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.