2.6. Determination of 5-hydroxymethylfurfural

Extraction and analysis of HMF were performed by the method described by Delgado-Andrade et al. [15] with some modifications. 1 g of homogenized sample (Section 2.3) was weighted in a 40 mL centrifuge tube ad 19 mL distilled water was added. Samples were vortexed for 1 min (1600 rpm), 0.5 mL Carrez I solution was added and the mixture was vortexed for 10 seconds, 0.5 mL Carrez II solution was added and the mixture was vortexed for 10 seconds. The tubes were then centrifuged at 14,000 g at 4 °C for 10 min. Supernatants were filtered (Whatman N. 4), then microfiltered (0.45 μm acetate filters) and samples injected in the HPLC system (Pump L-7100, UV detector L-7400, Merck, Darmstadt, Germany). A Spherisorb ODS-2 column (5 μm, 4.6 mm × 250 mm) was used, the elution phase was water:acetonitrile (95:5) 1 mL/min and elution temperature was 32 °C. Chromatograms were recorded at 280 nm and HMF was identified and quantified through a calibration curve built with the pure standard compound (Sigma-Aldrich Italia). Each sample was extracted in triplicate and each extract was singly analysed (n = 3). Data are expressed as mg/kg dw.