Effect of pH on the metabolism of aconitine under rat intestinal bacteria and analysis of metabolites using HPLC/MS-MSn technique

Article abstract of DOI:10.1002/cjoc.201190028

A semi-quantitative method of mass spectrometry (MS) has been described for the analysis of metabolites of aconitine by rat intestinal bacteria at different pH. At pH 7.0, the rat intestinal bacteria exhibit optimal activity for the metabolism of aconitine. A high-performance liquid chromatography- electrospray ionization multiple-stage mass spectrometry (HPLC/ESI-MS ⁿ) method has been applied to investigate the characteristic product ions of metabolites. Then, the logical fragmentation pathways of metabolites have been proposed. By comparing the retention time (t_R) of HPLC and the ESI-MSⁿ data with the data of standard compounds and reports from literature, ten metabolites have been identified and a distinctive metabolite (15-deoxyaconitine) has been deduced first time. The experimental results demonstrate that HPLC/ESI-MSⁿ is a specific and useful method for the identification of metabolites of aconitine. Also, in the present paper, the HPLC-MS method was introduced to determine the synthetical metabolite prior to the study of the toxicity by the method of Bliss. HPLC chromatogram of metabolites of aconitine at pH 7. 1. benzoylaconine, 2. 15-deoxybenzoylaconine, 3. 8-octenoyl-benzoylaconine, 4. 10-OH-aconitine, 5. 15-deoxyaconine, 6. aconitine, 7. 8-hexenoyl-benzoylaconine, 8. 8-propionyl-benzoylaconine, 9. 8-butyryl-benzoylaconine, 10. deoxyaconitine, 11. 8(3-hydroxyl)-butyryl- benzoylaconine.

Full text of DOI:10.1002/cjoc.201190028

FULL PAPER
Effect of pH on the Metabolism of Aconitine under Rat
Intestinal Bacteria and Analysis of Metabolites
Using HPLC/MS-MSn Technique
Wang, Xiyea,b(王曦烨)
Pi, Zifenga(皮子风)
Liu, Wenlonga,b(刘文龙)
Zhao, Yufenga,c(赵宇峰)
Liu, Shuying*,a(刘淑莹)
a Changchun Center of Mass Spectrometry, Changchun Institute of Applied Chemistry, Chinese Academy of
Sciences, Changchun, Jilin 130022, China
b Graduate School of the Chinese Academy of Sciences, Beijing 100039, China
c Shanghai Center of System Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China
A semi-quantitative method of mass spectrometry (MS) has been described for the analysis of metabolites of
aconitine by rat intestinal bacteria at different pH. At pH 7.0, the rat intestinal bacteria exhibit optimal activity for
the metabolism of aconitine. A high-performance liquid chromatography-electrospray ionization multiple-stage
mass spectrometry (HPLC/ESI-MSn) method has been applied to investigate the characteristic product ions of me-
tabolites. Then, the logical fragmentation pathways of metabolites have been proposed. By comparing the retention
time (tR) of HPLC and the ESI-MSn data with the data of standard compounds and reports from literature, ten me-
tabolites have been identified and a distinctive metabolite (15-deoxyaconitine) has been deduced first time. The ex-
perimental results demonstrate that HPLC/ESI-MSn is a specific and useful method for the identification of metabo-
lites of aconitine. Also, in the present paper, the HPLC-MS method was introduced to determine the synthetical
metabolite prior to the study of the toxicity by the method of Bliss.
Keywords aconitine, metabolism, intestinal bacteria, mass spectrometry, 8-butyryl-benzoylmesaconine
Introduction
the elimination rules of substituents in ESI-MSn for six
DDAs.11
Aconite tuber is an important and toxic crude drug
used in traditional Chinese medicine. Aconitine-type
alkaloids isolated from aconite have been shown to have
potential toxicity and wide bioactivity. The majority of
aconite alkaloids have an aconitane skeleton where aco-
nitine is the most important representative alkaloid. As
an analgesic and anaesthetic agent for the treatment of
rheumatism and neuralgia, it has been reported that the
toxicity of this herb apparently decreases after process-
ing because the fatally toxic alkaloids transform to less
toxic alkaloids, such as aconine, benzoylaconine and
lipo-aconitine in processing.1-5 Lipomesaconitine, one
of the lipo-alkaloids, was found to exhibit anti-inflam-
matory and analgesic activities.4 Deoxyaconitine is also
one of the toxic and active ingredient in aconite tuber.6
Studies of fragmentation pathways and esterified re-
actions of aconitine-type alkaloids had been published
by means of ESI-MSn several yeas ago.7-9 A method for
the direct determination of alkaloid profiling in plant
tissue by using MALDI-TOFMS has been developed.10
Quantum chemistry calculations were carried out to
theoretically analyze the stability of fragment ions and
Aconitine was found to be converted to
lipo-aconitine by GC-MS method after incubation with
human intestinal bacteria.12 In our laboratory, the bio-
transformation of aconitine in human intestinal bacteria
has been explored by using electrospray ionization mass
spectrometry.13,14 However, as the most previous studies,
the metabolism of aconitine was conducted at a fixed
pH. As a matter of fact, the pH of the intestinal contents
always changes with the different regions in rat.15,16 On
the other hand, although HPLC, LC/MS method are the
most common tools for the quantitative analysis of
natural products or metabolites in blood and urine,17-22
the analysis of lipo-alkaloids is difficult by chromatog-
raphy owing to their structural similarity.
In the present paper, the effect of pH on the metabo-
lism of aconitine under rat intestinal bacteria has been
explored by the semi-quantitative MS method. The ref-
erence metabolites of aconitine have been determined
by LC/MSn in the positive ion mode. The LC/MSn study
of the retention time and ESI-MSn data of aconitine and
its metabolites have been carried out. Also, two isomers
*
E-mail: syliu19@yahoo.com.cn, mslab21@ciac.jl.cn; Tel.: 0086-431-85262886; Fax: 0086-431-85262886
Received December 15, 2009; revised August 24, 2010; accepted September 19, 2010.
Project supported by the Major State Basic Research Development Program of China (973 program) (No. 2006CB504706) and the National Natural
Science Foundation of China (Nos. 30672600, 30772721).
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Analysis of Metabolites Using HPLC/MS-MSn Technique
have been detected by HPLC/ESI-MSn. The application
of HPLC-MS method was illustrated in the determina-
tion of synthetical metabolite prior to the study of the
toxicity by the method of Bliss.
residue was dissolved in ether (10 mL×3). The reaction
mixture was evaporated to dryness, and dissolved in
15% NaHCO3 and H2O (2.5 mL each), the pH of which
was adjusted to 7.0. Then the solution was extracted
with CHCl3 (5 mL×3). After CHCl3 was evaporated to
dryness, the residue was dissolved in acetonitrile (10 µL)
for HPLC-ESI/MSn analysis and the measurement of
LD50.
Experimental
Instrumentations and reagents
Anaerobic Chamber system (Thermo, America). The
standard sample of reserpine was purchased from Sigma
Corporation (USA). Methanol and acetonitrile were of
HPLC grade (Fisher). 28% ammonia solution, diethyl
ether, chloroform and dichloromethane were of analyti-
cal grade, and butyric acid, toluene and pyridine were of
chemical pure. Deionized water was prepared using a
Millipore (Billerica, MA, USA) water purification sys-
tem. General anaerobic medium (GAM) was purchased
from Nissui Co. (Tokyo, Japan). In the experiments of
rat intestinal bacteria, 1.29 mg of aconitine was dis-
solved in 200 µL of acetonitrile (0.02 mmol/L), and 1
mg of reserpine was dissolved in 100 mL of methanol.
Apparatus
The high performance liquid chromatograph (HPLC)
system consisted of a Waters (Milford, MA, USA) 2695
HPLC with a photodiode-array detector Waters 2996 set
at 235 nm. The chromatographic conditions were as
follows: column, Agilent Extent C18, 150 mm×4.6 mm,
5 µm; eluent: (A) water (containing 35 mmol ammo-
nium acetate and adjusted with 28% ammonia solution
to pH 10.5); (B) acetonitrile; (C) methanol. Gradient
elution was with 65%—55% A and 17.5%—22.5% B
and 17.5%—22.5% C from 0 to 20 min, 55%—40% A
and 22.5%—30% B and 22.5%—30% C from 20 to 40
min, 40%—10% A and 30%—0% B and 30%—90% C
from 40 to 60 min, 10%—5% A and 90%—95% C from
60 to 120 min, 5%—0% A and 95%—100% C from
120 to 150 min. The flow-rate was 0.5 mL/min and the
temperature was 30 ℃. Mass spectrometry instrument
was connected to the HPLC system via the UV cell out-
let. Table 1 shows the linear gradient of mobile phase.
Animals
Wister rats and Kunming mice were purchased from
Laboratory Animal Center of Jilin University. They
were housed under normal laboratory conditions (21±2
℃, 12 h light-dark cycle) with free access to standard
rodent chow and water. The experiments were con-
ducted in accordance with the guidelines and with the
approval of Animal Research Ethics Committee of Jilin
University.
Table 1 The linear gradient of mobile phase
Mobile phase/%
Time/min
Methods of metabolism
A
B
C
Rat feces (0.2 g) was anaerobically cultured in GAM
broth (10 mL×4) for 24 h at 37 ℃ and then centrifuged
at 5000 r/min for 10 min. The precipitate of four broths
0— 20
20— 40
65— 55
55— 40
40— 10
17.5— 22.5
22.5— 30
17.5— 22.5
22.5— 30
30— 90
were suspended in 0.2 mol•L－ phosphate buffer ac-
1
40— 60
30—
0—
0
cording to pH 5.8, 6.2, 6.6 and 7.0, respectively. 1.29
mg of aconitine was added to the suspension of different
pH, respectively, and the mixture was anaerobically
incubated at 37 ℃ for 5 d. 200 µL of the incubation
mixture was extracted with dichloromethane (1 mL,
twice). 5 µL of the dichloromethane extract was diluted
to 500 µL with chloroform and methanol (95∶5, V∶V)
and then 70 µL of reserpine (Mr 608.7) was added to the
500 µL of mixture . The mixture was filtrated and ana-
lyzed by ESI-MS/MSn. In addition, 2 mL of the di-
chloromethane extracted from pH 7.0 phosphate buffer
was evaporated to give a residue. The residue was dis-
solved in 200 µL of acetonitrile, then was filtered
through a 0.45 µm membrane and 10 µL of the filter
solution was used for the HPLC/ESI-MSn analysis.
60— 120
120— 150
10—
5—
5
0
90— 95
0
0—
0
95— 100
Both MSn and LC/MSn injected by syringe pump and
LC/MSn were performed using an LCQ ion trap mass
spectrometer (Finnigan, San Jose, CA, USA) equipped
with an electrospray source and capable of analyzing
ions up to m/z 2000. The spray voltage was 5.0 kV in
the positive ion mode. The sheath gas flow rate was
fixed at 60 a.u. and capillary temperature was main-
tained at 240 ℃. Collision energies for the MSn analy-
ses ranged from 25% to 40% arbitrary units depending
on the mass of the precursor ion, the collision gas used
for MS/MS was He, and isolation width was 2.
Synthetical methods of metabolite
Results and discussion
Butyric acid (1 mL) was added to a solution of aco-
nitine (40 mg) in toluene (50 mL) and pyridine (0.5 mL)
was used as catalyst. The mixture was refluxed at 100
℃ for 10 h. After the vacuum distillation at 60 ℃, the
Effect of pH on the metabolic activity of aconitine
Table 2 showed some possible structures of metabo-
lites of aconitine by rat intestinal bacteria.
Chin. J. Chem. 2010, 28, 2494— 2500
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Wang et al.
FULL PAPER
Table 2 Some possible structures of aconitine and its metabo-
lites by rat intestinal bacteriaa
pH 7.0.
It was interesting that some metabolites whose mo-
lecular mass was bigger than that of original aconitine
(m/z 646), such as, m/z 674 corresponding to
8-butyryl-benzoylaconine were produced at pH 7.0.
This may be generated by adding a group on the C-8.
Therefore the metabolism is a complicated process,
which involved not only decomposition but also addi-
tion reactions.
OH
OMe
OMe
R4
OCOC6H5
R1
R2
N
OH
OR3
OMe
CH2OMe
R1
C2H5 OH Ac
C2H5 Ac
C2H5 OH Ac OH
C2H5 OH
R2 R3 R4
Compound
Aconitine
m/z (MH＋)
646
One possible explanation is that the rat intestinal
bacteria can exhibit a better activity for the metabolism
of aconitine with the escalation of pH (in the range be-
tween 5.8 and 7.0), while the reduction of pH will result
in less metabolites quantitatively and qualitatively.
To confirm above point, we introduced the semi-
quantitative analysis of reserpine as internal standard.
As shown in Figure 2, the relative abundance of the re-
serpine ion was considered as 100% and we scaled th＋e
metabolites analyzed at 100×[metabolite]＋/[reserpine]
ratio. Thus, the relative abundance of the ion at m/z 646
according to residual aconitine standard after incubation
with rat intestinal flora for 5 d at pH 5.8, 6.2, 6.6 and
7.0 corresponded to 103.22, 81.09, 21.49 and 10.15,
respectively. In addition, with the escalation of pH, the
relative abundance of ion at m/z 604 corresponding to
benzoylaconine increased markedly from the values
16.04, 4.71, 9.84 to 39.72, and the relative abundance of
the ion at m/z 674 corresponding to 8-butyryl-benzoyl-
aconine increased from 0.66, 0.89, 1.04 to 6.13, respec-
tively.
H
H
H
Deoxyaconine
10-OH-Aconitine
Benzoylaconine
630
662
H
H
604
a If R3 are occupied by octenoyl, hexenoyl, propionyl, butyryl,
and 3-hyrdoxy-butyryl groups, the corresponding s-DDAs will be
formed.
The pH of the contents of the regions including
duodenum, upper small intestine, lower small intestine,
cecum and colon in rat corresponded to about 6.2, 6.6,
6.6, 6.2 and 6.6,15,16 respectively, and a pH of 7.0 was
also used because this was the pH of intestinal contents,
therefore, four spots were selected in the approximate
range 6.0—7.0.
Figure 1 was the ESI mass spectra in positive ion
mode for metabolites of aconitine at different pH. The
ion at m/z 646 corresponding to aconitine was detected
as the base peak at pH 5.8 and 6.2, which meant that
large amount of aconitine was not metabolized at lower
pH regions. While the base peaks were altered into the
586 and 604 at pH 6.6 and 7.0, respectively. Ion of 604
is the hydrolysis product, and 586 is the dissociating
product of losing an acetic acid from aconitine.7 In ad-
dition, although the relative abundance of the ion at m/z
604 was very low at pH 5.8, 6.2 and 6.6, m/z 604 be-
came the base peak, and 586 was a pretty large one at
Figure 2 The semi-quantitative analysis of aconitine and its
metabolites by reserpine.
The relative intensity of ion at m/z 586 correspond-
ing to deacetoxy aconitine, which in fact was a decom-
position product via losing a neutral acetic acid mole-
cule from aconitine, showed the change from 44.38,
49.36, 48.10 to 19.74 with the escalation of pH. At the
pH 5.8, 6.2 and 6.6, the relative abundances of deace-
toxy aconitine did not change obviously, but at pH 7.0,
the relative abundance of deacetoxy aconitine decreased
intensely. The dissociation reaction mentioned above
depends on pH and the reaction may be rather slow at
neutral condition under rat intestinal flora.
Figure 1 ESI mass spectra of metabolites of aconitine by rat
intestinal flora at pH 5.8 (A), 6.2 (B), 6.6 (C) and 7.0 (D).
The results provide a direct and credible evidence
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Chin. J. Chem. 2010, 28, 2494— 2500

Analysis of Metabolites Using HPLC/MS-MSn Technique
that the metabolic activity of rat intestinal bacteria is
highest for metabolism of aconitine, and most amounts
of metabolites can be obtained at pH 7.0. Furthermore,
from above study, it can be speculated that with the
prolongation of metabolic time, the acids released by rat
intestinal bacteria will increase the acidity of the phos-
phate buffer and negatively affect the conversion rate of
aconitine to its metabolites.12
CH3OH]＋ and 520 [F－H2O－CH3OH]＋ were de-
tected. If the hydrolytic reaction happened at C-13, the
ion at m/z 510 [F－CH3OH－CO]＋ could be detected
as base peak. But the MS3 spectrum of peak 5 did not
find m/z 510 ion at all. Therefore, this result strongly
suggested that the hydrolytic reaction occurred at C-15,
and the C＝C bond between C-8 and C-15 could not
conjugate with the OH group at C-15, resulting in the
impossible elimination of CO ion. Thus peak 5 was
identified as 15-deoxyaconitine and it was found in the
metabolites of aconitine for the first time.
HPLC/MS-MSn analysis of metabolites of aconitine
at pH 7.0
Figure 3 showed the HPLC spectrum of metabolites
of aconitine at pH 7.0.
Scheme 1 Proposed mechanism for successive losses of AcOH
and CO from protonated aconitine-type alkaloids
OH
OH
OMe
16
OCOC6H5
OMe
16
OCOC6H5
12
OMe
12
OMe
13
10
13
10
14
9
14
9
-AcOH
2
R1
2
1
1
15
15
11
11
R1
R2
8
8
5
N
5
N
OH
OH
6
6
3
3
R2
OAc
4
7
4
7
OMe
CH2OMe
OMe
CH2OMe
OH
OH
OMe
OCOC6H5
OMe
16
OCOC6H5
12
OMe
13
10
OMe
14
9
-CO
2
R1
1
15
11
R1
R2
N
8
5
N
Figure 3 HPLC chromatogram of metabolites of aconitine at
pH 7. 1. benzoylaconine, 2. 15-deoxybenzoylaconine, 3. 8-oct-
enoyl-benzoylaconine, 4. 10-OH-aconitine, 5. 15-deoxyaconine,
6. aconitine, 7. 8-hexenoyl-benzoylaconine, 8. 8-propionyl-benz-
oylaconine, 9. 8-butyryl-benzoylaconine, 10. deoxyaconitine, 11.
8(3-hydroxyl)-butyryl-benzoylaconine.
O
6
3
R2
4
7
OMe
CH2OMe
OMe
CH2OMe
The retention time (tR) of peak 6 was at 48.5 min and
it corresponded to the protonated molecule [M＋H]＋ at
m/z 646. The m/z 646 ion produced the fragment ion at
m/z 586 corresponding to [M＋H－AcOH]＋ as the base
peak in its MS2 spectrum. The further CID spectrum of
the fragment io＋n of m/z 586 included＋product ions at m/z
568 [F－H2O] , 554 [F－CH3OH] , 536 [F－H2O－
CH3OH]＋, and 526 [F－CH3OH－CO]＋. Comparing
above results with the data of standard component, peak
6 was confirmed as aconitine.7 (F stands for M＋H－
AcOH for the sake of convenience).
Peak 5 and peak 10 were eluted at 46.4 and 57.9 min,
respectively, and they both showed the [M＋H]＋ at m/z
630 corresponding to the loss of 16 Da of aconitine.
Furthermore, two ions both produced the fragment ions
[M＋H－AcOH]＋ at m/z 570 as the base peaks in the
MS2 spectra. In the MS2 spectrum of peak 10, it was
worth n＋oticing that there was no fragment ions [M＋H
－H2O] at m/z 612 (data not shown). In the MS3 spec-
trum of the ion (peak 10, showed in Figure 5), the data
were similar to deoxyaconitine.24 Thus, peak 10 was
identified as deoxyaconitine. It was sure that there were
three OH groups on the C-3, C-13 and C-15 position on
the skeleton of aconitine molecule, respectively. Since
C-3 hydrolysate of aconitine according to peak 10 was
identified as deoxyaconitine, estimating the hydrolytic
reaction to occurr at C-15 or C-13 became the next step.
As far as we know, the characteristic fragmentation
pathway of protonated aconitine-type alkaloids in the
MS3 spectra was shown in Scheme 17 and the [F－
CH3OH－CO]＋ ion was detected as the base peak for
almost all the aconitine-type alkaloids＋except the hy-
paconitine whose [F－CH3OH－CO] ion was ob-
served as the second major peak. But in the MS3 spec-
trum of the ion (peak 5), only the ions at m/z 538 [F－
From Figure 3, it could be seen that the major me-
tabolites were compounds 1, 7—10 and 2—5 were mi-
nor ones. The retention time of peak 1 was 19.2 min,
and it corresponded to the predominate ion [M＋H]＋ at
m/z 604 which was the hydrolysis product. The MS2
spectrum showed that the ion of m/z 604 produced
fragment ions at m/z 586, 572 and 554, corresponding to
neutral losses of H2O, CH3OH, and H2O＋CH3OH, re-
spectively. Thus, peak 1 was confirmed as benzoyla-
conine derived from the hydrolysis of aconitine.
Peak 9 was elu＋ted at 57.0 min and it showed the in-
tense ion [M＋H] at m/z 674. The m/z 586 ion from
neutral loss of 88 Da was observed as base peak in the
MS2 spectrum of the m/z 674 ion and the MS3 spectrum
of the 586 ion was consistent with that of aconitine
(showed in Figure 4), suggesting the presence of
8-butyryl-benzoylaconine23 corresponding to peak 9.
Chin. J. Chem. 2010, 28, 2494— 2500
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FULL PAPER
m/z 584 [F－H2O]＋, 570 [F－CH3OH]＋ and 542 [F－
CH3OH－CO]＋ were found. The difference between
peak 6 (aconitine) and peak 4 was the simultaneous
neutral losses of H2O and CH3OH in the MS3 spectra
and peak 4 was deduced as 10-OH-aconitine.
Peak 2 was eluted at 33.9 min and showed the pro-
tonated molecule [M＋H]＋ at m/z 588. The m/z 588 ion
produced the fragment ions at m/z 570 [M＋H－H2O]＋,
556 [M＋H－CH3OH]＋ and 538 [M＋H－CH3OH－
H2O]＋ and the ion at m/z 528 [M＋H－CH3OH－CO]＋
was not present in the MS2 spectrum. This result was
similar to the fragmentation pathways of peak 5 in the
MS3 spectrum and it was proposed that the m/z 588 ion
corresponding to neutral loss of 42 Da of
15-deoxyaconitine was identified as 15-deoxybenzoyl-
aconine.
Figure 4 The MS3 spectrum of the 586 ion of peak 9.
Proposed mechanism for all the metabolites of aco-
nitine was shown in Scheme 2. Table 3 showed the
HPLC/ESI-MSn data of aconitine and its metabolites in
the positive ion mode.
Figure 5 The MS3 spectra of peak 5 (A) and peak 10 (B).
Study of toxicity of the metabolite by HPLC-MS
method
In the same way as peak 9, peaks 3, 7, 8 and 11
eluted at 36.0, 52.8, 54.9 and 59.4 min, could be con-
firmed as 8-octenoyl-benzoylaconine, 8-hexenoyl-benz-
oylaconine, 8-propionyl-benzoylaconine and 8(3-hy-
droxyl)-butyryl-benzoylaconine,23 respectively.
Peak 4 was eluted at 44.1 min and showed the pro-
tonated molecular ion [M＋H]＋ at m/z 662. In the MS2
spectrum of the ion, the 662 ion ＋produced the fragment
ion at m/z 602 [M＋H－AcOH] as the base peak. In
the MS3 spectrum at m/z 662＞602, the product ions at
Based on above HPLC-MS/MSn analysis, it could be
speculated that aconitine could be converted into some
alkaloids in which the C-8 positions were occupied by
short-chain acid acyl groups, such as propionyl, butyryl,
but not by long-chain fatty acid acyl group, such as
oleoyl, stearoyl. Actually, the properties of these special
alkaloids were similar to the diester-diterpenoid alka-
loids (DDAs) and they were conveniently denoted
Scheme 2 Proposed mechanism for all the metabolites of aconitine (R denote octenoyl, hexenoyl, propionyl, butyryl, and
3-hydroxy-butyryl)
(1)
OH
OMe
(10)
OH
(4)
OMe
OCOC6H5
OH
OMe
OMe
C2H5
HO
N
OH
OMe
N
OMe
N
OCOC6H5
OH
OMe
OCOC6H5
OH
CH2OMe
C2H5
C2H5
HO
OH
OH
OAc
OMe
OAc
OMe
(6)
OH
CH2OMe
CH2OMe
OMe
OMe
N
OCOC6H5
C2H5
HO
OH
OAc
OMe
(3) (7) (8) (9) (11)
OH
CH2OMe
OMe
OMe
OCOC6H5
(2)
OH
(5)
OH
OMe
C2H5
HO
N
OMe
OH
OR
OMe
OMe
OMe
OCOC6H5
OCOC6H5
CH2OMe
C2H5
HO
N
C2H5
HO
N
OH
OMe
OAc
OMe
CH2OMe
CH2OMe
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Chin. J. Chem. 2010, 28, 2494— 2500

Analysis of Metabolites Using HPLC/MS-MSn Technique
Table 3 HPLC-MS/MSn data of aconitine and its metabolites in the positive ion mode
No.
1
tR/min MSn (m/z) and relative abundance (%)
Identification
MS: 604 (100)
19.2
Benzoylaconine
MS2 [604]: 586 (22), 572 (58), 554 (100)
MS: 588 (100)
2
3
33.9
15-Deoxybenzoylaconine
8-Octenoyl-benzoylaconine
MS2 [588]: 570 (36), 556 (72), 538 (100)
MS: 728 (100)
36.0
44.1
46.4
48.5
52.8
54.9
57.0
57.9
59.4
MS2 [728]: 586 (100)
MS3 [728→586]: 568 (1), 554 (43), 536 (13), 526 (100)
MS: 662 (100)
4
5
MS2 [662]: 602 (100)
10-OH-Aconitine
MS3 [662→602]: 584 (10), 570 (83), 542 (100)
MS: 630 (100)
MS2 [630]: 570 (100)
15-Deoxyaconine
MS3 [630→570]: 538 (94), 520 (100)
MS: 646 (100)
6
MS2 [646]: 586 (100)
Aconitine
MS3 [646→586]: 568 (2), 554 (36), 536 (13), 526 (100)
MS: 700 (100)
7
MS2 [700]: 586 (100)
8-Hexenoyl-benzoylaconine
8-Propionyl-benzoylaconine
8-Butyryl-benzoylaconine
Deoxyaconitine
MS3 [700→586]: 568 (2), 554 (37), 536 (12), 526 (100)
MS: 660 (100)
8
MS2 [660]: 586 (100)
MS3 [660→586]: 568 (1), 554 (43), 536 (13), 526 (100)
MS: 674 (100)
9
MS2 [674]: 586 (100)
MS3 [674→586]: 568 (1), 554 (41), 536 (12), 526 (100)
MS: 630 (100)
10
11
MS2 [630]: 570 (100)
MS3 [630→570]: 538 (64), 510 (100)
MS: 690 (100)
MS2 [690]: 586 (100)
8(3-Hydroxyl)-butyryl-benzoylaconine
MS3 [690→586]: 568 (1), 554 (40), 536 (12), 526 (100)
as s-DDAs. On the one hand, in order to investigate the
toxicity of these s-DDAs, it was necessary to chose one
of them to synthesize to study the LD50 which has not
been reported before. On the other hand, owing to the
comparability of structures and properties, mesaconitine
showed similar metabolism pathway and metabolites
with aconitine in the previous study.13,14 From newly
generated metabolites, we chose 8-butyryl-benzoyl-
mesaconine to synthesize and test the LD50.
converted into 8-butyryl-benzoylmesaconine. Volatile
butyric acid was eliminated in distillation, so it was ob-
vious that mesaconitine was converted into 8-butyryl-
benzoylmesaconine without impurity.
According to the method of Bliss, the LD50 of me-
saconitine was 2.86 mg/kg and 95% confidence interval
The synthetic method of 8-butyryl-benzoylmes-
aconine was improved on the basis of that by Yili
et al.25
Figure 6 showed the full-scan mass spectrum of
8-butyryl-benzoylmesaconine synthesized. Figure 7 (A)
and (B) showed the HPLC chromatograms of mesaco-
nitine and 8-butyryl-benzoylmesaconine under the same
HPLC condition, respectively. From Figure 7 (A), me-
saconitine was eluted at 48.5 min and in Figure 7 (B),
8-butyryl-benzoylmesaconine was eluted at 51.5 min
with nothing eluted at 48.5 min after mesaconitine was
Figure 6 The full-scan mass spectra of 8-butyryl-benzoyl-
mesaconine.
Chin. J. Chem. 2010, 28, 2494— 2500
© 2010 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
2499

Wang et al.
FULL PAPER
Yakugaku Zasshi 1984, 104, 848.
4
Kitagawa, I.; Chen, Z. L.; Yoshihara, M.; Kobayashi, K.;
Ono, N.; Yoshimura, Y.; Yoshikawa, M. Yakugaku Zasshi
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Kitagawa, I.; Chen, Z. L.; Yoshihara, M.; Yoshikawa, M.
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7
8
9
Wang, Y.; Liu, Z. Q.; Song, F. R.; Liu, S. Y. Rapid Com-
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Wang, Y.; Song, F. R.; Xu, Q. X.; Liu, Z. Q.; Liu, S. Y. J.
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Figure 7 The HPLC-UV chromatogram of mesaconitine (A)
and 8-butyryl-benzoylmesaconine (B).
10 Wu, W.; Liang, Z. T.; Zhao, Z. Z.; Cai, Z. W. J. Mass Spec-
trom. 2007, 42, 58.
was 2.38—3.43 mg/kg; the LD50 of 8-butyryl-benzoyl-
mesaconine was 15.78 mg/kg and 95% confidence in-
terval was 12.85—19.38 mg/kg.
11 Chen, L. H.; Jin, L. J.; Su, Z. M.; Chou, Y. Q.; Wang, Y.;
Liu, S. Y. Chem. J. Chin. Univ. 2005, 26, 2340 (in Chinese).
12 Yukio, K.; Norio, N.; Hong, W.; Masao, H.; Tsuneo, N.;
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13 Zhao, Y. F.; Song, F. R.; Yue, H.; Guo, X. H.; Li, H. L.; Liu,
Z. Q.; Liu, S. Y. Chem. J. Chin. Univ. 2007, 28, 2051 (in
Chinese).
It is demonstrated that although the LD50 of
8-butyryl-benzoylmesaconine is less than that of mes-
aconitine, it still possesses considerable toxicity. The
results suggest that HPLC along with MS method play a
significant role in the determination of metabolite syn-
thesized.
14 Zhao, Y. F.; Song, F. R.; Guo, X. H.; Liu, S. Y. Chem. J.
Chin. Univ. 2008, 29, 55 (in Chinese).
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Conclusion
In the present study, it was found that pH was an
important factor to affect the metabolic process and
metabolites of aconitine. By the semi-quantitative
analysis of ESI-MS with reserpine as internal standard,
it is concluded that the metabolic activity of rat intesti-
nal bacteria is the highest for aconitine at pH 7.0, which
is close to the pH condition of rat intestinal. The identi-
fication and elucidation of ten metabolites have been
carried out by HPLC/ESI-MSn. One pair of isomeric
compounds has been identified and 15-deoxyaconitine
has been found for the first time. Aconitine is able to be
converted into other diester-diterpenoid alkaloids,
monoester-diterpenoid alkaloids, s-DDAs by rat intesti-
nal bacteria and some of them still reveal great toxicity.
22 Yue, H.; Pi, Z. F.; Li, H. L.; Song, F. R.; Liu, Z. Q.; Liu, S.
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© 2010 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2010, 28, 2494— 2500