A new approach to detecting sugar syrup addition to honey: Stable isotope analysis of hexamethylenetetramine synthesised from honey monosaccharides (fructose and glucose)

•One of the most common types of adulteration of honey involves the addition of invert sugar syrups.•Intrinsic hydrogen stable isotope ratios in honey monosaccharides were measured after conversion to Hexamethylenetetramine.•The method offers distinct advantages in terms of ease of use, analysis time for δ2H and δ13C measurements.•Honey from higher latitudes and more inland areas have depleted δ2H values and offers potential to detect C3 rice invert syrup.•The method provides complementary information on the botanical source of sugars in honey. One of the most common types of adulteration of honey involves the addition of invert sugar syrups. A new method was developed to measure the stable isotope ratios of carbon and carbon-bound non-exchangeable (CBNE) hydrogen from specific molecular positions in fructose and glucose in honey. This was achieved through periodate oxidation of the sugars to produce formaldehyde, followed by reaction with ammonia to form hexamethylenetetramine (HMT). The preparation was simplified, optimized, and validated by isotopic analysis of replicate syntheses of HMT from fructose, glucose, sugar syrup and a representative authentic honey sample. The optimized method had a repeatability standard deviation from 1.5‰ to 3.0‰ and from 0.1‰ to 0.4‰ for δ2H and δ13C, respectively. This methodology has advantages over alternative isotopic methods, for measuring CBNE hydrogen isotope ratios in sugars, in terms of time, sensitivity and operability and offers a complementary method to differentiate authentic honey from invert sugar syrups.