Development of a Green Analytical Method for the Assessment of Honey Quality through Reducing Sugars, Hydroxymethylfurfural and Physicochemical Parameters

A low-cost and green spectrophotometric method was developed to be used as a routine quality control method of honey. The use of Carrez reagents, p-toluidine and Fehling's copper reagents was eliminated by simply filtering the solution through a membrane and measuring reducing sugars using a miniaturised alkaline hexacyanoferrate(III) reaction at 420 nm, and 5-hydroxymethylfurfural (HMF) at 284 nm against a sodium bisulfite reference. Moisture, pH, free acidity, electrical conductivity, ash and Pfund colour were measured with reagent-free or low-reagent procedures on the same samples. Under the optimum conditions (2.0 mmol L-1 K3[Fe(CN)6], 0.20 mol L-1 Na2CO3, 90 °C, 6 min) the sugar response was linear from 10 to 100 µg mL-1 (R2 = 0.9997), and the HMF response was linear from 1.0 to 20.0 mg L-1 (R2 = 0.9999). The limits of detection were 2.21 µg mL-1 and 0.21 mg L-1, respectively. Intra and Inter day RSD values were not more than 1.7% and 2.6% respectively and recovery ranged between 97.6% and 102.4%. Eighteen honeys from Iraqi markets and apiaries were analysed. Of the 15, all met the Codex Alimentarius and EU limits for all parameters, but three imported commercial blends failed the HMF limit (40 mg kg-1) and two of these also failed the moisture and sucrose limits. The results were in good agreement with the Lane – Eynon method and HPLC-UV method (paired t-test, p> 0.05). The procedure achieved a score of 89 on the Analytical Eco-Scale and 0.73 on AGREE in comparison to 0.49 to 0.54 for the conventional procedures. Two components accounted for 90.7% of the variance and principal component analysis discriminated heat-damaged blends from fresh local honeys.