Compound-specific hydrogen isotope analysis of lignin methoxy groups for tea provenance discrimination: Comparison with dual-water equilibration.

Hydrogen stable isotope compositions in plant tissues encode elevation-dependent climatic signals that can be exploited for the authentication of geographic origin. However, bulk hydrogen isotope measurements conflate exchangeable and non-exchangeable hydrogen pools, obscuring the climatic signal. Compound-specific analysis of lignin methoxy groups targets a chemically uniform, non-exchangeable hydrogen pool that preserves source-water isotopic signals. In this study, compound-specific gas chromatography-isotope ratio mass spectrometry (GC-IRMS) analysis of lignin methoxy δ2H was directly compared with non-exchangeable δ2H values derived independently using the dual-water-vapour equilibration method to evaluate methodological agreement and robustness. This approach was applied to 45 Sri Lankan orthodox black tea samples spanning low-grown, mid-grown, and high-grown elevation zones. Methoxy δ2H values decreased systematically with elevation, ranging from -123 ± 3‰ (low-grown) to -150 ± 2‰ (high-grown), yielding a 27‰ gradient across approximately 2000 m. Analysis of variance identified elevation as the dominant source of variation, accounting for 99.5% of the total variance (F = 4202, p < 0.001). Methodological performance was evaluated through direct comparison with non-exchangeable δ2H values obtained by dual-water vapour equilibration, which produced independent δ2Hnex estimates strongly correlated with GC-IRMS methoxy measurements (r = 0.9149, slope = 0.9601). The exchangeable hydrogen fraction remained consistent across all samples (18.49 ± 1%), indicating similar hydrogen-bonding environments at all elevations. Bland-Altman analysis demonstrated predictable inter-method agreement, with a systematic offset of -23‰. The strong proportional agreement, despite a consistent and predictable systematic offset, demonstrates that compound-specific methoxy δ2H analysis yields results consistent with established equilibration-based methods while providing a more direct, chemically defined measurement of non-exchangeable hydrogen. These findings highlight the potential of compound-specific methoxy δ2H analysis as a geochemically robust marker for elevation-based discrimination of Sri Lankan tea and provide a foundation for future multi-laboratory evaluation of isotope-based authentication frameworks.