**3. Discussion**

**2.3. Extraction procedure for phenolic compound analysis**

88 Honey Analysis

**Figure 2.** Total ion chromatograms (TIC) of phenolic compounds using UPLC-ESI-MS/MS.

Honey sample (10 g) is dissolved in ultrapure water (50 mL) and mixed 5 min via vortex. Then ethyl acetate (50 mL) is added into solution flask and the flask is placed on a shaker for 30 min. In the analysis of amino acids and phenolic compounds, an extraction procedure is important. Extraction of amino acids and phenolic compounds depends on their chemical properties such as natural matrix and molecular structure together with their polarity, concentration, aromatic chain number and variation in their hydroxyl groups, etc. Protein, carbohydrate and other complex structures are hindered to extract several phenolic compounds. Differences in the chemical structure of phenolics in the sample are related to concentration of the functional groups, simple and complex polyphenolic structures and phenolic acid and flavonoid in different proportions. In the literature, more than one methods and techniques are needed to be used for the extraction.

The extraction step of amino acids and phenolic compounds is the very critical step after sample preparation. Organic and inorganic solvents are commonly used in the extraction. The efficiency of the extraction is affected from including extraction temperature, time, solventsample ratio and solvent types.

Furthermore, treatment time and temperature together with a selection of solvent ratio is very crucial for optimum recovery of amino acids and phenolic compounds. Generally, increasing time and temperature is preferable for the solubility of analytes; however, undesirable enzymatic oxidation arising from high temperature and extended extraction time may cause degradation of amino acids and phenolic compounds. The solvent–sample ratio and repetition number of extraction affect the recovery of phenolic compounds for each sample [17].

Sample matrix and particle size are highly affected the extraction of amino acids and phenolics. Issue of diffusion is related to particle size. Diffusion becomes easier as particle size gets smaller and efficiency of extraction gets higher. However, this increasement continues to some level and after that point it stops or decreases. That situation shows up with the reduction of the mass transfer rate caused by small particles. More solvent is needed in this stage.

Phenolic substance can bound to organic bodies such as carbohydrate and protein in the sample materials. And thus, bounded phenolics can be liberated by hydrolysis with the addition of enzyme.

According to literature survey, there is a lack of knowledge about the profiles of amino acids and phenolic contents in honey to evaluate the quality of the product.

Several studies have been revealed that honey serves as a source of natural antioxidants with the anti-microbial, anti-inflammatory, anti-mutagenic, anti-tumour and anti-oxidative activity, which are effective in reducing the risk of heart disease, immune-system decline, different inflammatory processes, etc. [2]. Honey species also possess antibacterial activities and are scavengers of active oxygen radicals [15]. Among the components present in honey which are responsible for its anti-oxidative effect are phenolic compounds (flavonols, flavones, flavanones, benzoic and cinnamic acids) [17].

Thereby, as honey is a very complex product. Depending on the nectar-providing plant species, bee species, geographical area, season and a method of storage demand a comprehensive analysis of constituents, such as volatile compounds, phenolic acids, flavonoids, carbohydrates and amino acids, for its characterization [2, 18].

According to literature survey, arginine, tryptophan, phenylalanine, tyrosine and lysine are found in considerable amounts in honey. And also in various studies, they are qualified as a characteristic of some floral types of honey [18, 19].

Phenylalanine, proline, tyrosine, isoleucine, and leucine are revealed as the main amino acids [1]. The studies indicate, on the basis of honey activity, a better differentiation, considering free amino acid contents instead of physicochemical honey characteristics [19]. Moreover, amino acid composition may also be a suitable method to determine honey botanical origin [1, 20].

Around 200 substances have been reported in this complex natural liquid but the composition especially its secondary metabolites and quality of honey may be influenced by some external factors such as environmental and seasonal factors, processing, handling and storage [5, 6].

The determination and evaluation of phenolic constituents in honey appeal high attention by consumers and researchers owing to a health-promoting feature that is accompanied by bioactivity [21].

Botanical origin of honey is classified according to phenolic ingredients [21, 22] and this consequently implies that as honeybees collect nectar from plants which contain bioactive components. These phytochemical ingredients can be transferred to honey by honeybees [23, 24]. Numerous flavonoids (such as apigenin, kaempferol, quercetin, chrysin and luteolin) and phenolic acids (caffeic, gallic, cinnamic, protocatechuic, p-coumaric and chlorogenic acids) are identified in various honey samples [2].
