An integrated annotation strategy for the phytochemical characterization of Xie-Bai-San decoction based on UPLC-Q Exactive Orbitrap HRMS, multi-database screening, and feature-based molecular networking.

INTRODUCTION

Characterizing plant-derived compounds in complex botanical formulations remains challenging because of matrix complexity, structurally related analogues, and frequent isomerism. Xie-Bai-San decoction (XBSD), a traditional herbal formula composed of Cortex Mori, Cortex Lycii, and Radix et Rhizoma Glycyrrhizae, represents a complex botanical system requiring comprehensive phytochemical characterization.

METHODS

An integrated phytochemical annotation strategy was developed using UPLC-Q Exactive Orbitrap HRMS combined with an automated data processing workflow incorporating an in-house library, public databases, and feature-based molecular networking (FBMN). Compound characterization was further supported by reference standard validation, MS/MS spectral interpretation, literature comparison, and confidence-level assignment.

RESULTS

A total of 218 compounds were annotated or tentatively characterized in XBSD, including 80 flavonoids, 35 organic acids, 23 amides, 21 terpenoids, 17 coumarins, 8 saccharides, 6 alkaloids, 5 stilbenes, 3 cyclic peptides, and 20 other constituents. Among them, 74 compounds were reported from XBSD for the first time, and 13 compounds were unambiguously identified using authentic reference standards. Representative MS/MS fragmentation features were elucidated to guide the annotation of structurally related analogues. FBMN enabled the visual clustering of molecular families, facilitating rapid structural inference and improving confidence in analogue annotation, particularly for isomers and low-abundance compounds.

DISCUSSION: This study provides a comprehensive chemical profile of XBSD and demonstrates that the integrated UPLC-HRMS-based workflow improves the efficiency, reliability, and transparency of phytochemical annotation in complex traditional herbal formulations. The proposed strategy may serve as a practical analytical framework for the rapid characterization of structurally diverse constituents in other complex botanical systems.