Research Article

Received: 15 April 2011 Revised: 9 June 2011 Accepted: 12 June 2011 Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2011, 25, 2439–2447

(wileyonlinelibrary.com) DOI: 10.1002/rcm.5123

Chemical profiling of bioactive constituents in Sarcandra glabra

and its preparations using ultra-high-pressure liquid chromato-
graphy coupled with LTQ Orbitrap mass spectrometry
Xiong Li1, Yufeng Zhang2, Xing Zeng1, Liu Yang1* and Yuanhui Deng1
1
Second Affiliated Hospital, Guangzhou University of Traditional Chinese Medicine, Guangzhou, China
2
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China
In this study, a fast and reliable method based on an ultra-high-pressure liquid chromatography coupled with
photodiode-array detection (PDA) and a linear ion trap high-resolution mass spectrometer (LTQ-Orbitrap XL)
has been developed for the identification of bioactive constituents in the whole plant of Sarcandra glabra and
its related four preparations. By accurate mass measurements within 5ppm error for each molecular ion and subse-
quent fragment ions in routine analysis, 50 compounds, including organic acids, caffeoyl derivatives, flavonoids,
coumarins and terpenoids, were identified or tentatively characterized. Among them, 6,7,8-trihydroxycoumarin-O-
rhamnopyranoside (17), (2R)-naringenin-6-C-B-D-glucopyranosyl-(6!1)-apiose (25) and (2S)-naringenin-6-C-B-D-
glucopyranosyl-(6!1)-apiose (27) were tentatively identified as new compounds. Besides, 21 of these compounds
were coexisting in four preparations of Sarcandra glabra. Fragmentation behaviors of the four major categories of
compounds were also investigated. This established UPLC-PDA/ESI-MSn method was reliable and effective for
the separation and identification of the major constituents and would be the basis for quality control of Sarcandra
glabra and its related preparations. Copyright © 2011 John Wiley & Sons, Ltd.

Sarcandra glabra (Thunb) Nakai (Chloranthaceae family) is a
frequently used traditional Chinese medicine (TCM). The
traditional Chinese medicinal preparations (TCMPs) made
from the whole plant of Sarcandra glabra extract, such as
Qingrexiaoyanning capsules, Zhongjiefeng tablets, Xiekang
capsules and Zhongjiefeng injections, are mainly used to treat
acute influenza, pharyngolaryngitis, thrombocytopenia, pneu-
monia, cellulites, appendicitis, shigellosis, leukoderma vitiligo,
abscess and cancer, etc.[1] Many bioactive compounds have
been isolated from Sarcandra glabra, including caffeoylquinic
acids, rosmarinic acids, flavonoids, coumarins and sesquiter-
penoids, etc.[2,3] These types of components should be respon-
sible for its medicinal use, due to their wide range of
biological activities, including antioxidant, antiinflammatory,
antibacterial and antitumor properties.[4–7]
Several analytical methods, including high-performance
liquid chromatography ultraviolet (HPLC-UV),[8] capillary elec-
trophoresis (CE)[9] and CE with electrochemical detection,[10]
have been developed for the analysis of Sarcandra glabra and
its medicinal preparations. However, these methods proposed
in the literature are only limited to the analysis of one or several
classes of marker constituents, and these analytical methods are
inadequate for the comprehensive quality control of Sarcandra
glabra and its preparations. Nowadays, mass spectrometry
coupled to liquid chromatography (LC/MS) has proved
to be a very powerful analytical technique. To date, there is
only one report of the analysis of chemical constituents of
* Correspondence to: L. Yang, No. 111, Da De Road, Second
Affiliated Hospital, Guangzhou University of Traditional
Chinese Medicine, Guangzhou 510120, P.R. China.
Sarcandra glabra by HPLC/electrospray ionization tandem
mass spectrometry (ESI-MS/MS),[11] in which just five com-
pounds, namely protocatechuic acid, caffeic acid, isofraxidin,
rosmarinic acid and istanbulin A, were identified due to the
complexity of chemical components in this plant and the defi-
ciency of reference standards. So it is urgent to further
establish a rapid and effective LC/MS method for simulta-
neous separation and systematic characterization of the major
bioactive constituents of Sarcandra glabra and its related
preparations.
In this paper, we present a complete and systematically
analysis of major bioactive constituents from a 70% aqueous
acetonitrile extract of the whole plant of Sarcandra glabra
and its preparations using ultra-high-pressure LC coupled
to an LTQ Orbitrap mass spectrometer.
EXPERIMENTAL
Chemicals and standards
HPLC grade acetonitrile was purchased from Fisher Chemicals
(Fairlawn, NJ, USA). Formic acid was AR grade and purchased
from Guangzhou Chemical Reagent Corporation. Water used
in the experiment was deionized and further purified by a
Milli-Q Plus water purification system (Millipore Ltd.). Other
reagents and chemicals were of analytical grade.
4,5-Dicaffeoylquinic acid and caffeic acid (>99%) were
purchased from the National Institute for the Control of
Pharmaceutical and Biological Products (Beijing, China).
Fumaric acid (2), protocatechuic acid (3), 3-O-caffeoylquinic
acid or neochlorogenic acid (4), 5-O-caffeoylquinic acid
2439

E-mail: yangliume@yahoo.com.cn or chlorogenic acid (6), 4-O-caffeoylquinic acid or

Rapid Commun. Mass Spectrom. 2011, 25, 2439–2447 Copyright © 2011 John Wiley & Sons, Ltd.

cryptochlorogenic acid (7), isofraxidin-7-O-b-D-glucoside
(11), 5-O-caffeoylshikimic acid (12), 4-O-caffeoylshikimic acid
(13), drovomifoliol-O-B-D-glucopyranoside (14), 3-O-caf-
feoylshikimic acid (16), sarcaglaboside G (18), isofraxidin
(19), (2R)-naringenin-6-C-B-D-glucopyranoside (20), (2S)-nar-
ingenin- 6-C-B-D-glucopyranoside (23), (2R)-naringenin-6-C-
B-D-glucopyranosyl-(6!1)- apiose (25), (2S)-naringenin-6-C-
B-D-glucopyranosyl-(6!1)-apiose (27), neoastilbin (28),
chloranoside A (30), astilbin (31), neoisoastilbin (33), isoastil-
bin (34), rosmarinic acid 4-O-B-D-glucopyrannoside (36), ros-
marinic acid (41), ()-epiafzelechin 7-O-B-D-glucopyranoside
(44), rosmarinic acid methyl ester (48), and 8b,9a-dihydroxy-
lindan-(5),7(1)-ieb-8a,12-olide (50) were isolated from the
whole plant of Sarcandra glabra by the author. Their identities
were confirmed by 1H NMR, 13C NMR, DEPT, 1H-1H COSY,
HSQC, HMBC and MS spectral analysis, and their purities
were no less than 95% by HPLC analysis. The NMR data
of the isolated compounds are supplied as Supporting
Information.
Materials and sample preparation
The whole plant of Sarcandra glabra used to separate the
reference standards was supplied by Kangmei Pharmaceutical
Co. Ltd. and originated from Sichuan province, China;
Zhongjie feng injection (Batch number: 090706) was obtained
from Boluo Xianfeng Pharmaceutical Corporation, China;
Zhongjiefeng tablets (Batch number: 090201) were obtained
from Shanghai Xinyi Jiahua Pharmaceutical Corporation,
China; Qingre Xiao yanning capsules (Batch number:
N12015) were obtained from Guangzhou Jingxiutang Pharma-
ceutical Corporation, China; Jinsulan liniment (Batch number:
091204) was obtained from Guangdong Hospital of Traditional
Chinese Medicine; Xiekang capsules (Batch number: 09090801)
were obtained from Jiangxi Tianshikang Pharmaceutical
Corporation, China. All of the above samples were deposited
in the Center for Laboratory, Second Affiliated Hospital,
Guangzhou University of Traditional Chinese Medicine.
The dried whole plant of Sarcandra glabra sample (0.2 g, 60
mesh) was accurately weighed into a 50 mL flask and
extracted in an ultrasonic bath with 20 mL 70% aqueous
acetonitrile containing 0.5% formic acid for 30 min at room
temperature; then, the extract solutions were prepared by
the method of weight relief. As to Jinsulan liniment and
Zhongjiefeng injection, 1mL of liquid sample was diluted to
one-third and one-tenth with 70% aqueous acetonitrile,
respectively. All the solution was filtered through a 0.22 mm
nylon membrane filter prior to injection for LC/MS analysis.
HPLC-PAD/ESI-MSn analysis
Chromatographic separation was performed on a Accela™
ultra-high-pressure liquid chromatography (U-HPLC) system
(Thermo Fisher Scientific, San Jose, CA, USA) comprising an
U-HPLC pump, a PDA detector, scanning from 200 to 400
nm, and an autosampler settled to 30  C. The LC conditions
were as follows: column: Hypersil GOLD, 1002.1 mm,
1.9 mm particle size (Thermo Fisher Scientific, Bellefonte, PA,
USA); mobile phase: (A) water with 0.1% formic acid; (B)
acetonitrile; flow rate: 600 mL/min; injection volume: 10 mL;
2440
gradient: linear gradient of 100%–1% A over 20 min.
wileyonlinelibrary.com/journal/rcm Copyright © 2011 John Wiley & Sons, Ltd.
X. Li et al.
MS analysis was performed using an LTQ Orbitrap XL
hybrid mass spectrometer (Thermo Fisher Scientific, San Jose,
CA, USA), fitted with an ESI source, and operated in negative
ion mode, with a mass range of 100–1000 with resolution set
at 30000 using the normal scan rate. The data-dependent
MS/MS events were always performed on the most intense
ions detected in full scan MS. The MS/MS isolation width
was 1amu, and the normalized collision energy was 30% for
all compounds. Nitrogen was used as sheath gas and helium
served as the collision gas. The key optimized ESI parameters
were as follows: source voltage: 3.0 kV; sheath gas (nitrogen):
50 L/min; auxiliary gas flow: 10 L/min; capillary voltage:
–35.0V; capillary temperature: 300.0  C; tube lens: –110.0 V.
The ion injection time used was 50.0 ms. MS scan functions
and HPLC solvent gradients were controlled by the Xcalibur
data system (Thermo Fisher, San Jose, CA, USA). Data was
collected and analyzed with Xcalibur 2.07 (Thermo Scientific).
The Orbitrap mass analyzer was calibrated according to the
manufacturer's directions using a mixture of caffeine,
methionine-arginine-phenylalanine-alanine-acetate (MRFA),
sodium dodecyl sulfate, sodium taurocholate and Ultra-
mark 1621 in an acetonitrile/methanol/water solution
containing 1% acetic acid by direct injection at a flow rate
of 5 mL/min in negative mode.
RESULTS AND DISCUSSION
In this study, an LC/MS-based method was developed to
characterize the constituents of Sarcandra glabra and its
preparations, which gave the accurate molecular weights by
orbitrap analyzer and the fragmentation patterns acquired
from multi-stage mass fragmentation for comprehensive
understanding of their chemical structures. Both negative
and positive modes were examined. Generally, in positive
mode, low abundance of [M+H]+, [M+Na]+ ions and few
product ions were observed, while, in negative ion mode,
series of ions [MH] and/or adduct ions ([M+HCOOHH])
appeared with sufficient abundance; thus the negative ion
mode was chosen and the [MH]/([M+HCOOHH] ions
were further subjected to LC/MSn analysis. When a reference
standard was available, the compound was identified by
comparing the UPLC retention time, UV and mass spectral
data with those obtained for the standard, and some assigned
standards were further introduced into the ESI source by
direct infusion for MSn analysis. Table 1 showed the mass
spectra up to MS4, depending on the investigated com-
pounds. A total of 50 compounds were identified or tenta-
tively characterized from Sarcandra glabra. Figure 1 shows
the structures of the characterized compounds. The UPLC/
PAD and total ion current (TIC) profiles of the whole plant
of Sarcandra glabra are shown in Fig. 2. Among them, 6,7,8-
trihydroxycoumarin-7-O-rhamnopyranoside (17), (2R)-narin-
genin-8-C-B-D-glucopyranosyl-(6!1)-apiose (25) and (2S)-
naringenin-8-C-B-D-glucopyranosyl-(6!1)-apiose (27) were
tentatively identified as new compounds, three caffeoylquinic
acids (compounds 13, 16 and 45) and one flavonoid (compound
44) were reported for the first time. Besides, 21 of these compo-
nents (compounds 1, 2, 6–15, 18, 19, 22, 31, 37, 41, 42, 47 and
49) were coexisting in four different kinds of preparations made
from Sarcandra glabra (supplied as Supporting Information).
Rapid Commun. Mass Spectrom. 2011, 25, 2439–2447
 Table 1. Characterization of constituents of 70% aqueous acetonitrile extract of Sarcandra glabra and its four preparations by UPLC-PAD/ESI-MSn

tR UV max Observed Calculated Error LC/MSn data

No. (min) (nm) mass mass (ppm) Formula (% base peak) Identification
c 2
1 0.31  191.0556 191.0550 3.0 C7H11O6 MS [191]173.0453(25), 127.0401(34) quinic acid
2a 0.33 211 115.0036 115.0026 8.4 C4H3O4 MS2[115]71.0141(100) fumaric acid
3a 1.43 259, 293 153.0188 153.0182 3.5 C7H5O4 MS2[153]109.0295(100) protocatechuic acid
4a 2.68 220,324 353.0877 353.0867 0.2 C16H17O9 MS2[353]191.0548(100),179.0338(46),135.0443(7) 3-O-caffeoylquinic acid
5a 4.65 217,323 179.0332 179.0338 –3.2 C9H7O4 MS2[179]135.0451 (100) caffeic acid
6a 4.94 220,324 353.0875 353.0867 0.7 C16H17O9 MS2[353]191.0557(100),179.0343(7), 173. 0450 (5) 5-O-caffeoylquinic acid
7a 5.35 220,324 353.0875 353.0867 0.7 C16H17O9 MS2[353]191.0549(19),179.0338(57),173.0444(100) 4-O-caffeoylquinic acid
8 5.42 242,333 357.1182c 357.1180 0.6 C16H21O9 MS2[357]342.0944(51) , 195.0658(100) phenolic glycoside
9 5.65  369.0820c 369.0816 0.9 C16H17O10 MS2[359]207.0294(100) fraxin
10 5.67 243,287, 221.0452c 221.0444 3.4 C11H9O5 MS2[221]206.0215(100) fraxidin
312

Rapid Commun. Mass Spectrom. 2011, 25, 2439–2447

11a 5.67 292, 336 429.1029d 429.1028 0.3 C18H21O12 MS2[429]221.0450(100) isofraxidin-7-O-b-D-glucoside
12a 5.97 216, 327 335.0760c 335.0761 –0.5 C16H15O8 MS2[335]291.0867(24), 273.0762 (4), 247. 0971 5-O-caffeoylshikimic acid
(9),229.0867(4),179.0347(100),135.0451(13)
MS3[335!179] 135.0448 (100)
13a 6.03 216,327 335.0759c 335.0761 MS2[335]291.0867(27), 273.0761(1),247.0971 4-O-caffeoylshikimic acid
Constituents in Sarcandra glabra by UPLC LTQ Orbitrap

–0.8 C16H15O8
(4),229.0866(2),179.0347(100), 161.0423(87),135.0451
(32)
MS3[335!179] 135.0448 (100
14a 6.06  431.1912d 431.1912 0 C20H31O10 MS2[431]385.1846(100) drovomifoliol-O-B-D-
MS3[431!385]223.1325(92),205.1220(76), 153.0911 glucopyranoside
(100)
15b 6.19  433.2068d 433.2068 0 C20H33O10 MS2[433]387.2010(100) MS3[433!387]225.1481 dihydrovomifoliol-O-B-D-
(100),161.0445 (40) glucopyranoside
16a 6.29 217, 327 335.0770c 335.0761 0.5 C16H15O8 MS2[335]291.0862(1),179.0346(100),161.0242 3-O-caffeoylshikimic acid
(3),135.0450(23)
MS3[335!179] 135.0449 (100
17b 6.70 249,299 339.0712c 339.0710 0.5 MS2[339]205.0128(19),193.0129(100) 6,7,8-trihydroxycoumarin-7-O-a-

Copyright © 2011 John Wiley & Sons, Ltd.

C15H15O9
MS3[339!193]165.0183(52), 137.0236(100),121.0288 L-rhamnopyranoside
(50)
18a 7.04  471.1859d 471.1861 –0.4 C22H31O11 MS2[471]425.1804(100) sarcaglaboside G
MS3[471!425]263.1270(100)
19a 7.39 341 221.0441c 221.0445 –1.6 C11H9O5 MS2[221]206.0207(100) isofraxidin
MS3[221!206]190.9973(100)
MS4[221!206!191]163.0026(100)
20a 7.47 234,291 433.1129c 433.1129 0 C21H21O10 MS2[433]415.1019(4),397.0910(1), 343.0807 (2R/2S)-naringenin-6-C-B-D-
(18),313.0705(100) glucopyranoside
MS3[433!313]193.0137(100)
21 7.58 233,289 303.0503c 303.0499 1.3 C15H11O7 MS2[303]285.0396(100) (+)-3,5,7,3',4'-pentahydroflavonone
22 7.69  473.2017 473.2023 –0.1 C22H33O11 MS2[473]427.1960(100) sarcaglaboside H
23a 7.89 234,291 433.1130c 433.1135 0.1 C21H21O10 MS2[433]415.1016(4), 397.0913(1),343.0812(19), (2R/2S)-naringenin-6-C-B-D-
313.0703(100) glucopyranoside
(Continues)

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2441
 2442

Table 1. (Continued)

tR UV max Observed Calculated Error LC/MSn data

No. (min) (nm) mass mass (ppm) Formula (% base peak) Identification
3
MS [433!313]193.0136(100)
24 8.16  435.1285c 435.1286 –0.3 C21H23O10 MS2[435]389.1228(100) isoliquiritigenin7-O-b-D-
glucoside
25a 8.16 228,290 565.1548c 565.1552 –0.7 C26H29O14 MS2[565]547.1445(4),529.1342(1),397.0919(7),343.0813 (2R/2S)-naringenin-6-C-B-D-glu-
(36),313.0710(100) copyranosyl-(6!1)-apiose
MS3[565!313]193.0129(100)
26b 8.69 228 279.1231c 279.1233 1.4 C15H19O5 MS2[279]261.1116(40), 235.1323 (100), 217.1221 (50) sesquiterpenoid lactone
MS3[279!235]217.1219(100),207.1376(60),191.1428

wileyonlinelibrary.com/journal/rcm
(40),163.0754(40)
27 8.76 228, 290 565.1547c 565.1552 –0.8 C26H29O14 MS2[565]547.1447(4),529.1338(1),397.0919(7),343.0814 (2R/2S)-naringenin-6-C-B-D-glu-
(36),313.0710(100) copyranosyl-(6!1)-apiose
MS3[565!313]193.0129(100)
28a 8.78 289 449.1079c 449.1078 0.1 C21H21O11 MS2[449]303.0501(100),285.0396(78) neoastilbin
29 9.36 254,250 477.0665c 477.0669 0.3 C21H17O13 MS2[477]301.0344(100) quercetin-3-O-b-D-glucuronide
30a 9.36 280 469.1707d 469.1710 0.5 C22H29O11 MS2[469]423.1640 (100) Chloranoside A
MS3[469!423]261.1120 (100)
MS4[469!423!261]243.1017(32),233.1174
(100),231.1018(68),217.1226(80)
31a 9.60 289 449.1080c 449.1078 0.3 C21H21O11 MS2[449]303.0501(100),285.0396(86) astilbin
32 11.88 290 449.1077c 449.1078 –0.4 C21H21O11 MS2[449] 303.0499(100),285.0395(77) 3,5,7,3',5'-
pentahydroxyflavanonol-3-O-a-L-
rhamnopyranoside
33a 12.55 294 449.1078c 449.1078 –0.1 C21H21O11 MS2[449] 303.0501(100),285.0396(72) neoisoastilbin
34a 12.59 294 449.1070c 449.1078 –0.3 C21H21O11 MS2[449] 303.0501(100),285.0396(72) isoastilbin

Copyright © 2011 John Wiley & Sons, Ltd.

35b 13.16 447.0921c 447.0922 –0.1 C21H19O11 MS2[447]301.0333(100) quercetin-3-O-b-D-rhamnoside
36a 13.38 218,328 521.1289c 521.1290 –0.4 C24H25O13 MS2[521]359.0749(100) rosmarinic acid-4-O-b-D-
MS3[521!359]197.0442(100),179.0337(33), 161.0233 glucoside
(97)
37 13.49 217,327 515.1181c 515.1184 –0.6 C25H23O12 MS2[515] 353.0865(100) dicaffeoylquinic acid
38 14.03  587.2326d 587.2334 –1.0 C27H39O14 MS2[587]311.0974(100) sarcaglaboside D
39b 14.09 263 279.1231c 279.1227 1.4 C15H19O5 MS2[279]249.1115(17),235.0960(100),205.1220 sesquiterpene lactone
(19),139.0756(66)
40b 14.73 233,281 597.1608c 597.1603 –0.6 C30H29O13 MS2[597]451.1010 (100) glabraoside A
MS3[597!451]341.0644(100)
MS4[597!451!341]323.0539(15),297.0748
(22),217.0728(100)
41a 15.08 328 359.0763c 359.0761 0.4 C18H15O8 MS2[359]223.0229(12),197.0438(29), rosmarinic acid
179.0334(26), 161.0230(100),133.0285(5)
42 15.37  587.2332d 587.2334 –0.4 C27H39O14 MS2[587]541.2270 (100) sarcaglaboside E
43 15.46  455.1913d 455.1912 0.3 C22H31O10 MS2[455]409.1857(100) sarcaglaboside C
44a 15.48 232, 285 435.1286c 435.1286 0.1 C21H23O10 MS2[435]273.0767(100) (–)-epiafzelechin 7-O-B-D-
MS3[435!273]167.0351(100) glucopyranoside
(Continues)
X. Li et al.

Rapid Commun. Mass Spectrom. 2011, 25, 2439–2447

Constituents in Sarcandra glabra by UPLC LTQ Orbitrap

Characterization of caffeoyl derivatives

8b,9a-dihydroxylindan-(5),7(1)-
It was possible to detect a total of 12 caffeoyl derivatives.

rosmarinic acid methyl ester

Five peaks (compounds 4, 6, 7, 37 and 45) were identified
as caffeoylquinic acids based on their prominent [M–H]– ions
4,5-dicaffeoylquinic acid
Identification

sesquiterpene lactone
and characteristic fragment ions (m/z 515!353 for dicaffeoyl-
quinic acid; m/z 353!191, 179 for monocaffeoylquinic acid)

sarcaglaboside A
sarcaglaboside B
according to literature reports.[12,13] Compound 45 was further

ieb-8a,12-olide
identified as 4,5-dicaffeoylquinic acid by comparison with a
commercial standard. Three monocaffeoylquinic acids were
characterized as 3-O-caffeoylquinic acid (4), 5-O-caffeoylqui-
nic acid (6) and 4-O-caffeoylquinic acid (7), respectively,
according to the elution order and the relative abundance of
fragments. 4-O-Caffeoylquinic acid was easily distinguished
from 3-O- and 5-O-caffeoylquinic acid by its different MS2
MS3[423!279]249.1115(27),235.1322(24),205.1217(28),

base peak at m/z 173.0444. Although 3-O-caffeoylquinic acid

MS2[261]243.1015(18),217.1224(100) ,199.1120 (30),

and 5-O-caffeoylquinic acid yielded an identical MS2 base

MS2[373]197.0438(3)161.0242 (100), 179. 0347(60)

peak at m/z ~191.1, they could be distinguished by a com-

paratively intense ion at m/z ~179.0 [caffeic acidH] (mean
MS3[261!217]199.1119(100),189.1277(82)

intensity of 46% compared with 7%). These data were also

in accordance with the above reported literature. Besides,
three peaks (compounds 12, 13 and 16) showed their precur-
(% base peak)

MS4[261!217!199]184.0886(100)
LC/MSn data

sor [M–H]– ions at m/z ~335.1(C16H15O8), and the characteris-

tic MS2 base peak at m/z ~179.0, MS3 base peak ~135.0 for
169.0859(15),139.0756(100)

caffeic acid, suggested they might be dehydrated monocaf-

173.1329 (20),140.0111(46)

: Their UV spectra have not been properly observed due to low intensity or lack reference standards

feoylquinic acid derivatives. 5-O-Caffeoylshikimic acid has

MS2[515]353.0865(100)
MS2[453]407.1698(100)
MS2[423]279.1229(100)

MS2[455]409.1854(100)

been reported to be isolated from this plant.[14] The three caf-

feoylshikimic regioisomers could be distinguished by the MS2
fragment ion information according to known literature.[15]
For MS2 analysis of compound 13, the second abundant
product ion at m/z 161.0243 was a characteristic product ion
for 4-O-caffeoylshikimic acid. Besides, the product ions at
m/z ~291.1 ([MHCO2]), 273.1 ([MHCO2H2O]),
247.1 ([MH2CO2]) and 229.1 ([MH2CO22H2O])
for successive losses of CO2 and H2O were also characteristic
C25H23O12
C22H29O10

C22H31O10
C21H27O9

C19H17O8

C15H17O4
Formula

product ions for 5-O- and 4-O-caffeoylshikimic acid (Fig. 3),

while 3-O-caffeoylshikimic acid yielded relatively low
abundance for these ions, this could be useful to distinguish
5-O-caffeoylshikimic acid (12) from 3-O-caffeoylshikimic acid
(16), and the elution order of the three regioisomers (5>4>3)
(ppm)
Error

–0.3

0.7
0.1
1.4

were also in accordance with above reported literature. For

0
0

the MSn analysis of rosmarinic acid and its derivatives (com-

pounds 36, 41 and 48), except for the prominent product ions
Calculated

for caffeic acid at m/z 179.0337 and 161.0233, the characteristic

453.1755
423.1650

373.0918
455.1912
515.1184

261.1121

product ion at m/z 197.0438 [M–H–caffeoyl]– can also be

mass

observed.
Detected by the [M+HCOOHH] ion

Characterization of flavonoids
Observed

453.1755d

455.1922d

Compared with reference standards.

423.1650c

373.0920c
515.1182c

261.1125c
mass

A total of 15 flavonoids (compounds 20, 21, 2325, 2729,

3135, 40 and 44) were identified, including flavanonol,
Detected by the [MH] ion.

flavonone, flavonol, etc. The molecular formulae of the

flavonoids were deduced by their prominent deprotonated
UV max

245, 325

232,270

[M–H]– ions and the accurate molecular weights obtained

(nm)

263

330
Table 1. (Continued)

from high-resolution mass spectrometry (HRMS). The kinds

Obtained as trace.

of sugars conjugated with flavonoid aglycone were easily

deduced by their fragment ion information in the MS/MS
(min)

15.81
16.03
16.13

17.56
17.73
19.61

spectrum, which result from the losses of 146Da for a rham-

tR

nose, 162Da for a hexose (a glucose or galactose), 176Da for

a glucuronide and 132Da for a pentose (an apiose, arabinose
No.

or a xylose).[16] Both 6-C- and 8-C-flavanone glycosides were

47b
45a

48a

50a
46

49

2443
d
b
a

reported to be isolated from Sarcandra glabra according to

Rapid Commun. Mass Spectrom. 2011, 25, 2439–2447 Copyright © 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm

Figure 1. The structures of compounds identified in Sarcandra glabra.
previous literature.[17,18] In this study, two pairs of flavanone
C-glycosides (compounds 20 and 23, compounds 25 and 27)
were isolated. They both showed two peaks in UPLC chroma-
tography, and the two compounds could not be isolated
singly, because they were always converted into each other
during the preparative HPLC isolation process, and they
showed identical NMR data. Since both flavonoid-8-C-glyco-
sides and flavonoid- 6-C-glycosides can singly exist in nature,
the C-2 position of the flavonone moiety is a chiral center, and
the tested ORD (optical rotation dispersion) values of the two
pairs of mixtures were rather low at almost zero, they were
deduced to be two pairs of epimers. Compounds 20 and 23
were identified as naringenin C-glycosides by the [M–H]–
ions at m/z ~433.1 (C21H21O10), as well as the characteristic
neutral losses of 90Da and 120Da yielding prominent pro-
duct ions at m/z ~343.1 (0,3X–, [M–H–90]–) and ~313.1 (0,2X–,
[M–H–120]–) in the MS2 spectrum. Besides, both compounds
20 and 23 showed clear losses of H2O and 2H2O at m/z
~415.1 ([M–H–H2O]–) and ~397.1 ([M–H–2H2O]–) by LC/
2444
MS/MS analysis. These fragments were reported to only be
wileyonlinelibrary.com/journal/rcm Copyright © 2011 John Wiley & Sons, Ltd.
X. Li et al.
observed for flavonoids mono-6-C-glycoside in the negative
CID-MS/MS spectrum[19]; thus they were characterized as
mixtures of (2R/2S)-naringenin-6-C-B-D- glucosides. Com-
pounds 25 and 27 showed identical deprotonated ions at m/z
~565.15 (C26H29O14), and yielded similar prominent pro-
duct ions to compounds 20 and 23 at m/z ~313.1 and 343.1,
as well as neutral losses of H2O and 2H2O at m/z ~547.1
and ~529.1 in the MS2 spectrum, indicating they had the same
flavanone aglycone and they should be naringenin 6-C-glyco-
side conjugated with another pentose with a molecular
weight of 132 (M–H–433), which could be confirmed by the
characteristic product ion at m/z ~397.1 ([M–H–132–2H2O]–)
(Fig. 4), and they were further confirmed to be mixtures of
(2R/2S)-naringenin-6-C-B-D-glucopyranosyl-(6!1)-apiose.
The type of pentose sugar was confirmed to be apiose by
NMR data and the two epimers were concluded to be new
compounds, but the elution order of (2R/2S)-flavanone mix-
tures could not be determined. Another four isomers of flava-
nonols (compounds 28, 31, 33 and 34) were easily identified
by the same deduced molecular formula of C21H22O11, the
Rapid Commun. Mass Spectrom. 2011, 25, 2439–2447
Constituents in Sarcandra glabra by UPLC LTQ Orbitrap
Figure 2. UPLC-PAD/ESI-MSn analysis of the whole plant of Sarcandra glabra: (a) U-
HPLC-PAD chromatography and (b) ESI-MS total ion current profile of Sarcandra glabra.
Figure 3. Proposed fragmentation pathway for compound 13.
same fragment ions at m/z 303.0501 and 285.0396, and they
could be distinguished through their UV absorption and
elution order if compared to reference standards. Neoastilbin
(28) with 2S,3S configuration and astilbin (31) with2R,3R
configuration had the same UV maximal absorption at 289
nm, while neoisoastilbin (33) with 2S,3R configuration and
isoastilbin (34) with 2R,3S configuration also had the same
UV absorption at 294nm, the latter caused a red shift of about
5nm, and the elution order of the four flavanonol isomers
were 2S,3S >2R,3R> 2S,3R> 2R,3S (Table 1). A phenylpropa-
noid-substituted catechin glycoside (compound 40) could be
identified based on the MS2 characteristic fragment product
ions at m/z 451.1010 formed by loss of a rhamnose from the
[M–H]– ions at m/z 597.1608 (C30H29O13), and the base peak
in MS3 was [M–H–rhamnose–C6H6O2]– at m/z 341.0644, fol-
lowed by fragment ions at m/z 323.0539 [M–H–rhamnose–
C6H6O2–H2O]–, 297.0748 [M–H–rhamnose–C6H6O2–CO2]
and 217.0128 [M–H–rhamnose–C6H6O2–C7H8O2]– (Fig. 5).
Rapid Commun. Mass Spectrom. 2011, 25, 2439–2447 Copyright © 2011 John Wiley & Sons, Ltd.
The types of the flavonoid aglycones were deduced from their
characteristic deprotonated aglycone ion (Y–0) information,
and most of the flavonoids were further identified by com-
parison of retention time, UV spectrum and mass fragment
ions with those of isolated standards.
Characterization of coumarins
Five compounds (compounds 9, 10, 11, 17 and 19) were iden-
tified as coumarins. Generally, coumarins displayed promi-
nent [M–H]– ions in the negative ion spectrum, but
isofraxidin-7-O-b-D-glucoside (compound 11) exhibited an
[M–H+HCOOH]– ion as base peak at m/z 429.1029 and the
deprotonated ion was not observed. Successive losses of
CH3 (15Da) and CO (28Da) groups were the characteristic
product ions for methoxylated coumarins. For compound 17,
the [M–H]– ion at m/z 339.0712 (C15H15O9) yielded a promi-
nent [M–H–146]– product ion at m/z 193.0129 (C9H5O5), and
the MS3 fragmentation of the ion gave fragment ions at m/z
165.0183 and 137.0236 for the successive neutral losses of
CO (28Da), combined with its characteristic UV absorption
(249, 299nm) for a coumarin nucleus, compound 17 was
deduced to be 6,7,8-trihydroxycoumarin conjugated with a
rhamnose rather than a coumaroyl group, the sugar linkage
position to the aglycone could not be assigned, but any posi-
tion of its conjugation was reported to be a new compound,
and this is the first report of coumarin conjugated with a
rhamnose.
Characterization of terpenoids
Compounds 14, 15, 18, 22, 26, 30, 38, 39, 42, 43, 46, 47, 49 and
50 were identified as terpenoids with their retention times,
UV and mass data shown in Table 1. Most of the terpenoids
were identified as sesquiterpene lactones, or sesquiterpe-
noid glycosides, except for compounds 14 and 15. Generally,
terpenoids showed [M–H+HCOOH]– ions as their base peaks
in negative mode and few product ions such as [M–H–
HCOOH]– were obtained in LC/MS data-dependent scanning
2445
mode. When conjugated with diglycosides, such as
wileyonlinelibrary.com/journal/rcm

Figure 4. Proposed fragmentation pathway for compound 25.
Figure 5. Proposed fragmentation pathway for compound 40.
Figure 6. Proposed fragmentation pathway for compound 30.
compound 38, the MS/MS fragment ion at m/z 311.0974
[apiose+glucose–H]– was produced as base peak but the
nucleus aglycone ion [M–H–311]– was not found (Table 1).
MSn fragmentation analysis of the isolated authentic sesqui-
terpene lactone glycosides, like compound 30, yielded a
prominent [M–H+HCOOH]– ion at m/z 469.1707, its analysis
by MS2 fragmentation resulted in losses of HCOOH m/z
2446
423.1640 [MH] and its MS3 spectrum showed the aglycone
wileyonlinelibrary.com/journal/rcm Copyright © 2011 John Wiley & Sons, Ltd.
X. Li et al.
ion at m/z 261.1120, suggesting the presence of a hexoside
residue. Further MS4 fragmentation of m/z 261.1120 gave frag-
ment ions at m/z 243.1017, 233.1174, 231.1018 and 217.1226
due to the losses of H2O, CO, HCHO and CO2, respectively,
and these characteristic neutral loss fragments are useful for
rapid screening and identification of sesquiterpene lactones
(Fig. 6).
CONCLUSIONS
A reliable and effective method employing ultra-high-pressure
liquid chromatography (U-HPLC) coupled with linear trap
quadrupole (LTQ) and the high-resolution Orbitrap mass
analyzer (U-HPLC-LTQ-Orbitrap) was successfully devel-
oped for online identification of small molecular constituents
of Sarcandra glabra and its related preparations in this study.
A total of 50 compounds, including caffeoyl derivatives,
flavonoids, coumarins and sesquiterpenoids, were identified
or tentatively characterized based mainly on their accurate
molecular data from LTQ-Orbitrap, the fragment ions informa-
tion obtained by LC-MS/MS, as well as the mass data provided
by reference standards within 5ppm error in routine analysis.
Among them, compounds 17, 25 and 27 were tentatively identi-
fied as new compounds, and four of them were reported from
the Sarcandra genus for the first time. Besides, 21 of those com-
pounds, including organic acids, phenolic acids, caffeoylquinic
acids, coumarins and sesquiterpenoids, were coexisting as major
constituents in its four related preparations, Qingrexiaoyanning
capsules, Zhongjiefeng tablets, Jinsulan liniment, Xuekang cap-
sules and Zhongjiefeng injection, but flavonoids were not found.
The fragmentation behaviors of the four major categories of con-
stituents in Sarcandra glabra were also investigated, and the
fragmentation pathways of some types of compounds like
phenylpropanoid-substituted catechin glycoside and sesqui-
terpenoid glycoside were reported for the first time. This
research sets a good example for the rapid identification of
complicated polar constituents in the plant extracts and its
related preparations by high-resolution orbitrap mass spec-
trometry, and will be of great help in developing a high-
Rapid Commun. Mass Spectrom. 2011, 25, 2439–2447
Constituents in Sarcandra glabra by UPLC LTQ Orbitrap
quality fingerprint for the comprehensive quality control of
this herbal drug and identifying the metabolites in vivo to
better understand the mechanism of action of the herb.
SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article.
Acknowledgement
This work was supported by the National S&T Major Project
(No. 2009ZX09304-002).
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