药学学报

Research objective	Sample type	Compounds investigated	Separation column	MS model	Acquisition mode	Ref.
Class compounds identification	Citrus reticulata extract	Polymethoxylated flavonoids	Agilent Zorbax Extended C18 (4.6 mm×250mm, 5 μm)	Thermo LTQ-Orbitrap	Full scan & DDA MS/MS	[13]
	Lonicerae japonicae	Chlorogenic acids	Waters ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm)	Thermo LTQ-Orbitrap	Full scan & DDA MS/MS	[14]
	Citrus reticulata extract	Polymethoxylated flavonoids	Agilent Zorbax Extended C18 (4.6 mm×250 mm, 5 μm)	Thermo LTQ-Orbitrap	Full scan & DDA MS/MS	[17]
	Yin Chen Si Ni Tang	Aconitum alkaloids	Waters ACQUITY UPLC BEH C18 (2.1 mm×100 mm ID, 1.7 μm)	Waters SYNAPT HDMS Q-TOF	MS^E	[18]
	Uncariae ramulus extract	Indole alkaloids	Phenomenex Kinetex^® EVO C18 column (2.1 mm×150 mm, 2.6 μm)	Thermo LTQ-Orbitrap	Full scan & DDA MS/MS	[19]
	Panax notoginseng extract	Saponins	Agilent Eclipse XDB-C18 (4.6 mm×250 mm, 5 μm)	Agilent 6530 Q-TOF	Full scan & DDA MS/MS	[20]
	Scutellaria baicalensis extract	Flavonoids	Agilent ZORBAX Eclipse Plus C18 (2.1 mm×150mm, 1.8 um)	Thermo Q-Exactive hybrid quadrupole-orbitrap	Full scan & DDA MS/MS	[24]
	Lonicerae japonicae extract	Chlorogenic acids	Agilent Zorbax SB C18 (4.6 mm×250 mm, 5 μm)	Agilent MSD Trap XCT Plus	Full scan & DDA MS/MS	[31]
	Ainsliaea fragrans extract	Chlorogenic acids	Waters ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm)	AB Sciex Triple TOF^TM 5600+	Full scan & DDA MS/MS	[32]
	Curcuma longa extract	Terpene-conjugated curcuminoids	Waters Acquity UPLC HSS T3 (2.1 mm×150 mm, 1.8 μm)	Agilent 6495 QQQ Thermo Q-Exactive hybrid quadrupole-orbitrap	SIM full scan & DDA MS/MS	[33]
	Panax ginseng extract	Ginsenosides	ACE UltraCore 2.5 Super C18 (3.0 mm×150 mm, 2.5 μm)	ABSciex 5500 Qtrap	MRM	[37]
	Eleven Chinese patent medicines including Carthamus tinctorius	Quinochalcone C-glycosides	Waters BEH Shield RP 18 (2.1 mm×100 mm, 1.7 μm)	Waters Xevo®G2-S QTOF	SIM	[38]
	The popular medicinal used Pueraria species	Sulfur derivative of flavonoids	Agilent ZORBAX Eclipse Plus C18 (2.1 mm×100 mm, 1.8 um)	Thermo LTQ Orbitrap Velos Pro	Full scan & DDA MS/MS	[39]
	Ligusticum chuanxiong extract	Phthalides	Waters ACQUITY BEH C18 (2.1 mm×100 mm, 1.7 μm)	Waters SYNAPT G2-SI Q-TOF	MS^E	[40]
	Lotus plumule extract	Flavonoids	Thermo Hypersil GOLD column (2.1 mm×100 mm, 1.9 μm)	Thermo Q-Exactive hybrid quadrupole-orbitrap	Full scan & DDA MS/MS	[41]
	Sinomenii caulis extract	Alkaloid analogues	Waters Symmetry C18 analytical (2.1 mm×100 mm, 3.5 μm)	Agilent 6530 Q-TOF	Full scan & DDA MS/MS	[42]
Chemical profiling of all compounds	Artemisiae argyi extract	Flavonoids, chlorogenic acids	Thermo Hypersil GOLD column (2.1 mm×100 mm, 1.9 μm)	Thermo Q-Exactive hybrid quadrupole-orbitrap	Full scan & DDA MS/MS	[43]
	Yi Xin Shu capsule	Lignans, tanshinones, flavonoids, phenolic acids, saponins, and others	Agilent ZORBAX Eclipse Plus C18 (3.0 mm×100 mm, 1.8 μm)	Agilent 6540 Q-TOF	Full scan & DDA MS/MS	[44]
	Dachuanxiong decoction	Phthalides, ester of citric acid, chlorogenic acids, phenols, nucleosides, and others	Agilent ZORBAX Eclipse Plus C18 (2.1 mm×150 mm, 1.8 μm)	Agilent 6550 Q-TOF	Full scan & DDA MS/MS	[45]
	Qishen granule	Chlorogenic acids, tanshinones, flavonoids, iridoid glucosides, alkaloids, salvianolic acids, phenolic acids, triterpenoid saponins, and phenylpropanoids	ACQUITY TM UPLC HSS T3 (2.1 mm×100 mm, 1.8µm)	Agilent 6550 Q-TOF	Full scan & target MS/MS	[46]
	Kai xin san	Saponins, triterpenoid, phenolic acids, and others	ACQUITY TM UPLC HSS T3 (2.1 mm×100 mm, 1.8µm)	Waters Synapt^TM High Definition TOF	MS^E	[47]
	Yangxue Qingnao granule	Phenolic acids, monoterpenoid glycosides, quinones, alkaloids, phthalides and others	Waters ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm)	Waters Acquity UPLC-Synapt G2 Q-TOF	Full scan & DDA MS/MS	[48]
	Liujing toutong tablet	Isoflavonoids, iridoids, coumarins, phthalides, and others	Dikma Diamonsil C18 (4.6 mm×250 mm, 5 μm)	Bruker micrOTOF-Q Ⅱ	Full scan & DDA MS/MS	[50]
	Buqi Tongluo granule	Flavonoids, saponins, terpenoids, esters, amino acid, phenolic acids	Waters ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm)	Waters Q-TOF	MS^E	[51]
	Danhong injection	Phenolic acids, flavonoids, and others	Waters ACQUITY UHPLC HSS T3 (2.1 mm×100 mm, 1.8µm)	Thermo Q-Exactive hybrid quadrupole-orbitrap	Full scan & DDA MS/MS	[53]
	Anti-renal interstitial fibrosis I decoction	Sennosides, galloyl glycosides, flavonoids, lignans, iridoids, organic acids, phenylpropanoids	Thermo Hypersil GOLD column (2.1 mm×100 mm, 1.9 μm)	Thermo Q-Exactive hybrid quadrupole-orbitrap	Full scan & DDA MS/MS	[54]
	Lishukang capsule	Flavonoids, alkaloids, phenolic acids, saponins, and benzyl ester glycosides	Waters ACQUITY UHPLC HSS T3 (2.1 mm×100 mm, 1.8µm)	AB SCIEX 5600+Q-TOF	Full scan & DDA MS/MS	[56]
	Xiao Xu Ming decoction	Flavonoids, monoterpenoid glycosides, terpenoids, and others	Thermo BDS HYPERSIL C18 (2.1 mm×150 mm, 3µm)	Thermo LTQ-FT	Full scan & DDA MS/MS	[57]
Metabolites identification	Intestinal bacteria	Nonpolysaccharide fraction from Bletilla striata metabolites	Waters BEH C18 (2.1 mm×50 mm, 1.7 μm)	Waters XEVO G2‐XS Q‐TOF	MS^E	[62]
	Rat urine	Gandou decoction metabolites	Agilent Eclipse Plus C18 (2.1 mm×100 mm, 1.8 μm)	Waters Xevo G2 Q-TOF	MS^E	[63]
	Rat bio-samples	Qi-Lin pill metabolites	Waters ACQUITY UHPLC HSS T3 (2.1 mm×100 mm, 1.8 μm)	Waters SYNAPT™G2 Q-TOF	MS^E	[64]
	Human urine	Synthetic cannabinoids metabolites	Bellefonte Ultra Biphenyl HPLC (2.1 mm×100 mm, 3.0 μm)	AB Sciex Triple-TOF 5600+	SWATH MS/MS	[65]
	Liver microsomes, rat urine and bile	Ponicidin metabolites		AB Sciex Triple-TOF 5600+	SWATH MS/MS	[66]
	Liver microsomes, and rat bio-samples	Osthole metabolites	Agilent Poroshell 120 EC-C18 (2.1 mm×100 mm, 2.6 μm)	AB Sciex Triple-TOF 5600+	SWATH MS/MS	[67]
	Rat bio-samples	Berberine metabolites	Waters ACQUITY UPLC BEH Shield RP18 (2.1 mm×50 mm, 1.7 μm)	Waters SYNAPT^TM G2 Q-TOF	MS^E	[68]
	Rat bio-samples	Berberrubine metabolites	Waters ACQUITY BEH Shield RP18(2.1 mm×50 mm, 1.7 μm)	Waters SYNAPT^TM G2 Q-TOF	MS^E	[69]
	Rat urine and bile	Oridonin metabolites	Phenomenex Kinetex C18 (2.1 mm×100 mm, 2.6 μm)	AB Sciex Triple-TOF 5600+	Full scan & DDA MS/MS	[71]
	Rat urine and bile	Deoxyschizandrin metabolites	Phenomenex Kinetex C18 (2.1 mm×100 mm, 2.6 μm)	AB Sciex Triple-TOF 5600+	Full scan & DDA MS/MS	[72]
	Rat bio-samples	Gualou Xiebai Baijiu decoction metabolites	Agilent Acquity BEH C18 (2.1 mm×50 mm, 1.7 μm)	Waters SYNAPT^TM G2 Q-TOF	Full scan & DDA MS/MS	[73]
	Rat plasma and urine	Gegen-Qinlian decoction metabolites	Waters Atlantis T3 column (2.1 mm×150 mm, 3 μm)	Thermo Fisher Finnigan TSQ QQQ	Neutral loss scan, precursor ion scan, and SRM scan	[74]
	Rat plasma, urine and feces	Glycyrrhiza uralensis metabolites	Welch Ultimate XB-C18 (2.1 mm×100 mm, 1.8 μm)	AB Sciex API 4500 QTRAP	MIM-EPI and NL-EPI	[75]
	Rat bio-samples	Epimedium brevicornu metabolites	Agilent SB-C18 column (4.6 mm×250 mm, 5 μm)	Thermo LTQ-FT	Full scan & DDA MS/MS	[77]
	Rat bio-samples	Xiao Xu Ming decoction metabolites	Thermo BDS HYPERSIL C18 (2.1 mm×150 mm, 3 μm),	Thermo LTQ-FT	Full scan & DDA MS/MS	[78]
	Rat plasma	Fufang Xialian capsule metabolites	Waters ACQUITY UPLC BEH C18(2.1 mm×50 mm, 1.7 μm)	Waters SYNAPT^G2 Q-TOF	MS^E	[79]
	Rat bio-samples	Yi-Xin-Shu capsule metabolites	Agilent Eclipse Plus C18 (2.1 mm×100 mm, 1.8 μm)	Agilent 6540 Q-TOF	Full scan & DDA MS/MS	[80]
	Rat plasma	Zhikebao tablet metabolites	Waters Acquity UPLC HSS C18(2.1 mm×150 mm, 1.8 μm)	AB Sciex Triple-TOF 5600+	Full scan & DDA MS/MS	[81]

Research objective	Sample type	Compounds investigated	Separation column	MS model	Acquisition mode	Ref.
Class compounds identification	Citrus reticulata extract	Polymethoxylated flavonoids	Agilent Zorbax Extended C18 (4.6 mm×250mm, 5 μm)	Thermo LTQ-Orbitrap	Full scan & DDA MS/MS	[13]
	Lonicerae japonicae	Chlorogenic acids	Waters ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm)	Thermo LTQ-Orbitrap	Full scan & DDA MS/MS	[14]
	Citrus reticulata extract	Polymethoxylated flavonoids	Agilent Zorbax Extended C18 (4.6 mm×250 mm, 5 μm)	Thermo LTQ-Orbitrap	Full scan & DDA MS/MS	[17]
	Yin Chen Si Ni Tang	Aconitum alkaloids	Waters ACQUITY UPLC BEH C18 (2.1 mm×100 mm ID, 1.7 μm)	Waters SYNAPT HDMS Q-TOF	MS^E	[18]
	Uncariae ramulus extract	Indole alkaloids	Phenomenex Kinetex^® EVO C18 column (2.1 mm×150 mm, 2.6 μm)	Thermo LTQ-Orbitrap	Full scan & DDA MS/MS	[19]
	Panax notoginseng extract	Saponins	Agilent Eclipse XDB-C18 (4.6 mm×250 mm, 5 μm)	Agilent 6530 Q-TOF	Full scan & DDA MS/MS	[20]
	Scutellaria baicalensis extract	Flavonoids	Agilent ZORBAX Eclipse Plus C18 (2.1 mm×150mm, 1.8 um)	Thermo Q-Exactive hybrid quadrupole-orbitrap	Full scan & DDA MS/MS	[24]
	Lonicerae japonicae extract	Chlorogenic acids	Agilent Zorbax SB C18 (4.6 mm×250 mm, 5 μm)	Agilent MSD Trap XCT Plus	Full scan & DDA MS/MS	[31]
	Ainsliaea fragrans extract	Chlorogenic acids	Waters ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm)	AB Sciex Triple TOF^TM 5600+	Full scan & DDA MS/MS	[32]
	Curcuma longa extract	Terpene-conjugated curcuminoids	Waters Acquity UPLC HSS T3 (2.1 mm×150 mm, 1.8 μm)	Agilent 6495 QQQ Thermo Q-Exactive hybrid quadrupole-orbitrap	SIM full scan & DDA MS/MS	[33]
	Panax ginseng extract	Ginsenosides	ACE UltraCore 2.5 Super C18 (3.0 mm×150 mm, 2.5 μm)	ABSciex 5500 Qtrap	MRM	[37]
	Eleven Chinese patent medicines including Carthamus tinctorius	Quinochalcone C-glycosides	Waters BEH Shield RP 18 (2.1 mm×100 mm, 1.7 μm)	Waters Xevo®G2-S QTOF	SIM	[38]
	The popular medicinal used Pueraria species	Sulfur derivative of flavonoids	Agilent ZORBAX Eclipse Plus C18 (2.1 mm×100 mm, 1.8 um)	Thermo LTQ Orbitrap Velos Pro	Full scan & DDA MS/MS	[39]
	Ligusticum chuanxiong extract	Phthalides	Waters ACQUITY BEH C18 (2.1 mm×100 mm, 1.7 μm)	Waters SYNAPT G2-SI Q-TOF	MS^E	[40]
	Lotus plumule extract	Flavonoids	Thermo Hypersil GOLD column (2.1 mm×100 mm, 1.9 μm)	Thermo Q-Exactive hybrid quadrupole-orbitrap	Full scan & DDA MS/MS	[41]
	Sinomenii caulis extract	Alkaloid analogues	Waters Symmetry C18 analytical (2.1 mm×100 mm, 3.5 μm)	Agilent 6530 Q-TOF	Full scan & DDA MS/MS	[42]
Chemical profiling of all compounds	Artemisiae argyi extract	Flavonoids, chlorogenic acids	Thermo Hypersil GOLD column (2.1 mm×100 mm, 1.9 μm)	Thermo Q-Exactive hybrid quadrupole-orbitrap	Full scan & DDA MS/MS	[43]
	Yi Xin Shu capsule	Lignans, tanshinones, flavonoids, phenolic acids, saponins, and others	Agilent ZORBAX Eclipse Plus C18 (3.0 mm×100 mm, 1.8 μm)	Agilent 6540 Q-TOF	Full scan & DDA MS/MS	[44]
	Dachuanxiong decoction	Phthalides, ester of citric acid, chlorogenic acids, phenols, nucleosides, and others	Agilent ZORBAX Eclipse Plus C18 (2.1 mm×150 mm, 1.8 μm)	Agilent 6550 Q-TOF	Full scan & DDA MS/MS	[45]
	Qishen granule	Chlorogenic acids, tanshinones, flavonoids, iridoid glucosides, alkaloids, salvianolic acids, phenolic acids, triterpenoid saponins, and phenylpropanoids	ACQUITY TM UPLC HSS T3 (2.1 mm×100 mm, 1.8µm)	Agilent 6550 Q-TOF	Full scan & target MS/MS	[46]
	Kai xin san	Saponins, triterpenoid, phenolic acids, and others	ACQUITY TM UPLC HSS T3 (2.1 mm×100 mm, 1.8µm)	Waters Synapt^TM High Definition TOF	MS^E	[47]
	Yangxue Qingnao granule	Phenolic acids, monoterpenoid glycosides, quinones, alkaloids, phthalides and others	Waters ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm)	Waters Acquity UPLC-Synapt G2 Q-TOF	Full scan & DDA MS/MS	[48]
	Liujing toutong tablet	Isoflavonoids, iridoids, coumarins, phthalides, and others	Dikma Diamonsil C18 (4.6 mm×250 mm, 5 μm)	Bruker micrOTOF-Q Ⅱ	Full scan & DDA MS/MS	[50]
	Buqi Tongluo granule	Flavonoids, saponins, terpenoids, esters, amino acid, phenolic acids	Waters ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm)	Waters Q-TOF	MS^E	[51]
	Danhong injection	Phenolic acids, flavonoids, and others	Waters ACQUITY UHPLC HSS T3 (2.1 mm×100 mm, 1.8µm)	Thermo Q-Exactive hybrid quadrupole-orbitrap	Full scan & DDA MS/MS	[53]
	Anti-renal interstitial fibrosis I decoction	Sennosides, galloyl glycosides, flavonoids, lignans, iridoids, organic acids, phenylpropanoids	Thermo Hypersil GOLD column (2.1 mm×100 mm, 1.9 μm)	Thermo Q-Exactive hybrid quadrupole-orbitrap	Full scan & DDA MS/MS	[54]
	Lishukang capsule	Flavonoids, alkaloids, phenolic acids, saponins, and benzyl ester glycosides	Waters ACQUITY UHPLC HSS T3 (2.1 mm×100 mm, 1.8µm)	AB SCIEX 5600+Q-TOF	Full scan & DDA MS/MS	[56]
	Xiao Xu Ming decoction	Flavonoids, monoterpenoid glycosides, terpenoids, and others	Thermo BDS HYPERSIL C18 (2.1 mm×150 mm, 3µm)	Thermo LTQ-FT	Full scan & DDA MS/MS	[57]
Metabolites identification	Intestinal bacteria	Nonpolysaccharide fraction from Bletilla striata metabolites	Waters BEH C18 (2.1 mm×50 mm, 1.7 μm)	Waters XEVO G2‐XS Q‐TOF	MS^E	[62]
	Rat urine	Gandou decoction metabolites	Agilent Eclipse Plus C18 (2.1 mm×100 mm, 1.8 μm)	Waters Xevo G2 Q-TOF	MS^E	[63]
	Rat bio-samples	Qi-Lin pill metabolites	Waters ACQUITY UHPLC HSS T3 (2.1 mm×100 mm, 1.8 μm)	Waters SYNAPT™G2 Q-TOF	MS^E	[64]
	Human urine	Synthetic cannabinoids metabolites	Bellefonte Ultra Biphenyl HPLC (2.1 mm×100 mm, 3.0 μm)	AB Sciex Triple-TOF 5600+	SWATH MS/MS	[65]
	Liver microsomes, rat urine and bile	Ponicidin metabolites		AB Sciex Triple-TOF 5600+	SWATH MS/MS	[66]
	Liver microsomes, and rat bio-samples	Osthole metabolites	Agilent Poroshell 120 EC-C18 (2.1 mm×100 mm, 2.6 μm)	AB Sciex Triple-TOF 5600+	SWATH MS/MS	[67]
	Rat bio-samples	Berberine metabolites	Waters ACQUITY UPLC BEH Shield RP18 (2.1 mm×50 mm, 1.7 μm)	Waters SYNAPT^TM G2 Q-TOF	MS^E	[68]
	Rat bio-samples	Berberrubine metabolites	Waters ACQUITY BEH Shield RP18(2.1 mm×50 mm, 1.7 μm)	Waters SYNAPT^TM G2 Q-TOF	MS^E	[69]
	Rat urine and bile	Oridonin metabolites	Phenomenex Kinetex C18 (2.1 mm×100 mm, 2.6 μm)	AB Sciex Triple-TOF 5600+	Full scan & DDA MS/MS	[71]
	Rat urine and bile	Deoxyschizandrin metabolites	Phenomenex Kinetex C18 (2.1 mm×100 mm, 2.6 μm)	AB Sciex Triple-TOF 5600+	Full scan & DDA MS/MS	[72]
	Rat bio-samples	Gualou Xiebai Baijiu decoction metabolites	Agilent Acquity BEH C18 (2.1 mm×50 mm, 1.7 μm)	Waters SYNAPT^TM G2 Q-TOF	Full scan & DDA MS/MS	[73]
	Rat plasma and urine	Gegen-Qinlian decoction metabolites	Waters Atlantis T3 column (2.1 mm×150 mm, 3 μm)	Thermo Fisher Finnigan TSQ QQQ	Neutral loss scan, precursor ion scan, and SRM scan	[74]
	Rat plasma, urine and feces	Glycyrrhiza uralensis metabolites	Welch Ultimate XB-C18 (2.1 mm×100 mm, 1.8 μm)	AB Sciex API 4500 QTRAP	MIM-EPI and NL-EPI	[75]
	Rat bio-samples	Epimedium brevicornu metabolites	Agilent SB-C18 column (4.6 mm×250 mm, 5 μm)	Thermo LTQ-FT	Full scan & DDA MS/MS	[77]
	Rat bio-samples	Xiao Xu Ming decoction metabolites	Thermo BDS HYPERSIL C18 (2.1 mm×150 mm, 3 μm),	Thermo LTQ-FT	Full scan & DDA MS/MS	[78]
	Rat plasma	Fufang Xialian capsule metabolites	Waters ACQUITY UPLC BEH C18(2.1 mm×50 mm, 1.7 μm)	Waters SYNAPT^G2 Q-TOF	MS^E	[79]
	Rat bio-samples	Yi-Xin-Shu capsule metabolites	Agilent Eclipse Plus C18 (2.1 mm×100 mm, 1.8 μm)	Agilent 6540 Q-TOF	Full scan & DDA MS/MS	[80]
	Rat plasma	Zhikebao tablet metabolites	Waters Acquity UPLC HSS C18(2.1 mm×150 mm, 1.8 μm)	AB Sciex Triple-TOF 5600+	Full scan & DDA MS/MS	[81]

高分辨质谱数据处理策略在中药体内外成分检测和表征中的应用进展

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刘洁 ¹^,² , 李月婷 ²^,³ , 陈奕君 ²^,⁴ , 肖红斌 ^*^,¹^,²

药学学报 | 综述 2021,56(1): 113-129

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药学学报 | 综述 2021, 56(1): 113-129

高分辨质谱数据处理策略在中药体内外成分检测和表征中的应用进展

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刘洁¹^,², 李月婷²^,³, 陈奕君²^,⁴, 肖红斌^*^,¹^,²

作者信息

1.北京中医药大学北京中医药研究院, 北京 100029

2.北京中医药大学中药分析与转化研究中心, 北京 100029

3.北京中医药大学中药学院, 北京 100029

4.北京中医药大学中医学院, 北京 100029

通讯作者:

*肖红斌, Tel/Fax: 86-10-64286490, E-mail: hbxiao69@163.com

Advances in high-resolution mass spectrometric-based data-mining technologies for detecting and characterizing the components and metabolites of Chinese materia medica

Jie LIU¹^,², Yue-ting LI²^,³, Yi-jun CHEN²^,⁴, Hong-bin XIAO^*^,¹^,²

Affiliations

1. Beijing Research Institute of Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China

2. Research Center for Chinese Medicine Analysis and Transformation, Beijing University of Chinese Medicine, Beijing 100029, China

3. School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China

4. School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China

出版时间: 2021-02-12 doi: 10.16438/j.0513-4870.2020-1385

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摘要

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中药物质基础研究是中药现代化研究的基础。现代高分辨质谱(HRMS)已成为成分定性分析的有力手段。近年来, 一些基于高分辨质谱的数据处理策略, 如全信息串联质谱扫描采集策略、基于MS/MSALL的所有理论碎片离子的连续窗口采集技术、质量亏损过滤技术、特征离子过滤技术、质谱树状图过滤技术等极大地推动了中药物质基础的阐明。本文分别从中药类成分、全成分和中药体内成分三个方面综述高分辨质谱数据处理策略在中药体内外成分检测和表征中的应用进展, 为中药成分和体内代谢物的快速发现和结构表征提供方法参考。

关键词

中药 / 高分辨质谱 / 数据处理策略 / 类成分 / 全成分 / 体内成分

Abstract

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Research on the material basis of Chinese materia medica(CMM) is the basis for modernization of CMM. High-resolution mass spectrometry(HRMS) has become a powerful tool for the qualitative analysis of the components of CMM. Some data-mining strategies based on high-resolution mass spectrometry, such as full-information tandem mass spectrometry scanning acquisition strategy, sequential windowed acquisition of all theoretical fragment ions, mass detect filter, characteristic ion filter, mass spectral tree similarity filter, etc. have greatly promoted the elucidation of the qualitative basis of CMM. In order to provide methods for the rapid discovery and structural characterization of components and metabolites of CMM, this review summarized the advances in HRMS-based data-mining technologies for detecting and characterizing the compounds and metabolites of CMM, which includes class compounds, all compounds and metabolites.

Key words

Chinese materia medica / high-resolution mass spectrometry / data mining strategy / class compound / total compounds / metabolite

引用本文

刘洁, 李月婷, 陈奕君, 肖红斌. 高分辨质谱数据处理策略在中药体内外成分检测和表征中的应用进展. 药学学报, 2021 , 56 (1) : 113 -129 . DOI: 10.16438/j.0513-4870.2020-1385

Jie LIU, Yue-ting LI, Yi-jun CHEN, Hong-bin XIAO. Advances in high-resolution mass spectrometric-based data-mining technologies for detecting and characterizing the components and metabolites of Chinese materia medica[J]. Acta Pharmaceutica Sinica, 2021 , 56 (1) : 113 -129 . DOI: 10.16438/j.0513-4870.2020-1385

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中药化学成分通常由负责其功效的多成分组成, 因此, 系统鉴定中药中含有的化学成分对于阐明中药药效物质基础和发挥功效的药理作用十分重要。对中药中化学成分的定性鉴定包括已知化合物的确认和未知结构的阐明。前期研究者大多数致力于从中药提取物中分离和纯化足够量的纯化合物(纯度 > 90%), 然后采用IR、MS和NMR确定单体化合物的结构。由于中药化学成分的多样性, 整个分离、鉴定过程繁琐, 费时费力且昂贵, 而且很可能使用IR、MS和NMR技术鉴定出的化合物是已知的或无功效的。高分辨质谱能提供所有离子的高分辨质谱图、一级精确质量和丰富的二级或多级碎片信息。目前主要通过精确质量数、特征性质谱裂解碎片/中心丢失、参考标准物的裂解行为以及从保留时间数据、在线UV光谱获得的有关极性的信息, 来自文献或标准品的数据以及化合物的生物来源等快速有效地推断、鉴定中药中的化学成分。此外, 精确的质量数能给出母体离子和碎片离子的元素组成, 用于鉴定未知物和更多地区分同量异位素(两个不同的化合物具有相同的质量数, 但元素组成也不同, 确切的质量数也不同)^{[1, 2]}。因此, 高分辨率质谱(high-resolution mass spectrometry, HRMS)不仅具有高灵敏度、出色的分离度和宽检测动态范围的特点, 且能提供有关中药化学成分的有用结构信息, 还具有与色谱技术结合使用以在线鉴定每个单峰的可能性, 其在现代中药成分研究中应用日益广泛^{[3, 4]}, 为中药体内外成分表征提供强有力的支撑。

然而, 由于高分辨质谱原始数据中包含了海量信息, 且样品基质或内源性成分干扰的普遍存在, 使得中药全成分表征、微量组分的识别以及体内成分变得更加困难。如何快速、高效地阐明中药物质基础, 高分辨质谱数据的深度后处理策略是近年来国内学者不断探索的热点问题。快速识别并获得精确母离子信息对于复杂样品中成分的识别异常重要。近年来, 为了提高获取MS/MS或MSⁿ数据的灵敏度和选择性, 研究者们发展出一些新的采集策略, 如基于分子设计的、分子特征的、离子强度以及基于化合物生源关联等, 依据这些设定的选择标准(如设计的分子精确质量数、离子丰度、分子特征、氮原则等)触发二级质谱扫描。这些采集策略的发展为后期研究者在样品一级质谱数据的采集上提供了丰富的技术支撑, 为鉴定中药体内外化学成分奠定了基础。此外, 许多质谱数据鉴定策略也相应得到了较大程度的开发和发展, 如提取离子色谱技术^[5]、空白扣除技术^[6]、中性丢失过滤技术^[7]、产物离子过滤技术^[8]等。近年来随着研究者的不断挖掘, 在中药成分表征和体内成分鉴定研究中发展了一些其他新颖灵敏的数据鉴定策略, 如线性梯度质量亏损过滤技术、栅状质量亏损过滤技术、多点筛查质量亏损过滤技术、定量结构-保留相关性分析策略、MS网络分析模式等。

虽然已有研究者^[9-12]对前期报道的数据处理策略进行了综述, 但其主要从药物代谢物的发现和鉴定方面综述相关的数据采集和处理策略。中药成分种类丰富、数量繁多、含量悬殊, 根据成分的种类、数量、来源的不同, 其数据采集和鉴定策略亦有区别和针对性。因此, 本文主要对目前应用于中药体内外成分表征的数据处理策略进行综述, 包括采集和鉴定策略两大层面, 并从中药体外类成分、全成分以及体内中药成分表征三个方面阐述相应的数据鉴定策略及其优缺点, 为后期研究中药体内外成分提供方法参考。

1 中药中类成分的数据处理策略

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1.1 基于分子设计的数据采集策略

为了获取更多的母离子, 排除干扰离子, 研究者提出可以根据化合物的结构或生源途径进行预测, 设计尽可能多的化合物, 从而基于设计出的分子列表进行靶向二级质谱数据的采集, 鉴定中药提取物中的化学成分。如Zhang等^[13]建立基于分子设计的全扫描-母离子列表-动态排除采集策略(full scan-parent ions list-dynamic exclusion acquisition, FS-PIL-DE), 利用该采集策略结合诊断产物离子从柑橘的叶中鉴定了135个多甲氧基黄酮(polymethoxylated flavonoids, PMFs)。该策略首先对黄酮母核上2位至7位羟基和甲氧基取代位置及数量进行了预测, 归纳得到32个[M+H]⁺母离子, 然后针对这些母离子触发二级或多级质谱的采集, 并在此基础上进行动态排除, 以便触发更多的母离子(尤其是低丰度的母离子)碎裂以获取二级或多级质谱。该FS-PIL-DE策略比FS、FS-PIL和FS-DE策略能够靶向采集更多的成分, 从而增加成分筛选的覆盖范围。Zhang等^[14]还开发了基于分子设计的多离子监测(multiple ion monitoring, MIM)采集策略, 并结合靶向筛选及质谱树状图过滤鉴定策略共表征了金银花中由18个类别组成的115个绿原酸类成分。该策略首先设计并预测了70种绿原酸及其糖苷的分子式, 从而将78个[M-H]^-的目标离子导入SIM (selected ion monitoring)模式。为了充分考虑检测效率和灵敏度, 在每次SIM运行中, 将约20个目标母离子添加到MS/MS列表中以获得高质量的MS数据。虽然MIM与FS、FS-PIL的总离子色谱图的组成分布彼此相似, 但MIM采集中检测到的峰强度(139个候选成分)明显高于FS (54个候选成分)和FS-PIL (81个候选成分)获得的强度。

1.2 数据鉴定策略

目前, 对于中药提取物化学成分鉴定, 研究者主要集中于与功能相关的一类或两类成分, 且近年来发展到了类成分的深度分析。对于具有相同碳骨架的类成分, 近年来报道的可用于鉴定类成分的策略主要为质量亏损过滤技术、关键产物离子/中性丢失过滤、多反应/选择性反应监测技术、质谱树状过滤技术、定量结构-保留时间关联分析方法等。

1.2.1 质量亏损过滤技术

质量亏损过滤技术(mass defect filter, MDF)是基于高分辨质谱数据的过滤技术, 其根据化合物与类似物具有相近的质量亏损这一规律, 对采集的高分辨质谱数据进行处理和识别, 通过一次或有限几次进样可从复杂背景中发现和鉴定药物及其代谢产物, 显现出独特的优势。因此, MDF技术常用于中药中同类型化合物的鉴定或单体化合物的体内代谢物的鉴定。中药含有大量繁杂的化学成分, 而采用常规MDF技术会导致筛选结果偏差大、干扰性化合物多等问题。因此, 近年来研究者对数据挖掘技术进行了不断发展, 目前已在常规MDF技术的基础上发展了多重MDF技术、逐级MDF技术、线性梯度MDF技术、多点筛查MDF技术等新技术^[15]。

1.2.1.1 常规MDF技术

常规MDF技术一般选择单一的母体化合物为模板, 设置一定的质量亏损范围, 对其衍生物进行识别鉴定。Xie等^[16]利用MDF技术对麦冬提取物中的化学成分进行深入地挖掘, 选取麦冬皂苷和麦冬高异黄酮为模板, 从麦冬提取物中共发现和鉴定了50个麦冬皂苷类和27个麦冬高异黄酮类化合物。

1.2.1.2 逐级MDF技术

逐级MDF技术(stepwise MDF), 通过确定不同取代基化合物的母体结构, 以及取代基的数量, 选取多个质量亏损过滤窗口或多个质量范围, 可以筛选出更多的化合物。Zhang等^[17]总结了黄酮类化合物结构规律, 确定黄酮分子质量范围为282~436 Da, 取代基最多为5个甲基或5个羟基, 从而设置质量亏损范围为70~166 mDa, 利用逐级MDF技术, 分五段对柑橘中的多甲氧基黄酮进行筛选, 共鉴定出81个成分, 较常规MDF技术(鉴定30多个成分)检出了更多的化合物。

1.2.1.3 线性梯度MDF技术

线性梯度MDF (linear gradient MDF)技术, 以多个化合物为模板, 用曲线连接各模板化合物的质量数, 形成一个动态质量过滤曲线, 将曲线两端平行延长50 Da, 质量亏损值设为一定的区间范围, 则认为分子质量落在这一区域内的化合物可能是代谢物或结构类似物。该技术利用Waters公司UPLC/Q-TOF-MS的MS^E扫描模式进行数据采集, 然后采用Metabolynx XS软件中的MDF工具对数据进行处理。Yan等^[18]利用线性梯度MDF技术, 对茵陈四逆汤中的乌头类生物碱进行检测, 以准噶尔乌头碱、森布星A、次乌头碱3个化合物为模板, 将质量亏损范围设定在-38~23 mDa之间, 共筛选识别145个化合物通过假阳性筛选剔除55个化合物, 通过元素分析和质谱裂解规律剔除27个化合物, 最终鉴定了62个乌头类生物碱; 而采用传统的气质或液质方法, 只能鉴定出15个乌头类生物碱。

1.2.1.4 栅状MDF技术

Pan等^[19]提出一种新颖的栅状MDF (raster MDF)过滤方法, 即结合理论的stepwise母离子列表和最优的母离子质量宽度(parent mass width, PMW), 在触发扫描的过程中实现栅状MDF过滤。这种方法包括: ①基于理论的步进前体离子列表(precursor ion list, PIL)对潜在目标成分进行全面覆盖; ②在数据采集过程中实行光栅-MDF筛选。在310~950 Da的质量范围内, 理论上逐步进行PIL, 步长为2 Da, 以选择性地触发碎裂并扩展潜在吲哚生物碱的覆盖范围。同时, 通过将阶梯式PIL的母质量宽度(PMW)定义为±55 mDa, 在获取过程中实现了光栅-MDF筛选。最后, 利用该策略从五种植物来源的钩藤中共表征了1 227个吲哚生物碱类成分。

1.2.1.5 多点筛查MDF技术

多点(n≥5)筛查MDF技术, 将MDF与数学中的边界理论相结合, 以多个化合物(a、b、c、d、e…)为模板, 连接这些点, 确定筛选范围, 它使筛选结果不再是一个开放的空间, 理论上能够将其他干扰类型的化合物排除在外。Lai等^[20]采用五点筛查MDF (five-point screening MDF)技术来鉴定中药三七中的皂苷类成分, 其五点的选择原则如下: a点为分子质量最低的化合物; b、c点为小的取代基连接小的糖配基的化合物, b、c两点相连确定质量亏损的下界限; d、e点为大的取代基连接大的糖配基的化合物, d、e两点相连确定质量亏损的上界限; 上下界限斜率与取代基的数量和种类相关。因此, 选取最小质量数的B7型人参皂苷为a点, 3个氧(负贡献最小)取代的齐墩果酸型皂苷为b点, 5个木糖(正贡献第二大) 3个二甲酰基(正贡献最小)取代的齐墩果酸型皂苷为c点, 7个鼠李糖取代(正贡献最大)的去氢A1型人参皂苷为d点, 1个鼠李糖取代的去氢A1型人参皂苷为e点, 五点顺次连接确定皂苷类化合物的筛选范围及边界斜率, 结合诊断离子分析和视觉同位素技术, 共从三七中鉴定了234个皂苷类成分, 其中67个为潜在的新化合物。

此外, Pan等^[21]提出了集成多点质量亏损过滤的新颖分类和前体离子列表-触发采集的高分辨率质谱复制分析策略。首先, 该策略发展了基于知情的植物化学信息和分子设计开发的内部8点MDF算法, 该算法能更准确地筛选目标生物碱, 并从三个新颖的水平: 已知的(known, KN)、未知但预测的(unknown-butpredicted, UP)和意外的(unexpected, UN)区分成分。进而采用混合数据采集方法, 即PIL触发的碰撞诱导解离-MS²和高能C-trap解离-MS³, 在线性离子阱/Orbitrap质谱仪上动态排除, 促进多种产物离子的获取, 以便用于结构解析吲哚生物碱和N-氧化物。最后, 利用该策略从中华绒螯蟹的茎、叶和花中快速鉴定出158个潜在的新生物碱, 包括10个UP和108个UN。

1.2.2 特征离子过滤技术

特征的产物离子或中性丢失分子可以提供特征性的结构信息。产物离子过滤(product ion filter, PIF)和中性丢失过滤(neutral loss filter, NLF)是依赖化合物MS/MS谱的有效性, 以及利用裂解规律相似性识别同类物的技术。一般通过对中药中代表性对照成分的质谱裂解规律进行分析和总结, 获取同类型成分共有的子离子信息(产物离子或中性丢失), 然后对中药样品中同类物进行搜索与归类, 或使用多个产物离子或者中性丢失进行数据挖掘检测发现较多的未知物。近年来研究者常用特征离子过滤的策略来鉴定中药中具有相同碳骨架的成分如黄酮类、绿原酸类、萜烯类、生物碱类、皂苷类等成分。

黄酮类化合物是广泛存在于自然界的一大类化合物, 数量繁多, 结构类型繁杂, 其根据A环和B环中间的三碳链的氧化程度、三碳链是否构成环状结构、3位是否有羟基取代、B环(苯基)连接的位置、以及糖苷配基的连接位置等特点分为多个亚型, 不同亚型结构的黄酮类化合物产生不同特征的产物离子或中性丢失^{[22, 23]}。如Qiao等^[24]利用特征离子(产物离子和中性丢失)过滤策略从黄芩提取物中共鉴定了7类132个化合物, 包括9个苯乙醇苷、20个黄酮碳苷、11个黄烷酮氧苷、14个黄烷酮苷元、33个黄酮氧苷、42个黄酮苷元、3个其他类, 其中59个化合物为首次从黄芩中报道。此外, 近年来Yao等^[25]构建了一个增强目标识别策略, 共从红花中鉴定了107个黄酮-O-糖苷类成分, 其中80个成分为首次从红花中报道的。该策略整合离线二维液相色谱-线性离子阱-Orbitrap质谱-高分辨诊断产物离子/中性丢失过滤和固相萃取-核磁共振, 离线二维液相色谱使共洗脱组分获得更好地分离, 碰撞诱导裂解(collision induced dissociation, CID)和高能C-trap裂解(high-energy collision dissociation, HCD)的组合使用可以产生互补的碎片信息, 糖苷配基亚型和表征糖基化模式可通过HCD-MS²光谱中糖苷配基离子种类的强度来识别, 五种糖苷配基结构进一步通过LC-SPE-NMR的¹H NMR数据得到了确认。此外, 其中的一个新化合物被分离并完全鉴定, 这从某种程度上验证了MS导向鉴定的可靠性。一方面, CID通过消除糖配基以提供更丰富的高质量产物离子, 而HCD产生更多丰富的与糖苷配基相关的产物离子。HCD-MS²光谱中糖苷配基离子种类([Y₀-H]^-/Y₀^-和[Y₀-2H]^-/Y₀^-)的强度对比可以用来区分糖苷配基亚型和表征糖基化模式。根据特征的产物离子, 黄酮类成分中不同亚型的同分异构体能够得到鉴别。

绿原酸类成分是奎宁酸和某些肉桂酸(最常见的是咖啡酸、对-香豆酸和阿魏酸, 或为苯丙酸、3, 4-三羟基-咖啡酸、葡萄糖苷、丙二酸)的常见骨架之间形成的一大酯类成分^[26-29]。Guo等^[30]发现并总结了咖啡酸类成分在正离子模式下的中性丢失规律, 并利用该策略分析鉴定了咖啡酸衍生物。此外, 由于奎宁酸上相同取代基的取代位置不同, 绿原酸类成分通常以异构体形式存在。Zhang等^[31]在总结了绿原酸类成分在负离子模式下的特征产物离子和利用诊断产物离子过滤技术从金银花中鉴定出41个绿原酸类化合物的基础上, 根据诊断产物离子的丰度对其中的同分异构体进行了区分。研究^[32]进一步总结了更多绿原酸类成分的特征产物离子, 利用产物离子过滤策略从苦艾茶提取物中快速发现并鉴定了88个成分, 包括14种绿原酸类型成分, 同时也区分鉴定了同分异构体。

Qiao等^[33]根据前期分离得到的萜烯类成分对其质谱碎裂规律进行总结归纳, 确定了3个特征的中性丢失和9个产物离子信息, 进而基于多中性丢失/前体离子(multiple neutral losses/precursor ions, MNL/PI)扫描采集策略进行数据采集, 然后结合全局分析及多元统计学策略对姜黄中的萜烯类成分进行靶向及非靶向分析, 共从中药姜黄中快速识别出846个萜烯类成分, 其中包括大量的潜在新化合物, 作者进一步分离制备得到其中的两个潜在新化合物, 并通过核磁共振技术确定了它们的结构。

Li等^[34]首次根据对照品的结构特征和通过UHPLC-QTOF-MS/MS对4个亚型石蒜碱型生物碱对照品的质谱裂解规律进行总结归纳, 确定了4个不同亚型的特征碎片离子和中性丢失信息, 进而使用不同亚型中出现较多且质荷比恒定的5个特征离子筛选所有候选化合物, 在此基础上, 通过特定的23个诊断离子对候选化合物4个亚型进行归属和表征, 以及通过8个中性丢失分子过滤对结构进行合理化。最终, 利用多个特征离子过滤策略从6个石蒜科植物中共鉴定出37个石蒜碱型生物碱, 其中16个可能为未经报道的新化合物。Guo等^[35]利用特征碎片离子从白鲜皮组分中鉴定了8个喹啉类生物碱, 并在此基础上, 基于质谱导向策略追踪分离得到2个喹啉类生物碱。

Ma等^[36]通过归纳总结三萜皂苷对照品的裂解规律, 进而利用诊断离子、特征产物离子和中性丢失过滤策略, 从预知子提取物中共鉴定出85种三萜皂苷类似物。

1.2.3 离子对监测技术

中药中某些成分在质谱中的响应较弱, 为了更灵敏地监测一些特征、难电离的化合物, 近年来研究者亦发展了多反应/选择性离子对监测技术。该技术为某些难电离化合物或微量成分提供了支撑。Song等^[37]构建了步进甲酸根阴离子-去质子化离子的离子对监测筛选方法, 用于人参皂苷类成分的检测。当引入甲酸作为流动相添加剂时, 人参皂苷类成分容易形成甲酸根阴离子, 进而碎裂成去质子化离子。在最佳碰撞能量为-32 eV, 以([M+HCOO]^- > [M-H]^-)作为离子对监测人参皂苷类成分时, 人参皂苷类成分响应最强。因此, 采用[M+HCOO]^- > [M-H]^-离子对的步进多反应监测(multiple reaction monitor, MRM)方法, 从分子量为501~1 250 Da (Q1)中以2 Da为步长大小进行筛选, 进而在混合三重四极杆-线性离子阱质谱仪上触发MS²光谱采集的增强产物离子扫描; 然后根据明确定义的人参皂苷质量裂解模式, 对筛选的人参皂苷进行鉴定。同样地, Si等^[38]构建了一种选择性离子监测采集策略, 用于11种含有红花的中成药中的醌式查尔酮C-糖苷的标记。红花含有特征的醌式查尔酮C-糖苷(quinochalcone C-glycosides, QCGs)。27种QCG类似物(涉及16种潜在的新类似物)通过产物离子过滤(m/z 119.05)和负模式MS^E的综合分析进行了选择性表征。

1.2.4 同位素过滤技术

同位素过滤(isotope pattern filter, IPF)技术一般用于筛选含S、Cl和Br的化合物。由于³⁴S同位素离子受¹³C₂和¹⁸O的影响显著, 目前的算法几乎不被用于筛选含S化合物, 特别是当与色谱分析结合时。目前该技术常用于筛查含有Cl、Br药物的同位素模型的代谢产物。近年来, Yang等^[39]在基于高分辨质谱的基础上提出了一种用于筛选传统中药中的硫衍生物的新方法, 也就是一种基于分离的M+2离子(¹²C_x¹H_y¹⁶O_z³²S¹³C₂¹⁸O, ¹²C_x+2¹H_y¹⁶O_z³₊₁⁴S, 暂定为M+2OC和M+2S)的超精确同位素模式过滤(FIPF)方法。这种更精细的算法主要通过便捷的过滤器进行操作, 包括M+2OC和M+2S与M的精确质量偏移以及它们相对于M的相对强度。最后, 应用该策略在流行的药用葛根种中发现了12个S-衍生物, 且其中9个成分通过高分辨多级质谱(HR MSⁿ)进行初步鉴定。

1.2.5 定量结构-色谱保留关联技术

化合物的定量结构-色谱保留相关分析(quantita‐tive structure retention relationship analysis, QSRR)将被分析物的色谱保留数据与其分子结构特征相关联, 其在研究分子结构参数与色谱保留性质的递变规律、实现色谱过程中保留数据的预测、选择分离条件及探索色谱保留机制等方面有重要意义, 如今越来越受到重视。Zhang等^[40]利用异构体结构预测结合QSRR模型策略从川芎中共检出81个峰以及鉴定其可能的结构, 并对26种潜在的新型苯酞类化合物进行了结构表征。该策略包括三个步骤。首先, 根据结构特征和MS/MS碎片模式合理预测苯酞类化合物的结构: ①利用内部数据库初步筛选原始HRMS数据; ②总结类似化合物的MS/MS碎裂模式; ③鉴定已报道的苯酞类化合物, 并合理预测异构体的结构。其次, 建立QSRR模型并使用代表性的苯酞类化合物标准品进行验证。最后, 通过QSRR模型计算预测异构体的保留时间, 并通过匹配检测的色谱峰和预测异构体的保留时间合理地表征这些峰的结构。共使用23个苯酞标准品建立了多元线性回归QSRR模型, 其中筛选了6个物理化学变量。通过QSRR模型结合合理的结构预测(R²=0.955), 可以很好地预测川芎中苯酞类异构体的保留时间。该策略可以提高复杂基质中同系物的鉴定效率和可靠性。

1.2.6 “钓鱼”策略

Liu等^[41]在自建质量数据“池”和非目标质量数据采集方法的基础上, 提出了一种有效的“钓鱼”策略, 用于从莲子水提取物中鉴定非目标黄酮类成分。该策略中“分子诱饵”的特征为分子特征、碎片模式、特征性碎片离子、相对丰度和保留时间。最后, 成功利用该策略从莲子水提取物中表征了29个黄酮类成分, 其中3个化合物是首次从莲子提取物中识别的。

1.2.7 多种策略整合

Jiang等^[42]在构建已知成分数据库的基础上, 整合利用质量亏损过滤策略、特征离子过滤策略从清风藤提取物中鉴定91个生物碱类化合物。

1.3 小结

目前应用于中药类成分的数据鉴定策略研究较多, 其在数据采集策略发展较局限。基于分子设计的数据采集策略主要依赖于研究者预测、设计尽可能多的化合物, 根据母离子的精确分子质量数筛选目标母离子, 进而鉴定化合物, 但其可能会漏掉对未预测和设计出的已知或未知化合物的鉴定。对于中药类成分的采集, 应用该数据采集策略利于发现中药中更多可能的类成分。

以上用于鉴定中药中类成分的数据鉴定策略各有优缺点。如质量亏损过滤技术不需要总结化合物的裂解规律, 就能实现已知化合物的多个衍生物的识别, 其中常规MDF识别衍生物的数量有限, 多重MDF、线性梯度MDF、栅状MDF以及多点MDF等新技术则相对能识别出多的衍生物, 但目前不能通过该策略鉴定衍生物中取代基的位置或鉴别同分异构体或区分不同亚型结构。利用特征产物离子/中性丢失过滤策略能鉴定具有相同碳骨架的类成分, 能够检测出MDF无法检测到的类成分, 且可在一定程度上识别位置异构的同分异构体, 如绿原酸、不同类型的黄酮类化合物、不同类型的生物碱, 而对于手性异构的同分异构体, 该策略均无法实现结构的确定。该策略前期需要研究者对类成分裂解规律进行总结, 确定特征产物离子和中性丢失片段, 多用于含有相似亚型结构的类成分发现。多反应/选择性监测离子对方法多用于不易电离或响应比较低的类成分鉴定, 但其鉴定能力相较特征产物离子/中性丢失过滤策略较弱一些。同位素过滤技术仅适用含有同位素原子的S、Cl、Br等化合物的鉴定。定量结构-保留相关性分析策略则是根据类成分的结构裂解规律以及保留时间的相关性, 鉴定过程相对复杂, 但其能合理预测异构体的结构, 提高类成分的鉴定效率和可靠性。这些数据鉴定策略虽能通过一个化合物或多个同类化合物识别出更多已知的以及潜在未知/新的类成分, 但对于化合物的准确结构仍需经过IR、NMR、X-ray等技术进一步确认, 尤其是未知或新化合物的结构。

2 中药全成分的数据采集和鉴定策略

收起

中药含有的化学成分数量繁多, 种类复杂。目前中药化学成分鉴定从主成分和常量成分的分析逐渐过渡到全成分系统分析。中药全成分的系统表征对其物质基础的阐明十分重要。为了发现更多的中药全成分, 研究者在数据采集策略方面进行了多方面的探索, 发展了不少的策略, 如基于氮规则的前体离子选择采集策略、基于分子特征的前体离子定向扫描数据采集策略、基于离子丰度的前体离子选择采集策略、基于化合物生源关联的采集策略。然而目前对于中药全成分的鉴定策略相对于类成分的分析鉴定策略少, 常用于鉴定中药全成分有多重产物离子/中性丢失过滤技术、新发展的MS网络分析模式以及多种采集和鉴定策略的综合联用。

2.1 数据采集策略

2.1.1 基于氮规则策略

为了从原始UPLC-HRMS数据中去除不相关或干扰MS¹离子, 有助于保留目标组分的MS特征并尽可能有效地暴露目标化合物。天然小分子化合物只有生物碱类成分含有N原子, 而其他类成分则不含氮原子。因此, Ren等^[43]在精确分子质量数、保留时间、离子丰度的基础上提出了基于氮原则的前体离子选择策略, 并结合质量缺陷和中性丢失/诊断碎片离子过滤等鉴定策略, 从艾叶原始UPLC-HRMS数据集快速筛选和鉴定了16种甲氧基化黄酮和55种绿原酸类似物。运用该策略成功检测到了艾叶中的几种微量成分, 表明其具有明显的优势。

2.1.2 基于分子特征策略

中药及其复方含有多种类型的化学成分, 其结构、分子质量和保留时间存在一定的内在特征。基于此, 本课题组Wang等^[44]构建了基于分子特征的前体离子定向扫描数据采集策略(precursor ion selection, PIS-MS/MS), 该策略根据各类成分在色谱和质谱上的分子特征, 运用安捷伦Mass Hunter数据处理软件中的“分子特征查找”功能, 提取各类型化合物的前体离子, 最后对前体离子进行靶向MS/MS分析。整个操作流程分为以下三步: ①寻找分子特征; ②定位感兴趣的化合物并构建前体离子列表; ③对前体离子进行靶向MS/MS分析并进行结构推断。结果表明, 在给定的保留时间和m/z范围内, 使用PIS-MS/MS获得的前体离子数量是auto-MS/MS方式的1.3倍, 有利于进一步地获取低丰度化合物的碎片信息, 其可实现高、低含量甚至微量化合物的同步鉴定。最后, 成功利用该策略在20 min内从益心舒胶囊中辨识出276种化学成分。

2.1.3 基于离子丰度策略

针对色谱峰中离子响应丰度低的共流出化合物难以被检测的问题, 本课题组Liu等^[45]提出了基于前体离子选择采集(precursor ions selection, PIS)的策略。该策略对选择前体离子的多种参数(动态、静态排除、丰度限值等)进行优化, 在同一色谱峰中获取多个母离子, 保证丰度高、低的共流出化合物均能采集到, 进而对获取的母离子进行靶向MS/MS采集。在相同的保留时间和m/z范围内, 负离子模式下PIS方式采集得到的母离子数为873, 而在自动采集中得到的母离子仅为613, 表明PIS方式获得的前体离子数量比自动采集方式多, 而引起二者数量的差异的原因, 主要是同一循环中母离子个数的增加的贡献。此外, PIS采集的6种诊断产物离子发现的化合物数量较自动采集的多。以上结果说明通过PIS-MS/MS分析策略, 共流出色谱峰中无论是含量高的化合物还是含量低的化合物或者微量化合物都可以被鉴定。

2.1.4 基于化合物生源关联策略

植物体内尤其是同一植物所含的同一类型化合物之间存在内部的生源途径关联, 这些类似物之间存在诸如甲基化/去甲基化、羟基化/去羟基化、糖苷化/去糖苷化等生物转化关系, 因此, 当给定一个中心化合物, 并总结与之相关的化合物生源关联, 就可以形成以该化合物为中心, 向四周辐射的生源关系网, 以此为基础建立一个可以预测植物体内某一给定类型化合物的所有类似物的拟靶向数据库, 在MS的基础上, 进行拟靶向筛选得到目标前体离子, 再进行目标前体离子的tar‐get-MS/MS数据采集, 将大大加快化合物的鉴定效率和数量。本课题组Li等^[46]提出了基于化合物生源关联的采集策略, 将该策略成功应用于中药复方芪参颗粒化学成分的定性分析中, 共定性鉴别了213个化合物。

2.1.5 基于全信息串联质谱策略

全信息串联质谱技术(MS^E)是采用两个全扫描MS功能的数据相关采集方法, 在一次液质剖析中同时获得高精确的母离子及碎片离子信息的串联质谱方法。它由“低碰撞能”与“高碰撞能”两种扫描交替形成, 分别记录母离子及碎片信息, 来自低碰撞能量的质谱提供完整的分子离子信息, 而来自高碰撞能量的质谱包含碎片离子信息。并通过母离子与其碎片离子具有相同色谱行为的特性进行母-子离子的关联归属。Sun等^[47]利用UPLC-Q-TOF的自动MS^E技术结合Me‐taboLynx^TM数据分析软件从中药复方开心散中快速鉴定了107个化合物。Yang等^[48]采用UPLC-ESI-Q-TOF/MS^E技术对养血清脑颗粒的化学成分进行分析, 通过Q-TOF-MS^E提供化合物的准确相对分子质量、质谱碎片离子信息, 与对照品的保留时间和质谱数据比对, 结合参考文献, 共鉴定出5类142个化合物。

2.2 数据鉴定策略

2.2.1 基于本地数据库筛选策略

前期对中药全成分鉴定时, 常根据文献报道及标准品数据构建本地数据库进行鉴定。Zhang等^[49]利用HPLC-ESI IT-MS/MS建立了含有600多个化合物的液相色谱-质谱-数据库, 并对其谱库检索结果的可信度进行了验证, 并从千里香药材中快速鉴定了15个多甲氧基黄酮类化合物。You等^[50]利用HPLC-Q-TOF/MS对六经头痛片进行分析, 通过自建的八味组方药材的化学成分库信息结合质谱裂解规律, 共鉴定出95个化合物, 主要包括异黄酮类、香豆素类、环烯醚萜类和苯酞类等成分, 并对化合物的药材来源进行了归属。Huang等^[51]应用UPLC-Q-TOF的MS^E技术结合UNIFI数据库平台从补气通络颗粒中快速定性鉴定了72个化合物。

2.2.2 多重特征离子过滤技术

前面已介绍特征离子(产物离子和中性丢失)过滤技术常用于中药类成分的鉴定, 而中药全成分是由多个类型的化合物组成的, 因此, 通过多个产物离子和中性丢失过滤技术也能实现中药全成分的系统分析。如He等^[52]总结了木脂素类、有机酸类、苯丙素类、环形醚萜类的多个特征产物离子和中性丢失数据库, 采用过滤技术从杜仲提取物中共鉴定了5类113个化合物; Xu等^[53]利用产物离子和中性丢失过滤技术从丹红注射液中鉴定了3类117个化合物; Jiang等^[54]利用多重产物离子过滤技术从抗肾间质纤维化I号方中鉴定了10类360个化合物。Pan等^[55]利用代表性成分的诊断离子进行搜寻, 并结合质谱偶电子规律、氮规律、不饱和度和同位素峰等信息, 以及参阅文献, 从虎杖根中鉴定出2类34个化合物。

2.2.3 MS网络分析策略

近年来, Li等^[56]开发了一种新颖的MS网络分析模式, 该模式整合了植物化学化合物的内在结构相关性和源自质谱的结构信息, 从原始数据流中识别相同类型的化合物。初步建立了利舒康胶囊中黄酮和黄酮苷、生物碱、酚酸、皂苷和苄基酯糖苷的5个MS网络, 基于MS网络鉴定或暂定分配278个化合物, 包括9个潜在的新化合物。

2.2.4 质谱树状图过滤技术

近年来, 研究^[57]利用质谱树状图过滤技术(mass spectral tree similarity filter, MTSF)于中药复方小续命汤的化学成分表征中, 从高分辨质谱自动检测到的3 362个化合物中共鉴定了68个化合物。

2.2.5 多种策略整合联用

中药全成分类型多样、数量繁多, 研究者常通过多种数据处理策略对单味中药或复方中的全成分进行鉴定。Chen等^[58]利用多种采集和鉴定策略联用从紫菀中共鉴定了8类132个化合物; Pang等^[59]利用诊断产物离子过滤、质量亏损过滤、中性丢失过滤等技术联用从银丹心脑通软胶囊中共鉴定了6类124个化合物; Pan等^[60]在二维液相分离富集多个流分的基础上利用质量亏损过滤、内部数据库、诊断片段、分子网络等综合鉴定策略从金银花中鉴定出4类537个化合物; Feng等^[61]利用已知化合物数据库、诊断离子过滤、质量亏损过滤等多种策略联用从紫草中鉴定出6类90个化合物。

2.3 小结

对于中药全成分的表征, 在数据采集方面, 基于分子特征的采集策略则需要研究者熟练掌握化合物的结构特点和物理性质。在后期研究中, 也可以通过优化前体离子选择的参数获取更多的前体离子以及根据氮原则选择采集中药不同类型化合物的前体离子。在数据鉴定方面, 根据文献报道及标准品数据构建本地数据库的鉴定策略可以快速初步确定未知化合物的归属, 但还需要结合对照品和MS/MS裂解规律进行准确的结构鉴定, 因此, 应尽可能地采用多种数据鉴定策略联用(如多重产物离子/中性丢失过滤技术、多重质量亏损过滤等)。

3 中药体内成分的数据采集和鉴定策略

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由于内源性成分的干扰, 中药体内成分鉴定仍是难点工作。近年来报道常用于体内中药成分鉴定的数据采集和鉴定策略主要有以下几种。

3.1 数据采集策略

通常, 动物或人口服中药后, 在其血浆、尿液或粪便中会发现数十种具有一定结构的中药母体成分(原形)和代谢物成分。因此, 基于预测结构信息的目标数据依赖型采集方法(例如基于分子列表、质量缺陷、同位素模式和依赖中性丢失的MS/MS采集)不适合中药体内成分分析。近年来发展了许多基于非靶向的数据非依赖型采集(DIA)策略, 如MS^E、SWATH, 用于中药体内成分的表征。

3.1.1 MS^E采集技术

Yang等^[62]利用UPLC-Q-TOF的MS^E采集模式从白芨非糖部位的肠道细菌模型中分析鉴定出8个成分(5个原形成分和3个代谢物)。Zhang等^[63]采用UPLC-Q-TOF-MS^E技术结合外源性代谢组学方法从肝豆汤给药后正常和铜负荷模型大鼠尿液中共检测到77个外源性成分(45个原形成分和32个代谢产物)。同样地, Tang等^[64]利用UPLC-Q-TOF的MS^E采集模式结合特征片段或中性丢失等策略从麒麟丸的给药血浆、尿液、胆汁、粪便中鉴定了203个成分(76个原形成分和127个代谢物)。

3.1.2 SWATH采集技术

基于MS/MS^ALL的所有理论碎片离子的连续窗口采集技术(sequential windowed acquisition of all theo‐retical fragment ions, SWATH)是一种新型的MS/MS扫描技术, 它将扫描范围划分为以几十个Da为间隔的一系列区间, 通过超高速扫描来获得扫描范围内全部离子的所有碎片信息, 是MS/MS^ALL技术的扩展。与传统的信息依赖采集方法相比, SWATH™可以获取所有MS/MS光谱, 因此显著提高了低含量或痕量代谢物的命中率。适用于结构多样、含量较少的代谢物的定性与定量研究。Scheidweiler等^[65]采用LC-Q-TOF/MS结合SWATH技术成功地鉴定了人的尿液中47个合成大麻素代谢物。研究^[66]采用UHPLC-Q-TOF-MS结合SWATH鉴别了冬凌草乙素的20个代谢物。此外, Liao等^[67]利用SWATH技术结合关键产物离子过滤(KPIs)技术从蛇床子中的特征成分蛇床子素的大鼠肝微粒体和给药大鼠体液中共检测到72种代谢物, 包括大鼠肝微粒体中的39个代谢物、血浆中的20个代谢物、胆汁中的32个代谢物、尿中的32个代谢物和粪便中的37个代谢物。

3.2 数据鉴定策略

3.2.1 质量亏损过滤技术

成分进入体内一般发生Ⅰ相和Ⅱ相代谢, 原形成分与代谢物存在一定的质量亏损, 因此, 其十分适用于体内中药成分的鉴定。目前常应用MDF技术于中药中单一化合物的体内血液、尿液、胆汁、粪便中代谢物的检测。Wang等^{[68, 69]}采用UPLC-Q-TOF/MS结合MDF技术从小檗碱、9-去甲小檗碱给药大鼠体内样品中分别发现和鉴定了97、57个代谢物, 其中尿液中有68、54个代谢物, 血浆中有45、39个代谢物, 胆汁中有44、28个代谢物, 粪便中含有41、18个代谢物。同时, 该研究发现去甲基化、去亚甲基化、还原、羟基化和随后的葡萄糖醛酸化、硫酸化和甲基化是小檗碱和9-去甲小檗碱在体内的主要代谢途径。此外, 近年来研究者发展了多重质量亏损过滤技术(multiple mass defect filtering, MMDF)。如Zhou等^[70]通过UPLC-Q-TOF/MS结合质量亏损过滤两步法共从栀子-连翘药对给药血浆鉴定出36个原形成分和16个代谢产物。第一步通过UPLC-Q-TOF/MS结合质量亏损过滤从不同结构类型的代表性化合物中快速鉴定了未知类似物, 并进一步通过标准物质和已发表的文献进行了确认。类似地, 在第二步中, 通过质量亏损过滤从第一步中鉴定的化合物中快速发现其他未知组分。Tian等^[71]利用该策略与动态背景扣除(dynamic background subtraction, DBS)相结合的在线数据采集方法, 以及结合提取离子色谱、产物离子过滤和中性丢失过滤等多种数据挖掘技术, 追踪冬凌草甲素的所有潜在代谢物。在口服冬凌草甲素后的大鼠血浆中检测到16个Ⅰ相和2个Ⅱ相代谢物。MMDF与DBS相结合的策略可以触发信息依赖的采集扫描, 该策略有利于复杂基质中的背景噪音和内源性成分掩盖的低丰度代谢物信息的采集。Liu等^[72]也利用该策略从脱氧五味子素的给药体内大鼠中鉴定到49个Ⅰ相和2个Ⅱ相代谢物。此外, Lin等^[73]在MDF技术的基础上提出基于UPLC-Q-TOF/MS的可靠的“基于代表性结构的同源代谢物识别”(RSBHXI)策略来研究瓜蒌薤白汤提取物中的化学成分。RSBHXI策略主要是将整个瓜蒌薤白汤转换为几种代表性化学成分的数据类型进行数据处理, 实现复杂代谢研究的优势分析。其将几种主要的原形成分作为母体化合物, 进行质量亏损分析。利用RSBHXI策略从口服瓜蒌薤白汤提取物的给药大鼠体液中共发现并鉴定了138种瓜蒌薤白汤相关的代谢物。

3.2.2 产物离子/中性丢失过滤技术

研究^[74]将中性丢失和前体离子扫描和选择性反应监测等技术应用于中药复方体内成分的深度分析, 从葛根芩连汤给药大鼠体内检测到131个代谢物, 包括46个原形成分, 85个新生成的代谢产物, 其体内代谢途径主要为甲基化、乙酰化、脱水、脱糖基以及葡萄糖醛酸化、硫酸化等。

此外, Huang等^[75]开发了灵敏的基于QTRAP的多离子监测(multiple ion monitor, MIM)-增强产物离子(enhanced product ion, EPI)和中性丢失(NL)-EPI扫描方法, 该方法通常用于分别检测由Ⅰ相(氧化、还原和水解)和Ⅱ相(结合)反应产生的非靶向代谢物^[76]。它们与靶向MRM-EPI扫描互补, 因此MIM-EPI和NL-EPI最初用于检测代表性的单一化合物的体外代谢物。在此基础上, 根据体外结果和预测的代谢物分析体内代谢物。其使用该方法从甘草水提取物的临床给药剂量大鼠体内样品中共检测并鉴定57个代谢物, 其中尿液中有45种代谢物、粪便中有21个代谢物和血浆中有35个代谢物。在这些代谢物中, 18个痕量代谢物为首次报道的, 6个次要代谢物为首次在体内检测到的。

3.2.3 质谱树状图过滤技术

质谱树状图过滤技术(MTSF)是近年来由张金兰课题组^[77]提出的。利用该技术鉴定化合物需要进行以下步骤: ①通过全扫描模式获得化合物的一级高分辨质谱图, 将其作为质谱树状图的树干; ②通过数据依赖型扫描模式获得化合物的多级质谱图, 将其作为树枝, 构建被检测化合物的质谱树状图; ③将已知结构的化合物质谱树状图作为模板导入自建的质谱树状图数据库; ④将被检测样品的质谱树状图与自建的质谱树状图库进行相似度比较, 根据设置相似度匹配得分阈值获得一定可信度的成分信息, 从而发现潜在代谢产物。最后, 张金兰课题组成功利用MTSF技术从淫羊藿提取物的大鼠给药血浆、尿液、粪便及胆汁样品中辨识出11类115个代谢物, 且发现MTSF技术在发现代谢产物方面优于NLF和PIF技术, 与MDF技术互补。此外, 张金兰课题组^[78]应用该技术从中药复方小续命汤活性组分的给药大鼠体内成分鉴定了247个代谢物。该技术可用于中药体内外成分的鉴定, 然而目前常依托LTQ-FT和Orbitrap质谱仪上进行数据采集分析, 其他质谱仪上暂未应用此策略。

3.2.4 基于目标基团变化策略

Li等^[79]利用目标基团变化(target group change, TGC)策略来分析复方夏莲胶囊体内代谢物及其代谢途径。该策略主要通过Metabolynx XS软件推测原形成分代谢后结构中潜在的基团变化, 以及通过比较代谢物与其原形成分的串联质谱裂解行为确定获得的目标基团变化, 从而推断出每个原形成分经代谢后基团发生变化的位置和代谢途径。基于TGC策略, 鉴定了复方夏莲胶囊中11个原形成分中经代谢转化为33个代谢物, 这11个原形成分为4种黄酮、5种生物碱和2种人参皂苷, 原形成分经代谢后的主要基团变化主要包括去甲基化、甲基化、羟化和葡萄糖醛酸化结合。

3.2.5 多技术整合策略

中药复方进入体内的成分数量多, 其鉴定过程更为复杂, 目前研究者常采用多种技术整合策略进行体内中药成分识别和鉴定。本课题组Wang等^[80]通过整合质量亏损过滤、提取离子色谱、同位素模式匹配和生物转化补偿分子式等综合鉴定策略从益心舒胶囊给药血浆、尿液和粪便中共鉴定了184种代谢物(122个原形成分和62个代谢产物), 首次发现木脂素类成分体内代谢途径主要为脱掉CH₂或OCH₂、氧化、二氧化、酮基化和甲酸化等反应。Qiao等^[81]采用UHPLC-Q-TOF-MS技术分析大鼠灌胃给予止咳宝片后的血浆生物样本, 在采集时进行实时多重质量亏损和动态背景扣除, 并结合子离子扫描信息以及应用Metabolite Pilot 2.0软件中质量亏损、同位素类型、特征子离子和特征中性丢失等功能进行数据处理, 从血浆样本中鉴定5个阿片类生物碱、甘草苷和甘草酸, 及其相关代谢产物31个。

3.3 小结

对于中药复方体内成分的表征, 由于其进入体内的成分数量多, 且受内源性成分的干扰, 其鉴定过程十分复杂, 因此, 常用多种数据处理策略整合联用来鉴定中药体内成分。

4 总结和展望

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LC-HRMS技术, 由于其具有高灵敏度、高质量准确度和分析复杂混合物的优势, 是用于快速定性确定已知化合物以及从中药提取物或体内生物样本中鉴定未知化合物的最具选择性的首选方法。表 1总结了近年来质谱在中药体内外成分检测和表征中的相关应用, 发现Q-TOF质谱仪在中药体内外成分表征研究中发挥着重要作用。基于此, 本文归纳并梳理了基于Q-TOF的数据采集和处理策略在中药成分辨识中的解析流程(图 1)。

文中提到的数据采集和鉴定策略(图 2), 为中药体内外成分识别提供了很好的方法参考。然而中药含有的物质成分复杂, 往往由几大类不同成分构成, 对于中药化学成分鉴定, 近年来发展的数据鉴定策略大多应用于中药中类成分鉴定, 仅有少量鉴定策略应用于中药或复方中全成分鉴定。中药全成分识别鉴定对于中药物质基础的阐明至关重要, 因此, 还需要发展更多新的技术策略。此外, 进入体内的中药成分具有数量多、含量低、部分成分信号响应低等特点, 目前中药体内成分所取得的一些研究成果主要是高含量、易测成分, 其中大量含量低、质谱信号响应差的体内成分还没有被系统检测及测定, 如何排除高含量及内源性成分干扰, 高覆盖度地检测中药体内成分仍是面临的难点, 因此, 需要开发更多新的技术和研究策略来应对这一难点问题。

虽然高分辨质谱技术对于快速鉴定中药化学成分或发现中药新化合物提供了可能, 但其化学成分的结构仍需进一步通过核磁共振技术确认, 尤其是对于同分/手性异构体、新化合物的结构, 需通过¹H NMR、¹³C NMR、HSQC、HMBC、¹H, ¹H-COSY等才能确定化合物的结构。通过核磁共振技术能确定化合物的整体结构母核、支链的连接位置、取代基团位置、手性构型等, 从而准确确定化合物的结构。

作者贡献: 刘洁进行了全文撰写及图表绘制; 李月婷、陈奕君提供部分材料并做了辅助修改; 肖红斌提供了文章思路并负责主要的审阅与校对修改工作。

利益冲突: 所有作者不存在利益冲突。

基金

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国家自然科学基金项目(81803703)
国家自然科学基金项目(81774155)
重大新药创制国家科技重大专项(2019ZX09201004)
中国博士后科学基金面上项目(2018M630119)
北京中医药大学新教师启动基金项目(2020-JYB-XJSJJ-022)

参考文献

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文献

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2021年第56卷第1期

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doi: 10.16438/j.0513-4870.2020-1385

接收时间：2020-08-24
首发时间：2025-12-18
出版时间：2021-02-12

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收稿日期：2020-08-24
修回日期：2020-09-21

基金

国家自然科学基金项目(81803703)

国家自然科学基金项目(81774155)

重大新药创制国家科技重大专项(2019ZX09201004)

中国博士后科学基金面上项目(2018M630119)

北京中医药大学新教师启动基金项目(2020-JYB-XJSJJ-022)

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1.北京中医药大学北京中医药研究院, 北京 100029

2.北京中医药大学中药分析与转化研究中心, 北京 100029

3.北京中医药大学中药学院, 北京 100029

4.北京中医药大学中医学院, 北京 100029

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*肖红斌, Tel/Fax: 86-10-64286490, E-mail: hbxiao69@163.com

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Research objective	Sample type	Compounds investigated	Separation column	MS model	Acquisition mode	Ref.
Class compounds identification	Citrus reticulata extract	Polymethoxylated flavonoids	Agilent Zorbax Extended C18 (4.6 mm×250mm, 5 μm)	Thermo LTQ-Orbitrap	Full scan & DDA MS/MS	[13]
	Lonicerae japonicae	Chlorogenic acids	Waters ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm)	Thermo LTQ-Orbitrap	Full scan & DDA MS/MS	[14]
	Citrus reticulata extract	Polymethoxylated flavonoids	Agilent Zorbax Extended C18 (4.6 mm×250 mm, 5 μm)	Thermo LTQ-Orbitrap	Full scan & DDA MS/MS	[17]
	Yin Chen Si Ni Tang	Aconitum alkaloids	Waters ACQUITY UPLC BEH C18 (2.1 mm×100 mm ID, 1.7 μm)	Waters SYNAPT HDMS Q-TOF	MS^E	[18]
	Uncariae ramulus extract	Indole alkaloids	Phenomenex Kinetex^® EVO C18 column (2.1 mm×150 mm, 2.6 μm)	Thermo LTQ-Orbitrap	Full scan & DDA MS/MS	[19]
	Panax notoginseng extract	Saponins	Agilent Eclipse XDB-C18 (4.6 mm×250 mm, 5 μm)	Agilent 6530 Q-TOF	Full scan & DDA MS/MS	[20]
	Scutellaria baicalensis extract	Flavonoids	Agilent ZORBAX Eclipse Plus C18 (2.1 mm×150mm, 1.8 um)	Thermo Q-Exactive hybrid quadrupole-orbitrap	Full scan & DDA MS/MS	[24]
	Lonicerae japonicae extract	Chlorogenic acids	Agilent Zorbax SB C18 (4.6 mm×250 mm, 5 μm)	Agilent MSD Trap XCT Plus	Full scan & DDA MS/MS	[31]
	Ainsliaea fragrans extract	Chlorogenic acids	Waters ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm)	AB Sciex Triple TOF^TM 5600+	Full scan & DDA MS/MS	[32]
	Curcuma longa extract	Terpene-conjugated curcuminoids	Waters Acquity UPLC HSS T3 (2.1 mm×150 mm, 1.8 μm)	Agilent 6495 QQQ Thermo Q-Exactive hybrid quadrupole-orbitrap	SIM full scan & DDA MS/MS	[33]
	Panax ginseng extract	Ginsenosides	ACE UltraCore 2.5 Super C18 (3.0 mm×150 mm, 2.5 μm)	ABSciex 5500 Qtrap	MRM	[37]
	Eleven Chinese patent medicines including Carthamus tinctorius	Quinochalcone C-glycosides	Waters BEH Shield RP 18 (2.1 mm×100 mm, 1.7 μm)	Waters Xevo®G2-S QTOF	SIM	[38]
	The popular medicinal used Pueraria species	Sulfur derivative of flavonoids	Agilent ZORBAX Eclipse Plus C18 (2.1 mm×100 mm, 1.8 um)	Thermo LTQ Orbitrap Velos Pro	Full scan & DDA MS/MS	[39]
	Ligusticum chuanxiong extract	Phthalides	Waters ACQUITY BEH C18 (2.1 mm×100 mm, 1.7 μm)	Waters SYNAPT G2-SI Q-TOF	MS^E	[40]
	Lotus plumule extract	Flavonoids	Thermo Hypersil GOLD column (2.1 mm×100 mm, 1.9 μm)	Thermo Q-Exactive hybrid quadrupole-orbitrap	Full scan & DDA MS/MS	[41]
	Sinomenii caulis extract	Alkaloid analogues	Waters Symmetry C18 analytical (2.1 mm×100 mm, 3.5 μm)	Agilent 6530 Q-TOF	Full scan & DDA MS/MS	[42]
Chemical profiling of all compounds	Artemisiae argyi extract	Flavonoids, chlorogenic acids	Thermo Hypersil GOLD column (2.1 mm×100 mm, 1.9 μm)	Thermo Q-Exactive hybrid quadrupole-orbitrap	Full scan & DDA MS/MS	[43]
	Yi Xin Shu capsule	Lignans, tanshinones, flavonoids, phenolic acids, saponins, and others	Agilent ZORBAX Eclipse Plus C18 (3.0 mm×100 mm, 1.8 μm)	Agilent 6540 Q-TOF	Full scan & DDA MS/MS	[44]
	Dachuanxiong decoction	Phthalides, ester of citric acid, chlorogenic acids, phenols, nucleosides, and others	Agilent ZORBAX Eclipse Plus C18 (2.1 mm×150 mm, 1.8 μm)	Agilent 6550 Q-TOF	Full scan & DDA MS/MS	[45]
	Qishen granule	Chlorogenic acids, tanshinones, flavonoids, iridoid glucosides, alkaloids, salvianolic acids, phenolic acids, triterpenoid saponins, and phenylpropanoids	ACQUITY TM UPLC HSS T3 (2.1 mm×100 mm, 1.8µm)	Agilent 6550 Q-TOF	Full scan & target MS/MS	[46]
	Kai xin san	Saponins, triterpenoid, phenolic acids, and others	ACQUITY TM UPLC HSS T3 (2.1 mm×100 mm, 1.8µm)	Waters Synapt^TM High Definition TOF	MS^E	[47]
	Yangxue Qingnao granule	Phenolic acids, monoterpenoid glycosides, quinones, alkaloids, phthalides and others	Waters ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm)	Waters Acquity UPLC-Synapt G2 Q-TOF	Full scan & DDA MS/MS	[48]
	Liujing toutong tablet	Isoflavonoids, iridoids, coumarins, phthalides, and others	Dikma Diamonsil C18 (4.6 mm×250 mm, 5 μm)	Bruker micrOTOF-Q Ⅱ	Full scan & DDA MS/MS	[50]
	Buqi Tongluo granule	Flavonoids, saponins, terpenoids, esters, amino acid, phenolic acids	Waters ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm)	Waters Q-TOF	MS^E	[51]
	Danhong injection	Phenolic acids, flavonoids, and others	Waters ACQUITY UHPLC HSS T3 (2.1 mm×100 mm, 1.8µm)	Thermo Q-Exactive hybrid quadrupole-orbitrap	Full scan & DDA MS/MS	[53]
	Anti-renal interstitial fibrosis I decoction	Sennosides, galloyl glycosides, flavonoids, lignans, iridoids, organic acids, phenylpropanoids	Thermo Hypersil GOLD column (2.1 mm×100 mm, 1.9 μm)	Thermo Q-Exactive hybrid quadrupole-orbitrap	Full scan & DDA MS/MS	[54]
	Lishukang capsule	Flavonoids, alkaloids, phenolic acids, saponins, and benzyl ester glycosides	Waters ACQUITY UHPLC HSS T3 (2.1 mm×100 mm, 1.8µm)	AB SCIEX 5600+Q-TOF	Full scan & DDA MS/MS	[56]
	Xiao Xu Ming decoction	Flavonoids, monoterpenoid glycosides, terpenoids, and others	Thermo BDS HYPERSIL C18 (2.1 mm×150 mm, 3µm)	Thermo LTQ-FT	Full scan & DDA MS/MS	[57]
Metabolites identification	Intestinal bacteria	Nonpolysaccharide fraction from Bletilla striata metabolites	Waters BEH C18 (2.1 mm×50 mm, 1.7 μm)	Waters XEVO G2‐XS Q‐TOF	MS^E	[62]
	Rat urine	Gandou decoction metabolites	Agilent Eclipse Plus C18 (2.1 mm×100 mm, 1.8 μm)	Waters Xevo G2 Q-TOF	MS^E	[63]
	Rat bio-samples	Qi-Lin pill metabolites	Waters ACQUITY UHPLC HSS T3 (2.1 mm×100 mm, 1.8 μm)	Waters SYNAPT™G2 Q-TOF	MS^E	[64]
	Human urine	Synthetic cannabinoids metabolites	Bellefonte Ultra Biphenyl HPLC (2.1 mm×100 mm, 3.0 μm)	AB Sciex Triple-TOF 5600+	SWATH MS/MS	[65]
	Liver microsomes, rat urine and bile	Ponicidin metabolites		AB Sciex Triple-TOF 5600+	SWATH MS/MS	[66]
	Liver microsomes, and rat bio-samples	Osthole metabolites	Agilent Poroshell 120 EC-C18 (2.1 mm×100 mm, 2.6 μm)	AB Sciex Triple-TOF 5600+	SWATH MS/MS	[67]
	Rat bio-samples	Berberine metabolites	Waters ACQUITY UPLC BEH Shield RP18 (2.1 mm×50 mm, 1.7 μm)	Waters SYNAPT^TM G2 Q-TOF	MS^E	[68]
	Rat bio-samples	Berberrubine metabolites	Waters ACQUITY BEH Shield RP18(2.1 mm×50 mm, 1.7 μm)	Waters SYNAPT^TM G2 Q-TOF	MS^E	[69]
	Rat urine and bile	Oridonin metabolites	Phenomenex Kinetex C18 (2.1 mm×100 mm, 2.6 μm)	AB Sciex Triple-TOF 5600+	Full scan & DDA MS/MS	[71]
	Rat urine and bile	Deoxyschizandrin metabolites	Phenomenex Kinetex C18 (2.1 mm×100 mm, 2.6 μm)	AB Sciex Triple-TOF 5600+	Full scan & DDA MS/MS	[72]
	Rat bio-samples	Gualou Xiebai Baijiu decoction metabolites	Agilent Acquity BEH C18 (2.1 mm×50 mm, 1.7 μm)	Waters SYNAPT^TM G2 Q-TOF	Full scan & DDA MS/MS	[73]
	Rat plasma and urine	Gegen-Qinlian decoction metabolites	Waters Atlantis T3 column (2.1 mm×150 mm, 3 μm)	Thermo Fisher Finnigan TSQ QQQ	Neutral loss scan, precursor ion scan, and SRM scan	[74]
	Rat plasma, urine and feces	Glycyrrhiza uralensis metabolites	Welch Ultimate XB-C18 (2.1 mm×100 mm, 1.8 μm)	AB Sciex API 4500 QTRAP	MIM-EPI and NL-EPI	[75]
	Rat bio-samples	Epimedium brevicornu metabolites	Agilent SB-C18 column (4.6 mm×250 mm, 5 μm)	Thermo LTQ-FT	Full scan & DDA MS/MS	[77]
	Rat bio-samples	Xiao Xu Ming decoction metabolites	Thermo BDS HYPERSIL C18 (2.1 mm×150 mm, 3 μm),	Thermo LTQ-FT	Full scan & DDA MS/MS	[78]
	Rat plasma	Fufang Xialian capsule metabolites	Waters ACQUITY UPLC BEH C18(2.1 mm×50 mm, 1.7 μm)	Waters SYNAPT^G2 Q-TOF	MS^E	[79]
	Rat bio-samples	Yi-Xin-Shu capsule metabolites	Agilent Eclipse Plus C18 (2.1 mm×100 mm, 1.8 μm)	Agilent 6540 Q-TOF	Full scan & DDA MS/MS	[80]
	Rat plasma	Zhikebao tablet metabolites	Waters Acquity UPLC HSS C18(2.1 mm×150 mm, 1.8 μm)	AB Sciex Triple-TOF 5600+	Full scan & DDA MS/MS	[81]

Research objective

Sample type

Compounds investigated

Separation column

MS model

Acquisition mode

Ref.

Class compounds
identification

Citrus reticulata extract

Polymethoxylated
flavonoids

Agilent Zorbax Extended C18
(4.6 mm×250mm, 5 μm)

Thermo LTQ-Orbitrap

Full scan & DDA MS/MS

[13]

Lonicerae japonicae

Chlorogenic acids

Waters ACQUITY UPLC BEH C18
(2.1 mm×100 mm, 1.7 μm)

Thermo LTQ-Orbitrap

Full scan & DDA MS/MS

[14]

Citrus reticulata extract

Polymethoxylated
flavonoids

Agilent Zorbax Extended C18
(4.6 mm×250 mm, 5 μm)

Thermo LTQ-Orbitrap

Full scan & DDA MS/MS

[17]

Yin Chen Si Ni Tang

Aconitum alkaloids

Waters ACQUITY UPLC BEH C18
(2.1 mm×100 mm ID, 1.7 μm)

Waters SYNAPT HDMS Q-TOF

MS^E

[18]

Uncariae ramulus extract

Indole alkaloids

Phenomenex Kinetex^® EVO C18
column (2.1 mm×150 mm, 2.6 μm)

Thermo LTQ-Orbitrap

Full scan & DDA MS/MS

[19]

Panax notoginseng extract

Saponins

Agilent Eclipse XDB-C18
(4.6 mm×250 mm, 5 μm)

Agilent 6530 Q-TOF

Full scan & DDA MS/MS

[20]

Scutellaria baicalensis extract

Flavonoids

Agilent ZORBAX Eclipse Plus C18
(2.1 mm×150mm, 1.8 um)

Thermo Q-Exactive hybrid
quadrupole-orbitrap

Full scan & DDA MS/MS

[24]

Lonicerae japonicae extract

Chlorogenic acids

Agilent Zorbax SB C18
(4.6 mm×250 mm, 5 μm)

Agilent MSD Trap XCT Plus

Full scan & DDA MS/MS

[31]

Ainsliaea fragrans extract

Chlorogenic acids

Waters ACQUITY UPLC BEH C18
(2.1 mm×100 mm, 1.7 μm)

AB Sciex Triple TOF^TM 5600+

Full scan & DDA MS/MS

[32]

Curcuma longa extract

Terpene-conjugated
curcuminoids

Waters Acquity UPLC HSS T3
(2.1 mm×150 mm, 1.8 μm)

Agilent 6495 QQQ Thermo Q-Exactive hybrid quadrupole-orbitrap

SIM full scan & DDA MS/MS

[33]

Panax ginseng extract

Ginsenosides

ACE UltraCore 2.5 Super C18
(3.0 mm×150 mm, 2.5 μm)

ABSciex 5500 Qtrap

MRM

[37]

Eleven Chinese patent medicines
including Carthamus tinctorius

Quinochalcone
C-glycosides

Waters BEH Shield RP 18
(2.1 mm×100 mm, 1.7 μm)

Waters Xevo®G2-S QTOF

SIM

[38]

The popular medicinal used
Pueraria species

Sulfur derivative of
flavonoids

Agilent ZORBAX Eclipse Plus C18
(2.1 mm×100 mm, 1.8 um)

Thermo LTQ Orbitrap Velos Pro

Full scan & DDA MS/MS

[39]

Ligusticum chuanxiong extract

Phthalides

Waters ACQUITY BEH C18
(2.1 mm×100 mm, 1.7 μm)

Waters SYNAPT G2-SI Q-TOF

MS^E

[40]

Lotus plumule extract

Flavonoids

Thermo Hypersil GOLD column
(2.1 mm×100 mm, 1.9 μm)

Thermo Q-Exactive hybrid
quadrupole-orbitrap

Full scan & DDA MS/MS

[41]

Sinomenii caulis extract

Alkaloid analogues

Waters Symmetry C18 analytical
(2.1 mm×100 mm, 3.5 μm)

Agilent 6530 Q-TOF

Full scan & DDA MS/MS

[42]

Chemical profiling of all compounds

Artemisiae argyi extract

Flavonoids, chlorogenic
acids

Thermo Hypersil GOLD column
(2.1 mm×100 mm, 1.9 μm)

Thermo Q-Exactive hybrid
quadrupole-orbitrap

Full scan & DDA MS/MS

[43]

Yi Xin Shu capsule

Lignans, tanshinones,
flavonoids, phenolic acids,
saponins, and others

Agilent ZORBAX Eclipse Plus C18
(3.0 mm×100 mm, 1.8 μm)

Agilent 6540 Q-TOF

Full scan & DDA MS/MS

[44]

Dachuanxiong decoction

Phthalides, ester of citric acid, chlorogenic acids, phenols,
nucleosides, and others

Agilent ZORBAX Eclipse Plus C18
(2.1 mm×150 mm, 1.8 μm)

Agilent 6550 Q-TOF

Full scan & DDA MS/MS

[45]

Qishen granule

Chlorogenic acids, tanshinones, flavonoids, iridoid glucosides,
alkaloids, salvianolic acids,
phenolic acids, triterpenoid
saponins, and phenylpropanoids

ACQUITY TM UPLC HSS T3
(2.1 mm×100 mm, 1.8µm)

Agilent 6550 Q-TOF

Full scan & target MS/MS

[46]

Kai xin san

Saponins, triterpenoid, phenolic acids, and others

ACQUITY TM UPLC HSS T3
(2.1 mm×100 mm, 1.8µm)

Waters Synapt^TM High
Definition TOF

MS^E

[47]

Yangxue Qingnao granule

Phenolic acids, monoterpenoid glycosides, quinones, alkaloids, phthalides and others

Waters ACQUITY UPLC BEH C18
(2.1 mm×100 mm, 1.7 μm)

Waters Acquity
UPLC-Synapt G2 Q-TOF

Full scan & DDA MS/MS

[48]

Liujing toutong tablet

Isoflavonoids, iridoids, coumarins, phthalides, and others

Dikma Diamonsil C18
(4.6 mm×250 mm, 5 μm)

Bruker micrOTOF-Q Ⅱ

Full scan & DDA MS/MS

[50]

Buqi Tongluo granule

Flavonoids, saponins, terpenoids, esters, amino acid, phenolic acids

Waters ACQUITY UPLC BEH C18
(2.1 mm×100 mm, 1.7 μm)

Waters Q-TOF

MS^E

[51]

Danhong injection

Phenolic acids, flavonoids, and others

Waters ACQUITY UHPLC HSS T3
(2.1 mm×100 mm, 1.8µm)

Thermo Q-Exactive hybrid
quadrupole-orbitrap

Full scan & DDA MS/MS

[53]

Anti-renal interstitial
fibrosis I decoction

Sennosides, galloyl glycosides, flavonoids, lignans, iridoids,
organic acids, phenylpropanoids

Thermo Hypersil GOLD column
(2.1 mm×100 mm, 1.9 μm)

Thermo Q-Exactive hybrid
quadrupole-orbitrap

Full scan & DDA MS/MS

[54]

Lishukang capsule

Flavonoids, alkaloids, phenolic acids, saponins, and benzyl ester glycosides

Waters ACQUITY UHPLC HSS T3
(2.1 mm×100 mm, 1.8µm)

AB SCIEX 5600+Q-TOF

Full scan & DDA MS/MS

[56]

Xiao Xu Ming decoction

Flavonoids, monoterpenoid
glycosides, terpenoids, and others

Thermo BDS HYPERSIL C18
(2.1 mm×150 mm, 3µm)

Thermo LTQ-FT

Full scan & DDA MS/MS

[57]

Metabolites
identification

Intestinal bacteria

Nonpolysaccharide fraction from Bletilla striata metabolites

Waters BEH C18
(2.1 mm×50 mm, 1.7 μm)

Waters XEVO G2‐XS Q‐TOF

MS^E

[62]

Rat urine

Gandou decoction metabolites

Agilent Eclipse Plus C18
(2.1 mm×100 mm, 1.8 μm)

Waters Xevo G2 Q-TOF

MS^E

[63]

Rat bio-samples

Qi-Lin pill metabolites

Waters ACQUITY UHPLC HSS T3
(2.1 mm×100 mm, 1.8 μm)

Waters SYNAPT™G2 Q-TOF

MS^E

[64]

Human urine

Synthetic cannabinoids
metabolites

Bellefonte Ultra Biphenyl HPLC
(2.1 mm×100 mm, 3.0 μm)

AB Sciex Triple-TOF 5600+

SWATH MS/MS

[65]

Liver microsomes, rat urine
and bile

Ponicidin metabolites

AB Sciex Triple-TOF 5600+

SWATH MS/MS

[66]

Liver microsomes, and rat
bio-samples

Osthole metabolites

Agilent Poroshell 120 EC-C18
(2.1 mm×100 mm, 2.6 μm)

AB Sciex Triple-TOF 5600+

SWATH MS/MS

[67]

Rat bio-samples

Berberine metabolites

Waters ACQUITY UPLC BEH Shield RP18
(2.1 mm×50 mm, 1.7 μm)

Waters SYNAPT^TM G2 Q-TOF

MS^E

[68]

Rat bio-samples

Berberrubine metabolites

Waters ACQUITY BEH Shield
RP18(2.1 mm×50 mm, 1.7 μm)

Waters SYNAPT^TM G2 Q-TOF

MS^E

[69]

Rat urine and bile

Oridonin metabolites

Phenomenex Kinetex C18
(2.1 mm×100 mm, 2.6 μm)

AB Sciex Triple-TOF 5600+

Full scan & DDA MS/MS

[71]

Rat urine and bile

Deoxyschizandrin
metabolites

Phenomenex Kinetex C18
(2.1 mm×100 mm, 2.6 μm)

AB Sciex Triple-TOF 5600+

Full scan & DDA MS/MS

[72]

Rat bio-samples

Gualou Xiebai Baijiu
decoction metabolites

Agilent Acquity BEH C18
(2.1 mm×50 mm, 1.7 μm)

Waters SYNAPT^TM G2 Q-TOF

Full scan & DDA MS/MS

[73]

Rat plasma and urine

Gegen-Qinlian decoction
metabolites

Waters Atlantis T3 column
(2.1 mm×150 mm, 3 μm)

Thermo Fisher Finnigan
TSQ QQQ

Neutral loss scan, precursor ion scan, and SRM scan

[74]

Rat plasma, urine and feces

Glycyrrhiza uralensis
metabolites

Welch Ultimate XB-C18
(2.1 mm×100 mm, 1.8 μm)

AB Sciex API 4500 QTRAP

MIM-EPI and NL-EPI

[75]

Rat bio-samples

Epimedium brevicornu
metabolites

Agilent SB-C18 column
(4.6 mm×250 mm, 5 μm)

Thermo LTQ-FT

Full scan & DDA MS/MS

[77]

Rat bio-samples

Xiao Xu Ming decoction
metabolites

Thermo BDS HYPERSIL C18
(2.1 mm×150 mm, 3 μm),

Thermo LTQ-FT

Full scan & DDA MS/MS

[78]

Rat plasma

Fufang Xialian capsule
metabolites

Waters ACQUITY UPLC BEH
C18(2.1 mm×50 mm, 1.7 μm)

Waters SYNAPT^G2 Q-TOF

MS^E

[79]

Rat bio-samples

Yi-Xin-Shu capsule
metabolites

Agilent Eclipse Plus C18
(2.1 mm×100 mm, 1.8 μm)

Agilent 6540 Q-TOF

Full scan & DDA MS/MS

[80]

Rat plasma

Zhikebao tablet metabolites

Waters Acquity UPLC HSS
C18(2.1 mm×150 mm, 1.8 μm)

AB Sciex Triple-TOF 5600+

Full scan & DDA MS/MS

[81]