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RAPID COMMUNICATIONS IN MASS SPECTROMETRY
Rapid Commun. Mass Spectrom. 2009; 23: 2724–2732
) DOI: 10.1002/rcm.4179
Published online in Wiley InterScience (www.interscience.wiley.comIdentification of active compounds and their metabolites
by high-performance liquid chromatography/electrospray
ionization Fourier transform ion cyclotron resonance
mass spectrometry from Xiao-xu-ming decoction (XXMD)
Yilin Wang1, Chunguang Ding1, Kehe Du1,2, Yao Xiao1, Caisheng Wu1, Jinlan Zhang1,3*,
Hailin Qin1 and Guanhua Du1
1Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, P.R. China2Liaoning Shihua University, Liaoning 11300, P.R. China3Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Institute of Materia Medica, Chinese Academy
of Medical Sciences & Peking Union Medical College, Beijing 100050, P.R. China
Received 13 April 2009; Revised 25 June 2009; Accepted 27 June 2009
*CorrespoChinese ACollege, BE-mail: zContract/contract/
Xiao-xu-ming decoction (XXMD) prescription is a traditional Chinese prescription that has been
widely used to treat theoplegia and the sequela of theoplegia. Modern pharmacological research
has also indicated that the active fraction from XXMD is able to treat cardiovascular diseases
and Alzheimer’s disease. In the study reported here, high-performance liquid chromatography
coupled with Fourier transform ion cyclotron resonance mass spectrometry (HPLC/FTICR-MS)
was developed to identify active compounds and their metabolites after oral administration of
active fraction from Xiao-xu-ming decoction to rats, using parent mass list triggered data-dependent
multiple-stage mass analysis at a resolving power of 100 000 in the external calibration mode. The
mass accuracies obtained for full-scan MS were within 2ppm in most cases. Fifteen constituents
were identified in the active fraction from XXMD and the biological samples of rats. The fragmenta-
tion behaviors of these constituents were summarized which would be helpful for structural
characterization. The profiles of the constituents in the active fraction and biological samples
of rats were obtained which provided us with much information for a better understanding
of the chemical basis of the pharmacologic actions of XXMD. Copyright # 2009 John Wiley & Sons,
Ltd.
Xiao Xu Ming-Decoction (XXMD) prescription is a tradi-
tional Chinese prescription that was firstly recorded in
‘QianJinYaoFang’ which was written by Chinese ancient
Si-Miao Sun of the Tang Dynasty. The formula consists of
twelve crude drugs including Saposhnikovia divaricata
(Turcz.) Schischk., Scutellaria baicalensis Georgi, Paeonia
lactiflora Pall., Glycyrrhiza uralensis Fisch., Zingiber officinale
Rosc., Stephania tetrandra S. Moore, Panax ginseng C. A. Mey.,
Cinnamomum cassia Presl, Prunus armeniaca L. var. ansu
Maxim., Ephedra sinica Staph, Ligusticum chuanxiong Hort.,
Aconitum carmichaeli Debx. in a ratio of 3:3:3:3:3:3:6:6:6:9:9:9
on a dry weight basis. The prescription has attracted a great
deal of attention for its pharmaceutical and therapeutic uses.
The active fraction was screened byWang et al.1 and showed
similar pharmacological effects to XXMD. It is effective in
ndence to: J. L. Zhang, Institute of Materia Medica,cademy of Medical Sciences & Peking Union Medicaleijing 100050, P.R. China.
[email protected] sponsor: National Nature Science Foundation;grant number: 30630073.
treating theoplegia and the sequela of theoplegia. Modern
pharmacological work has also indicated that the active
fraction is able to treat cardiovascular diseases and
Alzheimer’s disease.2,3
Fourier transform ion cyclotron resonance mass spec-
trometry (FTICR-MS) is a mass spectrometry technique
which has been developed rapidly in the last 30 years. In
1974, Comisarow and Marshall4,5 adapted Fourier transform
methods to ICR spectrometry and built the first FTMS
instrument. Since that time, FTMS mass spectrometers with
the highest mass accuracy and mass resolving power6 have
received increasingly attention and become efficient tools for
various analyses including proteomics analyses,7 accurate
mass measurements for drug discovery,8 drug mixture
analyses,9 analyses of trace impurities in a drug substance,10
toxicology and forensic sciences,11 microbiology,12 and most
recently, metabonomics.13–17
As the active fraction of XXMD is prepared from twelve
medicinal herbs and each of them contains very complex
chemical components, it is very important to identify and
characterize the constituents present in the active fraction
Copyright # 2009 John Wiley & Sons, Ltd.
Active compounds and their metabolites in XXMD 2725
and the metabolites in vivo for a better understanding of
its mechanism of action. In the work reported herein, a
high-performance liquid chromatography (HPLC) separ-
ation prior to LTQ/FTICR-MS was employed to profile
parent constituents in the active fraction from XXMD, as well
as the metabolites after oral administration of the active
fraction to rats. The accuracy of HPLC/FTICR-MS m/z value
measurements were within 2ppm in most cases. Besides,
multistage MS/MS analysis (MSn) was applied to provide
further information about the structures of the compounds
under analysis. So a novel method for the rapid identification
and characterization of constituents in complex extract and
their metabolites was developed using the combination of
high mass accuracy and fragmentation behavior. As a result,
15 constituents were totally identified in the active fraction
from XXMD and biological samples from rats.
EXPERIMENTAL
Chemicals, reagents, and materialsPaeoniflorin (1), prim-o-glucosylcimifugin (2), cimifugin (3),
40-O-b-D-glucosyl-5-O-methylvisamminol (4), baicalin (5),
baicalein (10), glycyrrhizic acid (11), glycyrrhetinic acid (12),
wogonin (13), and chrysin (14) were purchased from the
National Institute for Control of Pharmaceutical and
Biological Products (Beijing,China). Liquiritigenin (6) was
obtained from the Dalian Fusheng Pharmaceutical Co., Ltd.
Wogonoside (9) was ordered from the Shanghai Usea Biotech
Co., Ltd. Oroxylin A-7-O-glucuronide (8) and oroxylin A (15)
were gifts from Professor Hailin Qin. 5-O-Methylvisammiol
(7) was isolated and purified in our laboratory. The purity of
all compounds was greater than 99% (by HPLC). The
structures of these materials were confirmed by UV, MS,1H NMR, and 13C NMR analyses. The structures are shown
in Scheme 1.
Acetonitrile (LC/MS reagent grade) was supplied by
Mallinckrodt Baker, Inc. (Phillipsburg, NJ, USA). Deionized
water was purified using a water purification system
(Millipore, Billerica, MA, USA). Analytical grade ethyl
acetate, methanol and acetic acid were purchased from
Beijing Chemical Corp. (Beijing, China). The active fraction of
XXMD prescription was also a kind gift from Professor
Hailin Qin.
Preparation of the dosed solution ofthe active fractionThe orally administrated solution of the active fraction
powder was dissolved in deionized water at the concen-
tration of 250mg/mL, adding 0.5% Tween 80 as solution
adjuvant.
ChromatographyA Thermo Scientific Surveyor LC Plus system equipped with
a Surveyor MS pump plus and a Surveyor autosampler was
used to carry out the assay. The samples were separated on a
HYPERSIL C18 column (100� 2.1mm, 5mm) protected by a
ZORBAX SB-C18 guard column (12.5� 2.1mm, 3.5mm). The
mobile phase consisted of acetonitrile (A) and 0.4% (v/v)
acetic acid (B) delivered at a flow rate of 0.8mL/min with the
Copyright # 2009 John Wiley & Sons, Ltd.
following gradient program: starting with 15% A, then
reaching 25%A at 20min, reaching 30%A at 35min, reaching
40% A at 40min and maintaining 40% A until 50min, then
reaching 60% A at 60min and maintaining 60% A until
70min. The systemwas then returned to the initial conditions
within 30 s, and the column was reconditioned for 9.5min.
The column temperature was maintained at 308C and the
sample injection volume was 10mL.
Mass spectrometryA Thermo Scientific LTQ FT mass spectrometer was
connected to the Thermo Scientific Surveyor LC Plus system
via an electrospray ionization (ESI) interface. The column
effluent was split in a ratio of 3:1, so that 200mL/min entered
the source of the mass spectrometer. The operating
parameters in the positive ion mode were as follows:
collision gas, ultrahigh-purity helium (He); nebulizing gas,
high-purity nitrogen (N2); ion spray voltage, 3.5 kV; capillary
temperature, 3008C; capillary voltage, 40V; sheath gas flow
rate, 35 (arbitrary units); auxiliary gas flow rate, 10 (arbitrary
units); sweep gas flow rate, 5 (arbitrary units); and tube lens,
120V. Mass spectra were recorded in a mass range ofm/z 100
to 1500 at a resolving power of 100 000 with data-dependent
MS/MS analysis triggered by the most abundant ions from
the parent mass list of predicted compounds (mass list of the
positive ion mode: 823, 503, 471, 469, 461, 453, 447, 307, 291,
285, 271, 257, 255), followed by MS/MS/MS analysis on the
most abundant product ions. Collision-induced dissociation
(CID) was conducted with an isolation width of 1 Da.
Xcalibur software (version 2.0; Thermo Scientific) was
employed for data acquisition and reduction after HPLC/
FTICR-MS analysis.
AnimalsMale Wistar rats (190� 20 g) were obtained from the
Laboratory Animal Center of the Chinese Academy of
Medical Sciences & Peking Union Medical College (Beijing,
China). The animals were kept in a fully acclimatized room
for 3 days before starting the experiment and had free access
to routine diet and water. Prior to the experiment rats were
fasted in ametabolic cage and given only physiological saline
for 24 h. Then they were orally given the active fraction from
Xiao-xu-ming decoction at a dose of 400mg/200 g body
weight. All protocols and procedures involving animals
were approved by the Animal Care and Welfare Committee
of Institute of Materia Medica, Chinese Academy of Medical
Sciences & Peking Union Medical College (Beijing, China).
Urine and feces were collected after administration during
different periods thereafter (0–12, 12–24, and 24–48 h). The
amounts of urine and feces during each period were
recorded, and samples were stored at �208C until assay.
Blood samples were obtained from the abdominal aorta
according to the specific schedule (5, 20, 45, 60, 90, 120, 180,
240, 300, 360, 420, 450, 480, 510, 540, 600, 720, 960, 1440min)
after the oral administration of the active fraction to rats.
Plasmawas separated by centrifugation at 765 g for 5min. All
plasma samples were stored at �208C until the assay was
performed.
Rapid Commun. Mass Spectrom. 2009; 23: 2724–2732
DOI: 10.1002/rcm
Scheme 1. Chemical structures of the 15 identified constituents in the active fraction from XXMD
and their metabolites in rats.
2726 Y. L. Wang et al.
Sample preparation
Sample preparation from plasma and urineAll plasma or urine samples were centrifuged at 1721 g for
10min and then divided into two portions, respectively. For
one portion, aliquots of 1mL of plasma or urine supernatants
were placed into 5mL conical plastic test tubes and 1mL
amounts of ethyl acetate were added. Next, the test tubes
were vortex-mixed vigorously for 90 s and centrifuged at
1721 g for 10min. The extraction was repeated twice. The
supernatants were combined and evaporated to dryness at
308C under reduced pressure. For another portion, aliquots
of 1mL of urine or plasma supernatants were placed into
10mL conical plastic test tubes and 6mL amounts of
methanol were added. Next, the test tubes were vortex-
Copyright # 2009 John Wiley & Sons, Ltd.
mixed vigorously for 90 s and centrifuged at 1721 g for
10min. The supernatants were evaporated to dryness at 308Cunder reduced pressure. All residues were reconstituted in
0.2mL amounts of methanol. Each sample was then filtered
through a 0.45mm nylon filter film. Aliquots of 10mL were
injected into the HPLC/FTICR-MS system.
Sample preparation from fecesAll feces samples were weighed and completely ground
with ten times their volume of physiological saline and
the mixtures centrifuged at 1721 g for 10min. Then the
supernatants were divided into two portions which
were treated with ethyl acetate and methanol, respectively.
The subsequent procedures were as described above for
plasma and urine.
Rapid Commun. Mass Spectrom. 2009; 23: 2724–2732
DOI: 10.1002/rcm
Active compounds and their metabolites in XXMD 2727
RESULTS AND DISCUSSION
Structural characterization of constituents in theactive fraction from XXMD
The MS and MSn experiments using LTQ/FTICR-MS were
first performed to investigate the fragmentation character-
istics of the constituents in the active fraction from XXMD
and the relative standards. These fragmentation patterns and
the corresponding masses of fragment ions are sufficient to
determine the chemical structures of the compounds in the
active fraction, and furthermore to identity the constituents
of the biological specimens from rats.
In this study, a Thermo LTQ FTICR-MS systemwas chosen
to carry out the analysis. The Thermo LTQ FTICR-MS system
combines a linear ion trap (IT) mass spectrometer with an
FTICR analyzer.18 The linear IT, with greater ion storage
capacity than conventional three-dimensional (3D) IT
devices, is a fully operational MS detector, which can store,
isolate, and fragment ions. And all the ion handling, selection
and excitation capabilities of the IT can be used to prepare
ions for analysis in the ICR cell. The combination can
enhance ion accumulation and improve the duty cycle,
therefore making it well suited for LC analysis and
meanwhile providing excellent performance and capabilities
to the FT mass spectrometers. In this paper, the mass
accuracies of the compounds in the active fraction obtained
for full-scan MS spectra were within 4ppm. In most cases,
the errors were within 1 ppm, as listed in Table 1.
The HPLC/FTICR-MS analysis of the active fraction is
shown in Fig. 1. Constituents in the active fraction were
profiled in one chromatographic run with parent mass list
triggered data-dependent multiple-stage mass analysis. By
comparing with the reference compounds, 14 constituents
including eight flavonoids (peak nos. 5, 6, 8, 9, 10, 13, 14, 15),
Table 1. Mass data from the fifteen compounds detected in the a
No.Rt
(min) [MþH]þProposedformula
Error(ppm) Identified compoun
1 8.28 503.15182� C23H28O11Naþ �1.12 paeoniflorin2 8.76 469.17093 C22H29O
þ11 1.05 prim-o-glucosylcimi
3 13.45 307.11761 C16H19Oþ6 �0.02 cimifugin
4 15.95 453.17578 C22H29Oþ10 0.57 40-O-b-D-glucosyl-5
methylvisamminol5 20.96 447.09256 C21H19O
þ11 0.83 baicalin
6 24.55 257.08084 C15H13Oþ4 �0.02 liquiritigenin
7 24.7 291.12286 C16H19Oþ5 0.55 5-O-methylvisammi
8 26.60 461.10706 C22H21Oþ11 �1.69 oroxylin A 7-O-gluc
9 29.48 461.10776 C22H21Oþ11 �0.17 wogonoside
10 40.97 271.05951 C15H11Oþ5 �2.18 baicalein
11 46.06 823.41156 C42H63Oþ16 0.60 glycyrrhizic acid
12 48.14 471.34833 C30H47Oþ4 3.06 glycyrrhetinic acid
13 48.64 285.07523 C16H13Oþ5 �1.82 wogonin
14 49.92 255.06522 C15H11Oþ4 0.14 chrysin
15 51.49 285.07565 C16H13Oþ5 �0.35 oroxylin A
�The ions at m/z 503.15182 and 503.15238 were [MþNa]þ.a XX, PL, UR and FE represent XXMD, plasma, urine and feces, respectivND: not detected, þ: detected. Rt: retention time.
Copyright # 2009 John Wiley & Sons, Ltd.
three chromones (peak nos. 2, 3, 4), two triterpenes (peak nos.
11, 12) and one monoterpene (peak no. 1) were identified, as
listed in Tables 1 and 2, respectively.
HPLC/LTQ-MSn investigation of compounds 5, 6,8, 9, 10, 13, 14, 15These eight compounds are all flavonoids. The common
features of ESI-MS/MS data of [MþH]þ ions observed in this
work were the loss of CO (28 Da) and H2O (18 Da). The
compounds containing methoxyl groups could also give an
ion loss of CH3 (15 Da). Also, the retro-Diels-Alder (RDA)
fragmentation reaction could be observed. In previous
studies,19–23 flavonoids were mostly investigated by MS in
the negative mode. However, the common features of these
reported data are similar to that obtained in the positive
mode in our work.
Baicalin (5) displayed an [MþH]þ ion atm/z 447.09256 and
gave an ion at m/z 271 in the MS2 spectrum as the base peak
from the neutral loss of a glucuronic acid. This neutral loss
can be used for the identification of O-glucuronides.
Baicalein (10) gave an [MþH]þ ion at m/z 271.05951. The
MS2 spectrum of baicalein yielded product ions at m/z 253
(base peak) and 225, which were from the loss of H2O and
CO. Besides, the ion atm/z 169 and 103 was produced from a
RDA fragmentation reaction cleaved at the C-ring. Lliquir-
itigenin (6) exhibited an [MþH]þ ion at m/z 257.08084. In the
MS2 spectrum, the product ions at m/z 239 and 137 were
formed by the loss of H2O and the RDA fragmentation
reaction, respectively. The fragment ion at m/z 211 in the
MS3 spectrumwas attributed to the loss of CO from the ion at
m/z 239, whichwas themost abundant product ion in theMS2
spectrum. Oroxylin A 7-O-glucuronide (8) and wogonoside
(9) are a pair of isomers, both having glucuronic acid
groups on the A-ring and showing an [MþH]þ ion atm/z 461
ctive fraction from XXMD, plasma, urine and feces
ds Plant source
Distributiona
XX PL UR FE
Paeonia lactiflora Pall. þ þ þ NDfugin Saposhnikovia divaricata (Turcz.)
Schischk.þ þ þ ND
Saposhnikovia divaricata (Turcz.)Schischk.
þ þ þ þ
-O- Saposhnikovia divaricata (Turcz.)Schischk.
þ ND ND ND
Scutellaria baicalensis Georgi þ þ ND ND
Glycyrrhiza uralensis Fisch. þ þ þ þol Saposhnikovia divaricata (Turcz.)
Schischk.ND þ þ þ
uronide Scutellaria baicalensis Georgi þ þ þ ND
Scutellaria baicalensis Georgi þ þ þ ND
Scutellaria baicalensis Georgi þ ND þ þGlycyrrhiza uralensis Fisch. þ þ þ þGlycyrrhiza uralensis Fisch. þ þ þ þScutellaria baicalensis Georgi þ þ þ þScutellaria baicalensis Georgi þ ND þ þScutellaria baicalensis Georgi þ þ þ þ
ely.
Rapid Commun. Mass Spectrom. 2009; 23: 2724–2732
DOI: 10.1002/rcm
Figure 1. The extracted ion chromatograms of the active fraction fromXXMD. The numbers assigned to compounds are the same
as in Scheme 1.
Copyright # 2009 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2009; 23: 2724–2732
DOI: 10.1002/rcm
2728 Y. L. Wang et al.
Table 2. HPLC/LTQ-MSn data from the compounds detected in the active fraction from XXMD, plasma, urine and feces
No. Name HPLC/LTQ-MSn Ref.
1 paeoniflorin MS2[503]: 381, 341, 307, 219, 185 26,27MS3[381]: 363
2 prim-o-glucosylcimifugin MS2[469]: 307, 289, 259, 235 —3 cimifugin MS2[307]: 289, 259, 235 24
MS3[235]: 217, 2074 40-O-b-D-glucosyl-5-O-methylvisamminol MS2[453]: 291, 273 —
MS3[291]: 273, 2435 baicalin MS2[447]: 271 19,216 liquiritigenin MS2[257]: 239, 211, 163, 147, 137 19,22
MS3[239]: 211, 183, 165MS3[147]: 119
8 oroxylin A 7-O-glucuronide MS2[461]: 285, 270 —MS3[285]: 270
9 wogonoside MS2[461]: 285, 270 20,21MS3[285]: 270
10 baicalein MS2[271]: 253, 225, 169, 103 19,2011 glycyrrhizic acid MS2[823]: 471 2512 glycyrrhetinic acid MS2[471]: 453, 435, 389 —13 wogonin MS2[285]: 270, 183, 103 20
MS3[270]: 252, 242, 22414 chrysin MS2[255]: 237, 227, 153, 103 19,2015 oroxylin A MS2[285]: 270, 183, 103 19,23
MS3[270]: 252, 242, 224
Active compounds and their metabolites in XXMD 2729
(8:m/z 461.10706, 9:m/z 461.10776). The MS2 andMS3 spectra
of the two compounds were highly similar to each other,
except for the relative abundance of ions. The ion at m/z 285
in the MS2 spectrum was generated by the loss of glucuronic
acid from the molecular ion atm/z 461, and the ion atm/z 270
in the MS3 spectrum was formed by the loss of CH3 from the
ion at m/z 285. It was hard to distinguish these two isomers
only by MS spectra, but it was easier to identify them by
comparing their retention behaviors with the help of the
available reference standards. Similarly, wogonin (13) and
oroxylin A (15) are also a pair of isomerswhich gave the same
[MþH]þ ion at m/z 285 and showed an ion at m/z 270 as the
base peak from the loss of CH3. They were finally identified
by comparing the two compounds with the reference
standards. Chrysin (14) displayed an [MþH]þ ion at m/z
255.06522. In theMS2 experiment the [MþH]þ ion underwent
the RDA fragmentation reaction to yield product ions at m/z
153 and 103.
HPLC/LTQ-MSn investigation of compounds 2,3, 4prim-o-Glucosylcimifugin (2), cimifugin (3) and 40-O-b-D-
glucosyl-5-O-methylvisamminol (4) are all chromones. prim-
o-Glucosylcimifugin (2) gave an [MþH]þ ion at m/z
469.17093. In the MS2 spectrum, the [MþH]þ ion lost a
glucose group and transformed into cimifugin (3), giving a
product ion at m/z 307. Cimifugin (3) displayed an [MþH]þ
ion atm/z 307.11761. In theMS2 spectrum, the product ions at
m/z 289, 259 and 235 (base peak) were formed by the loss
of H2O, HCHO and C3H4O2 (72 Da) from the [MþH]þ ion at
m/z 307, respectively. The ion at m/z 235 then lost H2O and
CO to produce the ions at m/z 217 and 207 in the MS3
spectrum. 40-O-b-D-Glucosyl-5-O-methylvisamminol (4) dis-
played an [MþH]þ ion at m/z 453.17578. Its fragmentation
Copyright # 2009 John Wiley & Sons, Ltd.
patterns were similar to that of cimifugin (3) and displayed
the characteristic product ions at m/z 291 and 273 in the MS2
spectrum.
HPLC/LTQ-MSn investigation of compounds11, 12Glycyrrhizic acid (11) and glycyrrhetinic acid (12) are both
triterpenes. Glycyrrhizic acid (11) showed an [MþH]þ ion at
m/z 823.41156 and can successively lose two glucuronic acid
groups and then transform into glycyrrhetinic acid (12).
Glycyrrhetinic acid (12) afforded an [MþH]þ ion at m/z
471.34833. In the MS2 spectrum of glycyrrhetinic acid (12),
the ions at m/z 453 and 435 were formed by the successive
loss of H2O and 2 H2O.
HPLC/LTQ-MSn investigation of compound 1Paeoniflorin (1) is a monoterpene compound which gave an
[MþNa]þ ion at m/z 503.15265. The ions at m/z 381 (base
peak), 341 and 307 in the MS2 spectrum were produced from
the losses of a benzoic acid, a glucose and a parent nucleus,
respectively. The fragment ion at m/z 363 was then produced
from the loss of H2O from the ion at m/z 381 in the MS3
spectrum.
Identification of constituents in the plasma ofratsThe MS and MSn data of the plasma which were obtained
from the rats treated with the active fraction from XXMD
(Tables 1 and 2) were firstly compared to the blank plasma
samples to ensure that the constituents detected did not exist
in the blank samples.
Then the data of the plasma were compared with that of
the active fraction and the standards, and a total of eleven
constituents were detected in the plasma of rats, as can be
Rapid Commun. Mass Spectrom. 2009; 23: 2724–2732
DOI: 10.1002/rcm
Figure 2. The extracted ion chromatograms of the plasma of rats treated with ethyl acetate. The
numbers assigned to compounds are the same as in Scheme 1.
2730 Y. L. Wang et al.
seen from Figs. 2 and 3. The mass accuracies of the
constituents in the plasma obtained for full-scan MS spectra
were within 1ppm in most cases. Glycyrrhetinic acid (12),
however, showed an error of 5.59 ppm as a special case. The
main differences between the compounds found in the
plasma and the active fractionwere that 40-O-b-D-glucosyl-5-
O-methylvisamminol (4), baicalin (5), baicalein (10) and
chrysin (14) were not detected in the plasma. However, 5-O-
methylvisammiol (7), which was formed by the loss of a
glucose from 40-O-b-D-glucosyl-5-O-methylvisamminol (4)
and gave an [MþH]þ ion at m/z 291.12286, caught our
attention. We also found that the compounds detected both
in plasma and the active fraction had different relative
amounts, which indicated that these compounds had
different abilities to be absorbed into the plasma attributed
to their different physicochemical characteristics.
Identification of constituents in the urine ofratsThe strategy used for the study of urine of rats was the same
as that of plasma. By comparing the MS and MSn data of the
urine (Tables 1 and 2) with that of the blank urine samples
and the active fraction, a total of 14 constituents were
Copyright # 2009 John Wiley & Sons, Ltd.
detected in the urine of rats (Table 1). The mass accuracies of
the constituents detected in the urine obtained for full-scan
MS spectra were within 1 ppm in most cases, except for
cimifugin (3, error �7.54 ppm) and glycyrrhizic acid (11,
error 6.83 ppm). The reason for this high error may be due to
theweak peak intensity of cimifugin (3) and glycyrrhizic acid
(11) in rat urine.
Identification of constituents in the feces of ratsBy comparing the MS and MSn data of the feces (Tables 1
and 2) with that of the blank feces samples and the active
fraction, a total of nine constituents were detected in the feces
of rats (Table 1). The mass accuracies of the constituents
detected in the feces obtained for full-scan MS spectra were
within 3ppm in most cases, except for glycyrrhizic acid (11,
error�12.59 ppm). The reason for this high error may be also
due to the weak peak intensity of glycyrrhizic acid (11) in rat
feces. Compared with the analytical results from the urine, it
was found that some glycosides with higher polarity, such as
cimifugin (3), baicalin (5) and wogonoside (9) were all not
detected in the feces. The result demonstrated that the
compounds that were present in the feces were mainly the
compounds with lower polarity.
Rapid Commun. Mass Spectrom. 2009; 23: 2724–2732
DOI: 10.1002/rcm
Figure 3. The extracted ion chromatograms of the plasma of rats treated with methanol. The
numbers assigned to compounds are the same as in Scheme 1.
Active compounds and their metabolites in XXMD 2731
CONCLUSIONS
In this paper, an HPLC/FTICR-MS method was developed
to profile parent constituents in the active fraction from
XXMD, as well as biological samples after oral adminis-
tration of the active fraction to rats. Accurate mass
measurement at a resolving power of 100 000 indicated that
the technique was capable of providing mass accuracy
within 2ppm in most cases in the external calibration mode.
The results suggest that HPLC/FTICR-MS is a valuable
analytical tool in compound identification because it can
provide robust accurate mass andMS/MS determinations of
the compounds. The valuable information is helpful for the
rapid confirmation of expected metabolites and elucidation
of the structures of unusual or unexpected metabolites. As a
result, 15 constituents were totally identified in the active
fraction from XXMD and the biological samples from rats.
The fragmentation behaviors of these constituents were
summarized which would be helpful for structural charac-
terization. The profiles of the constituents in the active
fraction and the biological samples of rats were obtained
which could provide us with much more information for
better understanding of pharmacologic actions of XXMD
from the chemical viewpoint.
Copyright # 2009 John Wiley & Sons, Ltd.
AcknowledgementsWe are grateful for financial support from the National
Nature Science Foundation of China (No. 30630073).
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