Hydroxyacetone

Chemical analyses of honey samples were performed using methods adapted from Windsor et al. [27 , 35 ]. Briefly, this method involved accurately weighing 0.30–0.40 g of honeys in duplicate into 75 mm test tubes. hydroxyacetone internal standard (0.250 ml, 0.528 mg/ml) was weighed into the test tubes and the entire sample dissolved in 1.2 ml of O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine (PFBHA) reagent (0.2 g per 10 ml 0.1 M citrate buffer at pH 4). Derivatisation of MGO, DHA and 5-hydroxymethylfurfural (HMF) with PFBHA took 75 minutes. The samples were diluted with water and acetonitrile until homogeneous, and quantified in duplicate by Reverse Phase-High Performance Liquid Chromatography (RP-HPLC; Perkin Elmer^TM Series 200 HPLC and Autosampler, Binary Pump and Flexar® Photodiode Array Detector with a ZORBAC Eclipse Plus column (75 mm x 4.6 mm, 3.5 μm) utilising a 75:25 water: acetonitrile to 100% acetonitrile mobile phase program).
MGO, DHA, HMF (Sigma-Aldrich, St Louis, MO, USA) standards were used for six point calibration, and hydroxyacetone (Sigma-Aldrich) was used as the internal standard for quantitation. All standard solutions were stored at -20°C. Quantitation standards were prepared by dissolving known concentrations of the standard solutions in six aliquots of clover honey (which has no detectable MGO, DHA or HMF unless spiked).