H/D Exchange Coupled with 2H‑labeled Stable Isotope-Assisted Metabolomics Discover Transformation Products of Contaminants of Emerging Concern

Stable isotope-assisted metabolomics (SIAM) is a powerful tool for discovering transformation products (TPs) of contaminants. Nevertheless, the high cost or lack of isotope-labeled analytes limits its application. In-house H/D (hydrogen/deuterium) exchange reactions enable direct 2H labeling to target analytes with favorable reaction conditions, providing intuitive and easy-to-handle approaches for environmentally relevant laboratories to obtain cost-effective 2H-labeled contaminants of emerging concern (CECs). We first combined the use of in-house H/D exchange and 2H-SIAM to discover potential TPs of 6PPD (N-1,3-dimethylbutyl-N′-phenyl-p-phenylenediamine), providing a new strategy for finding TPs of CECs. 6PPD-d9 was obtained by in-house H/D exchange with favorable reaction conditions, and the impurities were carefully studied. Incomplete deuteride, for instance, 6PPD-d8 in this study, constitutes a major part of the impurities. Nevertheless, it has few adverse effects on the 2H-SIAM pipeline in discovering TPs of 6PPD. The 2H-SIAM pipeline annotated 9 TPs of 6PPD, and commercial standards further confirmed the annotated 6PPDQ (2-anilino-5-(4-methylpentan-2-ylamino)cyclohexa-2,5-diene-1,4-dione) and PPPD (N-phenyl-p-phenylenediamine). Additionally, a possible new formation mechanism for 6PPDQ was proposed, highlighting the performance of the strategy. In summary, this study highlighted a new strategy for discovering the TPs of CECs and broadening the application of SIAM in environmental studies.稳定同位素辅助代谢组学（Stable isotope-assisted metabolomics, SIAM）是发掘污染物转化产物（transformation products, TPs）的有力工具。然而，同位素标记分析物的高昂成本或供应匮乏限制了其应用范围。自主开展的氢（H）/氘（D）交换反应可在适宜反应条件下直接对目标分析物进行氘标记，为环境相关研究实验室获取高性价比的新出现关注污染物（contaminants of emerging concern, CECs）的氘标记形式提供了直观且易于操作的技术途径。本研究首次将自主氢氘交换技术与氘标记SIAM相结合，用于发掘N-(1,3-二甲基丁基)-N′-苯基对苯二胺（6PPD）的潜在转化产物，为CECs的转化产物发掘提供了全新策略。本研究通过自主氢氘交换反应以优异的反应条件制备得到氘代9位6PPD（6PPD-d9），并对其中的杂质进行了细致表征：例如未完全氘代的6PPD-d8是该样品中杂质的主要组分。尽管如此，其对用于6PPD转化产物发掘的氘标记SIAM工作流程几乎无不良影响。该氘标记SIAM工作流程共注释得到6PPD的9种转化产物，市售标准品进一步验证了注释得到的2-苯胺基-5-(4-甲基戊烷-2-基氨基)环己-2,5-二烯-1,4-二酮（6PPDQ）与N-苯基对苯二胺（PPPD）。此外，本研究提出了6PPDQ一种可能的全新生成机制，凸显了该策略的应用性能。综上，本研究提出了一种发掘CECs转化产物的全新策略，并拓展了SIAM在环境研究领域的应用边界。