Comprehensive honey analysis: A novel HPLC-DAD method for hydroxymethylfurfural quantification and quality evaluation via diastase activity and sugar profile determination

Ensuring the authenticity and quality of honey is essential to safeguarding consumer health and combating food fraud. The primary analyses for honey quality involve determining diastase activity (DA), 5-hydroxymethylfurfural (HMF), and carbohydrate composition. A new high-performance liquid chromatography coupled with diode array detection (HPLC-DAD) method was developed, for the precise quantification of HMF, a key marker of honey quality and degradation. Utilizing a Phenomenex Kinetex RP-18 column (5 µm, 150 × 4.6 mm i.d.), with a mobile phase containing water and methanol (95:5, v/v) in gradient mode, at a flow rate of 1 mL/min, and UV detection at 284 nm, the method achieved rapid and efficient separation within 10 minutes, with a low detection limit of 0.03 mg L⁻¹, and demonstrated good linearity, precision and recovery rates. The carbohydrate composition of honey, specifically glucose, fructose, saccharose, and maltose, was analysed using a HPLC with refractive index detection (HPLC-RI) method, using a Phenomenex Luna Omega Sugar column (3 µm, 250 ×4.6 mm i.d.), with a mobile phase composed of acetonitrile and water (85:15, v/v). This analytical protocol, incorporating the quantification of maltose, a critical yet often overlooked sugar, significantly bolsters the standard evaluation framework for honey authenticity. DA, a crucial enzymatic indicator, was assessed via the Schade method, providing additional insights into the enzymatic integrity and freshness of the honey samples. The methods were applied to analyse 65 commercial Spanish honey samples, revealing significant compliance with EU regulatory standards, yet also uncovering situations of potential adulteration. This research adds to the ongoing discussions on food safety by offering improved and dependable techniques for thoroughly assessing honey samples.