Determination of HMF in honey:

Weigh 5 g of honey in an analytical balance using a properly labeled 50 mL beaker, dissolve

the sample by adding 25 mL of distilled water and then transfer it to a 50 mL volumetric flask.

Add 0.5 mL of Carrez solution I and 0.5 mL of Carrez solution II and fill the volumetric flask

to the meniscus with distilled water.

Add two drops of ethanol to prevent foaming. Mix the solution and filter using filter paper;

discarding the first 10 mL filtered.

Label two test tubes and pipette 5 mL of the filtrate over 5 mL of distilled water in the first

(sample) and 5 mL of the filtrate added to 5 mL of 0.2% sodium bisulfite solution in the second

tube (blank). Shake the tubes using a vortex mixer. Measure the absorbance in a UV-vis

spectrophotometer at wavelengths of 284 and 336 nm using quartz cuvettes.

Before the readings, calibrate the spectrophotometer with a blank reference for each sample

evaluated. If absorbance at 284 nm exceeds 0.6 the sample is diluted with water and the blank

reference with sodium bisulfite 0.2%, in the same proportions, and the reading is repeated.

The HMF content in honey is calculated with Eq. (9) .

HMF = ( A284_  -_ A__33_6_ )_  ×_ 1_4_9_.7_ ×_ _5 Sample weight ( g ) ,