JOURNAL OF MASS SPECTROMETRY

J. Mass Spectrom. 2004; 39: 13561365

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.727

Structural elucidation and identification of alkaloids in

Rhizoma Coptidis by electrospray ionization tandem
mass spectrometry
Daowu Wang, Zhiqiang Liu, Mingquan Guo and Shuying Liu
Laboratory of New Drug Research and Mass Spectrometry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun
130022, China

Received 6 May 2004; Accepted 16 August 2004

Simple, convenient, sensitive and accurate analytical methods are needed for the structural characterization
and identification of alkaloid components in Rhizoma Coptidis in traditional Chinese herbal medicine,
which has important bioactivity. In this work, the identification of alkaloid compounds in Rhizoma
Coptidis was investigated by obtaining molecular mass information using electrospray ionization mass
spectrometry (ESI-MS). Multi-stage tandem mass spectrometric (ESI-MSn ) data for the alkaloid compounds
were used for detailed structural characterization, then structure information was obtained by comparison
of the fragmentation mechanisms of both alkaloids in Rhizoma Coptidis and standard samples of
berberine, palmatine, coptisine and jatrorrhizine by MS. Based on the results obtained, the structure of a
novel compound was elucidated. The results of the experiments demonstrate that ESI-MSn is a sensitive,
selective and effective tool for the rapid determination of alkaloids in Rhizoma Coptidis. Copyright
2004 John Wiley & Sons, Ltd.

KEYWORDS: alkaloid; Rhizoma Coptidis; structure; electrospray ionization tandem mass spectrometry

INTRODUCTION chromatography,10,11 micellar chromatography,12 electron

Traditional Chinese herbal medicine (TCHM) is the oldest
continuously practised system of herbal medicine in the
world, dealing with many essential problems that have not
yet been solved by means of modern scientific techniques.
Modern scientific investigations demonstrate that alkaloids
are a large group of phytochemicals that can be found
primarily in the roots, petals and foliage of many plants.
Rhizoma Coptidis is commonly used in TCHM. The
alkaloids in Rhizoma Coptidis,1 6 including berberine,
palmatine, coptisine, jatrorrhizine and berberastine, are the
major bioactive components,7 which can treat a variety of
diseases, such as headache, fever, gnostids, constipation
and diarrhoea.8 In particular, the alkaloids have a good,
efficient and safe effect of curing chronic atrophy gastritis,9
so the availability of effective methods for the structural
characterization and identification of alkaloids in Rhizoma
Coptidis is very important.
Several methods have been reported for the determi-
nation and analysis of the alkaloids, including thin-layer
Correspondence to: Shuying Liu and Zhiqiang Liu, Laboratory of
New Drug Research and Mass Spectrometry, Changchun Institute
of Applied Chemistry, Chinese Academy of Sciences, Changchun
130022, China. E-mail: liuzq@ns.ciac.jl.cn
Contract/grant sponsor: National Natural Science Foundation of
China; Contract/grant number: 20173057.
Contract/grant sponsor: Great Research Project of Chinese
Academy of Sciences; Contract/grant number: KGCX2-SW-213-06.
Contract/grant sponsor: Natural Science Foundation of Jilin
Province; Contract/grant number: 20030916-1.
microscopic analysis,13 15 high-performance liquid chro-
matography (HPLC),16 21 capillary electrophoresis (CE)22
and capillary electrophoresis/ionspray mass spectrometry
(CE/MS).23 However, rigorous structural elucidation meth-
ods for the alkaloids, such as x-ray crystallography and
NMR spectroscopy, are time consuming, because the pure
compounds to be analysed and characterized must be avail-
able in order to obtain their structure information, that
is, the isolation and the purification of the alkaloids are
necessary. The alkaloids in Rhizoma Coptidis are very
complex, hence their isolation and the purification are diffi-
cult.
Electrospray ionization mass spectrometry (ESI-MS) is a
soft ionization technique that forms mainly molecular ion
peaks, and multi-stage tandem mass spectrometry (MSn )
data on the molecular ions are used for detailed structural
analysis, which can provide structural information on the
alkaloids in Rhizoma Coptidis without purification.
In the present work, all of alkaloid compounds in Rhi-
zoma Coptidis were identified by ESI-MS, and structural
information was obtained by comparison of the fragmen-
tation mechanisms of both alkaloids in Rhizoma Coptidis
and standard samples of berberine, palmatine, coptisine
and jatrorrhizine by MS and, as a result, the structure of
a novel compound present in Rhizoma Coptidis was eluci-
dated.

Copyright 2004 John Wiley & Sons, Ltd.

 Structure of alkaloids in Rhizoma coptidis by ESI-MSn 1357

EXPERIMENTAL for 24 h and then dissolved in methanol to give a final

solution for ESI-MSn analysis.
Chemicals and reagents
The TCHM Coptidis Rhizoma was purchased from Chang-
Mass spectrometry
chun Drugstore. All solvents were of analytical purity.
The structural characterization and identification of alkaloids
Standard samples of berberine, palmatine, coptisine and
in Rhizoma Coptidis were performed using a Finnigan
jatrorrhizine were purchased from the Chinese Authenticat-
MAT (San Jose, CA, USA) LCQ ion trap mass spectrometer
ing Institute of Material Medical and Biological Products
equipped with an electrospray source over the mass range
(Beijing, China).
from m/z 50 to 2000. The mass spectrometer was operated
Extraction and isolation in the positive ion mode. The samples were dissolved in
methanol and introduced into the ESI source by continuous
Dry Rhizoma Coptidis was crushed to a powder. About
infusion at a rate of 3 l min
1 by a syringe pump. The spray
100 g of the powder was weighed into a Soxhlet extractor
voltage was set to 4.5 kV and the capillary temperature
and extracted with 500 ml of 95% ethanol. The solvent was
to 185 C. Nitrogen was used as the sheath gas. The mass
evaporated and removed by reduced pressure distillation
scale was calibrated using standard compounds (caffeine,
and the sticky brown residue was collected in a beaker
peptide, MRFA, which is abbreviation of L-methionyl-
and then dissolved in 300 ml of 0.2% HCl; this acidification
arginyl-phenylalanyl-alanine and Ultramark) provided by
converted the alkaloid compounds to their corresponding
the instrument manufacturer according to the standard
salts, which dissolved in the aqueous phase. The aqueous
phase containing the corresponding salts of the alkaloids calibration procedure. The isolation width for MSn was
was collected and filtered and the filtrate was basified with 1.0 Da and the collision energy was as follows: MS2 , 23;
ammonia and the acidity was adjusted to pH 10, which MS3 , 18; MS4 , 22; and MS5 , 20%.
restored the alkaloid salts to the alkaloid compounds.
The basified aqueous solution was extracted three times RESULTS AND DISCUSSION
with 300 ml of chloroform. The extracted solution was Identification of alkaloid compounds in Rhizoma
collected and evaporated by reduced pressure distillation. Coptidis by ESI-MS
The evaporated sample was dried in a vacuum drying oven
Structural data for the most of the protoberberine alkaloid
compounds in Rhizoma Coptidis taken from the literature are
Table 1. Protoberberine alkaloids in Rhizoma Coptidis given in Table 1, which demonstrates that they have the same
framework structure. Under the ESI-MS conditions without
the additional voltage of collision-induced dissociation
(CID), the alkaloid compounds in Rhizoma Coptidis are
easily ionized and show strong molecular species in the
positive ion mode. The full-scan ESI mass spectrum in the
positive ion mode of the protoberberine alkaloid mixture in
the methanol extract of Rhizoma Coptidis is shown in Fig. 1,
with a series of peaks between m/z 300 and 400, i.e. ions at
m/z 320, 336, 338, 350, 352 and 366. By comparison with the
Compound R1 R2 R3 R4 R5 Mn literature and the molecular masses of known alkaloids in
Berberine CH2 CH3 CH3 H 336
Coptidis Rhizoma (Table 1), we can assume that the first five
Jatrorrhizine H CH3 CH3 CH3 H 338
peaks represent coptisine, berberine, jatrorrhizine, palmatine
Coptisine CH2 CH2 H 320
and 13-methylberberine, respectively. In addition, a novel
Palmatine CH3 CH3 CH3 CH3 H 352
compound, represented by the ion at m/z 366, is present that
13-Methylberberine CH2 CH3 CH3 CH3 350
has not been reported previously in Rhizoma Coptidis. Its
possible structure was elucidated by means of ESI-MSn .

352.3
100

80
336.3
60

40

20 350.3
320.3 338.3
366.3
0
270 280 290 300 310 320 330 340 350 360 370 380
m/z

Figure 1. Full-scan ESI mass spectrum of the mixture extracted from Coptidis Rhizoma.

Copyright 2004 John Wiley & Sons, Ltd. J. Mass Spectrom. 2004; 39: 13561365
1358 D.-W. Wang et al.
Structural characterization of alkaloid compounds
in Rhizoma Coptidis by ESI-MSn
It is well known that the structures of the alkaloid compounds
in Rhizoma Coptidis cannot be identified solely by means
of ESI-MS. Multiple tandem mass spectrometry (MSn ) can
select any ion from ESI-MS to obtain considerable structural
information, which has been used successfully for the
structure elucidation of saponins24,25 and flavonoids26 in
some herbal plants in our laboratory. To confirm the structure
of the alkaloid compounds of Rhizoma Coptidis by ESI-MS,
and as to demonstrate the power of MSn to characterize
the structure of alkaloids, ESI-MSn studies of the alkaloid
compounds in Fig. 1 and the corresponding standards were
performed in order to obtain structural information.
ESI-MSn investigation of the fragmentation mechanism
of standard samples of alkaloids in Rhizoma Coptidis
Standard samples of the alkaloids in Rhizoma Coptidis,
namely berberine, palmatine, jatrorrhizine and coptisine,
were studied by means of ESI-MSn , and the results are
shown in Figs 25. The fragmentation data for the ESI-MSn
experiments are given in Table 2.
In ESI-MS2 of standard samples of berberine (Fig. 2(a))
and palmatine (Fig. 3(a)), the predominant ions appeared
at m/z 321 and 337, respectively, which correspond to the
elimination of the methyl radical of the methoxy group at
C-9 or C-10 derived from the ions at m/z 336 and 352,
respectively, which is in agreement with the literature.27
Furthermore, the interesting fragment ions are observed at
m/z 292, 308 (Figs 2(a) and 3(a)) corresponding to the loss of
a neutral fragment with molecule mass 44 Da from berberine
and palmatine, respectively, which can be considered to be
due either to the loss of a methyl radical followed by the
elimination of CO, or to the loss of a C3 H8 neutral fragment
after a rearrangement in one or several steps. MS2 gives two
321.1
100
Relative Abundance
80
60
40
336.1
20 292.1
0 0
240 260 280 300 320 340
a m/z
318.2
100
Relative Abundance
80
60
40 320.2
20
0 0
240 260 280 300 320 340
c m/z
Figure 2. ESI-MSn of the standard sample of berberine. (a) MS2 of the standard of berberine; (b) MS3 of the ion at m/z 321 from
MS2 ; (c) MS4 of the ion at m/z 320 from MS3 ; (d) MS5 of the ion at m/z 318 from MS4 .
Copyright 2004 John Wiley & Sons, Ltd.
kinds of possible mechanisms, and it is necessary to establish
which one is correct.
In the MS3 experiment, the ion (M
15) at m/z 321 for
berberine and at m/z 337 for palmatine in MS2 was chosen
as the precursor ion; fragment ions at m/z M
15
29 (m/z
292 in Fig. 2(b) and m/z 308 in Fig. 3(b)) are observed, which
are the same as the ions at m/z M
44 (m/z 292 in Fig. 2(a)
and m/z 308 in Fig. 3(a)). The loss of 29 Da is in agreement
with the fragmentation pathway from the ion at m/z 321 to
the ion at m/z 292 for berberine, and from the ion at m/z
337 to ion at m/z 308 for pamadine (Figs 2(b) and 3(b)). On
the other hand, the fragment ions at m/z M
15
1 (m/z
320 for berberine and m/z 336 for palmatine) correspond
to the loss of a hydrogen radical from the C-10 methoxy
radical of the ion at m/z M
15 (Figs 2(b) and 3(b)) and to
the reaction of ring closure, so the fragmentation pathway of
the loss of a fragment of 29 Da possibly includes two steps:
the first step is the loss of a hydrogen radical and the second
is the loss of a neutral CO fragment. Summarizing the above,
the fragmentation mechanism of the ions at m/z M
44
(m/z 292 in Fig. 2(a) and m/z 308 in Fig. 3(a)) corresponds
to a threestep process, which are interpreted in Schemes 1
and 2.
In the first step, the loss of 15 Da corresponds to the
loss of a methyl radical; in the second step, a hydrogen
radical is lost; in the last step, the neutral fragment of
CO is eliminated. In the MS4 experiment, the ions at m/z
M
15
1 (m/z 320 in Fig. 2(b) and m/z 336 in Fig. 3(b))
are selected as the parent ions, and the loss of 2 Da is
unexpectedly found, which is corresponds to the loss of two
hydrogens (Figs 2(c) and 3(c)). This is due to the particular
structure of the protoberberine alkaloids; all of the rings are
benzene conjugated except for ring B (Table 1) and, as a
result, the whole molecule trends to turn into the stable state
of a conjugated structure by means of the loss of the two
292.2
100
320.2
Relative Abundance
80
60
40 321.1
20
360 380 240 260 280 300 320 340 360 380
b m/z
318.1
100
Relative Abundance
80 290.2
60
40
20
360 380 240 260 280 300 320 340 360 380
d m/z
J. Mass Spectrom. 2004; 39: 13561365
 Structure of alkaloids in Rhizoma coptidis by ESI-MSn 1359

337.0 308.1 336.1

100 100

Relative Abundance
Relative Abundance

80 80
337.2
60 60

40 40

20 352.1 20
308.1
0 0
300 310 320 330 340 350 360 290 300 310 320 330 340 350
a m/z b m/z

334.1
100 321.2 318.2
336.2 100
Relative Abundance

319.2

Relative Abundance
80 80
320.2
60 60
40 40
334.1
20 20

0 0
280 290 300 310 320 330 340 280 290 300 310 320 330 340
c m/z d m/z

Figure 3. ESI-MSn of the standard sample of palmatine. (a) MS2 of the standard sample of palmatine; (b) MS3 of the ion at m/z 337
from MS2 ; (c) MS4 of the ion at m/z 336 from MS3 ; (d) MS5 of the ion at m/z 334 from MS4 .

323.1
F:
294.1
F: 322.1
Relative Abundance

Relative Abundance

338.1

323.1
294.2
0 0
220 260 300 340 380 420 460 500 250 270 290 310 330 350 370 390
a m/z b m/z
307.2 320.1
F: F:
Relative Abundance

Relative Abundance

305.1
322.2

320.1

0 0
240 260 280 300 320 340 360 380 240 260 280 300 320 340 360 380
c m/z d m/z

Figure 4. ESI-MSn of the standard sample of jatrorrhizine. (a) MS2 of the ion at m/z 338 of jatrorrhizine; (b) MS3 of the ion at m/z 323
from MS2 ; (c) MS4 of the ion at m/z 322 from MS3 ; (d) MS5 of the ion at m/z 320 from MS4 .

hydrogen radicals at C-5 and C-6 in ring B (see Schemes 1 The fragmentation pathway of berberine and palmatine
and 2 and Figs 2(c) and 3(c)), which can be demonstrated by can be distinguished by the ring closure reaction. In MS4 ,
means of the fragmentation pathway of the standard sample the ions at m/z 321 (M
CH3
H
CH3 ) and m/z 320 (M

of copstine discussed below. CH3
H
CH4 ) can be observed for palmatine (Scheme 2,

Copyright 2004 John Wiley & Sons, Ltd. J. Mass Spectrom. 2004; 39: 13561365
1360 D.-W. Wang et al.

290.3
292.1 100
100 318.1
318.2

Relative Abundance
Relative Abundance

320.1

0 0
240 260 280 300 320 340 360 200 250 300 350 400 450
a m/z b m/z

262.2
100
Relative Abundance

290.2

0
210 230 250 270 290 310 330
c m/z

Figure 5. ESI-MSn of the standard sample of coptisine. (a) MS2 of the ion at m/z 320 of coptisine; (b) MS3 of the ion at m/z 318 from
MS2 ; (c) MS4 of the ion at m/z 290 from MS3 .

Fig. 3(c)), whereas the same fragmentation pathway does selected as the parent ion and the ion at m/z 320 was found,
not occur for berberine (Scheme 1, Fig. 2(c)). Moreover, In which corresponds to the loss of two hydrogens (Fig. 3(c)). In
MS5 , the ions at m/z 290 for berberine and m/z 318 and the MS5 experiment, the ion at m/z 320 formed the ion at m/z
319 for palmatine correspond to the ions produced by losing 305 by loss of CH3 (Fig. 4(d)). It lost a hydrogen radical of
neutral fragments of 28, 15 and 16 Da from the precursor the C-10 methoxy radical, proceeding with a reaction of ring
ions in MS4 , respectively (Figs 2(d) and 3(d)). According to closure between C-9 and C-10 in ESI-MS3 , continued with the
the spectrometric data, it can be speculated that the groups loss of two hydrogen radicals (C-6 and C-5) in ESI-MS4 , and
at C-9 and C-10 turned into the stable methylenedioxy group then the methoxy group at C-3 lost a methyl radical group
after ring closure reactions at C-9 and C-10 (Schemes 1 and and underwent the ring closure reaction.
2) had taken place. The difference between the groups at In ESI-MSn of the standard sample of coptisine (Fig. 5(a)),
C-2 and C-3 for berberine and palmatine (Schemes 1 and 2) the loss of methyl and H radicals and the ring closure reaction
makes the fragmentation pathway in MS5 different (Figs 2(d) between C-2 (or C-9) and C-3 (or C-10) were not observed
and 3(d)). Therefore, the parent ions of M
CH3
H
2H because of the existence of the ring structure between C-2 (or
(at m/z 318 for berberine and m/z 334 for palmatine) lose CO C-9) and C-3 (or C-10); only the elimination of two hydrogen
for berberine and CH3 and CH4 for palmatine in MS5 , which radicals and the loss of a neutral CO fragment were found
is due to the presence of the methylenedioxy group at C-2 in MS2 and the ions at m/z 318 and 292 were produced (see
and C-3 for the former and two methoxy groups at C-2 and Scheme 1 and Fig. 5). In MS3 of the ion at m/z 318, the ion at
m/z 290 was observed by loss of a neutral CO fragment from
C-3 for the latter, respectively.
methylenedioxy (see Scheme 4), and in MS4 the ion at m/z
The ESI-MS2 spectrum of jatrorrhizine is also dominated
262 was produced by the loss of another neutral CO fragment.
by the predominant ion at m/z 323 in Fig. 4(a) formed
by elimination of the methyl radical of methoxy13 as for ESI-MSn investigation of the structural characterization
berberine and palmatine.12 In the ESI-MS3 experiment, the of alkaloid compounds in Rhizoma Coptidis
ion at m/z 323 was chosen as the precursor, and the ion The ions at m/z 320, 336, 338 and 352 in Fig. 1 were also
at m/z 294 was observed in Fig. 4(b). The fragmentation studied using ESI-MSn . The ESI-MSn data are summarized in
mechanism of the ion at m/z 338 in Fig. 4 is summarized in Table 2 (including the standard alkaloid samples, berberine,
Scheme 3. In the first step, the loss of 15 Da corresponds to palmatine, jatrorrhizine and coptisine). It is notable that
the loss a methyl radical, and in the second step, the neutral the fragmentation pathways of the ions at m/z 320, 336,
fragment CO is eliminated. In the MS3 experiment, the ion at 338 and 352 are identical with those of the standard
m/z 322 formed by the loss of one H is found except the ion samples of coptisine, berberine, jatrorrhizine and palmatine,
at m/z 294 (Fig. 4(b)), and in ESI-MS4 , the ion at m/z 322 was respectively, confirming their identification.

Copyright 2004 John Wiley & Sons, Ltd. J. Mass Spectrom. 2004; 39: 13561365
 Structure of alkaloids in Rhizoma coptidis by ESI-MSn 1361

Table 2. Data for the ESI-MSn experiment on the ions at m/z 336, 352, 350
and 366 and standard samples of berberine and palmatine

Species Mr MS2 MS3 MS4 MS5

Berberine 336 321 320 318 290

292 292

Palmatine 352 337 336 334 319

308 308 321 318

320

Jatrorrhizine 338 323 322 320 305

294 294 307

Coptisine 320 318 290 262

292

Ion at m/z 338 323 322 320 305

294 307
294

Ion at m/z 320 318 290 262

292

Ion at m/z 336 321 320 318 290

292 292

Ion at m/z 352 337 336 334 319

308 308 321 318

320

Ion at m/z 350 335 334 332 304

306 306

Ion at m/z 366 351 350 348 333

322 322 335

334

The ESI-MSn data for the ion at m/z 350 in Fig. 1 are given
in Fig. 6, These data indicate the loss of a methyl radical in
MS2 (Fig. 6(a)) and then one hydrogen in MS3 (Fig. 6(b)),
two hydrogens in MS4 (Fig. 6(c)) and finally the ion at
m/z 350
15
1
2 lost 28 Da, corresponding to berberine;
the corresponding ESI-MSn data are given in Table 2. It is
interesting that the fragmentation pathway of the ion at m/z
350 is similar to that of the standard sample of berberine. In
other words, the mass differences of the fragment ions in the
corresponding MSn spectra are all 14 Da, corresponding to
the molecular mass difference between the ions at m/z 350
and the standard sample of berberine (m/z 336). Comparing
the fragmentation pathway of the ion at m/z 350 with that of
the standard sample of berberine (m/z 336), it can be deduced
that the structure of the compound at m/z 350 is mostly
analogous to that of the standard sample of berberine. Also,
the mass of the compound at m/z 350 corresponds to that
of 13-methylberberine, which was reported recently,28 and
its structure is given in Table 1. Comparison of the ESI-MSn
data for the ion at m/z 350 and those for 13-methylberberine
confirms that the alkaloid compound corresponding to the
ion at m/z 350 is 13-methylberberine.
ESI-MSn investigation of the structural characterization
of unknown alkaloid compounds in Rhizoma Coptidis
The ion at m/z 366 in Fig. 1 has not been reported previously.
The ion at m/z 366 possibly corresponds to an unknown
compound, which can be identified from the ESI-MSn data.
As mentioned above, the ESI-MSn data for the ion at m/z
366 match those for the ion at m/z 352(corresponding to

Copyright 2004 John Wiley & Sons, Ltd. J. Mass Spectrom. 2004; 39: 13561365
1362 D.-W. Wang et al.

O 4 5 CH3O 4 5
3 6 3
6
CH2 A B A B
2 N+ 8 2 N+
O 1 8
C OCH3 CH3O 1 C
9 OCH3
13 9
m/z 336 D 10 13
12 m/z 352 D 10
11 OCH3 12
11 OCH3
-CH3-H-CO 2 MS2 -CH3
MS -CH3-H-CO -CH3
MS2 MS2
O
O
CH2 CH3O
N+ -H-CO CH2 CH3O
+
O N
MS3 O N + -H-CO
m/z 292 O N+
CH3O
MS3
OCH3 CH3O
m/z 308 O
O OCH3 m/z 337
-H OCH3
m/z 321
CH2 MS3
N+
CH3O -H OCH3
O O
3
m/z 320 MS
CH2
N+
O
CH3O O O
4
MS -H-H m/z 336
CH2 N+
O O CH3O
N+ O O
CH2 N + O MS4 -H-H m/z 319
CH2
O O CH2
-CO CH3O -CH3 O
O O
m/z 318 CH2 MS5 m/z 290 MS5
N+

O
Scheme 1. Fragmentation pathway of berberine.
palmatine). The MS data indicate the loss of a methyl radical
in MS2 (Fig. 7(a)) and then the loss of one hydrogen in MS3
(Fig. 7(b)), two hydrogens in MS4 (Fig. 7(c)) and finally the
ion at m/z 366
15
1
2 lost 15 Da, which is similar to that
of palmatine (Fig. 7(d)). Comparison of the fragmentation
pathways of the ion at m/z 366 and of the standard sample
of palmatine (m/z 352) indicates that the structure of the
compound at m/z 366 is mostly analogous to that of the
standard sample of palmatine. The MS data demonstrate
that the difference between ion at m/z 350 and berberine
(m/z 336) is the group at C-13, the former being CH3 and
the latter H. Hence it can be deduced that in the structure
of the ion at m/z 366, the H at C-13 in palmatine (m/z 352)
is replaced by CH3 , because its ESI-MSn data are similar to
those of palmatine. If the CH3 group is at C-5 or C-6, the
corresponding ion will not involve the loss of two hydrogens,
and if the CH3 group is at a position other than C-13, C-5
or C-6, it will effect the loss of a methyl radical in the MS2
experiment owing to steric hindrance and conjugation effects.
As a result, the novel alkaloid compound corresponding to
the ion at m/z 366 in Rhizoma Coptidis can be confirmed as
13-methylpalmatine.
As is well known, some components in Rhizoma Cory-
dalis, such as dehydrocorybubline, dehydrothalictrifoline
and dehydrocorydaline, have a similar structural framework
to those in Rhizoma Coptidis, in which dehydrocorybubline
has the same molecular mass as palmatine but with a methyl
group as R5 instead of R3 , and dehydeothalictrifoline has
the same molecular mass as 13-methylberberine but with
CH2 between R4 and R5 instead of between R1 and
-CH2
CH3O N+
O
O
O
CH2 -CH4
m/z 334 CH2
O MS5 m/z 318
O
Scheme 2. Fragmentation pathway of palmatine.
R2 . Interestingly, the difference in molecule mass between
dehydrocorydaline (366) and palmatine (352) is 14 Da, i.e.
corresponding to a methyl group. Furthermore, according
to the literature, the structure of dehydrocorydaline is iden-
tical with that of 13-methylpalmatine mentioned above, so
a pure sample of dehydrocorydaline (13-methylpalmatine)
was isolated and purified from Rhizoma Corydalis in our lab-
oratory, and then the fragmentation pathway in ESI-MSn of
dehydrocorydaline (13-methylpalmatine) was investigated,
as shown in Fig. 8.
The experimental results demonstrate that the compound
corresponding to the ion at m/z 366 in Rhizoma Coptidis has
the same fragmentation pathway in ESI-MSn as dehydro-
corydaline (13-methylpalmatine) in Rhizoma Corydalis, so
the compound at m/z 366 in Rhizoma Coptidis is initially
speculated to be dehydrocorydaline (13-methylpalmatine).
The exact structure of the compound corresponding to the
ion at m/z 366 in Rhizoma Coptidis will be confirmed by
NMR spectroscopy in future work.
CONCLUSIONS
ESI-MS combined with tandem mass spectrometry (MSn )
has been proved to be a fast and effective method for the
investigation of the protoberberine alkaloid mixtures, avoid-
ing the tedious and difficult tasks of isolation, separation

Copyright 2004 John Wiley & Sons, Ltd. J. Mass Spectrom. 2004; 39: 13561365
 Structure of alkaloids in Rhizoma coptidis by ESI-MSn 1363

HO 3 4 5 O 4 5
3
6 6
A B CH2 A B
2 N+ 2 N+
8 8
H3CO 1 C O 1
OCH3 C
9 9 O
13 13
m/z 338 D 10 D CH2
m/z 320 10
12
11 OCH3 12
11 O

-CH3
-CH4-CO MS2 2H
MS2 MS2 -CO
MS2

HO
O O
O
N+ -H-CO CH
2 CH2
N+ N+
CH2
H3CO MS3 N+
O O
O O
m/z 294 O
OCH3 CH2
m/z 318 O
HO -H OCH3 O
MS3 m/z 323
m/z 292
N+ 3
H3CO -CO MS
O
m/z 322 CH2 O

O CH2 O
N+ N+
MS4
MS4 O
-H-H HO
-CO
HO O O
N+
O O m/z 262
N+ m/z 290

H3CO O -CH3 CH2 Scheme 4. Fragmentation pathway of coptisine.

O
CH2 MS5 m/z 305
m/z 320
O

Scheme 3. Fragmentation pathway of jatrorrhizine.

335.0 334.1
100 100
Relative Abundance
Relative Abundance

80 80
306.1
60 60
1
350.0
40 40
335.1
20 306.1 20

0 0
300 310 320 330 340 350 360 370 380 300 310 320 330 340 350 360 370 380
a m/z b m/z

332.0
100 304.1
100
Relative Abundance

Relative Abundance

80 80
334.2
60 60 332.2

40 40

20 20

0 0
300 310 320 330 340 350 360 370 380 220 260 300 340 380 420
c m/z d m/z

Figure 6. (a) MS2 of the ion at m/z 350 from figure 1; (b) MS3 of the ion at m/z 335 from MS2 ; (C) MS4 of the ion at m/z 334 from
MS3 ; (d) MS5 of the ion at m/z 332 from MS4 .

Copyright 2004 John Wiley & Sons, Ltd. J. Mass Spectrom. 2004; 39: 13561365
1364 D.-W. Wang et al.

351.0 350.1
100 100

Relative Abundance
Relative Abundance

80 80 322.0

60 366.0 60
351.0
40 40

20 322.0 20

0 0 .
280 300 320 340 360 380 400 420 280 300 320 340 360 380 400 420
a m/z b m/z

350.1
100
100 333.1 348.3
Relative Abundance

80

Relative Abundance
335.1 80
60 334.1
348.1 60
40 40
20 20

0 0
260 280 300 320 340 360 380 280 320 360 400 440 480
c m/z d m/z

Figure 7. (a) MS2 of the ion at m/z 366 from Fig. 1; (b) MS3 of the ion at m/z 351 from MS2 ; (c) MS4 of the ion at m/z 350 from MS3 ;
(d) MS5 of the ion at m/z 348 from MS4 .

100 351.2 100 350.2

Relative Abundance

Relative Abundance

322.2
80 80

60 60
366.2
40 40
351.3
20 20
322.3
0 0
250 300 350 400 450 500 550 300 320 340 360 380 400
a m/z b m/z

334.2 100 348.2

100 333.2
Relative Abundance
Relative Abundance

80 80

60 60

40 350.3 40 332.2
335.3 348.3
20 20

0 0
310 320 330 340 350 360 370 380 310 320 330 340 350 360 370
c m/z d m/z

Figure 8. (a) MS2 of the ion at m/z 366 of dehydrocorydaline; (b) MS3 of the ion at m/z 351 from MS2 ; (c) MS4 of the ion at m/z 350
from MS3 ; (d) MS5 of the ion at m/z 348 from MS4 .

and purification of derivatives. The considerable fragmen- indicate that the fragmentation pathways of protoberberine
tation information obtained by ESI-MSn is very useful for alkaloid compounds and their structural characteristics can
the elucidation of the structure of known and unknown be used for their structural elucidation and identification.
compounds. The novel alkaloid compound dehydrocoryda-
Acknowledgements
line (13-methylpalmatine) has been found for the first time This work was supported by the National Natural Science Foun-
in Rhizoma Coptidis by ESI-MSn . The experimental results dation of China (No. 20173057), the Great Research Project of the

Copyright 2004 John Wiley & Sons, Ltd. J. Mass Spectrom. 2004; 39: 13561365

Chinese Academy of Sciences (No. KGCX2-SW-213-06) and the
Natural Science Foundation of Jilin Province (No. 20030916-1).
REFERENCES
1. Tomita M, Kura S. Ingredients of aristolochiaceous plants. I.
Ingredients of Aristolochia Kaempferi and A.debilis. J. Pharm.
Soc. Jpn. 1956; 76: 1425.
2. Yahara S, Satoshiro N. Isolation and Characterization of
phenolic compounds from coptidis rhizome. Chem. Pharm. Bull.
1985; 33: 527.
3. Chen CY, Maclean DB. Mass Spectra and proton magnetic
resonance spectra of some tetrahydroprotoberberine alkaloids.
Canadian Journal of Chemistry 1968; 46: 25.
4. Chatterjee R. Plant alkaloids Part I Berberis Floribanda Wall. J.
India. Chem. Soc. 1951; 28: 225.
5. Sawada T. Chemistry of Coptidis. Gendai. Toyo. Igaku. 1981; 2:
48.
6. Sakai T, Ohno N, Sasaki H. Extraction-spectrophotometric
determination of berberine in crude drugs by the formation
of a new ion assiciate. Anal. Sci. 1991; 7: 39.
7. Chuang W-C, Young D-S, Liu L-K, Sheu S-J. Liquid chromato-
graphic-electrospray mass spectrometric analysis of Coptidis
Rhizoma. J. Chromatogr. A 1996; 755: 19.
8. Tanaka T, Metori K, Mineo Satoshi. Studies on collagenase
inhibitors .IV. Inhibitors of bacterial collagenase in Coptidis
rhizoma. Yakugaku Zasshi 1991; 111: 538.
9. Higaki Shuichi, Hasegawa Y, Morohashi M. The influence of
Coptidis Rhizoma on lipase activity of Propionibacterium anes.
Nippon Hifuka Gakkai Zasshi 1990; 100: 883.
10. Wu T-M, Lee Sh-F. The application of TLC-densitometry to the
quantilative analysis of berberine in Chinese herb preparations.
Tai-wan Yao Hsueh Tsa Chih 1975; 27: 17.
11. Noguchi M, Mochida K. Determination of alkaloids in Coptidis
rhizoma by thin-layer electrophoresis. Shoyakugaku Zasshi 1976;
30: 47.
12. Qiu X, Wu C, Chen B. Analysis of berberine-type alkaloids in
Rhizoma coptidis and Chinese patent medicines by micellar
chromatography. Yaoxue Xuebao 1986; 21: 458.
13. Namba T, Mikage M, Ushiyama T. Fundamental studies on the
evaluation of crude drugs. VI. Electron microscopic analysis of
crude drugs. 1. Determination of berberine in coptidis rhizoma.
Yakugaku Zasshi 1982; 102: 56.
14. Namba T, Mikage M. Analysis of coptidis rhizoma by electron
microscopicgraphy. Pharm. Tech. Jpn. 1987; 3: 227.
15. Mikage M, Ushiyama T, Namba T. Fundamental studies on the
evaluation of crude drug. X. Electron microscopic analysis of
crude drugs. 2. on the distribution of alkaloids in Coptidis
Rhizoma and Phellodendri Cortex. Yakugaku Zasshi 1987; 107:
690.
Copyright 2004 John Wiley & Sons, Ltd.
Structure of alkaloids in Rhizoma coptidis by ESI-MSn 1365
16. Ishikawa O, Hishimoto T, Nakajima T, Tanaka O, Itokawa H.
Application of high-speed liquid chromatography to the analysis
of crude drugs: quaternary alkaloids of Coptidis rhizoma.
Yakugaku Zasshi 1978; 98: 976.
17. Misaki T, Sagara K, Ojima M, Kakizawa S, Oshima T,
Yoshizawa H. Simultameous determination of berberine
,palmatine and coptisine in crude drugs and oriental
pharmaceutial preparations by ion-pair high-performance liquid
chromatography. Chem. Pharm Bull. 1982; 30: 354.
18. Hattori T, Kamiya N. Determiantion of berberine in Coptidis
rhizoma by high performance liquid chromatography. Yakugaku
Zasshi 1977; 97: 1305.
19. He X-G. On line identification of photochemical constituents
in botanical extracts by combined high-performance liquid
chromatographic detection mass spectrometric techniques.
Journal of Chromatography A 2000; 880: 203.
20. Wu T, Sheu, Shuenn J. Quantitative analysis of the eight
quaternary alkaloids in coptidis rhizoma by high-performance
liquid chromatography. Zhonghua Yaoxue Zazhi 1993; 45: 157.
21. Luo G, Wang Y, Zhou G, Yu Y. Determination of coptidis
Chinese franch and phellodendron amurense ruprin qingwei-
huanglian wan by pls-HPLC method. J. Liq. Chromatogr. 1990;
13: 3825.
22. Liu YM, Sheu SJ. Determination of quaternary alkaloids from
Coptidis Rhizoma by capillary electrophoresis. J. Chromatogr.
1992; 623: 196.
23. Henion JD, Mordehai AV, Cai J. Quantitative Capillary
Electrophoresis-Ion Spray Mass Spectrometry on a Benchtop
Ion Trap for the Determination of Isoquinoline Alkaloids. Anal.
Chem. 1994; 66: 2103.
24. Fang S-P, Hao C-Y, Sun W-X, Liu Z-Q, Liu S-Y. Rapid Analysis
of Steroidal Saponin Mixture Using Electrospray Ionization
Mass Spectrometry Combined with Sequential Tandem Mass
Spectrometry. Rapid Commun. Mass Spectrom. 1998; 12: 589.
25. Cui M, Song F-R, Zhou Y, Liu Z-Q, Liu S-Y. Rapid identification
of saponins in plant extracts by electrospray ionization multi-
stage tandem mass spectrometry and liquid chromatography.
Rapid Commun. Mass Spectrom. 2000; 14: 1280.
26. Chen M-L, Song F-R, Guo M-Q, Liu Z-P, Liu S-Y. Analysis of
flavonoid constituents from leaves of Acanthopanax senticosus
Harms by electrospray tandem mass spectrometry. Rapid
Commun. Mass Spectrom. 2002; 16: 264.
27. Chen Y-R, Wen K-C, Her G-R. Analysis of coptisine ,berberine
and palmatine in adulterated Chinese medicine by capillary
electrophoresis-electrospray ion trap mass spectrometry. J.
Chromatogr. A 2000; 866: 273.
28. Jun L-L, Abliz Z, Yun X, Guo W-A, Zhang F-X, Bin L, Yi H-W.
Application of Tandem Mass Spetrometry Techniques for the
analysis of Flavonoid Glycosides. Fenxi Ceshi Xuebao 2001; 20: 63.
J. Mass Spectrom. 2004; 39: 13561365