Please wait a minute...
TMR Modern Herbal Medicine  2018, Vol. 1 Issue (4): 209-219    DOI: 10.12032/TMRmhm2017B32
Application of LC-MS based glutathione-trapped reactive metabolites in the discovery of toxicity of traditional Chinese medicine
Liu Xiao-Mei1#, Lv Hong1#, Wang Xiao-Ming1,*(), Guo Ya-Qing1, Li Ting-Ting1, Pan Gui-Xiang2,*()
1 Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, China.
2 Second Affiliated hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China.
Download: HTML     PDF(523KB)
Export: BibTeX | EndNote (RIS)      

We summarized the discovery of liquid chromatography mass spectrometry (LC-MS) based glutathione (GSH) capture of reactive metabolites in traditional Chinese medicine (TCM), which provides scientific basis for further research and clinical application of Chinese medicine toxicity discovery. This dissertation overviews relevant literatures of GSH capture of reactive metabolites in recent years. And then we review the principles and methods of LC-MS based GSH capture of reactive metabolites, as well as the research progress in the discovery of toxicity of TCM including pyrrolizidine alkaloids, furans and quinoid species. The review shows that the representative compounds of TCM includes adonifoline, lasiocarpine, diosbulbin B and safrol are well dctected by LC-MS based GSH capture technique. And the main analytic systems of LC-MS are triple quadrupole and Q-Trap mass spectrometer. Constant neutral loss scan (CNLS), precursor ion scan (PIS) and multiple reaction monitor (MRM) are main detection methods to monitor the characteristic GSH conjugate fragmentations. The approach of LC-MS based GSH-trapped metabolites has a good application prospect in the discovery of toxic components of TCM.


Metabolic bioactivation of certain drugs can generate reactive metabolites (RM) that are capable of covalently modifying cellular biomolecules and are implicated in drug induced toxicity. These electrophilic metabolites, also known as intermediates, cannot easily be detected due to the chemical instability and shorter half-life. As a natural nucleophilic agent, glutathione (GSH) can capture these electrophilic RM, forming GSH covalent conjugates. GSH conjugates screening by liquid chromatography-mass spectrometry (LC-MS) can be applied for rapid characterization and detection of potential toxicity of traditional Chinese medicine (TCM). In this paper, we briefly introduced the principle of RM trapped by GSH, as well as mentioned some qualitative and quantitative methods by LC-MS for GSH conjugates screening over recent years. Moreover, the application of this technology in the discovery of toxicity of TCM, especially those containing pyrrolizidine alkaloids, furans and quinoid species, were also reviewed. In short, the approach of LC-MS based GSH-trapped RM can be employed for rapid and sensitive detection of potential toxic RM in TCM, with a good prospect in application.

Key wordsglutathione conjugation      Liquid Chromatography Mass Spectrometry      Reactive metabolites      Toxicity      traditional Chinese medicine     
Published: 25 October 2018
Fund:  This work was supported by grants from the National Natural Science Foundation of China (81303182, 81173523).
Corresponding Authors: Wang Xiao-Ming,Pan Gui-Xiang     E-mail:;
About author:

# These authors contributed equally to this study.

Cite this article:

Liu Xiao-Mei, Lv Hong, Wang Xiao-Ming, Guo Ya-Qing, Li Ting-Ting, Pan Gui-Xiang. Application of LC-MS based glutathione-trapped reactive metabolites in the discovery of toxicity of traditional Chinese medicine. TMR Modern Herbal Medicine, 2018, 1(4): 209-219. doi: 10.12032/TMRmhm2017B32

URL:     OR

Figure 1. Different conjugation sites of GSH with reactive species
Figure 2. Qualitative and quantitative analysis techniques by LC-MS for capturing toxic substances based on GSH.
Figure 3. Characteristic fragment ions of glutathione conjugates under collision-induced dissociation [15]
Compound type Representative compound Compound structure Trapping agents Analytic system Detection method Characteristic ions Plant origin Reference

Pyrrolizidine alkaloids




1. PIS
2. polarity switch mode
308 Da
290 Da
138 Da
120 Da

Senecio scandens;
S. dolichodoryius.





504 Da
666 Da
360 Da
130 Da

Symphytum officinale


Furan-containing compounds

diosbulbin B


Q-Trap MS
1.polarity switch mode
4.MRM scan
861 Da
863 Da
129 Da
272 Da
169 Da
171 Da
79 Da
81 Da

Dioscorea bulbifera L.


Quinoid species





456 Da
130 Da
307 Da

Sassafras albidum (Nutt.) Nees

Table 1 Summarized characteristic of the adonifoline, lasiocarpine, diosbulbin B and safrole were detected by LC-MS.
1.   Park K, Williams DP, Naisbitt DJ,et a1. Investigation of toxic metabolites during drug development. Toxicol Appl Pharmacol 2005, 207: 425-434.
doi: 10.1016/j.taap.2005.02.029 pmid: 15996699
2.   Jia Z, Dan W, You S,et a1. Progress in research of glutathione. ShenyangYao Ke Da Xue Xue Bao 2009, 26: 238-242.
3.   Ma S, Subramanian R.Detecting and characterizing reactive metabolites by liquid chromatography tandem mass spectrometry. J Mass Spectrom 2006, 41: 1121-1139.
doi: 10.1002/(ISSN)1096-9888
4.   Chen WG, Zhang C, Avery MJ,et a1. Reactive metabolite screen for reducing candidate attrition in drug discovery. Adv Exp Med Biol 2001, 500: 521-524.
doi: 10.1002/0471743984.vse8459
5.   Evans DC, Watt AP, Nicoll-Griffith DA,et a1. Drug-protein adducts: an industry perspective on minimizing the potential for drug bioactivation in drug discovery and development. Chem Res Toxicol 2004, 17: 3-16.
doi: 10.1021/tx034170b
6.   Zhou S, Chan E, Duan W,et a1. Drug bioactivation covalent binding to target proteins and toxicity relevance. Drug Metab Rev 2005, 37: 205-213.
7.   Pohl LR, Branchflower RV.Covalent binding of electrophilic metabolites to macromolecules. Methods Enzymol 1981, 77: 43-50.
doi: 10.1016/S0076-6879(81)77009-5
8.   Blair IA.Endogenous glutathione adducts. Curr Drug Meta 2006, 7: 853-872.
doi: 10.2174/138920006779010601
9.   Waldon DJ, Teffera Y, Colletti AE,et a1. Identification of quinone imine containing glutathione conjugates of diclofenac in rat bile. Chem Res Toxicol 2010, 23: 1947-1953.
doi: 10.1021/tx100296v pmid: 21053927
10.   Zhang XY, Elfarra AA.Toxicity mechanism-based prodrugs: glutathione-dependent bioactivation as a strategy for anticancer prodrug design. Expert Opin Drug Discov 2018, 13: 1-10.
doi: 10.1080/17460441.2018.1394839
11.   Cao L, Waldon D, Teffera Y,et a1. Ratios of biliary glutathione disulfide (GSSG) to glutathione (GSH): a potential index to screen drug-induced hepatic oxidative stress in rats and mice. Anal Bioanal Chem 2013, 405: 2635-2642.
doi: 10.1007/s00216-012-6661-8
12.   Xie W, Zhong DF, Chen XY,et a1. Determination of Reactive Metabolites by Liquid Chromatography-Tandem Mass Spectrometry. J Chin Mass Spectrom Soc 2011, 32: 1-12.
13.   Yan Z, Caldwell GW, Maher N,et a1. Unbiased high-throughput screening of reactive metabolites on the linear ion trap mass spectrometer using polarity switch and mass tag triggered data-dependent acquisition. Anal Chem 2008, 80: 6410-6422.
doi: 10.1021/ac800887h
14.   Dieckhaus CM, Fernández-Metzler CL, King R,et a1. Negative ion tandem mass spectrometry for the detection of glutathione conjugates. Chem Res Toxicol 2005, 18: 630-638.
doi: 10.1021/tx049741u pmid: 15833023
15.   Wen B, Ma L, Nelson SD,et a1. High-throughput screening and characterization of reactive metabolites using polarity switching of hybrid triple quadrupole linear ion trap mass spectrometry. Anal Chem 2008, 80: 1788-1799.
doi: 10.1021/ac702232r
16.   Zheng J, Ma L, Xin B,et a1. Screening and identification of GSH-trapped reactive metabolites using hybrid triple quadruple linear ion trap mass spectrometry. Chem Res Toxicol 2007, 20: 757-766.
doi: 10.1021/tx600277y
17.   Lim HK, Chen J, Cook K,et a1. A generic method to detect electrophilic intermediates using isotopic pattern triggered data-dependent high-resolution accurate mass spectrometry. Rapid Commun Mass Spectrom 2008, 22: 1295-1311.
doi: 10.1002/(ISSN)1097-0231
18.   Castro-Perez J, Plumb R, Liang L,et a1. A high-throughput liquid chromatography/tandem mass spectrometry method for screening glutathione conjugates using exact mass neutral loss acquisition. Rapid Commun Mass Spectrom 2005, 19: 798-804.
doi: 10.1002/(ISSN)1097-0231
19.   Zhu X, Kalyanaraman N, Subramanian R.Enhanced screening of glutathione-trapped reactive metabolites by in-source collision-induced dissociation and extraction of product ion using UHPLC-high resolution mass spectrometry. Anal Chem 2011, 83: 9516-9523.
doi: 10.1021/ac202280f
20.   Tang C, Zhang W, Dai C,et a1. Identification and quantification of adducts between oxidized rosmarinic acid and thiol compounds by UHPLC-LTQ-Orbitrap and MALDI-TOF/TOF tandem mass spectrometry. J Agric Food Chem 2015, 63: 902-911.
doi: 10.1021/jf5044713
21.   Zhu M, Ma L, Zhang H,et a1. Detection and Structural Characterization of Glutathione-Trapped Reactive Metabolites Using Liquid Chromatography-High-Resolution Mass Spectrometry and Mass Defect Filtering. Anal Chem 2007, 79: 8333-8341.
doi: 10.1021/ac071119u
22.   Ruan Q, Peterman S, Szewc MA,et a1. An integrated method for metabolite detection and identification using a linear ion trap/Orbitrap mass spectrometer and multiple data processing techniques: application to indinavir metabolite detection. J Mass spectrom 2008, 43: 251-261.
doi: 10.1002/(ISSN)1096-9888
23.   Prasad B, Garg A, Takwani H,et a1. Metabolite identification by liquid chromatography-mass spectrometry. TrAC Trends Anal Chem 2011, 30: 360-387.
doi: 10.1016/j.trac.2010.10.014
24.   Gan J, Harper TW, Hsueh M M,et a1. Dansyl glutathione as a trapping agent for the quantitative estimation and identification of reactive metabolites. Chem Res Toxicol 2005, 18: 896-903.
doi: 10.1021/tx0496791
25.   Li P, Li Z, Beck W D,et a1. Bio-generation of stable isotope-labeled internal standards for absolute and relative quantitation of phase II drug metabolites in plasma samples using LC-MS/MS. Anal Bioanal Chem 2015, 407: 4053-4063.
doi: 10.1007/s00216-015-8614-5
26.   MacDonald C, Smith C, Michopoulos F, et al. Identification and quantification of glutathione adducts of clozapine using ultra-high-performance liquid chromatography with orthogonal acceleration time-of-flight mass spectrometry and inductively coupled plasma mass spectrometry. Rapid Commun Mass Spectrom 2011, 25: 1787-1793.
doi: 10.1002/rcm.5043
27.   Wang J, Wang CH, Wang YT, ,et al. Progress in the Cytotoxicity. Progress in the Cytotoxicity and Toxicity Mechanism of Pyrrolizidine Alkaloids. Int J Pharm Res 2007, 34: 246-249+258.
28.   Gan J, Ruan Q, He B,et a1. In Vitro Screening of 50 Highly Prescribed Drugs for Thiol Adduct Formation Comparison of Potential for Drug-Induced Toxicity and Extent of Adduct Formation. Chem Res Toxicol 2009, 22: 690-698.
doi: 10.1021/tx800368n
29.   Tamta H, Pawar RS, Wamer WG, et al. Comparison of metabolism-mediated effects of pyrrolizidine alkaloids in a HepG2/C3A cell-S9 co-incubation system and quantification of their glutathione conjugates. Xenobiot 2012, 42: 1038-1048.
doi: 10.3109/00498254.2012.679978
30.   Cheng M, Tang J, Gao QF,et a1. Analysis of the main alkaloids in the extract of Aster sinensis Clivorine and its preliminary study on hepatotoxicity in rats. Chinese Tradi Herb Drugs 2011, 42: 2507-2511.
31.   Mei N, Guo L, Fu PP,et a1. Metabolism, genotoxicity, and carcinogenicity of comfrey. Environ Health Toxicol 2010, 13: 509-526.
doi: 10.1080/10937404.2010.509013 pmid: 21170807
32.   Lu D, Peterson LA.Identification of Furan Metabolites Derived from Cysteine-cis-2-Butene-1, 4-dial-Lysine Cross-Links. Chem Res Toxicol 2009, 23: 142-151.
33.   Li C, Lin D, Gao H,et a1. N-Acetyl lysine/glutathione-derived pyrroles as potential Ex Vivo biomarkers of bioactivated furan-containing compounds. Chem Res Toxicol 2014, 28: 384-393.
34.   Phillips MB, Sullivan MM, Villalta PW,et a1. Covalent modification of cytochrome c by reactive metabolites of furan. Chem Res Toxicol 2013, 27: 129-135
doi: 10.1021/tx400368r pmid: 24364757
35.   Zhang KE, Naue JA, Arison B,et a1. Microsomal metabolism of the 5-lipoxygenase inhibitor L-739,010: evidence for furan bioactivation. Chem Res Toxicol 1996, 9(2): 547-554.
doi: 10.1021/tx950183g
36.   Wang K, Zheng L, Peng Y,et a1. Selective and sensitive platform for function-based screening of potentially harmful furans. Anal Chem 2014, 86: 10755-10762.
doi: 10.1021/ac502796x pmid: 25279953
37.   Van Breemen RB, Nikolic D, Bolton JL.Metabolic screening using on-line ultrafiltration mass spectrometry. Drug Metab Dispos 1998, 26: 85-90.
doi: 10.1016/S1359-6446(97)01147-1 pmid: 9456292
38.   Thompson DC, Barhoumi R, Burghardt RC.Comparative toxicity of eugenol and its quinone methide metabolite in cultured liver cells using kinetic fluorescence bioassays. Toxicol Appl Pharmacol 1998, 149: 55-63.
doi: 10.1006/taap.1997.8348
39.   Thompson D, Constantin-Teodosiu D, Egestad B,et a1. Formation of glutathione conjugates during oxidation of eugenol by microsomal fractions of rat liver and lung. Biochem Pharmacol 1990, 39(10): 1587-1595.
doi: 10.1016/0006-2952(90)90525-P
40.   Yang AH, Zhang L, Zhi DX, et al. Identification and analysis of the reactive metabolites related to the hepatotoxicity of safrole. Xenobiot 2018, 48: 11164-1172.
41.   Bolton JL, Acay NM, Vukomanovic V.Evidence that 4-allyl-o-quinones spontaneously rearrange to their more electrophilic quinone methides: potential bioactivation mechanism for the hepatocarcinogen safrole. Chem Research Toxicol 1994, 7: 443-450.
doi: 10.1021/tx00039a024
42.   Johnson BM, Bolton JL, van Breemen RB. Screening botanical extracts for quinoid metabolites. Chem Res Toxicol 2001, 14: 1546-1551.
doi: 10.1021/tx010106n pmid: 11712913
43.   Wu H, Zhong RL, Xia Z,et a1. Progress in the study of components of potential hepatotoxic Chinese medicines. Chin J Tradi Chin Med 2016, 41: 3209-3217.
[1] Man-Ke Guan, Jia-Yuan Hu, Xiao-Yu Zhang, Yang Sun, Hong-Cai Shang. Clinical research progress of ischemic heart failure and prospect of traditional Chinese medicine[J]. TMR Modern Herbal Medicine, 2019, 2(2): 115-120.
[2] Yu-hui Ma, Li-na Zhang, Qian Cui, Lin-feng Li, Guo-wei Zhang. Mechanism analysis of Traditional Chinese Medicine in treatment of Diabetic Nephropathy based on network pharmacology and Traditional Chinese Medicine inheritance support system[J]. TMR Modern Herbal Medicine, 2019, 2(2): 64-73.
[3] Jin Xiao-Ming, Cui Qian, Ma Yu-Hui, Gao Jing, Li Yuan. An introduction of the experience in treating Ventricular premature beat with stasis heat[J]. TMR Modern Herbal Medicine, 2019, 2(1): 19-24.
[4] Zhang Li-Hui, Zhao Yong-Chen. Systematic evaluation and Meta-analysis of acupuncture and collaterals cupping therapy for Knee Osteoarthritis[J]. TMR Modern Herbal Medicine, 2019, 2(1): 36-47.
[5] Kao Hsiu-Ying, Jiang Yong. Survey of dose-effect relationship in Chinese materia medica[J]. TMR Modern Herbal Medicine, 2018, 1(4): 249-258.
[6] Cui Qian, Ma Yu-Hui, Jin Xiao-Ming, Liu Jing-Jing, Zhang Yan-Hong, Zhang Guo-wei. Toxic effects of BuGuZhi and feasibility analysis of ways to synergistic and attenuated[J]. TMR Modern Herbal Medicine, 2018, 1(4): 241-248.
[7] Wang Jian-hui, Guo Jie-mei, Li Bao-lin, Teng Fang-zhou, Zhu Ya-ju, Lin Jian-ping, Xiao Yan, Mao Xiao, Huang Lu-lu, Su You-xin. Effect of TongFengNing Decoction on Uric Acid Levels and Xanthine Oxidase Activity in Hyperuricemia Rats[J]. TMR Modern Herbal Medicine, 2018, 1(4): 189-197.
[8] Zhao Shao-Yang, Dong Xin, Tu Peng-Fei, Zeng Ke-Wu, Wang Xue-Mei. Neuroprotective Effect of Bu-Shen-Huo-Xue Extract against High Glucose-induced Apoptosis in PC12 Cells[J]. TMR Modern Herbal Medicine, 2018, 1(3): 143-154.
[9] Chen Qiang, Shang Hong-Cai, Han Bing-Kai, Jiao Jun. Perspectives in application of biosensors for the modernization of traditional Chinese medicine[J]. TMR Modern Herbal Medicine, 2018, 1(1): 39-46.