Extraction and Quantification of a Carcinogenic and Cytotoxic compound: 5-hydroxymethyl-2-furfural (HMF) in honey using HPLC

Abstract

As there is a rise in adulteration of honey which leads to the formation of a carcinogenic and cytotoxic compound, 5-hydroxymethyl-2-furfural (HMF) in honey, it has become very essential to test honey for the presence of this compound. Honey is a natural sweet product that is produced by honey bees (Apis mellifera). This carcinogenic compound, 5-hydroxymethyl-2-furfural is developed on dehydration of reducing sugars present in honey in acidic conditions. The concentration of this toxic compound in fresh honey is mostly absent or is present in very low amounts, but its concentration increases during processing, upon storage for longer periods, and upon heating/storing at higher temperatures. This compound causes toxic fatal effects (mutagenic, chromosomal aberrations, genotoxic, organotoxic, carcinogenic, cytotoxicity towards mucous membranes, the skin, the eyes, and the upper respiratory tract). An accurate and specific liquid chromatographic method was developed due to the urgent requirement for an analytical method to estimate the quantity of 5-hydroxymethylfurfural in honey. A mobile phase with a combination of Acetonitrile and Water with 0.2% Formic Acid was used with a flow rate of 0.6 ml/minute and the separation was done on Phenomenex Non-Polar C18 Column with dimensions of 150 mm X 4.6 mm X 3 µm at a wavelength of 282 nm. The Calibration curve with R2 value of 0.999 was obtained. These results were repeated two times in the laboratory, hence Repeatability Precision was performed to validate the results. The amount of HMF in honey in the samples were found to be within the limit of 40 mg/kg as per FSSAI. The amount of HMF present in Sample 1 and Sample 2 was found to be 3.80 mg/kg and 6.50 mg/kg, respectively. This new HPLC method developed would be economical, reproducible and repeatable.