Oligosaccharides (OSs) are abundant in natural products (NPs), yet their rapid and effective characterization, especially for absorbed OSs, remains challenging. In this study, an enhanced data acquisition and analysis strategy using three derivative labels was proposed and successfully applied to characterize the in vitro and in vivo profiles of OSs in Mori Fructus fruit (MF). For data acquisition, Data Independent Acquisition (DIA) was performed to maximize the MS information of OSs, followed by Mass Defect Filtering (MDF) using Metabolynx XS™ to automatically exclude irrelevant ions and generate Precursor Ion List (PIL) of potential OSs. A PIL-based Data Dependent Acquisition (DDA) was subsequently implemented to obtain clear MS2 spectra. For data analysis, fragmentation behaviors of OS reference standards were evaluated to obtain Characteristic Fragment Ions (CFIs) and Neutral Losses (NLs) that served as criteria for eliminating false-positives. Ultimately, 79 OSs (44 neutral-reducing OSs, 11 nitrogen-reducing OSs, 20 acid-reducing OSs, and 4 neutral non-reducing OSs) were characterized in MF extract. In addition, 68 OS prototypes were detected in intestinal contents and 15 OS prototypes were observed in plasma. In conclusion, this novel strategy enabled stepwise screening, classification and systematic characterization of OSs in NPs in vitro and in vivo.
