Integrated UHPLC-MS/MS and HRMS Strategy for Screening Potentially Hepatotoxic Furan-Containing Compounds in Dioscorea bulbifera L.

Summary

This study developed an integrated strategy combining UHPLC–MS/MS and HRMS to screen for potentially hepatotoxic furan-containing compounds in Dioscorea bulbifera L.

Abstract

Dioscorea bulbifera L. (DBL), a traditional herbal medicine used to treat thyroid disorders and tumors, has raised considerable safety concerns due to its potential hepatotoxic effects. Previous research suggests that this liver injury may be associated with the metabolic activation of furan-containing compounds (FCCs) present in DBL. Here, we systematically characterized FCCs and their reactive metabolites in DBL extract through an integrated analytical approach combining ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) and ultra-high performance liquid chromatography coupled with tandem high-resolution mass spectrometry (UHPLC-HRMS). Employing both targeted and untargeted mass spectrometric analyses, we identified a total of 849 constituents in the aqueous extract of DBL, including 10 furanoditerpenoids and 17 additional FCCs. To elucidate the metabolic activation pathways, we used glutathione (GSH) and 4-bromobenzylamine (BBA) as dual trapping agents within a mouse liver microsomal (MLM) incubation system. Through P450-mediated metabolism, seven corresponding adducts derived from diosbulbin B (DSB), C (DSC), D (DSD), E (DSE), L (DSL), N (DSN), and 2-pentylfuran were successfully trapped and characterized. This study establishes a sensitive and specific approach for the comprehensive profiling of potentially hepatotoxic furan compounds in DBL, and provides valuable insights into the formation of reactive metabolites and toxicity mechanisms related to furan-containing herbal medicine.

Introduction

Safety concerns related to traditional Chinese medicine (TCM), especially drug-induced liver injury (DILI), hinder their widespread clinical utility and modernization^1,2,3. Growing evidence suggests that most constituents of TCM are not intrinsically toxic; instead, their hepatotoxicity results from metabolic activation in vivo^1,4,5. Furan-containing compounds (FCCs) are well recognized as prominent hepatotoxic precursors, undergoing P450–mediated metabolic activation ....

Protocol

NOTE: The reagents and the equipment used in this study are listed in the Table of Materials.

1. Preparation of the DBL test solution

1. Collect and authenticate three batches of the tubers of Dioscorea bulbifera L. Following the National Specification for Processing of TCM, remove fibrous roots from the tubers, wash them to remove dirt, slice them into 1 cm thick circular slices while fresh, dry the slices in a forced air oven at 60 °C, then remove and cool them to room temperature²⁰.
2. Weigh 200 g of DBL from each batch and grind them into a coarse po....

Results

Identified FCCs in DBL
To characterize the chemical profile of the DBL, a combination of triple quadrupole-based WTM and HRMS profiling was employed. This dual approach allowed for the comprehensive analysis of extracted ion pairs, generating highly informative extracted ion chromatograms (EICs, Figure 3A) and total ion chromatograms (TICs, Figure 3B). Under the optimized chromatographic and detection conditions, a total of 849 compounds spa.......

Discussion

Previous investigations into the hepatotoxicity of DBL have primarily focused on the metabolic intermediates of individual FCCs using targeted capture strategies^9,22. Although these approaches provide valuable mechanistic insights, their effectiveness in complex herbal matrices is often limited by the poor detectability of low-abundance bioactivated intermediates. In the present study, we developed an upgraded analytical workflow for the systematic identification.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4-Bromobenzylamine	Jiuding Chemical (Shanghai) Technology Co., Ltd	X114214A
Acetonitrile	Sigma-Aldrich (Shanghai) Trading Co., Ltd	1000294000
Centrifuge	Wuxi BaiTaKe Biotechnology Co., Ltd	CR3180
Chromatographic sample bottles	Zhejiang ALWSCI Technology Co., Ltd	C0000008
diosbulbin B	Chengdu PufeiDe Biotech Co., Ltd	18022603
Dioscorea bulbifera L.	-	DBL-1; DBL-2; DBL-3	The plant materials were authenticated by Prof. Weike Jiang (Guizhou University of Traditional Chinese Medicine, Guiyang, China), with voucher specimens deposited at the Key Laboratory of Basic Pharmacology of Guizhou Province.
Electronic Balance	Shanghai Anting Electronic Instrument Factory	FA1004B
Formic acid	Thermo Fisher Scientific Co., Ltd	195715
Glutathione	Sigma-Aldrich (Shanghai) Trading Co., Ltd	SLBX5462
High speed  crusher	Yongkang Biao Hardware Products Co., Ltd	XC-2000Y
Kunming mice Liver Microsomes (male)	Dalian Meilun Biotechnology Co., Ltd.	MB12496-1
Magnesium Chloride	Beijing Solarbio Science & Technology Co., Ltd	M8161
Methanol	Sigma-Aldrich (Shanghai) Trading Co., Ltd	646377
millipore filter	Tianjin Jinteng Experimental Equipment Co., Ltd	Nylon66
NADPH Na4	Beijing Solarbio Science & Technology Co., Ltd	102X022
Oven	Tianjin Laboratorial Instrument & Equipment Co., Ltd	GFL125
Phosphate-Buffered Saline	Beyotime Biotechnology  Co., Ltd	C0221B
Thermostatic water bath oscillator	Shanghai Zijia Instrument Co., Ltd	THZ-82A
Ultra-freezer	Thermo Fisher Scientific Co., Ltd	TDE60086FV-ULTS
Ultrasonic Cleaner	Shanghai Zijia Instrument Co., Ltd	L10-250A
Vortex Mixer	Jiangsu Haimen Medical Instrument Factory	VM-D