COMPARISON OF ANALYTICAL METHODS FOR DETERMINING NON- EXCHANGEABLE STABLE HYDROGEN RATIOS IN TEA

Abstract

Determining the δ2H value of organic compounds is challenging due to the presence of moisture traces from ambient humidity, hygroscopic compounds, and exchangeable non-bound hydrogen atoms. Several approaches have been developed to avoid the interference of exchangeable hydrogen. However, these approaches are comparatively difficult to use for the δ2H analysis of tea samples due to a lack of suitable solid reference materials for two-point calibration and time-consuming procedures. This study adapts and scales the method for lignin analysis, targeting methoxy groups to measure non-exchangeable hydrogen for the analysis of tea samples using gas chromatography coupled with isotope ratio mass spectrometry (GC-IRMS). It aims to compare the mentioned method with bulk δ²H analysis of tea samples using equilibration techniques that involve different isotopic compositions and laboratory moisture. A total of 45 orthodox black tea samples were collected from three different tea growing regions with varying elevations in Sri Lanka: Nuwara Eliya (n=15), Kandy (n=15), and Ruhuna (n=15), and δ2H values were measured using three different methods: GC-IRMS analysis of methoxy group, water vapor equilibration (enriched δ²H) and laboratory moisture equilibration. One-way analysis of variance (ANOVA) was performed, and the results indicated a statistically significant difference in the averages of δ²H values between the three methods (p < 0.01). The GC-IRMS method for analysing δ²H in the methoxy groups of tea samples was the most reliable and precise method for analysing δ²H in tea, as it offered consistent δ²H values with low variability. There was a slight variability due to natural variations in the δ²H of the non-exchangeable hydrogen in different elevations. It provided a clear separation between regions with minimal overlap. The laboratory moisture equilibrium and water vapor equilibration methods demonstrated high variability and lower precision, which may be due to environmental factors such as temperature and humidity. In addition, these methods were time-consuming compared to the GC-IRMS method for analysing the δ2H in methoxy groups. Therefore, GC-IRMS methoxy group analysis is a robust method for δ²H determination in tea with precision and consistency.