Spectrophotometric Assessment of 5-HMF in Thermally Treated Honey: Analytical Performance Comparison of Classical and Modified Methods

To ensure high-quality honey, and to delay crystallization or fermentation, honey is subjected to heat treatment. The heating process, as well as the duration and storage conditions, can lead to an increased level of 5-HMF (5-hydroxymethylfurfural), which exhibits numerous adverse effects on human health. The objective of the present study was to evaluate the evolution of 5-HMF levels in different varieties of honey from Romania, thermally treated at different temperatures between 45 and 75 °C. Both classical spectrophotometric methods, such as White and Winkler methods, and two modified simpler methods were used. The methods were compared based on their analytical reactions and analytical parameters such as linearity, LOD and LOQ, sensitivity, accuracy, and precision. The best-performing method was the one that employed thiobarbituric acid. The level of 5-HMF in the control samples and samples treated at 45 °C are below the limits accepted by the legislation (40 mg/kg). At higher temperatures, such as 55–65 °C, the values in some honey species, especially polyfloral species, already exceed the accepted threshold (57.5 ± 0.7 mg/kg; 98.8 ± 4.6 mg/kg) whereas at 75 °C, the 5-HMF level is very high (142.8 ± 8.7 mg/kg; 453.8 ± 51.3 mg/kg). The results obtained indicate that 5-HMF level increases gradually with temperature and is variety dependent.