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The simultaneous determination of berberine,

palmatine, coptisine, epiberberine and jatrorrhizine
Cite this: Anal. Methods, 2014, 6, 2998
in rat plasma by LC-MS/MS and a pharmacokinetic
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comparison after the oral administration of

Rhizoma coptidis and Jiao-Tai-Wan extract
Guanghui Liu,a Wei He,a Hao Cai,a Xiuman Sun,a Waner Hou,a Manna Lin,a
Zhiyong Xie*b and Qiongfeng Liao*a

Received 13th November 2013
Accepted 7th February 2014
DOI: 10.1039/c3ay42000k
www.rsc.org/methods
A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was
developed to simultaneously determine berberine, palmatine, coptisine, epiberberine and jatrorrhizine in
rat plasma, using tetrahydropalmatine as an internal standard. The separation of the five compounds was
carried out on a C18 column using a mobile phase consisting of acetonitrile and water (containing 5
mmol ammonium acetate adjusted to pH 5.0 with formic acid). The detection was performed in the
multiple reaction monitoring (MRM) mode via the electrospray ionization (ESI) source operating in the
positive ionization mode. Linear calibration curves were obtained over the concentration range of 0.20–
200.0 ng ml
1 for berberine, and 0.20–100.0 ng ml
1 for palmatine, coptisine, epiberberine and
jatrorrhizine. The accuracy, precision and extraction recovery of the five analytes were all satisfactory.
The validated method was successfully applied to study the pharmacokinetic differences of the five
protoberberine-type alkaloids after the oral administration of Rhizoma coptidis (RC) and Jiao-Tai-Wan
(JTW) extract. It was found that the Tmax values of the five ingredients in the JTW extract were
significantly increased (P < 0.05) when compared with those in RC. In addition, the AUC0–t of palmatine,
coptisine and epiberberine were significantly increased (P < 0.05).

Cinnamon cassia granules could increase the plasma concen-

1 Introduction
Jiao-Tai-Wan (JTW), recorded in Han Shi Yi Tong, consists of
Rhizoma coptidis (RC) and Cinnamon cassia powder (10 : 1, g/g).
It has been used for centuries for the treatment of insomnia in
traditional chinese medicine (TCM). Many investigations have
focused on the pharmacokinetics of the alkaloids contained in
RC, while there were few reports on the pharmacokinetics of
JTW. For example, Wang et al. have investigated the kinetic
distribution of berberine in rat thalamus aer the intravenous
administration of RC extract;1 Li et al. have investigated the
pharmacokinetics of berberine and palmatine aer the oral
administration of coptidis and coptidis–Gardeniae couple
extract.2 But so far, only Huang et al. have reported the effects of
Cinnamon cassia granules on the pharmacokinetics of berberine
in Rhizoma coptidis granules in healthy male volunteers.3 The
results indicated that Rhizoma coptidis granules combined with
a
School of Chinese Materia Medica, Guangzhou University of Chinese Medicine,
Guangzhou, 510006, China. E-mail: liaoqf2075@yahoo.com; Fax: +86-20-39358081;
Tel: +86-20-39358081
b
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006,
China. E-mail: xiezy2074@yahoo.com; Fax: +86-20-39943047; Tel: +86-20-39943047
tration of berberine, promote its absorption and lengthen the
detention time in healthy male volunteers. The main active
constituents in RC are protoberberine-type alkaloids, including
berberine, palmatine, coptisine, epiberberine and jatrorrhizine.
The chemical structures of them are shown in Fig. 1. The
alkaloids might have a synergistic effect.4 Berberine is the major
alkaloid which accounted for 39.66% of the RC extract.
However, the total proportion (30.94%) of the other four pro-
toberberine-type alkaloids (palmatine 6.75%, coptisine 8.55%,
epiberberine 10.46% and jatrorrhizine 5.17%) also makes up a
considerable proportion. Therefore, in pharmacokinetic studies
of the ve ingredients it would be a better strategy to represent
the pharmacokinetics of JTW in vivo. Moreover, there is little
precedence for comparing the pharmacokinetic behaviors of
the marker compounds of JTW acting individually in single-
herb extracts versus together in a compound combination
prescription. Whether the combination with Cinnamon cassia
would change the pharmacokinetics of the ingredients con-
tained in RC was still unclear. Therefore, to make a comparison
of the ve protoberberine-type alkaloid pharmacokinetic
differences between RC and JTW it may help to explore potential
herb–herb interactions between RC and Cinnamon cassia.

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Paper Analytical Methods

them can simultaneously determine the ve alkaloids. As far as

we know, only Yu et al. have reported an LC-MS method to
determine the ve alkaloid concentrations in rat plasma.16
The method incorporated a sample preparation procedure
including solid phase extraction and gradient elution with 8.0
min. In this method, 100 ml plasma was used. With so much
plasma, it is hard to get a completed drug–concentration–time
curve on the same rat. It is known that the LC-MS/MS method
possesses a better selectivity when compared to the LC-MS
method. Therefore, an alternative validated method with a
simpler sample preparation, excellent selectivity, using less
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plasma and high throughput will be an advance for a greater

Fig. 1 The chemical structures of berberine (I), palmatine (II), coptisine
(III), epiberberine (IV), jatrorrhizine (V) and tetrahydropalmatine (IS, VI).
In order to make the pharmacokinetic comparison of RC and
JTW, a method to simultaneously determine the ve alkaloids
was required. In our previous study, we have developed an
LC-MS/MS method to determine berberine, palmatine and
jatrorrhizine in rat plasma aer the oral administration of
Zoujinwan.5 But the method could not be sufficient to deter-
mine coptisine and epiberberine. Thus, the method should be
improved in this investigation. Meanwhile, earlier publications
have described some methods to determine protoberberine-
type alkaloids contained in RC in biological samples utilizing
HPLC-UV,6 HPLC-ECD7,8 and liquid chromatographic tandem
mass spectrometry.5,9–16 The LC-MS and LC-MS/MS methods are
listed in Table 1. Most of them use LLE or SPE sample prepa-
ration, which needs a larger sample volume. Moreover, few of
efficiency in pre-clinical pharmacokinetics.
In this study, a rapid and sensitive LC-MS/MS method which
was based on our previous study was developed and validated
for the simultaneous determination of berberine, palmatine,
coptisine, epiberberine and jatrorrhizine in rat plasma. In
addition, the method was applied to the pharmacokinetic
comparisons of the ve alkaloids aer the oral administration
of RC and JTW extract to rats.
2 Experimental and methods
2.1 Materials
RC and Cinnamon cassia were purchased from Zhixin Medicine
Health Co. Ltd. (Guangzhou, China). The reference standards of
berberine (no. 110713-200208, purity 98%), palmatine (no.
110732-200907, purity 98.0%) and tetrahydropalmatine (no.
110726-200610, purity 98.0%) were all purchased from the
National Institute for the Control of Pharmaceutical and Bio-
logical Products (Beijing, China). Jatrorrhizine (no. 111022,
purity 98.0%) and coptisine (no. 110718, purity 98.0%) were
purchased from Weikeqi Biological Technology Co. Ltd.
(Sichuan, China). Epiberberine (no. 12022503, purity 98.0%)
was purchased from Chengdumust Biological Technology Co.
Ltd. (Chengdu, China). HPLC-grade ammonium acetate was
purchased from Aladdin (Shanghai, China). HPLC-grade

Table 1 The list of the LC-MS and LC-MS/MS methods

Sample Analysis
Components Method Sample type volume/ml Sample preparation LLOQ/ng ml
1 time/min References

Berberine, palmatine LC-MS Rat plasma 100 Liquid–liquid 0.31 for each 5.5 10
extraction
Berberine LC-MS Human 1000 Liquid–liquid 0.02 for each 12 11
plasma extraction
Berberine, palmatine, LC-MS/ Rat plasma 20 Protein 1 for each 2.5 12
jatrorrhizine MS precipitation
Berberine, palmatine, LC-MS/ Rat plasma 50 Protein 0.6 for each 10 5
jatrorrhizine MS precipitation
Berberine, palmatine, LC-MS/ Rabbit 100 Protein 0.1 for each 2.4 13
jatrorrhizine MS plasma precipitation
Jatrorrhizine, berberine, coptisine, LC-MS/ Rat plasma 100 Liquid–liquid 0.44, 0.48, 0.52, 0.42 5 14
palmatine MS extraction
Berberine, coptisine, LC-MS/ Rat plasma 100 Protein 1.01, 0.0996, 0.0501, 7 15
jatrorrhizine, palmatine MS precipitation 0.0889
Berberine, palmatine, coptisine, LC-MS Rat plasma 100 Solid phase 0.31 for each 5.5 16
epiberberine, jatrorrhizine extraction

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Analytical Methods
acetonitrile and methanol purchased from Merck (Darmstadt,
Germany) were used for the HPLC analysis and plasma sample
preparation. Deionized water was produced by Heal Force-
PWVF Reagent Water System (Shanghai CanREX Analyses
Instrument Corporation Limited, China). All other reagents
were of analytical grade.
2.2 Liquid chromatography-tandem mass spectrometry
analysis
2.2.1 Equipment. The LC-MS/MS system consisted of an
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Agilent 1260 liquid chromatograph and a 6460 triple quadru-
pole mass spectrometer with an electrospray ionization (ESI)
source. Data acquisition was performed with MassHunter B 05
soware.
2.2.2 Chromatographic conditions. The chromatographic
separation was achieved at 30  C on a BDS Hypersil C18
column (50  2.1 mm i.d., 2.4 mm, Thermo Scientic) with a
Phenomenex C18 guard column (4  2 mm i.d.) by gradient
elution with 0–2.5 min, 70% / 30% mobile phase A; 2.5–4.0
min, 30% mobile phase A, owing at 0.25 ml min
1. Aer
changing back to 70% solvent A at 4.1 min, the mobile phase
gradient was maintained at this composition from 4.1 to 6.0
min for column equilibration. Eluent A was water containing
5 mmol ammonium acetate adjusted to pH 5.0 with formic
acid, and B was acetonitrile. The injection volume was 5 ml.
The initial 1.3 min was switched to the waste. The column
was fully eluted aer a daily analysis cycle (around 100
samples).
2.2.3 Mass spectrometric conditions. The mass spectrom-
eter was operated in the positive ion mode using multiple
reaction monitoring (MRM) to detect the mass transitions.
Both analytes and IS were detected by tandem mass spec-
trometry using the MRM of the precursor–product ion transi-
tions at m/z 336.0 / 320.1 and m/z 336.0 / 292.1 for
berberine, at m/z 352.0 / 336.1 and m/z 352.0 / 308.1 for
palmatine, at m/z 320.0 / 292.1 and m/z 320.0 / 277.1 for
coptisine, at m/z 336.0 / 320.1 and m/z 336.0 / 292.1 for
epiberberine, at m/z 338.0 / 322.1 and m/z 338.0 / 294.1 for
jatrorrhizine, and at m/z 356.4 / 192.1 and m/z 356.4 / 176.1
for tetrahydropalmatine. The MRM transitions m/z 336.0 /
320.1, m/z 352.0 / 336.1, m/z 338.0 / 322.1, m/z 336.0 /
320.1, m/z 320.0 / 292.1 and m/z 356.4 / 192.1were selected
for determining berberine, palmatine, jatrorrhizine, epi-
berberine, coptisine and tetrahydropalmatine, respectively.
The other one (m/z 336.0 / 292.1, m/z 352.0 / 308.1, m/z
338.0 / 294.1, m/z 336.0 / 292.1, m/z 320.0 / 277.1 and m/z
356.4 / 176.1 for the analysis of berberine, palmatine,
jatrorrhizine, epiberberine, coptisine and tetrahy-
dropalmatine, respectively) was used as a qualitative channel
to conrm the identity of berberine, palmatine, jatrorrhizine,
epiberberine, coptisine and tetrahydropalmatine. High purity
nitrogen served as both a nebulizing and drying gas.
Compound-dependent parameters are listed in Table 2. The
other parameters of the mass spectrometer were set as follows:
drying gas ow 10 l min
1; drying gas temperature 350  C;
nebulizer pressure 40 psi; capillary voltage 3500 V.
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2.3 The preparation of Rhizoma coptidis extract and
Cinnamon cassia oil
RC (1000 g) was extracted two times by reuxing with 60%
ethanol (1 : 10, w/v) for 1 hour each time. The extraction solu-
tions were combined for ltration, and the ethanol was
removed under reduced pressure. Then it was dried into powder
by spray drying. The Cinnamon cassia oil was extracted by
supercritical carbon dioxide extraction with the temperature
being 45  C, the pressure being 35 MPa, the time being 2 h and
the ultrasonic frequency being 2 kW.
To calculate the administered dose, the amounts of ve
protoberberine-type alkaloids in the extract were quantitatively
determined with the same method described in this paper. The
amounts of berberine, palmatine, coptisine, epiberberine and
jatrorrhizine were 396.59, 67.45, 85.54, 104.63 and 51.74 mg g
1
extract. The amounts of cinnamic aldehyde were quantitatively
determined with a HPLC method described in our previous
study.17 The HPLC analysis was performed on a Dalian Elite BDS
Hypersil C18 column (250  4.6 mm, 5 mm) using a mobile
phase consisting of 0.1% phosphoric acid–acetonitrile
(70 : 30, v/v) at 25  C with the ow rate of 1.0 ml min
1. The
injection volume was 10 ml and the detection wavelength was
254 nm. The content of cinnamic aldehyde was 197.9 mg g
1
Cinnamon Cassia oil.
2.4 The preparation of the standard solution and quality
control samples
The stock solutions I of berberine, palmatine, jatrorrhizine,
epiberberine and coptisine at the concentration of 200 mg ml
1
for each were prepared respectively by dissolving the accu-
rately weighed reference compounds in methanol. The stock
solutions II containing ve substances at the concentration of
20 mg ml
1 for berberine and 10 mg ml
1 for palmatine,
jatrorrhizine, epiberberine and coptisine, were obtained by
mixing and diluting the stock solutions I. The solutions II were
then successively diluted with methanol to prepare working
solutions in the concentration range 4.0–4000.0 ng ml
1 for
berberine and 4.0–2000.0 ng ml
1 for the other four
compounds. A 2 mg ml
1 stock solution of tetrahy-
dropalmatine (IS) was also prepared in methanol and then
diluted to obtain a working solution of 100 ng ml
1. All the
solutions were stored at
20  C.
The calibration standards were prepared by spiking blank
plasma with appropriate amounts of working solutions (5% of
the total plasma sample volume) and vortex mixing for 30
s. Then they were extracted as described in the “The preparation
of the plasma sample” section below. Finally the calibration
samples were made at concentrations of 0.2, 0.5, 1.6, 4.0, 10.0,
32.0, 80.0 and 200.0 ng ml
1 for berberine; 0.2, 0.5, 0.8, 1.6, 4.0,
10.0, 32.0, and 100.0 ng ml
1 for palmatine, jatrorrhizine, epi-
berberine and coptisine, respectively. The quality control (QC)
samples used in the validation and during the pharmacokinetic
study had been prepared in the same way as the calibration
standards with a separated stock solution: berberine (0.5, 10.0,
and 160.0 ng ml
1), palmatine, jatrorrhizine, epiberberine and
coptisine (0.5, 4.0, and 80.0 ng ml
1).
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Table 2 The MS/MS transitions and parameters for the detection of the analytes and internal standards
Compound Fragmentor/V
Berberine 154
Palmatine 155
Jatrorrhizine 155
Epiberberine 175
Coptisine 180
Tetrahydropalmatine 160
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2.5 The preparation of the plasma samples
Rat plasma (30 ml) and IS solution (10 ml) were placed in an
Eppendorf tube. Aer mixing, 50 ml of acetonitrile was added.
Then the mixture was vortex mixed for 3 min and centrifuged at
13 000 g for 10 min. Subsequently, the supernatant was
transferred to another Eppendorf tube and centrifuged at
13 000 g for 5 min. Finally, 5 ml of the supernatant liquor was
injected into the LC-MS/MS system for analysis.
2.6 The validation of the method
The method was validated according to the currently accepted
USA Food and Drug Administration (FDA) bioanalytical method
validation guidance.18–20
2.6.1 Selectivity. The selectivity of the method was tested by
comparing the chromatograms of six individual rat blank
samples, samples at the concentrations of the lower limit of
quantication (LLOQ) and the rat plasma samples aer
administration. The analyte area in the blank plasma should
not exceed 20% of that from the samples at the LLOQ.
2.6.2 Linearity and sensitivity. The calibration curve con-
sisted of eight concentration levels. Each of these samples was
prepared and assayed in triplicate on 3 separate days. The linear
regression of the ratio of the areas of the analytes and the IS
peaks versus the concentration were weighted with weighting
factor 1/x2 (where x ¼ concentration). The concentrations of the
analytes in unknown samples were determined by interpolation
from the calibration curve. The LLOQ of the assay can be
measured with an accuracy (relative error: RE) and precision
(relative standard deviation: RSD) less than 20%.
2.6.3 Precision and accuracy. Three validation batches,
each containing six QC samples of three concentration levels
were analyzed to assess the precision and accuracy of this
method on three consecutive days. The concentrations were
calculated with calibration curves obtained daily. The inter- and
intra-day precision is expressed as the relative standard devia-
tion (RSD). Accuracy is dened as the relative error (RE) and is
calculated using the formula RE% ¼ [(measured value
theo-
retical value)]/theoretical value  100%.
2.6.4 Extraction recovery and matrix effect. The extraction
recovery of the analytes was determined by comparing the areas
of the QC samples at low, medium and high levels (n ¼ 6) with
those of the spike-aer-extraction samples (representing 100%
recovery).
The matrix effect on the ionization of the analytes was eval-
uated by comparing the peak areas of the analytes resolved in
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Precursor ion Product ion Collision energy/V
336.0 320.1 30
352.0 336.1 30
338.0 322.1 30
336.0 320.1 34
320.0 292.1 30
356.4 192.1 58
the blank sample (the nal solution of blank plasma aer
extraction and reconstitution) with that resolved in the mobile
phase. Three different concentration levels of berberine, pal-
matine, epiberberine, jatrorrhizine, coptisine and 20 ng ml
1 of
the IS were evaluated by analyzing the six samples at each level.
The blank plasma used in this study were six different batches
of blank rat plasmas. If the ratio is <85% or >115%, an exoge-
nous matrix effect is implied.
2.6.5 Cross-analyte interference. To investigate the
possible cross-interference between the analytes and IS, a cross-
interference check was performed. Blank plasma was spiked
with each of the six ingredients at the ULOQ level (200 ng ml
1
for berberine, 100 ng ml
1 for palmatine, jatrorrhizine, epi-
berberine and coptisine, respectively) and was processed
without IS. In addition, blank plasma with only IS was pro-
cessed. The response of any interfering peak with the same
retention time as the analytes should be less than 20% of the
response of a LLOQ sample.
2.6.6 Carryover. Carryover was tested by injecting two pro-
cessed blank matrix samples sequentially aer injecting a
ULOQ sample. The response in the rst blank matrix at the
retention times of the analytes and IS should be less than 20%
of the response of a LLOQ sample.
2.6.7 Stability. The purpose of the stability study was to
evaluate the optimal storage conditions of the samples. The
stability of the analytes in rat plasma was investigated by
analyzing stability of the samples at three concentrations (the
same levels as the QC samples) (n ¼ 6) that were exposed to
different conditions. They were prepared by spiking blank
plasma with appropriate amounts of the working solutions (5%
of the total plasma sample volume) to obtain the concentra-
tions. The short-term stability was determined during storage
for 4 hour at room temperature and the concentrations of the
analytes in the plasma deviated less than 10% from those in
the freshly spiked plasma. The post-treatment stability was
obtained by the extracted samples maintained in an autosam-
pler at 4  C for 24 h. The freeze–thaw stability was evaluated
aer three freeze (
20  C)–thaw (room temperature) cycles on 3
consecutive days. The long-term stability was assessed aer the
QC samples had been stored at
20  C for 30 days.
2.7 Application
The pharmacokinetic study was approved by the Animal Ethics
Committee of Guangzhou University of Chinese Medicine.
Twelve male Sprague Dawley rats (250  20 g) were supplied by
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the Lab Animal Center at Guangzhou University of Chinese
Medicine (Guangdong, China). The rats were housed in stan-
dard cages at room temperature with free access to food and
water until 12 h prior to experiments.
Rats were divided into two groups randomly (n ¼ 6) and
administrated an oral dose of RC extract (300 mg kg
1) and JTW
extract (300 mg kg
1 RC extract and 4.7 mg kg
1 Cinnamon
cassia oil). Blood samples of approximately 0.1 ml were
collected in heparinized centrifuge tubes at 0, 10, 30, 60, 90,
120, 180, 240, 300, 360, 480 and 600 min aer a single oral
administration. Then the samples were centrifuged at 5000 g
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292.1 for coptisine, at m/z 336.0 / 320.1 for epiberberine, at
m/z 338.0 / 322.1 for jatrorrhizine and at m/z 356.4 / 192.1
for tetrahydropalmatine. The MS/MS product ion spectra of the
analytes and IS are shown in Fig. 2.
The mass spectrometric parameters such as the drying gas
ow, the drying gas temperature, the nebulizer pressure, and
capillary voltage were all optimized to obtain a higher signal for
both the precursor ions and product ions mentioned above.
In the present study, a gradient elution was adopted. Our
previous investigation showed that berberine, palmatine and
jatrorrhizine can be quantitated rapidly with a mixture of water

for 10 min. The separated plasma samples were frozen in (containing 0.1% formic acid) and acetonitrile (30 : 70, v/v) as
polypropylene tubes at
20  C until analysis. the mobile phase.5 But in this study, it was found that berberine
and epiberberine could not be separated under these condi-
2.8 Data analysis tions. Since berberine and epiberberine have the same selective
product ions they cannot be accurately determined without
The non compartmental pharmacokinetic analysis of the
separation. To select a mobile phase, comparisons were made
concentration time data was performed using Winnonlin 5.0.1
among the changes caused by the water content (30, 40, 50%) at
soware. The pharmacokinetic parameters, such as maximum
the beginning of the gradient cycle. The berberine and epi-
plasma concentration (Cmax) and the time of the maximum
berberine can be separated with 30% water content at the
concentration (Tmax), were obtained directly from the plasma
beginning of the gradient cycle. To nd the optimized mobile
concentration–time plots. The elimination rate constants (k)
phase, we tested ammonium acetate (5 mmol l
1) with an
were determined by linear regression analysis of the loga-
adjusted pH to 3.0, 4.0 and 5.0 with formic acid. It was found
rithmic transformation of the last three or four data points of
that the responses were similar under different mobile phase
the curve. The elimination half-life (t1/2) was calculated using
conditions. But the mobile phase adjusted to pH 5.0 with formic
the following equation: t1/2 ¼ 0.693/k. The area under the
acid was found to be the best mobile phase to obtain a good
plasma concentration–time curve up to the last time (t) (AUC0–t)
peak shape. So the pH 5.0 mobile phase was adopted. Then,
was determined using the trapezoidal rule. An unpaired Stu-
concentrations of ammonium acetate (5, 10, 15 mmol L
1)
dent's t-test was used for comparisons with SPSS 13.0.

3 Results and discussion

3.1 Sample preparation
In the present study, a simple single-step protein precipitation
procedure was used. In our previous study, different kinds of
extraction procedures, including liquid–liquid extraction (LLE)
had been evaluated during our method development.5 Finally, a
simple single-step protein precipitation procedure had been
used. In this study, it was found that the extraction recoveries of
coptisine and epiberberine were also satisfactory with the
protein precipitation procedure. Therefore, it was appropriate
for the sample preparation in the determination of the ve
alkaloids.

3.2 LC-MS/MS optimization

The analytes and IS were at rst characterized by a MS2 scan and
the MS/MS product ion to ascertain their precursor ions and to
select product ions for use in the MRM mode, respectively. In
the full-scan spectra, the analytes formed predominantly
quaternary ammonium ions [M]+. The IS produced abundant
protonated molecular ions [M + H]+. To get the richest relative
abundance of the precursor and product ions, the parameters
for the fragmentor energy and collision energy were optimized
by Masshunter Optimizer soware. Both the analytes and IS
were detected by tandem mass spectrometry using the MRM of Fig. 2 The product ion mass spectra of the analytes and internal
the precursor–product ion transitions at m/z 336.0 / 320.1 for standards. (A) berberine, (B) palmatine, (C) coptisine, (D) epiberberine,
berberine, at m/z 352.0 / 336.1 for palmatine, at m/z 320.0 / (E) jatrorrhizine, (F) tetrahydropalmatine (IS).

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adjusted to pH 5.0 with formic acid were investigated. It was
found that the concentration of ammonium acetate did not
signicantly affect the sensitivity and retention time of each
analyte. So the concentration of 5 mmol L
1 adjusted to pH 5.0
with formic acid was selected as the mobile phase.
3.3 Method validation
3.3.1 Selectivity. Fig. 3 demonstrates the typical chro-
matograms of the blank plasma sample (A), the blank plasma
sample spiked with analytes at the LLOQ with IS (B), and the test
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plasma sample obtained aer the administration of RC extract
to the rats (C). No signicant interference by endogenous enti-
ties was observed under the current chromatographic and MS
conditions. The running time of each injection was only 6.0
min. The retention time of berberine, palmatine, jatrorrhizine,
epiberberine, coptisine and IS was 2.66 min, 2.30 min, 1.65 min,
1.73 min, 1.95 min and 2.73 min, respectively.
Fig. 3 The chromatograms of the six compounds in plasma: (A) blank plasma; (B) blank plasma spiked with the six components at the LLOQ;
(C) the plasma sample 40 min after a single oral administration of JTW (I ¼ coptisine, II ¼ berberine, III ¼ epiberberine, IV ¼ jatrorrhizine,
V ¼ palmatine and VI ¼ tetrahydropalmatine).
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Analytical Methods
3.3.2 Linearity and sensitivity. The assay was linear over
the validated concentration range from 0.20 to 200.0 ng ml
1 of
berberine and from 0.20 to 100.0 ng ml
1 for palmatine,
jatrorrhizine, epiberberine and coptisine, respectively. Table 3
shows the typical regression equations of the calibration curve.
As seen in Table 3, the LLOQ of berberine, palmatine, jatror-
rhizine, epiberberine and coptisine was proved to be 0.20 ng
ml
1 which was sufficient for the preclinical pharmacokinetic
study in rats following the oral administration of RC and JTW
extract. The precision and accuracy data corresponding to the
LLOQ are also shown in Table 4.
3.3.3 Precision and accuracy. Table 5 summarizes the
intra- and inter-day precisions and accuracies of berberine,
palmatine, jatrorrhizine, epiberberine and coptisine at three
concentration levels (low, medium and high). The intra- and
inter-day precision was less than 10.8% for each QC level of
three analytes. The accuracy, determined from the QC samples,
was within 10.5% for each QC level. These results were within
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Table 3 The regression equations and lower limit of quantification of the five analytes

Analytes Regression equation r Linear range (ng ml
1) LLOQ (ng ml
1)

Berberine Y ¼ 9.3552  10
2x + 7.1193  10
2 0.9992 0.20–200.0 0.20

Palmatine Y ¼ 1.7759  10
2x + 1.0093  10
1 0.9963 0.20–100.0 0.20
Jatrorrhizine Y ¼ 6.4770  10
4x + 6.2251  10
2 0.9986 0.20–100.0 0.20
Epiberberine Y ¼ 4.3148  10
3x
5.2032  10
2 0.9995 0.20–100.0 0.20
Coptisine Y ¼ 3.1119  10
3x + 4.1750  10
2 0.9992 0.20–100.0 0.20

the acceptance criteria and indicated that the method was 3.3.7 Stability. In our study, stability samples (at three
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accurate, reliable and reproducible.
3.3.4 Extraction recovery and matrix effect. At three
concentration levels of these analytes, the extraction recoveries
all were between 89.1% and 99.8%. These results demonstrated
that the values all were within the acceptable range and that the
method was accurate. The matrix effects were generally caused
by undetected matrix components that reduced or enhanced
the ion intensity of the analytes. An endogenous matrix effect is
implied if the ratio is less than 85% or greater than 115%. In the
current article, the mean absolute matrix effect values obtained
for palmatine, jatrorrhizine, epiberberine, berberine, coptisine
at three concentrations were between 98.7 and 113.5% (Table 6),
demonstrating that there was not a signicant matrix effect.
The extraction recoveries and matrix effects of berberine, pal-
matine, jatrorrhizine, epiberberine and coptisine in rat plasma
are shown in Table 6.
3.3.5 Cross-analyte interference. The cross-talk was
observed between berberine and epiberberine without separa-
tion. But in this investigation, they were fully separated. So
there was no cross-talk between berberine and epiberberine in
this study. Therefore, the developed analytical method was
reliable and suitable for this study.
3.3.6 Carryover. Carryover was tested by injecting two pro-
cessed blank plasma samples sequentially aer injecting an
ULOQ sample. The response in the rst blank matrix at the
retention times of each component were 24.5, 12.3, 8.4, 14.9 and
18.1% of the LLOQ for berberine, palmatine, jatrorrhizine,
epiberberine and coptisine, respectively. The carryover seemed
to arise from contamination of the autosampler needle. To solve
this carryover problem, a mixture of water (containing 0.1%
formic acid) and acetonitrile (40 : 60, v/v) was used as the needle
ush. In addition, the wash time was increased from 9 s to 15 s.
Carryover was reduced to 14.6, 7.3, 6.1, 8.7 and 11.3% of the
LLOQ for berberine, palmatine, jatrorrhizine, epiberberine and
coptisine, respectively in the present conditions. The carryover
was within the acceptable range.
levels) subjected to short-term storage, post-treatment storage,
three freeze–thaw cycles, and long-term storage were examined
for their stabilities, and the results are summarized in Table 7.
As can be seen from Table 7, there is no signicant reduction
(RE% between
11.49% and +9.52%) in the assay concentra-
tions at any QC level following the above conditions. This
demonstrated that berberine, palmatine, jatrorrhizine, epi-
berberine and coptisine have a good stability under the four
conditions in this method. There were no stability related
problems during the routine analysis of the samples for the
pharmacokinetics study.
3.4 The application of the analytical method in
pharmacokinetic studies
This study had investigated the pharmacokinetic comparisons of
berberine, palmatine, coptisine, epiberberine and jatrorrhizine
aer the oral administration of RC and JTW extract. The proto-
berberine-type alkaloids contained in JTW were considered as
the active ingredients among a variety of compounds. In previous
investigations on the pharmacokinetics of the TCM formula
containing RC, the main strategy was to choose one or multiple
alkaloids as the target components.3,5,10,12 For example, Lu et al.
have investigated the pharmacokinetics of berberine and pal-
matine aer the oral administration of Huang-Lian-Jie-Du
decoction.10 Feng et al. have investigated the pharmacokinetic of
baicalin, baicalein, wogonin, berberine, palmatine and jatror-
rhizine aer the oral administration of the Scutellaria–Coptis
herb couple.12 Yu et al. have investigated the pharmacokinetics of
berberine, palmatine, coptisine, epiberberine and jatrorrhizine
aer the oral administration of RC extract.16 For the complicated
system of TCM, there are large herb–herbs interactions. The
combination with other herbs may change the herb pharmaco-
kinetics.21–26 Lu et al. reported that the combination of Rhizoma
coptidis and Radix scutellariae would decrease the systematic
exposure level of baicalin and wogonoside, and this was probably

Table 4 The accuracy and precision results for the five analytes at the plasma concentrations of LLOQ (n ¼ 6)

Nominal plasma Concentration measured

Analytes concentration (ng ml
1) (mean  SD, ng ml
1) Relative error (%) RSD (%)

Berberine 0.20 0.18  0.01
12.4 6.54

Palmatine 0.20 0.19  0.02
3.18 11.80
Jatrorrhizine 0.20 0.17  0.01
12.9 5.98
Epiberberine 0.20 0.21  0.01 2.57 6.68
Coptisine 0.20 0.19  0.02
6.42 9.94

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Table 5 Intra-day and inter-day accuracy and precision of the five analytes in rat plasma at low, medium and high concentration levels

Analytes spiked Intraday (n ¼ 6) Interday (n ¼ 18)

Concentration measured Accuracy Precision Concentration measured Accuracy Precision

Concentration (ng ml
1) (mean  SD, ng ml
1) (%) (%) (mean  SD, ng ml
1) (%) (%)

Berberine
0.50 0.52  0.04 4.86 6.70 0.54  0.08 8.61 4.65
10.0 9.91  0.12
0.94 1.18 9.82  0.42
1.85 8.58
160.0 159.6  3.22
0.24 2.01 158.3  5.44
1.07 3.42

Palmatine
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0.50 0.50  0.02 0.89 4.65 0.51  0.02 1.64 4.80

4.0 3.93  0.09
1.76 2.26 3.89  0.18
2.69 8.58
80.0 78.14  2.04
2.32 2.61 79.52  5.7
0.60 3.42

Jatrorrhizine
0.50 0.48  0.02
4.19 3.74 0.51  0.04 2.72 4.65
4.0 3.93  0.22
1.81 5.74 3.98  0.27
0.60 8.58
80.0 77.18  3.03
3.52 3.93 81.62  6.08 2.02 3.42

Epiberberine
0.50 0.52  0.03 4.06 6.52 0.50  0.04 0.20 4.65
4.0 3.90  0.06
2.40 1.65 3.89  0.20
2.69 8.58
80.0 75.85  1.87
5.18 2.46 79.20  5.08
1.00 3.42

Coptisine
0.50 0.50  0.030
0.60 6.08 0.50  0.04
0.58 4.65
4.0 3.90  0.11
2.52 2.79 3.89  0.28
6.25 8.58
80.0 77.87  1.91
2.66 2.45 80.27  4.11 0.33 3.42

caused by the inhibition of bacterial b-glucuronidase activities by
Rhizoma coptidis and Cortex phellodendri or their main active
ingredients.22 Xu et al. reported that both the AUC0/tn and t1/2 of
schizandrin in Fructus schisandraeaqueous extract and in Sheng-
Mai-San (SMS) decoction were increased signicantly (P < 0.05)
when compared with that in the monomer.23
JTW is composed of RC and Cinnamon cassia, and the
Cinnamon cassia would affect the pharmacokinetics of the
alkaloids contained in RC. Huang et al. reported that RC gran-
ules combined with Cinnamon cassia granules could increase
the plasma concentration of berberine, promote berberine
absorption and lengthen the detention time of berberine in
healthy male volunteers.3 But whether combination with
Cinnamon cassia would affect the pharmacokinetics of other
alkaloids is still unclear. So in this study, berberine, palmatine,
coptisine, epiberberine and jatrorrhizine were chosen as the
target components to investigate the pharmacokinetics differ-
ences of RC and JTW.
Aer the oral administration of the RC and JTW extract to six
rats, the plasma concentrations of ve protoberberine-type

Table 6 The extraction recovery and matrix effect of the five analytes in rat plasma at low, medium, high concentration levels (n ¼ 6)

Spiked concentration Extraction recovery Matrix effect

Analytes (ng ml
1) (%, mean  SD) RSD (%) (%, mean  SD) RSD (%)

Berberine 0.5 99.13  6.04 6.09 113.51  3.10 2.73

10.0 98.98  7.21 7.27 112.19  7.56 6.74
160.0 92.30  9.52 10.09 102.33  2.22 2.17
Palmatine 0.5 99.79  3.07 3.08 107.46  6.16 5.72
4.0 98.08  6.26 6.36 108.08  5.26 4.87
80.0 91.45  10.06 10.76 101.45  2.97 2.93
Jatrorrhizine 0.5 99.12  8.09 8.16 103.99  8.21 7.90
4.0 93.90  4.22 4.49 102.60  1.92 1.87
80.0 89.12  8.61 9.52 98.73  4.71 4.77
Epiberberine 0.5 96.06  7.00 7.29 110.26  6.56 5.94
4.0 95.99  7.27 7.55 110.37  6.75 6.12
80.0 90.35  9.46 10.26 99.68  2.42 2.43
Coptisine 0.5 99.13  5.02 5.06 112.84  8.25 7.28
4.0 95.72  10.45 10.90 113.04  9.10 8.05
80.0 92.06  9.11 9.71 101.24  3.03 3.00

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Table 7 The stability of the three analytes in rat plasma (n ¼ 6)

At room temperature Post-treatment for Aer three freeze–thaw

for 4 h 24 h at 4  C cycles At
20  C for 30 days

Concentration Concentration Concentration Concentration

Concentration (mean  SD, RE (mean  SD, RE (mean  SD, RE (mean  SD, RE
(ng ml
1) ng ml
1) (%) ng ml
1) (%) ng ml
1) (%) ng ml
1) (%)

Berberine 0.5 0.53  0.03 6.08 0.53  0.012 4.86 0.53  0.03 6.97 0.53  0.03 6.97
10 10.97  0.23 9.71 9.47  0.521
5.26 11.27  0.29 12.7 9.63  2.26
3.73
160 176.75  3.05 10.50 163.60  1.820 2.25 173.38  4.34 8.36 154.04  10.61
3.72
Palmatine 0.5 0.50  0.03 0.19 0.45  0.018
10.8 0.45  0.05
10.2 0.47  0.15
6.87
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4 3.69  0.09
7.71 4.02  0.180 0.57 3.84  0.06
2.13 3.69  0.20
7.79
80 86.17  1.08 7.71 85.35  0.614 6.68 87.44  2.44 9.3 88.40  0.72 10.5
Jatrorrhizine 0.5 0.55  0.06 9.19 0.46  0.029
8.93 0.55  0.02 10.0 0.48  0.01
3.50
4 3.77  0.11
5.68 4.14  0.233 3.5 4.48  0.19 12.1 4.00  0.16
0.03
80 89.60  1.70 12.00 87.88  2.756 9.85 90.56  2.05 13.2 75.19  3.65
6.02
Epiberberine 0.5 0.51  0.04 1.59 0.45  0.019
10.3 0.45  0.02
9.62 0.49  0.06
2.97
4 3.66  0.08
8.51 4.00  0.238
0.10 3.73  0.06
6.75 3.69  0.22
7.75
80 85.81  0.360 7.26 85.46  0.611 6.82 83.49  0.87 4.36 85.69  2.30 7.11
Coptisine 0.5 0.52  0.04 3.73 0.47  0.018
5.9 0.47  0.03
6.64 0.48  0.04
3.90
4 3.66  0.06
8.56 4.03  0.170 0.77 3.92  0.15
1.95 3.68  0.20
7.94
80 85.41  1.23 6.77 84.92  1.737 6.15 86.99  4.00 8.74 75.10  4.83
6.13

alkaloids were simultaneously determined by the described LC- listed in Table 8. As shown in Table 8, the Cmax of the ve
MS/MS. The mean plasma concentration–time proles (n ¼ 6) protoberberine alkaloids were low aer the oral administration
are represented in Fig. 4. The pharmacokinetic parameters are of RC and JTW extract, which was consistent with previous

Fig. 4 The mean (SD, n ¼ 6) plasma concentration–time profiles of the five ingredients in rats after the oral administration of RC and JTW
extract. (A) Berberine; (B) palmatine; (C) coptisine; (D) epiberberine; (E) jatrorrhizine.

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Table 8 The pharmacokinetic parameters of the five analytes in rats after the single oral administration of RC and JTW extract (n ¼ 6)

Cmax /ng ml
1, AUC0–t

Compound Decoction form mean  S.D. Tmax/h T1/2/h k (ng
1$h
1 ml
1)

Berberine RC 12.27  3.30 1.90  0.58 3.96  0.92 0.18  0.05 92.71  15.03
JTW 10.19  3.96 5.00  1.79b 3.46  2.24 0.24  0.12 98.00  28.80
Palmatine RC 0.74  0.44 1.30  0.40 3.91  0.80 0.18  0.04 4.72  0.79
JTW 0.85  0.17 5.90  2.69a 2.62  0.52 0.25  0.07 7.87  1.90b
Coptisine RC 1.39  0.60 1.38  0.54 4.02  1.83 0.23  0.17 14.28  2.38
JTW 4.09  1.78 4.40  1.85b 2.31  0.18 0.27  0.07 44.76  19.23a
Epiberberine RC 1.05  0.75 1.38  0.54 5.28  1.44 0.14  0.04 6.63  1.70
JTW 2.86  1.81 4.60  1.74b 3.97  2.16 0.23  0.12 26.15  15.24a
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Jatrorrhizine RC 0.67  0.23 1.38  0.54 4.35  0.67 0.16  0.03 7.11  0.65
JTW 1.04  0.67 5.25  1.92b 4.17  1.64 0.20  0.08 10.36  4.28
a
P < 0.05. b P < 0.01; vs. RC extract.

ndings.16Among the parameters, the Tmax of the alkaloids in References

the plasma aer the oral administration of the two extracts were
considered to be signicant (P < 0.05) while the t1/2 and k were
considered to be not signicant aer the analysis of the
unpaired Student's t-test. The AUC0–t of palmatine, coptisine
and epiberberine were signicantly increased (P < 0.05). It could
be inferred that the co-administration of the Rhizoma coptidis
and Cinnamon cassia extract might delay the absorption of the
alkaloids without affecting the progress of elimination.
Furthermore it might increase the bioavailability of palmatine,
coptisine and epiberberine.
All of the data indicated that the combination of Cinnamon
cassia would affect the absorption of alkaloids contained in
Rhizoma coptidis.
4 Conclusion
A sensitive and selective LC-MS/MS method for the simulta-
neous determination of berberine, palmatine, coptisine,
epiberberine and jatrorrhizine in rat plasma was developed
and validated. The proposed method showed appropriate
accuracy and precision and was successfully applied to
pharmacokinetic comparisons of the ve ingredients aer
the oral administration of RC and JTW, which indicated that
the method is suitable for pharmacokinetics studies of the
ve compounds. The pharmacokinetic differences indicated
that the combination of Cinnamon cassia would delay the
absorption of the alkaloids and increase the bioavailability of
palmatine, coptisine and epiberberine contained in RC. But
the mechanism is still ambiguous and further profound
research is required.
Notes
The authors declare no competing nancial interest.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (grant no. 81173564 and 81274028).
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