Metabolite profile analysis of aconitine in rabbit stomach after oral administration by liquid chromatography/electrospray ionization/multiple-stage tandem mass spectrometry

Article abstract of DOI:10.3109/00498254.2012.753490

1. Aconitine (AC), an active and highly toxic constituent extracted from aconitum plants, is well known for its excellent effects against rheumatism and rheumatoid arthritis. The metabolism of AC in liver and intestine has been previously reported. However, little is known about the metabolism of AC in stomach. In this study, the metabolite profiling of AC in stomachs of rabbit and rat was performed by liquid chromatography/electrospray ionization/multiple- stage tandem mass spectrometry (LC/ESI/MSⁿ), for the first time. 2. The samples were purified by liquid-liquid extraction, separated using an Agilent extended C18 column following a linear gradient elution and then detected by ESI/MSⁿ in positive ion mode. Metabolites were identified by comparing their protonated molecules, fragmentation patterns and chromatographic behaviors with those of standard compounds and data from authorized literature works. 3. In conclusion, 14 metabolites were identified in animal stomach after oral administration of AC. The presentation of a large amount of metabolites of AC in stomach suggested that, for aconitum alkaloids, the stomach might play an important role in their metabolism.

Full text of DOI:10.3109/00498254.2012.753490

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Xenobiotica, 2013; 43(7): 628–635
2013 Informa UK Ltd DOI: 10.3109/00498254.2012.753490
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RESEARCH ARTICLE
Metabolite profile analysis of aconitine in rabbit stomach after oral
administration by liquid chromatography/electrospray ionization/
multiple-stage tandem mass spectrometry
Zhigang Sui1,2, Na Li2, Zhiqiang Liu3, Jun Yan1, and Zhongying Liu1
2
1College of Pharmacy, Jilin University, Changchun, China, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, and
3Chang Chun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, China
Abstract
Keywords
1. Aconitine (AC), an active and highly toxic constituent extracted from aconitum plants, is well
known for its excellent effects against rheumatism and rheumatoid arthritis. The metabolism
of AC in liver and intestine has been previously reported. However, little is known about the
metabolism of AC in stomach. In this study, the metabolite profiling of AC in stomachs of
rabbit and rat was performed by liquid chromatography/electrospray ionization/multiple-
stage tandem mass spectrometry (LC/ESI/MSn), for the first time.
2. The samples were purified by liquid–liquid extraction, separated using an Agilent extended
C18 column following a linear gradient elution and then detected by ESI/MSn in positive ion
mode. Metabolites were identified by comparing their protonated molecules, fragmentation
patterns and chromatographic behaviors with those of standard compounds and data from
authorized literature works.
Aconitine, fragmentation pathway,
LC/ESI/MSn, metabolites, stomach
History
Received 19 September 2012
Revised 21 November 2012
Accepted 23 November 2012
Published online 26 December 2012
3. In conclusion, 14 metabolites were identified in animal stomach after oral administration of
AC. The presentation of a large amount of metabolites of AC in stomach suggested that, for
aconitum alkaloids, the stomach might play an important role in their metabolism.
Introduction
other hand, aconitum alkaloids are unstable and undergo rapid
biotransformation in vivo (Zhang et al., 2005), which result in
the production of new metabolites with different levels of
toxicity or detoxificated metabolites. Therefore, the investiga-
tion on metabolism of AC, the representative aconitum
alkaloid, is valuable for the toxicological, pharmacological
and clinical research of aconitum plants.
In East Asia, there are some traditional aconitum plants
(Ranunculaceous family), such as Aconitum kusnezoffii and
Aconitum brachypodum (Singhuber et al., 2009), which are
widely used for more than a dozen of centuries because of
their analgetic and anti-inflammatory activities (Suzuki et al.,
1994), especially the excellent effects against rheumatosis and
rheumatoid arthritis. However, they also induce serious
arrhythmias with final result leading to death (Turabekova
et al., 2008). Some diester-diterpene alkaloids, such as
aconitine (AC), mesaconitine, hypaconitine and deoxyaconi-
tine, are considered to be the main, active and toxic
constituents (Yue et al., 2009) and share a common
C19-norditerpenoid skeleton (Wang et al., 2003). Every year,
fatal cases of poisoning from aconitum plants are reported
(Pullela et al., 2008; Strzelecki et al., 2010) due to the narrow
therapeutic index of aconitum alkaloids (Li et al., 2012). It is
reported that the median lethal dose of AC for mice (iv) is
0.270 ꢀ 0.002 mg/kg (Zhou et al., 1984), and its lethal dose
for humans is estimated to be 1–2 mg (single dose). On the
In general, the unprocessed aconitum herbs are too toxic
for internal use, but they can be used for external application
as anesthetics (Singhuber et al., 2009). The processed
aconitum herbs are commonly administrated via oral route
in the formulations of decoction or medicinal wine (Chan,
2011). Liver is the main site of drug metabolism, where
cytochrome P450 enzymes are significant phase-I drug
metabolizing enzymes involved in the metabolism of drugs.
Several literature works on metabolism of AC in liver
microsomes are available and several metabolites have been
identified (Tang et al., 2011; Wang et al., 2006). Because
patients are given aconitum herbs predominantly via oral
administration, AC, the main active compound in the herbs,
will unavoidably undergo biotransformation in alimentary
canal, which contains numerous microorganisms and diverse
enzymes, such as trypsin, chymotrypsin, elastase and
carboxypeptidases (Sarti et al., 2011), and produce metabo-
lites. Our previous study has found some metabolites in small
Address for correspondence: Zhongying Liu, College of Pharmacy, Jilin
University, 1266 FuJin Road, Changchun 130021, China. Tel: þ86 431
85619704. Fax: þ86 431 85262236. E-mail: Liuzy@jlu.edu.cn

DOI: 10.3109/00498254.2012.753490
Metabolite profile analysis of aconitine in rabbit stomach 629
Table 1. The LC gradient program.
intestine, cecum and rectum (Sui et al., 2009). However, there
has been no report about the metabolite profiling of AC in the
stomach. Compared with the intestine, the stomach is usually
believed to have minimal contribution to metabolism and
typically make a much more important contribution to
dissolution and absorption of drugs. Therefore, the biotrans-
formation of drugs in the stomach is usually ignored.
However, the endogenous factors (e.g. pepsin and bacteria)
in the stomach may also have effect on drug metabolism
(Delgado et al., 2011; Gupta et al., 2012). For example, arctiin
has been reported to be transformed into arctigenin in rat
stomach, where the concentration of arctigenin was signifi-
cantly higher than that of arctiin (He et al., 2012). Zhang et al.
(2008) found that polydatin could be transformed into
resveratrol by de-sugaring process in rat stomach. In addition,
baicalin, the main metabolite of baicalein, could be detected
in rat stomach 20 min after oral administration of baicalein
and maintained at a stable concentration for at least 16 h (Liu
et al., 2009). Herein, we focused on the biotransformation of
AC in the stomach, the first site of metabolism in the
alimentary canal.
Time (min)
Flow (mL/min)
A (%)
B (%)
C (%)
0
30
60
120
0.6
0.6
0.6
0.6
35
80
90
0
65
20
10
0
0
0
0
100
Apparatus and operation conditions
The high-performance LC system consisted of a Waters 996
photo-diode array detector and a Waters 2695 HPLC
(Milford, PA) equipped with a Millennium 32 software
program (Milford, PA) for data analysis. Separation of the
components was performed on an Agilent extend C18 column
(4.6 ꢁ 150 mm2, 5 mm; Agilent Technologies Inc, Santa Clara,
CA) with a column temperature of 30 ꢂC. The mobile phase
(Table 1) consisted of mixed organic phase (A), water phase
(B) and pure methanol phase (C). Elution solvent A consisted
of 50% methanol and 50% acetonitrile, and elution solvent B
consisted of 0.2% acetic acid and 2% ammonia, which was
adjusted to the desired pH value (10.50) using ammonia
solution (1%, v/v). The mobile phase flow rate was 0.6 mL/
min. Just before the eluent entered the MS, its flow rate was
reduced to 0.3 mL/min by a split-flow valve.
Owing to the high toxicity, AC should be orally
administered with low dose, which causes low concentrations
of AC and its metabolites in the biological samples.
Therefore, it is necessary to choose a sensitive and specific
analytical method. Liquid chromatography/electrospray ioni-
zation/multiple-stage tandem mass spectrometry (LC/ESI/
MSn) can meet the above-mentioned requirements and has
been widely used in pharmaceutical research, especially in
studies of drug metabolism. Because AC-type alkaloids have
a strong gaseous basicity and proton affinity, they are also
suitable for ESI/MSn analysis.
In this study, LC/ESI/MSn in positive ion mode was used
to elucidate and identify the metabolites of AC. To
investigate the metabolism of AC sufficiently, rat and
rabbit were chosen as the model animals. To determine if
the acid environment in stomach will ‘‘metabolize’’ the AC,
comparative data of AC incubated in acidic solution were
also provided. Totally, 14 metabolites were unambiguously
identified or tentatively deduced by comparing their
protonated molecules, fragmentation pathways and chroma-
tographic behaviors with those of standard compounds and
reports in literature works. The elemental compositions of
some product ions were also analyzed by Fourier transform
ion cyclotron resonance mass spectrometry (FT-ICR-MS).
Finally, a biotransformation pathway of AC in the stomach
was proposed.
MS was performed on a LCQ ion trap mass spectrometer
(Finnigan, San Jose, CA) with an electrospray source. The
spraying voltage was set at 5.00 kV, the tube voltage at 15 V,
the capillary voltage at 17 V and the capillary temperature at
200 ꢂC. The LC fluid was nebulized using high-purity
nitrogen (N2). The sheath gas and auxiliary gas flow rates
were set at 60 and 10 arbitrary units, respectively. The
collision gas used for MSn was helium (He). The collision
energies (%) ranged from 25% to 40%, and the isolation width
was 1.5 mass units. Data were collected and analyzed by
Xcalibur software (version 1.0; Thermo Fisher Scientific, San
Jose, CA). The high resolution mass data were obtained on an
Ionspec 7.0 T FT-ICR-MS (Ionspec Corporation, Irvine, CA)
with a Z-spray electrospray source. Negative-ion mass spectra
were acquired after the solutions were directly infused into
the source region with a flow rate of 3.0 mL/min. The
parameters were set as follows: the source temperature 80 ꢂC,
probe temperature 100 ꢂC, sample cone voltage 30 V,
extractor cone voltage 5 V, probe high voltage 3.5 kV,
desolvation gas voltage 0.1 V and cone gas voltage 0.85 V.
The spectrum was scanned from m/z 108 to 1000 at 1024 K
data points, with an ADC rate of 2 MHz.
As the concentrations of different metabolites varied
significantly, the metabolites at low concentrations can only
generate small peaks in the ion chromatogram, resulting in
too little time for MSn analysis. To solve this problem, a
channel-switching technique was applied to enrich the
metabolites at low concentrations. In the interface between
LC and MS, a six-port switch was installed. This device has
been commonly used to reduce the flow rate of eluate from
LC to make it suitable for MS. Furthermore, it can also be
used to enrich the metabolites which are of low abundance
and unable to be analyzed online by MSn analysis. The total
ion chromatogram of certain sample, showing the retention
time and peak width of the ion peak of each metabolite, was
Materials and methods
Chemicals and reagents
The standard compounds, AC, mesaconitine and
hypaconitine (purity >99%), were purchased from the
National Institute for the Control of Pharmaceutical and
Biological Products (Beijing, China). Methanol, acetonitrile
and acetic acid were of HPLC grade and were purchased from
Thermo Fisher Scientific, Inc. (Waltham, MA). Purified water
was produced using a Milli-Q ultra-pure water system
(Billerica, MA). All other reagents and chemicals were of
analytical grade.

630 Z. Sui et al.
Xenobiotica, 2013; 43(7): 628–635
Table 2. The structures of AC and its analogs.
first obtained in full scan mode. Subsequently, the same
sample was injected for the second time into the LC/MS
system for MSn analysis. Utilizing the channel switch, the
metabolites at low concentrations in the eluate were collected.
The start time and collection duration of each metabolite
depended on its retention time and peak width. After being
concentrated by nitrogen stream, the collected sample was
injected to MS spectrometer at 3 mL/min by a syringe pump.
If the metabolite concentration in the eluate was still not high
enough, the enrichment process could be performed
repeatedly.
OH
OCH3
OCO
13
16
12
H3CO
14
17
4'
1'
10
1
9
8
2
2'
3'
11
15
OH
R1
N
OCOCH3
3
5
6
7
R2
4
18
19
H3CO
OCH3
Animal experiment and sample preparation
Alkaloids
R1
R2
MW
The animal protocol was approved by the Jilin University
Animal Ethics Committee. Ten rabbits (six months,
2500 ꢀ 50 g) and 10 rats (four weeks, 200 ꢀ 20 g) were
purchased from the Laboratory Animal Center of Jilin
University (Changchun, China) and were housed individually
in a room with a controlled temperature (22 ꢀ 1 ꢂC) and
humidity (60 ꢀ 5%), 12 h dark/light cycle for one week before
the experiments. The rabbits and rats were divided into two
equal groups (n ¼ 5), respectively, and fasted for 12 h prior to
use, but had free access to water. The animals in the dosed
group were orally administered with aqueous suspension of
AC (0.5 mg/kg), while the animals in the control group were
orally administered with only water. It is reported that the half
value period of AC in rabbits is 56 min after administration
(Wang et al., 2004). In this study, for the sake of producing
more metabolites, the time of sacrifice was set at 4 h, 4 times
longer than the half value period. The endogastric contents
were collected immediately after sacrifice and stored at
ꢃ80 ꢂC until analysis.
AC
C2H5
CH3
CH3
OH
OH
H
645
631
615
629
Mesaconitine
Hypaconitine
Deoxyaconitine
C2H5
H
potential metabolites. To obtain more extensive and detailed
mass spectral patterns of AC, two of its analogs, mesaconitine
and hypoconitine, were analyzed as well. The structures of
AC and its analogs are presented in Table 2.
LC/ESI/MSn analysis of aconitine, mesaconitine and
hypaconitine
Prior to analysis, the LC and MS conditions were optimized
to improve separation efficiency and signal strength, and the
LC/ESI/MSn method was performed to determine the
retention time and obtain mass spectral data for the standard
compounds. The LC chromatogram of standard compounds is
shown in Supplementary Figure 1.
Five grams of endogastric contents were diluted with
20 mL of water and shaken for 60 s. Then, the suspension was
centrifuged at 4000 r/min for 10 min. After centrifugation, the
upper layer was separated and collected, 20 mL of ethyl
acetate was added and the resulting solution was shaken for
5 min. Then, the mixed solution was centrifuged at 4000 r/min
for 10 min. After extraction, the upper phase was evaporated
to dryness under a stream of nitrogen gas at 30 ꢂC, and the
residue was dissolved in 0.5 mL of acetonitrile. The solution
was finally passed through a 0.45 mm nylon membrane, and an
aliquot of 10 mL filtrate was injected into the LC/ESI/MSn
system for analysis.
AC eluted at 47.6 min and yielded a [M þ H]þ at m/z 646.
The base peak in the MS2 spectrum, [M þ H ꢃ 60]þ, appeared
at m/z 586, corresponding to the elimination of acetic acid
formed by the C8-acetyl group. The absence of the
[M þ H ꢃ 60]þ from the MS2 spectrum of an AC metabolite
indicates that it does not possess C8 acetyl. The base peak in
the MS3 spectrum of AC, [M þ H ꢃ 60 ꢃ 32 ꢃ 28]þ, appeared
at m/z 526, corresponding to the combined loss of CO and
methanol. The literature has shown that the hydroxyl of C15
plays a key role in this process (Wang et al., 2002). Detection
of the [M þ H ꢃ 60 ꢃ 32 ꢃ 28]þ in the MS3 spectrum of an
AC metabolite indicates that it has the C15 hydroxyl group. In
the MS4 spectrum of the [M þ H ꢃ 60 ꢃ 32 ꢃ 28]þ, a product
ion [M þ H ꢃ 60 ꢃ 32 ꢃ 28 ꢃ 122]þ could be found at m/z
404, corresponding to the loss of benzoic acid produced by
The acid solution was prepared according to the simulated
gastric fluid with modification (Consolini et al., 2012). In
brief, 1 mL of acid solution contained 0.084 N HCl and
35 mM NaCl, with pH1.2. 20 mL of 1 mM AC were prepared
in acid solution and supplemented with 10 mg of standard
oleic and stearic acid, and then placed in a 37 ꢂC shaking
water bath for 4 h. Then, the incubated samples (20 mL) were
collected and processed following the same protocol of
endogastric contents for further analysis.
C
hypaconitine are shown in Supplementary Figure 2.
14 benzoyl group. The MSn spectra of AC, mesaconitine and
Compared with AC, mesaconitine and hypaconitine also
produce product ions arising from successive neutral losses of
acetic acid, methanol, CO and benzoic acid. Of course, the
mass of these product ions will shift depending on the
difference between their molecular weight and that of AC. It
is clear that the fragmentation mechanisms of these three
compounds display common patterns because of their similar
structures. However, comparison of the MS2 spectra of AC,
Results and discussion
Most metabolites have structural similarities to the parent
drug, and this may yield common fragmentation pathways.
Therefore, it is logical to use the fragmentation pathways
of parent drugs to identify the structures of their
mesaconitine and hypaconitine reveals
[M þ H ꢃ 18]þ. This product ion with low intensity could
a special ion

DOI: 10.3109/00498254.2012.753490
Metabolite profile analysis of aconitine in rabbit stomach 631
Figure 1. The main fragmentation pathways of AC.
be detected in the spectra of AC (m/z 628) and mesaconitine
(m/z 614), but not in the spectrum of hypaconitine. AC and
mesaconitine possess a C3 hydroxyl group, whereas hypaco-
nitine does not, linking detection of the [M þ H ꢃ 18]þ to
the presence of C3 hydroxyl group. Therefore, if the
[M þ H ꢃ 18]þ were detected in the MS2 spectrum of an
AC metabolite, the C3 hydroxyl group should exist in that
metabolite.
molecule of CH3COOH. The second loss of 60 Da (m/z
482 ! 422) was proposed to be due to the loss of neutral
molecules of CH3OH and CO. Thus, H1 was identified to be
14-O-debenzoylaconitine. The retention time of H2 was
20.6 min and the [M þ H]þ exhibited at m/z 604. The MS2
spectrum displayed [M þ H ꢃ 18 ꢃ 32]þ as the base peak at
m/z 554. [M þ H ꢃ 60]þ was not detected, suggesting that the
C8 acetyl does not exist in H2. The 42 Da (m/z 646 ! 604)
decrease in molecular weight indicates that H2 might be
deacetylated from AC. The loss of 50 Da from the precursor
ion (m/z 604 ! 554) was proposed to arise via the combined
loss of neutral molecules of CH3OH and H2O. Therefore, H2
was deduced as 8-O-deacetylaconitine, also named benzoyla-
conitine. The MSn spectra of H1 and H2 are shown in
Supplementary Figure 4.
The fragmentation pathways of AC contain some special
ions, called diagnostic ions, which indicate the presence of
specific groups. On the contrary, the absence of such
diagnostic ions indicates that the corresponding groups do
not exist. Four diagnostic ions, [M þ H ꢃ 18]þ, [M þ H ꢃ
60]þ, [M þ H ꢃ 60 ꢃ 32 ꢃ 28]þ and [M þ H ꢃ 60 ꢃ 32 ꢃ
28 ꢃ 122]þ were found in this study. The detection of these
ions are related to the C3 hydroxyl, C8 acetyl, C15 hydroxyl
and C14 benzoyl group, respectively, and play an important
role in the identification of metabolites of AC. The main
fragmentation pathways of AC are shown in Figure 1.
Identification of metabolites of AC in rabbit
For identifying the metabolites in rabbit stomach, the base
peak ion chromatogram of the endogastric contents, obtained
by LC/MS in full scan mode, is shown in Figure 2. Compared
with the control, 14 metabolites of AC were detected except
unchanged AC and its hydrolysis products. Each metabolite
was identified on the basis of its retention time, mass spectra
and fragmentation patterns generated by LC/ESI/MSn
analysis.
M1, M3 and M6 had LC retention times of 12.5, 31.1 and
51.3 min, respectively. All of them showed [M þ H]þ at m/z
662 (data not shown), 16 Da (m/z 646 ! 662) higher than AC,
suggesting that they were isomers of oxidized metabolites of
AC. For M1 and M3, their MS2 spectra both contained
[M þ H ꢃ 60]þ as the base peak at m/z 602, and the MS3
spectra of precursor ions [M þ H ꢃ 60]þ both showed
[M þ H ꢃ 60 ꢃ 32 ꢃ 28]þ as the base peak at m/z 542. The
base peaks of M1 and M3 were all 16 Da higher than that of
AC in the corresponding spectra, indicating fragmentation
patterns of M1 and M3 are similar to that of AC. In addition,
the [M þ H ꢃ 60 ꢃ 32 ꢃ 28 ꢃ 138]þ at m/z 404 was detected
in their MS3 spectra. Compared with the corresponding
[M þ H ꢃ 60 ꢃ 32 ꢃ 28 ꢃ 122]þ of AC (also at m/z 404), the
[M þ H ꢃ 60 ꢃ 32 ꢃ 28 ꢃ 138]þ suggested the occurrence of a
hydroxylation reaction on the C14 group. Considering that
there are three positions of hydroxylation on the benzene ring,
the specific position is uncertain. However, the base peak in
Identification of hydrolysis products of AC in acid
solution
The base peak ion chromatogram for LC/MS analysis of an
incubation of 1 mM AC with standard fatty acid in acid
solution is shown in Supplementary Figure 3. The peak
eluting at 49.1 min displayed the [M þ H]þ at m/z 646 and
corresponded to unmodified AC. The MS2 and MS3 spectra of
the [M þ H ꢃ 60]þ provided a number of product ions, such
as ions at m/z 586, 526 and 404, which are characteristic
fragment ions of AC.
Except unchanged AC, two hydrolysis products were
identified. H1 eluted at 11.4 min and showed the [M þ H]þ
at m/z 542, showing a loss of 104 Da (m/z 646 ! 542)
from AC. The MS2 spectrum showed the dominant ion
[M þ H ꢃ 60]þ at m/z 482 while the MS3 spectrum of
[M þ H ꢃ 60]þ generated a dominant ion at m/z 422. The
two dominant ions were 104 Da lower than the corresponding
ions (at m/z 586 and 526) of AC, respectively. Thus, the
metabolite had a fragmentation mode similar to AC,
indicating that their diterpenoid skeleton were intact. The
reduction of 104 Da in molecular weight showed that H1 was
a debenzoylated metabolite of AC. The first loss of 60 Da (m/z
542 ! 482) was proposed to arise via the loss of a neutral

632 Z. Sui et al.
Xenobiotica, 2013; 43(7): 628–635
Figure 2. The base peak ion chromatogram of AC and its metabolites formed in rabbit stomach, as monitored by LC/MS.
the MS2 spectrum of M6 was detected at m/z 542, a loss of
120 Da (m/z 662 ! 542) from the precursor ion. Through FT-
ICR-MS analysis, the elemental composition of C7H4O2 was
obtained, and the unsaturation value was calculated to be 6.
We deduced that the neutral loss must contain a benzene ring,
which came from the C14 benzoyl. This never-before-reported
fragmentation pathway is elucidated in Supplementary Figure
5, in which the hydroxylation occurs at the para-position of
the benzene ring (C4 position). Therefore, M6 was identified
as 40-hydroxylaconitine, and M1 and M3 were deduced to be
20-hydroxylaconitine or 30-hydroxylaconitine. The LC–MSn
spectra of M1, M3 and M6 are shown in Supplementary
Figure 6.
M4 eluted at 36.4 min and its MS2 spectrum showed the
[M þ H]þ at m/z 632, a loss of 14 Da (m/z 646 ! 632) from
AC. The MS2 spectrum and MS3 spectrum of M4 exhibit
similar patterns to those of AC. The data suggest that M4 is a
demethylated metabolite of AC. If the demethylation occurred
on the ethyl group of the N atom, mesaconitine would be
produced and the retention time would be 37.6 min. However,
the retention time of M4 was less than that of mesaconitine.
Because the C16, C1, C6 and C18 positions all possess
methoxyl group, demethylation could occur at any of these
positions, making the specific position uncertain. Therefore,
M4 was deduced to be demethylaconitine.
M2 had a retention time of 28.7 min and showed the
[M þ H]þ at m/z 618. The MS2 spectrum of the [M þ H]þ
generated a base peak at m/z 558, and the base peak of the
MS3 spectrum was detected at m/z 526. The protonated
molecule of M2 was 28 Da (m/z 646 ! 618) lower than that
of AC and was 14 Da lower than the protonated molecule
of M4, suggesting that M2 was a further demethylated
derivative of M4 or a N-de-ethylated product of AC.
Therefore, M2 was identified as didemethylaconitine or
N-de-ethylaconitine.
M5 and M7 had retention times of 46.5 and 58.2 min,
respectively. Both of them exhibited protonated molecule at
m/z 630, a reduction of 16 Da (m/z 646 ! 632) from AC,
suggesting they were isomers of dehydroxylated derivatives
of AC. The difference between M5 and M7 lies in the position
of dehydroxylation in AC. Hydroxyl group existed at
positions C3, C13 and C15, suggesting dehydroxylation could
occur at any of them. According to the fragmentation
pathways discussed above, the [M þ H ꢃ 18]þ is generated
by the loss of water from the C3 hydroxyl group, and the
[M þ H ꢃ 60 ꢃ 32 ꢃ 28]þ is generated only when the C15
hydroxyl group exists. Comparing the MSn spectra of M5 and
M7, the [M þ H ꢃ 60 ꢃ 32 ꢃ 28]þ was not detected in the MS3
spectrum of M5, and the [M þ H ꢃ 18]þ was not detected in
the MS2 spectrum of M7. Hence, M5 was identified to be
indaconitine which lost the hydroxyl of C15, and M7 was
identified as deoxyaconitine which lost the hydroxyl of C3.
The LC–MSn spectra of M4, M2, M5 and M7 are shown in
Supplementary Figure 7.
All AC-type alkaloids share a common C19-norditerpe-
noid skeleton and they can be classified into several types
according to the groups at the C8 and C14 positions of the
skeleton. When an AC-type alkaloid has a benzoyl group at
the C14 position and a fatty acyl group at the C8 position, it
is called lipoaconitine. In this study, some metabolites of
lipoalkaloid type were also detected. M8 eluted with a
retention time of 70.4 min and showed the [M þ H]þat m/z
978. The MS2 spectrum contained a base peak at m/z 586,
which was selected for the MS3 analysis. Its MS3 spectrum
provided characteristic product ions at m/z 526 and 554,
showing the same fragmentation pathway as that of the m/z
586 ion produced by AC. So, we can deduce that the two
product ions at m/z 586 are the same ion. The only
difference between M8 and AC lies in the groups at the C8
position. The loss of 392 Da (m/z 978 ! 586) suggested that
M8 lost a hexacosandienoic acid from the C8 position.
Therefore, M8 was identified as 8-hcdo-benzoylaconine.
Here, ‘‘hcdo’’ represents the residue of hexacosandienoic
acid. According to the characteristic fragmentation patterns
of lipoaconitines, if a metabolite has a higher molecular
weight than AC, its MS2 spectrum shows main product ion
at m/z 586 and the MS3 spectrum of precursor ion (m/z
586) is identical to that of the [M þ H ꢃ 60]þ derived from
AC, it can be logically concluded to be a lipoaconitine.
Once the fatty acids are identified, their structures can be
elucidated. Then, other lipoaconitines can be identified
according to the identification process of M8. The MSn
spectra of these lipoaconitines are shown in Supplementary
Figure 8.
Based on their chromatographic behaviors and MSn data,
all the metabolites in the rabbit endogastric contents were
identified and are presented in Tables 3 and 4. Proposed
metabolism pathways of AC are shown in Figure 3.

DOI: 10.3109/00498254.2012.753490
Metabolite profile analysis of aconitine in rabbit stomach 633
Table 3. Data from AC, its hydrolysis products (H1 and H2) and its metabolites (M1–M7).
Compound
TR (min)
MS (m/z)
MS2 (m/z)
MS3 (m/z)
Identification
14-O-Debenzoylaconitine
H1
11.6
12.5
20.4
28.7
31.1
36.4
46.5
48.2
51.3
58.2
542
662
604
618
662
632
630
646
662
630
482
602
554
558
602
572
570
586
542
570
422
542
522
526
542
512
520
526
482
510
M1
H2
20-Hydroxylaconitine or 30-Hydroxylaconitine
8-O-Deacetylaconitine
M2
M3
M4
M5
AC
M6
M7
Didemethylaconitine or N-de-ethylaconitine
30-Hydroxylaconitine or 20-hydroxylaconitine
Demethylaconitine
Indaconitine
Aconitine
40-Hydroxylaconitine
Deoxyaconitine
Table 4. Data from AC’s metabolites (M8–M14).
Compound
TR (min)
Parent ion (m/z)
Neutral loss (Da)
Daughter ion (m/z)
Fatty acid
Identification
M8
70.4
84.3
89.5
91.2
98.9
100.7
114.6
978
864
828
866
842
868
870
392
278
242
280
256
282
284
586
586
586
586
586
586
586
Hexacosandienoic (hcdo)
Linolenic (lin)
Pentadecanoic (pdo)
Linoleic (lol)
Palmitic (pal)
Oleic (ole)
Stearic (ate)
8-Hcdo-benzoylaconine
8-Lin-benzoylaconine
8-Pdo-benzoylaconine
8-Lol-benzoylaconine
8-Pal-benzoylaconine
8-Ole-benzoylaconine
8-Ste-benzoylaconine
M9
M10
M11
M12
M13
M14
Figure 3. The proposed biotransformation process of AC in rabbit stomach.
Identification of metabolites of AC in rat
Up to now, some in vivo/in vitro studies have been reported
on metabolism of AC in human and animal species. However,
there has been no report available on the metabolism of AC in
stomach, which is generally considered an organ with little
contribution on drug metabolism. In this paper, we focused on
investigating the metabolism of AC in stomach and found a
total of 14 metabolites in the stomachs of rabbits and rats,
indicating that the metabolism of AC dose exists in animal
stomachs. Among these metabolites, didemethylaconitine or
N-de-ethylaconitine (m/z at 618) and demethylaconitine (m/z
at 632) have been detected in human liver microsomes
The base peak ion chromatogram of the endogastric
contents of rat, obtained by LC/MS in full scan mode, is
shown in Figure 4. Totally, 10 metabolites were detected
compared with the control and all of them have been
detected in endogastric contents of rabbit. The metabolite
profiling of AC obtained from stomachs of rabbit and rat is
very similar except that the metabolites in rat are fewer. All
the metabolites in the rat endogastric contents were
identified and are summarized in Supplementary Tables 1
and 2.

634 Z. Sui et al.
Xenobiotica, 2013; 43(7): 628–635
Figure 4. The base peak ion chromatogram of AC and its metabolites formed in rat stomach, as monitored by LC/MS.
(HLMs) (Tang et al., 2011; Wang et al., 2006). Three isomers
of hydroxylaconitine (m/z at 662) and their deduced structures
are reported in the present study for the first time. Although
one isomer of hydroxylaconitine has been detected in HLMs
by Tang et al. (2011), the exact position of hydroxylation was
not been clarified. For the two metabolites at m/z 630,
deoxyaconitine has been identified when AC was incubated
with human intestinal bacteria in vitro (Zhao et al., 2007)
while indaconitine was firstly reported to be transformed from
AC in this work. With regard to the identified lipoaconitines,
most of them have been detected previously in human
intestinal bacteria incubation system except for 8-hcdo-
benzoylaconine and 8-lin-benzoylaconine (Zhao et al.,
2008). Meanwhile, for the two hydrolyzates, 8-O-deacetyla-
conitine (m/z at 604) was reported by many previous studies
(Kentaro et al., 2005; Zhang et al., 2005) whereas 14-O-
debenzoylaconitine (m/z at 542) was scarcely mentioned.
It is generally considered that few metabolites would be
produced in stomach for its minimal contribution to
metabolism. However, for AC, its metabolites found in
stomachs of animals (rats and rabbits) are abundant. This
indicates that the stomach, commonly considered a digestive
organ, also plays an important role in metabolism of some
compounds, such as diterpenoid alkaloids. Maybe it is
because that some components in stomach, such as gastric
enzymes, can catalyze the transformation of AC. In addition,
it was reported that AC could be metabolized into some
lipoaconitines by incubation with intestinal bacteria (Zhao
et al., 2008), suggesting that the bacteria in stomach may also
play a role in the metabolism of AC.
development of orally administrated drug, such as diterpenoid
alkaloids, the investigation on their stability and metabolite
profiling in stomach should not be overlooked.
Conclusion
A sensitive and specific LC/ESI/MSn method has been
achieved for the general analysis of AC and its metabolites
in animal stomach. Elucidation and identification of these
metabolites were performed by comparing the protonated
molecules, fragmentation pathways and retention times with
those obtained from standard compounds and authorized
literature works. In the fragmentation pathways of AC-type
alkaloids, there are some diagnostic ions, such as
[M þ H ꢃ 18]þ, [M þ H ꢃ 60]þ, [M þ H ꢃ 60 ꢃ 32 ꢃ 28]þ
and
[M þ H ꢃ 60 ꢃ 32 ꢃ 28 ꢃ 122]þ/[M þ H ꢃ 60 ꢃ 32 ꢃ
28 ꢃ 138]þ, which are closely related to specific groups and
play an important role in structural identification. AC
biotransformation in the stomach occurred mainly by
demethylation, hydrolysis, hydroxylation, dehydroxylation
and ester-exchange reactions. Among the 14 metabolites,
three hydroxylated metabolites have never been reported, and
seven lipoaconitines were detected in vivo for the first time.
The metabolite profiling of AC in stomach suggested that
stomach might play an important role in the metabolism of
aconitum alkaloids.
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of this article.
This work was supported by the 973 Program (Nos
2011CB505300 and 2011CB505305) and the National
Natural Sciences Foundation of China (No. 81173507).
It is reported that lipoaconitines and monoester ACs have
lower toxicity than AC (Wang et al., 2003), so do indaconitine
and deoxyaconitine. Furthermore, lipoalkaloids are reported
to have obvious anti-inflammatory effect, which is signifi-
cantly influenced by the grade of unsaturation and length of
the fatty acid chain (Borcsa et al., 2011). Therefore, the
metabolism of AC in stomach can change its intrinsic
activities to some extent.
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