Carbon Stable Isotope Analysis for Glucose in Sake: Simple Freeze-Dried Sake Can Substitute for Glucose Following HPLC Isolation

Carbon stable isotope analysis of ethanol is an established method for determining the authenticity of alcoholic beverages, but there is no accepted method for measurements of glucose carbon stable isotopic composition (delta C-13). This study aims to establish two methods to determine accurately the delta C-13 value of glucose isolated from sake by high-performance liquid chromatography (HPLC) after three clean-up steps using ion exchange chromatography and in simple freeze-dried sake (total nonvolatile matter) and to examine two methods that yield different delta C-13 values. In the glucose isolation procedure, the delta C-13 values of glucose were not significantly affected by the glucose concentration, solvent (water or aqueous acetonitrile), drying method (freeze-dry or nitrogen-spraying), the clean-up step using ion exchange chromatography (weakly acidic cation-exchange, strongly acidic cation-exchange, and weakly basic anion-exchange resins), or HPLC isolation. Glucose nitrogen spray-dried after isolation by ion exchange chromatography and HPLC showed a carbon isotope discrimination value of < 0.1 parts per thousand. The glucose delta C-13 values ranged from -26.8 to -25.5 parts per thousand in authentic sake and from -27.0 to -11.0 parts per thousand in commercial sake samples. This HPLC isolation approach may provide a first step toward visualizing the brewing process via isotopic carbon flow during fermentation. Additionally, there was no significant difference between the delta C-13 values of glucose and total nonvolatile matter from sake. The delta C-13 measurement of simple freeze-dried sake can substitute for glucose delta C-13 measurement using HPLC isolation, thus aiding high-throughput detection of carbon derived from C-4 plants in sake.