Quinones are pivotal redox-reactive organic substances in natural organic matter with key roles in biogeochemical cycles of critical elements and reactions of pollutants. Despite their well-recognized importance, the chemical nature of quinones in environmental samples (such as biochars) are still largely unknown. This knowledge gap persisted over a decade due to intricate nature of environmental samples and diversity of quinones. To tackle this, our research aimed to develop a method for identifying quinones in complex environmental media by coupling novel chemical tagging with high resolution mass spectrometry (HRMS) and metabolomic analysis. Michael addition reaction with the thiol group-containing compounds, i.e., cysteine (Cys) and cysteine-containing peptides (CCPs), were used for tagging quinones selectively. Model quinone reactions were validated by UV spectroscopic analysis and time-of-flight mass spectrometry (TOF-MS). Quinone identificaiton in aqueous extractions of biochars was achieved by metabolomic data processing based on HRMS data analysis. Through analysis of Cys or CCP tagged samples, 73 and 397 quinones were identified with average molecular weights of 228.1031 and 262.4232. Presence of benzoquinone was validated with authentic standard and structures were predicted for top five high-confidence candidates (C₇H₆O₂, C₇H₆O₅, C₁₁H₁₀O₃, C₁₂H₁₂O₃, and C₁₂H₁₂O₄), showing triplet spectra including apo-quinones, Cys tagged quinone, and CCP tagged quinone. Molecular networking analysis revealed three additional candidates. Majority of the quinone candidates lied in shikhimates and phenylpropanoid biosynthetic pathways. Chromatography separation coupled with redox reactivity screening and HRMS analysis-based metabolomic interrogation was used for further isolation of critical quinones. Non-aromatic CCP tagging method was also developed for quantifying total quinones in complex environmental samples. Overall, this study developed innovative methods for identifying quinones within intricate environmental samples and accurately quantifying their concentrations. Such efforts can lead to the replacement of the enigmatic 'black box' of redox-reactive natural organic carbon in biogeochemistry modeling and engineering application design with a spectrum of distinct compounds.

Srinidhi Lokesh

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