Method dependency of exchangeable hydrogen determination in amino acids: a cautionary comparison of liquid- and vapor-phase equilibration

Stable hydrogen isotope analysis (δ²H) of organic compounds requires accurate correction for exchangeable hydrogen, yet this remains a major analytical challenge, particularly for amino acids. In this study, we evaluated the determination of exchangeable hydrogen fractions fex in a suite of amino acids using a multiple equilibration approach, comparing room-temperature liquid-water-phase equilibration with 90 °C online vapor equilibration using the UniPrep2 system. Theoretical fex values for the amino acids were derived from their molecular structure and compared with the experimentally determined values. Liquid equilibration yielded fex values that were generally consistent with theoretical expectations for most amino acids, indicating effective access to the exchangeable organic hydrogen pools. In contrast, online vapor equilibration frequently resulted in near-zero fex for several amino acids, particularly the hydrophobic and crystalline compounds, consistent with limited accessibility of exchangeable sites under heated, low-pressure equilibration conditions. Only a subset of amino acids (e.g. glycine, threonine) showed comparable results between the methods, while others exhibited clear method-dependent discrepancies or analytical artefacts (e.g. persistent water background for proline). A soluble protein (BSA) exhibited low but consistent exchangeability (∼ 4 %) with both approaches, highlighting the role of structural protection and accessibility in governing hydrogen exchange. Our findings demonstrate that experimentally derived fex values are likely to be significantly method- and compound-dependent and should be interpreted as operational parameters rather than intrinsic molecular constants. The observed variability has important implications for interlaboratory comparability and underscores the need for standardised methodologies and improved reference materials for compound-specific δ²H analysis of amino acids.