Comprehensive molecular profiling of lignin-derived products from reductive catalytic fractionation by HPLC-UV/MSn: from monomers to heavy oligomers.

Reductive catalytic fractionation (RCF) has emerged as a leading lignin-first biorefinery strategy for producing valuable phenolic chemicals. However, molecular-level characterization of its highly complex product mixtures remains challenging due to extensive structural isomerism, severe ion suppression, and the limited capability of conventional mass spectrometry to resolve higher molecular weight species. Herein, we present a comprehensive analytical platform based on high-performance liquid chromatography coupled with ultraviolet detection and multi-stage mass spectrometry (HPLC-UV/MSn) for direct characterization of RCF-derived phenolic products without further derivatization or fractionation after centrifugation. By integrating chromatographic separation with tandem mass spectrometry, the approach transforms congested one-dimensional mass spectra into two-dimensional molecular landscapes, enabling the differentiation of structural isomers and the putative assignment of more than 50 phenolic species through retention behavior and MSn fragmentation patterns. The chromatographic dimension also improves the detectability of some weak signals, extending molecular coverage from monomers to heavy oligomers with molecular weights up to approximately 1050 Da. In addition, a preliminary concentration-response assessment was conducted using six representative model compounds spanning oxidized phenolics, a non-oxidized phenolic monomer, reduced propyl G/S monomers, and a C-C-linked dimer. By bridging the long-standing analytical gap between volatile monomers and complex oligomeric fractions, this work provides a powerful and broadly applicable platform for elucidating lignin depolymerization pathways, tracking structural evolution during RCF, and accelerating the rational optimization of biomass valorization processes.