Stable Isotope Analysis of Non-exchangeable Hydrogen in Sugars by Oxidation/Derivatisation to hexamethylenetetramine and Elemental Analyzer-Chromium/High Temperature Conversion- Isotope Ratio Mass Spectrometry

Stable hydrogen isotope may provide evidence to identify the botanical origin of exogenous sugars added to honey, due to a combination of factors such as the systematic global variation of meteorological waters and morphological differences affecting rates of evapotranspiration prior to water incorporation into plant carbohydrate prior to photosynthesis. Here we report an simplified method, which involves the preparation of hexamethylenetetramine (HMT), produced through chemical transformation of sugar molecules, to allow direct analysis of the carbon-bound non-exchangeable hydrogen (CBNE) by elemental analyser high temperature chromium reduction coupled to Isotope Ratio Mass Spectrometry (EA-Cr/HTC-IRMS). We have optimized the reaction conditions for oxidation and derivatisation in terms of reaction time and pH of solution. A two month-monitoring for of the repeatability of δ²H values of an HMT standard  has demonstrated stable and reliable results of this technique. The procedure is relatively straightforward, convenient and easy to apply compared with alternative reported CBNE analysis methods for foodstuffs, particularly those where isolating sugars is challenging and consensus over dual-water equilibration conditions are contentious.  Moreover, it has the advantage over other methods by avoiding the use of corrosive and explosive reagents such as sugar nitro-derivatives.  This study has also demonstrated the potential to identify from the presence of rice syrup, which is currently a significant challenge for the honey industry.