Natural products have been used for ages as dietary supplements. Natural products like Honey are nutritional and beneficial for our body. Honey is a natural medicinal product that is produced by honey bees (Apis mellifera) after they collect the nectar from flowers. Honey is a natural sweetener that is added to food to give flavor of sweetness [1]. Honey has many secondary metabolites like Flavonoids, Alkaloids, Terpenoids, Phenolics, etc which can heal various diseases related to gastrointestinal and respiratory tracts [2, 3, 4]. Honey is used in the treatment of Cancer. Honey is useful for the skin as it gives an anti-oxidant effect, promotes the development of new tissue, cures wounds, acne, eczema, dandruff, tinea and psoriasis as it is anti-inflammatory, anti-fungal and anti-bacterial properties [2, 4, 5]. Honey contains amino acids and reducing sugars like glucose and fructose in majority and other sugars like trehalose, melibiose, turanose and raffinose in low amounts [1, 2, 3, 4, 6, 7].
An organic compound 5-hydroxymethyl-2-furfural (HMF) is produced from the dehydration of reducing sugars in honey in acidic environments (amino acids in honey) when heated. This is called as Maillard reaction which is a non-enzymatic browning reaction. [1, 3, 7, 8, 9]. This adulteration [3] leads to toxic effects. HMF has carcinogenic, mutagenic, genotoxic, chromosomal aberrations, organotoxic and cytotoxic on mucous membranes, the skin, the eyes and the upper respiratory tract [1, 7, 8]. HMF is a heterocyclic compound containing 6 carbon atoms comprising of a furan ring on which two functional groups i.e., formyl and hydroxymethyl groups are attached at the second and fifth positions, respectively (Fig. 1). HMF is a white-colored compound that is highly soluble in water [1, 8]. HMF concentration indicates honey’s freshness and quality. The concentration of HMF is negligible in fresh honey but increases during its processing or heating, upon storage for longer periods or storing in higher temperatures [1, 7, 10].
The FSSAI (The Food Safety and Standards Authority of India) also marks honey’s quality as degraded if HMF in honey exceeds the limit of 40 mg/kg. According to Codex Alimentarius, HMF in Honey must not exceed the limit of 40 mg/kg after processing and blending, except for honey obtained from tropical climatic region [10].
Due to this adulteration and processing of honey, there is an urgent need to develop an analytical method to quantify the amount of HMF present in honey as this leads to serious health issues and can lead to the death of an individual if the quality of honey (marketed and sold to the public) is not checked properly [1, 3, 7, 8, 10].
In this research work, a new analytical method to quantify the amount of HMF in honey was developed on HPLC (High-Performance Liquid Chromatography) by using the Calibration curve method as there are very few articles on HPLC analysis of HMF in honey were reported before, but they were not reproducible in the laboratory and were uneconomical [10–21], few articles reported for LC-MS/MS analysis of HMF in pharmaceutical products [22, 23, 24], few articles reported for detection of adulterants using NMR [25]. And there is no official method in FSSAI too for the quantification of HMF in honey. Hence, this new analytical method was developed. First, the Extraction of HMF from honey was carried out and then it was quantified on HPLC using the reference standard for HMF with 99% purity, obtained from Sigma-Aldrich.
These results were repeated twice in the same lab on the same instrument to validate the results for the Repeatability Precision Validation parameter.