A high-temperature water vapor equilibration method to determine non-exchangeable hydrogen isotope ratios of sugar,...

The analysis of the non-exchangeable hydrogen isotope ratio (δ² H_ne ) in carbohydrates is mostly limited to the structural component cellulose, while simple high-throughput methods for δ² H_ne values of non-structural carbohydrates (NSC) such as sugar and starch do not yet exist. Here, we tested if the hot vapor equilibration method originally developed for cellulose is applicable for NSC, verified by comparison with the traditional nitration method. We set up a detailed analytical protocol and applied the method to plant extracts of leaves from species with different photosynthetic pathways (i.e., C₃ , C₄ and CAM). δ² H_ne of commercial sugars and starch from different classes and sources, ranging from -157.8 to +6.4‰, were reproducibly analysed with precision between 0.2‰ and 7.7‰. Mean δ² H_ne values of sugar are lowest in C₃ (-92.0‰), intermediate in C₄ (-32.5‰) and highest in CAM plants (6.0‰), with NSC being ² H-depleted compared to cellulose and sugar being generally more ² H-enriched than starch. Our results suggest that our method can be used in future studies to disentangle ² H-fractionation processes, for improving mechanistic δ² H_ne models for leaf and tree-ring cellulose and for further development of δ² H_ne in plant carbohydrates as a potential proxy for climate, hydrology, plant metabolism and physiology.