may also be in conjugated form between themselves or with other organic compounds. This is the case of chlorogenic acid, which is the combination of quinic acid and caffeic acid [49, 50]. All cited phenolic acids have been described in honey samples in different concentrations according to the flora collected by honeybees [40, 46].

Flavonoids are compounds that possess the diphenylpropane skeleton: two benzene rings linked through oxygen containing a pyran or pyrone ring [46] (**Figure 4**). Flavonoids are a group of substances comprising classes of flavonols, flavones, flavonones, isoflavones, antho‐ cyanins and catechins. In plants, flavonoids are involved in pigmentation of fruits and flow‐ ers and the regulation of plant growth and plant protection against oxidative agents [32, 52]. In samples of honey and propolis naringenin, chrysin, rutin, morin, kaempferol, myricetin, hesperidin, apigenin, among others [40, 45, 46, 51] are found.

**Figure 4.** Structure of the major chemical types of flavonoids [51].
