基于UPLC-Q-Orbitrap MS/MS研究人参皂苷在发酵过程中的生物转化

doi:10.7503/cjcu20170385

摘要/Abstract

摘要：

应用超高效液相色谱-四极杆-静电场轨道阱串联质谱(UPLC-Q-Orbitrap MS/MS)联用技术分析鉴别人参发酵前后的皂苷类化合物, 研究了整体人参皂苷在自然发酵过程中的生物转化规律. 通过鉴定45种人参皂苷, 比较了生晒参和发酵人参中人参皂苷种类及含量的差异, 在发酵人参中检测到20(S)-Rg₃和20(R)-Rg₃, 20(S)-Rh₁和20(R)-Rh₁等异构体人参皂苷, 其中10余种人参皂苷在自然人参状态下不存在或未被检出. 本研究对于人参发酵前后的化学成分变化进行了高通量的精准分析, 对同类天然产物发酵研究具有借鉴意义, 可指导人参炮制品生晒参和发酵人参的安全有效使用.

关键词: 发酵, 人参皂苷, 生物转化, 液相色谱-质谱联用

Abstract:

This work aims to analyze and compare the ginsenosides contents from white ginseng and fermented black ginseng(FBG) using UPLC-Q-Orbitrap MS/MS(ultra performance liquid chromatography coupled with quadrupole orbitrap tandem mass spectrometry). FBG was processed by spontaneous short-term fermentation, along with the biotransformation of ginsenosides. The samples were separated by liquid chromatography using a Thermo Syncronis C₁₈ column at 30 ℃. Moreover, 0.1%(volume fraction) formic acid and acetonitrile were used as mobile phases A and B, respectively. The flow rate was 0.2 mL/min, and the injected sample volume was 0.5 μL. By the use of the UPLC-Q-Orbitrap MS/MS, 52 kinds of ginsenosides were identified. The contents of main ginsenosides, especially in malonylated ginsenosides, were decreased, because of hydrolyzed. The method of spontaneous short-term fermentationdid doesn’t require any additives, and provides the theoretical basis for spontaneous short-term fermentprocessed ginseng.

Key words: Fermentation, Ginsenoside, Biotransformation, Liquid chromatography-mass spectrometer

中图分类号:

O657

TrendMD:

乔梦丹, 刘尚, 张琰, 李晶, 郑飞, 戴雨霖, 越皓. 基于UPLC-Q-Orbitrap MS/MS研究人参皂苷在发酵过程中的生物转化. 高等学校化学学报, 2018, 39(2): 219.

QIAO Mengdan, LIU Shang, ZHANG Yan, LI Jing, ZHENG Fei, DAI Yulin, YUE Hao. Biotransformation of Ginsenosides in Fermented Ginseng Using UPLC-Q-Orbitrap MS/MS^†. Chem. J. Chinese Universities, 2018, 39(2): 219.

图/表 8

参考文献 24

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Ginsenoside	Linear range/ (μg·mL^-1)	Regression equation	Correlation coefficient, r	LOD/ (μg·kg^-1)	Recovery(n=5, %)
Ginsenoside	Linear range/ (μg·mL^-1)	Regression equation	Correlation coefficient, r	LOD/ (μg·kg^-1)	Mean	RSD
20(S)-Rh₁	0.001—100	y=1190720x+2039850	0.9992	0.1	99.54	1.09
20(R)-Rh₁	0.00105—105	y=1705800x+1812820	0.9992	0.1	98.04	2.37

Ginsenoside	Linear range/ (μg·mL^-1)	Regression equation	Correlation coefficient, r	LOD/ (μg·kg^-1)	Recovery(n=5, %)
Ginsenoside	Linear range/ (μg·mL^-1)	Regression equation	Correlation coefficient, r	LOD/ (μg·kg^-1)	Mean	RSD
20(S)-Rh₁	0.001—100	y=1190720x+2039850	0.9992	0.1	99.54	1.09
20(R)-Rh₁	0.00105—105	y=1705800x+1812820	0.9992	0.1	98.04	2.37

No.	Retention time/min		Identity		Molecular formula		WG^a		FBG^b						[M-H]^-/[M+HCOOH-H]^- (m/z, mass accuracy<10^-5)				MS/MS fragment ion(m/z)
No.	Retention time/min		Identity		Molecular formula		WG^a		13 d								31 d		70 d
1^c	17.0		Ra₀		C₆₀H₁₀₂O₂₈		+^g		+		-^h		-		1269.6485/1315.654		1107,945,783,621,459
2^c	15.4		Ra₁		C₅₈H₉₈O₂₆		++ⁱ		-		-		-		1209.6274/1255.6328		1107,945,783,621,459
3^c	15.4		Ra₂		C₅₈H₉₈O₂₆		++		-		-		-		1209.6274/1255.6328		1107,945,783,621,459
4^c	19.1		Ra₃		C₅₉H₁₀₀O₂₇		++		+		-		-		1239.6379/1285.6434		1107,1077,945,783,621,459
5^c	18.2		Rb₁		C₅₄H₉₂O₂₃		++		++		+		-		1107.5957/1153.6011		945,783,621,459
6^c	19.4		Rb₂		C₅₃H₉₀O₂₂		++		+++^j		+		-		1077.5851/1123.5906		945,783,621,459
7^c	19.5		Rb₃		C₅₃H₉₀O₂₂		+++		+++		+		-		1077.5851/1123.5906		945,783,621,459
8^c	18.8		Rc		C₅₃H₉₀O₂₂		++		++		+		-		1077.5851/1123.5906		945,783,621,459
9^c	11.0		Re		C₄₈H₈₂O₁₈		+		+		+		-		945.5428/991.5483		783,621,459
10^c	25.0		20(S)-Rg₃		C₄₂H₇₂O₁₃		+		++		+++		++		783.49/829.4955		621,459
11^c	26.9		20(R)-Rg₃		C₄₂H₇₂O₁₃		-		++		+++		++		783.49/829.4955		621,459
12^c	19.2		Rs₁		C₅₅H₉₂O₂₃		++		+		-		-		1119.5957/1165.6011		1077,1059,945,783,621,459
13^c	19.7		Rs₂		C₅₅H₉₂O₂₃		++		+		-		-		1119.5957/1165.6011		1077,1059,945,783,621,459
14^c	29.7		Rs₃		C₄₄H₇₄O₁₄		-		+		+		-		825.5006/871.5061		783,621,459
15^c	18.6		mRb₁		C₅₇H₉₄O₂₆		++		-		-		-		1193.5961/		1107,945,783,621,459
16^c	20.1		mRb₂		C₅₆H₉₂O₂₅		+++		-		-		-		1163.5855/		1077,915,783,621,459
17^c	21.1		mRb₃		C₅₆H₉₂O₂₅		+++		-		-		-		1163.5855/		1077,915,783,621,459
18^c	19.2		mRc		C₅₆H₉₂O₂₅		+++		-		-		-		1163.5855/		1077,915,783,621,459
19^c	21.2		mRd		C₅₁H₈₄O₂₁		+++		-		-		-		1031.5432/		945,783,765,621,459
20^c	18.5		F2		C₄₂H₇₂O₁₃		+		+		+		-		783.49/829.4955		621,459
21^c	31.0		Rh₂		C₃₆H₆₂O₈		-		+		+		+		621.4372/667.4427		459
22^c	31.2		CK		C₃₆H₆₂O₈		-		+		+		+		621.4372/667.4427		459
23^d	10.8		Rg₁		C₄₂H₇₂O₁₄		+++		+		-		-		/845.4904		637,475
24^d	17.0		Rf		C₄₂H₇₂O₁₄		+++		+++		++		-		799.4849/845.4904		637,475
25^d	16.2		Rg₂		C₄₂H₇₂O₁₃		+		+		+		+		783.49/829.4955		637,475
26^d	20.6		Rd		C₄₈H₈₂O₁₈		++		+		-		-		945.5428/		799,783,637,475
27^d	12.7		mRg₁		C₄₅H₇₄O₁₇		++		-		-		-		885.4853/		781,637,475
28^d	13.9		mRf		C₄₅H₇₄O₁₇		++		-		-		-		885.4853/		781,619,475
No.		Retention time/min		Identity		Molecular formula		WG^a		FBG^b						[M-H]^-/[M+HCOOH-H]^- (m/z, mass accuracy<10^-5)			MS/MS fragment ion(m/z)
No.		Retention time/min		Identity		Molecular formula		WG^a		13 d								31 d	70 d
29^d		21.0		mRe		C₅₁H₈₄O₂₁		+		-		-		-		1031.5432/—		945,799,637,475
30^d		8.5		20-Glc-Rf		C₄₈H₈₂O₁₉		+		+		-		-		961.5378/1007.5432		799,637,475
31^d		16.5		F3		C₄₁H₇₀O₁₃		-		+		-		+		769.4744/—		637,475
32^d		17.1		F1		C₃₆H₆₂O₉		-		+		+		+		637.4321/683.4376		475
33^d		18.0		20(S)-Rh₁		C₃₆H₆₂O₉		-		++		+++		+		637.4321/683.4376		475
34^d		19.6		20(R)-Rh₁		C₃₆H₆₂O₉		-		++		+++		+		637.4321/683.4376		475
35^d		30.3		PPT		C₃₀H₅₂O₄		-		-		-		+		475.3793/521.3848		475
36^e		19.1		Ro		C₄₈H₇₆O₁₉		++		+		+		+		955.4908/—		793,613,569,455
37^f		24.2		Rg₄/Rg₆		C₄₂H₇₀O₁₂		-		+		+		+		765.4795/811.4849		619,457
38^f		24.7		Rg₄/Rg₆		C₄₂H₇₀O₁₂		-		+		+		+		765.4795/811.4849		619,457
39^f		29.1		Rg₅/Rk₁		C₄₂H₇₀O₁₂		-		+		+		+		765.4795/811.4849		603,441
40^f		29.3		Rg₅/Rk₁		C₄₂H₇₀O₁₂		-		+		+		+		765.4795/811.4849		603,441
41^f		25.2		Rh₄/Rk₃		C₃₆H₆₀O₈		-		+		+		+		—/665.427		457
42^f		25.8		Rh₄/Rk₃		C₃₆H₆₀O₈		-		+		+		+		—/665.427		457
43^f		23.1		Rf₃		C₄₂H₇₄O₁₅		-		+		+		+		817.4955/863.501		477
44^f		18.0		Rg₈/Rg₉		C₄₂H₇₀O₁₃		-		+		+		-		781.4744/827.4798		457
45^f		17.7		Rg₈/Rg₉		C₄₂H₇₀O₁₃		-		+		+		-		781.4744/827.4798		457

No.	Retention time/min		Identity		Molecular formula		WG^a		FBG^b						[M-H]^-/[M+HCOOH-H]^- (m/z, mass accuracy<10^-5)				MS/MS fragment ion(m/z)
No.	Retention time/min		Identity		Molecular formula		WG^a		13 d								31 d		70 d
1^c	17.0		Ra₀		C₆₀H₁₀₂O₂₈		+^g		+		-^h		-		1269.6485/1315.654		1107,945,783,621,459
2^c	15.4		Ra₁		C₅₈H₉₈O₂₆		++ⁱ		-		-		-		1209.6274/1255.6328		1107,945,783,621,459
3^c	15.4		Ra₂		C₅₈H₉₈O₂₆		++		-		-		-		1209.6274/1255.6328		1107,945,783,621,459
4^c	19.1		Ra₃		C₅₉H₁₀₀O₂₇		++		+		-		-		1239.6379/1285.6434		1107,1077,945,783,621,459
5^c	18.2		Rb₁		C₅₄H₉₂O₂₃		++		++		+		-		1107.5957/1153.6011		945,783,621,459
6^c	19.4		Rb₂		C₅₃H₉₀O₂₂		++		+++^j		+		-		1077.5851/1123.5906		945,783,621,459
7^c	19.5		Rb₃		C₅₃H₉₀O₂₂		+++		+++		+		-		1077.5851/1123.5906		945,783,621,459
8^c	18.8		Rc		C₅₃H₉₀O₂₂		++		++		+		-		1077.5851/1123.5906		945,783,621,459
9^c	11.0		Re		C₄₈H₈₂O₁₈		+		+		+		-		945.5428/991.5483		783,621,459
10^c	25.0		20(S)-Rg₃		C₄₂H₇₂O₁₃		+		++		+++		++		783.49/829.4955		621,459
11^c	26.9		20(R)-Rg₃		C₄₂H₇₂O₁₃		-		++		+++		++		783.49/829.4955		621,459
12^c	19.2		Rs₁		C₅₅H₉₂O₂₃		++		+		-		-		1119.5957/1165.6011		1077,1059,945,783,621,459
13^c	19.7		Rs₂		C₅₅H₉₂O₂₃		++		+		-		-		1119.5957/1165.6011		1077,1059,945,783,621,459
14^c	29.7		Rs₃		C₄₄H₇₄O₁₄		-		+		+		-		825.5006/871.5061		783,621,459
15^c	18.6		mRb₁		C₅₇H₉₄O₂₆		++		-		-		-		1193.5961/		1107,945,783,621,459
16^c	20.1		mRb₂		C₅₆H₉₂O₂₅		+++		-		-		-		1163.5855/		1077,915,783,621,459
17^c	21.1		mRb₃		C₅₆H₉₂O₂₅		+++		-		-		-		1163.5855/		1077,915,783,621,459
18^c	19.2		mRc		C₅₆H₉₂O₂₅		+++		-		-		-		1163.5855/		1077,915,783,621,459
19^c	21.2		mRd		C₅₁H₈₄O₂₁		+++		-		-		-		1031.5432/		945,783,765,621,459
20^c	18.5		F2		C₄₂H₇₂O₁₃		+		+		+		-		783.49/829.4955		621,459
21^c	31.0		Rh₂		C₃₆H₆₂O₈		-		+		+		+		621.4372/667.4427		459
22^c	31.2		CK		C₃₆H₆₂O₈		-		+		+		+		621.4372/667.4427		459
23^d	10.8		Rg₁		C₄₂H₇₂O₁₄		+++		+		-		-		/845.4904		637,475
24^d	17.0		Rf		C₄₂H₇₂O₁₄		+++		+++		++		-		799.4849/845.4904		637,475
25^d	16.2		Rg₂		C₄₂H₇₂O₁₃		+		+		+		+		783.49/829.4955		637,475
26^d	20.6		Rd		C₄₈H₈₂O₁₈		++		+		-		-		945.5428/		799,783,637,475
27^d	12.7		mRg₁		C₄₅H₇₄O₁₇		++		-		-		-		885.4853/		781,637,475
28^d	13.9		mRf		C₄₅H₇₄O₁₇		++		-		-		-		885.4853/		781,619,475
No.		Retention time/min		Identity		Molecular formula		WG^a		FBG^b						[M-H]^-/[M+HCOOH-H]^- (m/z, mass accuracy<10^-5)			MS/MS fragment ion(m/z)
No.		Retention time/min		Identity		Molecular formula		WG^a		13 d								31 d	70 d
29^d		21.0		mRe		C₅₁H₈₄O₂₁		+		-		-		-		1031.5432/—		945,799,637,475
30^d		8.5		20-Glc-Rf		C₄₈H₈₂O₁₉		+		+		-		-		961.5378/1007.5432		799,637,475
31^d		16.5		F3		C₄₁H₇₀O₁₃		-		+		-		+		769.4744/—		637,475
32^d		17.1		F1		C₃₆H₆₂O₉		-		+		+		+		637.4321/683.4376		475
33^d		18.0		20(S)-Rh₁		C₃₆H₆₂O₉		-		++		+++		+		637.4321/683.4376		475
34^d		19.6		20(R)-Rh₁		C₃₆H₆₂O₉		-		++		+++		+		637.4321/683.4376		475
35^d		30.3		PPT		C₃₀H₅₂O₄		-		-		-		+		475.3793/521.3848		475
36^e		19.1		Ro		C₄₈H₇₆O₁₉		++		+		+		+		955.4908/—		793,613,569,455
37^f		24.2		Rg₄/Rg₆		C₄₂H₇₀O₁₂		-		+		+		+		765.4795/811.4849		619,457
38^f		24.7		Rg₄/Rg₆		C₄₂H₇₀O₁₂		-		+		+		+		765.4795/811.4849		619,457
39^f		29.1		Rg₅/Rk₁		C₄₂H₇₀O₁₂		-		+		+		+		765.4795/811.4849		603,441
40^f		29.3		Rg₅/Rk₁		C₄₂H₇₀O₁₂		-		+		+		+		765.4795/811.4849		603,441
41^f		25.2		Rh₄/Rk₃		C₃₆H₆₀O₈		-		+		+		+		—/665.427		457
42^f		25.8		Rh₄/Rk₃		C₃₆H₆₀O₈		-		+		+		+		—/665.427		457
43^f		23.1		Rf₃		C₄₂H₇₄O₁₅		-		+		+		+		817.4955/863.501		477
44^f		18.0		Rg₈/Rg₉		C₄₂H₇₀O₁₃		-		+		+		-		781.4744/827.4798		457
45^f		17.7		Rg₈/Rg₉		C₄₂H₇₀O₁₃		-		+		+		-		781.4744/827.4798		457