*2.5. Determination of Hydroxymethylfurfural (HMF)*

The HMF content was determined based on the UV absorbance of HMF at 284 nm using a UV-Visible spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) [32]. In order to avoid the interference of other components at this wavelength, the difference between the absorbance of a clear aqueous honey solution and the same solution after the addition of bisulfite was determined. The HMF content was calculated after the subtraction of the background absorbance at 336 nm. In a 50 mL volumetric flask containing 2 mg of honey dissolved in 25 mL of water, 0.5 mL of Carez I solution was added, followed by 0.5 mL of Carez II solution. Water was added to the flask to make up a volume of 50 mL, and the resulting solution was filtered. After discarding the first 5 mL of the filtrate, 5 mL of 0.2% sodium bisulfite solution was added to the test tube. In another test tube, 5 mL of pure water was added as a blank. A UV-visible spectrophotometer was used to measure the solution's absorbance at 284 and 336 nm in 10 mm quartz cells within 1 h. The calculation was performed using the formula below:

$$\text{HMF} \left( \text{mg/kg} \right) = \left( \text{A284} - \text{A336} \right) \times 149.7 \times 5 \times \frac{\text{D}}{\text{W}}$$

It should be noted that 149.7 is a constant, A284 is the absorbance at 284 nm, A336 is the absorbance at 336 nm, 5 is the theoretical nominal sample weight, D is the dilution factor (in case dilution is necessary), and W is the weight of honey taken.
