CRB-FCC: A Standardized Nontargeted Analysis for Formula Assignment and Structure Annotation

LC-MS-based nontargeted analysis is essential for identifying key chemicals in real-world samples. A significant technical bottleneck, however, arises in resolving the structures of unknown chemicals from their mass spectra. This challenge is exacerbated by the limited information available in existing databases and the poor quality of the acquired mass spectra. To address these issues, we developed the Fragmental Chain Characterization (FCC) method, which assigns formulas to unknown compounds based on their mass spectra. When integrated with the Chromatographic Retention Behavior (CRB) approach, the CRB-FCC method enables the identification of the same compound across different samples, leveraging consistent retention times and assigned formulas, even when sampling conditions cause significant variations in mass spectra. The CRB-FCC method is validated using a large set of 1,475 chemical standards. In comparison to conventional annotation methods, which rely on mass spectral matching, CRB-FCC has shown the ability to accurately annotate compounds even when their mass spectra are of poor quality or absent from the database. As a case in point, we combined CRB-FCC and NMR characterization to successfully identify unknown yellow-hue impurities in biosynthesized plastics sourced from local industrial sources, addressing a key barrier to their practical applications. We anticipate that CRB-FCC, a standardized protocol for data acquisition and interpretation in nontargeted analysis, will accelerate its adoption across both academia and industry.