Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 110

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Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.elsevier.com/locate/jpba

Simultaneous determination of four phenolic acids and seven

alkaloids in rat plasma after oral administration of traditional Chinese
medicinal preparation Jinqi Jiangtang Tablet by LC-ESIMS/MS
Yan-xu Chang a,b, , Ai-hua Ge a,b , Xie-an Yu a,b , Xiu-cheng Jiao a,b , Jin Li a , Jun He a,b ,
Ji Tian a,b , Wei Liu a,b , John Teye Azietaku a,b , Bo-li Zhang a , Xiu-mei Gao a
a
b

Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
Tianjin Key Laboratory of Phytochemistry and Pharmaceutical Analysis, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China

a r t i c l e

i n f o

Article history:
Received 9 May 2015
Received in revised form 18 August 2015
Accepted 19 August 2015
Available online 24 August 2015
Keywords:
Phenolic acids
Berberine
Chlorogenic acid
Jinqi Jiangtang tablet
LCMS

a b s t r a c t
A rapid, sensitive and selective high performance liquid chromatographytandem mass spectrometry
(LCMS/MS) method was developed and validated for the simultaneous determination of four phenolic
acids (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and ferulic acid) and seven alkaloids (berberine, epiberberine, coptisine, magnoorine, berberubine, palmatine and jatrorrhizine) in rat
plasma. After mixing with the internal standards tetrahydropalmatine (IS1 ) and rosmarinic acid (IS2 ),
plasma samples were pretreated by protein precipitation using acetonitrile. The HPLC analysis was performed on an Agilent Eclipse plus C18 (4.6 mm 100 mm, 1.8 m) column with mobile phase consisting
of 0.1% formic acid aqueous solution and acetonitrile at a ow rate of 0.3 mL min1 . The detection was
accomplished for the analytes and internal standards using positive electrospray ionization for the alkaloids and negative electrospray ionization for the phenolic acids in multiple-reaction monitoring mode.
The method showed a good linearity over a wide concentration range (r2 > 0.99). The lower limit of
quantication of seven alkaloids was lower than 2 ng mL1 and that of four phenolic acids was less than
20 ng mL1 . The developed method was applied to the pharmacokinetic study of 11 components after
oral administration of traditional Chinese medicinal preparation Jinqi Jiangtang Tablet in rats.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Traditional Chinese medicines (TCMs) have been used as an
important resource for the prevention and treatment of various diseases for more than 1000 years [1]. Typically, TCMs have different
kinds of components. It is well known that multiple components
act on multiple targets and exert synergistic therapeutic efcacies,
which are the unique mechanism of action of TCMs [2]. Nowadays,
more and more attention has been focused on the active components from TCMs. The pharmacokinetic study on active components
in TCM could have a great effect on evaluating the mechanism of
action to better elucidate the efcacy of TCMs.
Traditional Chinese medicinal preparation Jinqi Jiangtang (JJT)
Tablet consists of three herbal plants: Coptis chinensis (rhizome

Corresponding author at: Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, China.
Fax: +86 25 59596163.
E-mail address: Tcmcyx@126.com (Y.-x. Chang).
http://dx.doi.org/10.1016/j.jpba.2015.08.030
0731-7085/ 2015 Elsevier B.V. All rights reserved.

of C. chinensis Franch.), Astragalus membranaceus (root of A.

membranaceus Moench) and Lonicera japonica (ower buds of L.
japonica Thunb.). It has been used for the treatment of diabetes
and its complications clinically [34]. Modern pharmacological
researches demonstrated that JJT had various bioactivities such
as anti-inammation, gastrointestinal tract and radical scavenging effects [5]. Phenolic acids like neochlorogenic acid, chlorogenic
acid, cryptochlorogenic acid and ferulic acid are the main bioactive compounds in L. japonica [6], while the alkaloids berberine,
epiberberine, coptisine, magnoorine, berberubine, palmatine and
jatrorrhizine are the main bioactive components in Rhizome Coptidis [7]. In previous studies, it was found out that the total alkaloids
of C. chinensis had hypoglycemic effect and the phenolic acids had
free radical scavenging activity [8]. It was these components that
gave JJT tablet its pharmacological activity.
There are many studies on the simultaneous determination of
alkaloids in TCMs and their application in rat plasma using HPLCUV [9], HPLC-ED [10], LCMS/MS [11] and UHPLCMS/MS as the
analytical methods [12]. However, these methods only focused on
pharmacokinetics of the normal alkaloids with high concentrations

Y.-x. Chang et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 110

in TCMs and their preparations. Moreover, a UPLCMS/MS method

was reported to simultaneously determine phenolic acids after oral
administration of Flos Lonicerae prescriptions [13]. A method was
developed and validated to evaluate the pharmacokinetic properties of ferulic acid in normal and blood deciency rats [14].
However, there is currently no analytical method to assess the
pharmacokinetic study of JJT tablet, of which both alkaloids and
phenolic acids are the main components. The physicochemical differences between alkaloids and phenolic acids, the low contents
of the components and the low absorption of alkaloids after oral
administration may be the reasons why no method has been established for the simultaneous determination of these components in
rat plasma. Obviously, it is important to make a comprehensive
investigation into the pharmacokinetic study of the active components, which could help to determine the rational dose, avoid
undesirable interactions and minimize side effects in clinic. Therefore, it is necessary to develop a rapid and sensitive method for
the simultaneous determination of the alkaloids and the phenolic
acids which will help in effectively evaluating the pharmacokinetic
property of JJT tablet.
In this study, a simple, rapid and sensitive LC-ESIMS/MS
method was rstly developed and validated for the simultaneous determination of the four phenolic acids (neochlorogenic
acid,chlorogenic acid, cryptochlorogenic acid and ferulic acid) and
seven alkaloids (berberine, epiberberine, coptisine, magnoorine,
berberubine, palmatine and jatrorrhizine) after oral administration
of JJT tablet in rat plasma. The chemical structures of the 11 components and 2 internal standards are shown in Fig. 1. The method
was successfully applied to the pharmacokinetic study after oral
administration of the JJT tablet. To the best of our knowledge,
this is the rst report of a simultaneous determination of berberine, epiberberine, coptisine, magnoorine, berberubine, palmatine,
jatrorrhizine, chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid and ferulic acid after oral administration of JJT tablet.
This is also the rst study of the pharmacokinetic property of JJT
tablet and berberubine after oral administration.

spectrometer with an ESI source (Concord, Ontario, Canada). Data

were analyzed by Analyst 1.4.2 software (AB MDS Sciex).
The separation was achieved on an Agilent Eclipse plus C18
(4.6 mm 100 mm, 1.8 m) column. The mobile phases consisted
of acetonitrile (A) and 0.1% formic acid (B) using a gradient elution
of 1020% (v/v) A at 010 min; 2020% A at 1014 min; 2027% A
at 1417 min; 2750% A at 1720 min; 5070% A at 2025 min;
7080% A at 2530 min. The ow rate was set at 0.3 mL min1 .
The injection volume was 5 L. The column temperature was set
at 30 C. The ESIMS data was obtained in positive electrospray
ionization for alkaloids (berberine, epiberberine, coptisine, magnoorine, berberubine, palmatine and jatrorrhizine) and negative
electrospray ionization for the phenolic compounds (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and ferulic
acid) using multiple-reaction monitoring mode. The source parameters were optimized to obtain the best sensitivity and response of
every component. The results are listed in Table 1.
2.3. Preparation of standards and quality control (QC) samples
Stock solutions at 1.0 mg mL1 for each compound were
prepared in methanol. The mixture of IS stock solutions of tetrahydropalmatine (1.0 g mL1 ) and rosmarinic acid (1.0 g mL1 )
were also prepared in methanol and kept at 50 and 100 ng mL1
level in working solutions and samples. Appropriate volumes of
each compound stock solution were mixed together. Then, the
mixture was diluted serially by methanol to achieve the standard
working solutions. All the solutions were kept at 4 C until use.
Quality control (QC) samples of each compound were prepared
by spiking 10 L of the standard working solutions into 100 L of
blank rat plasma to provide the nal concentration in the ranges of
0.04, 0.12, 1.2, 12 ng mL1 for berberine and palmatine; 0.08, 0.24,
2.4, 24 ng mL1 for epiberberine and berberubine; 0.16, 0.48, 4.8,
48 ng mL1 for jatrorrhizin; 0.2, 0.6, 6, 60 ng mL1 for coptisine; 2,
6, 60, 600 ng mL1 for magnoorine; 0.4, 1.2, 12, 120 ng mL1 for
chlorogenic acid; 4, 12, 120, 1200 ng mL1 for ferulic acid and 20, 60,
600, 6000 ng mL1 for cryptochlorogenic acid and neochlorogenic
acid.

2. Experimental
2.4. Content of the 11 components in JJT tablet
2.1. Chemicals and reagents
Acetonitrile of MS-grade was purchased from Merck (Darmstadt, Germany) and methanol (Tianjin Concord Science Co. Ltd.,
Tianjin, China) were of HPLC grade. HPLC-grade formic acid was
purchased from Tedia Company Inc. (Tedia, Faireld, OH, USA).
Deionized water was puried with a Milli-Q Academic ultra-pure
water system (Millipore, Milford, MA, USA). Reference Standards
of chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid,
ferulic acid, magnoorine, coptisine, epiberberine, jatrorrhizin
hydrochloride, berberine hydrochloride, berberubine and palmatine hydrochloride (purity > 98%) were purchased from Chengdu
Must Biotechnology Co. Ltd. (Chengdu,China). traditional Chinese medicinal preparation Jinqi Jiangtang Tablet was obtained
from Tianjin pharmacy (Tianjin, China). Voucher specimens were
deposited at the Institute of Traditional Chinese Medicine (Tianjin university of Traditional Chinese Medicine). All other reagents
were of analytical grade and obtained commercially.

2.2. Equipment and LCMS/MS conditions

The LCMS/MS system was made up of an Agilent 1200
series HPLC system (Agilent Technologies, USA), consisting of a
G1312A binary pump, a vacuum degasser unit (G1322A), a HipALS auto-sampler (G13678) and an API3200 triple quadruple mass

The contents of berberine, epiberberine, coptisine, magnoorine, berberubine, palmatine, jatrorrhizine, cryptochlorogenic
acid, chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid
and ferulic acid in JJT tablet for oral administration were determined by the established LCMS/MS method. The powder of JJT
tablet (0.10 g) was extracted with 10 mL 70% methanol and ultrasonicated for 40 min according to previous research [15]. After
centrifugation at 14,000 rpm for 10 min, the supernatant was ltered through a 0.22 m membrane lter. At last, an aliquot of
2 L of supernatant was injected into the LC-ESIMS/MS system.
Finally, the contents of berberine, epiberberine, coptisine, magnoorine, berberubine, palmatine, jatrorrhizine, chlorogenic acid,
cryptochlorogenic acid, neochlorogenic acid and ferulic acid were
20.8, 5.5, 8.33, 0.22, 0.10, 3.48, 8.70, 27.4, 1.49, 1.17 and 0.19 mg g1
in traditional Chinese medicine JJT tablet, respectively.
2.5. Plasma sample preparation
Plasma samples were pretreated employing a simple protein
precipitation with acetonitrile. After thawing to room temperature,
100 L of the rat plasma was mixed with 10 L of the IS working
solutions containing tetrahydropalmatine (50 ng mL1 ) and rosmarinic acid (100 ng mL1 ) and 10 L of formic acid. The mixture
was vortex-mixed for 1 min and then 400 L acetonitrile was added
and centrifuged for 10 min at 14,000 rpm. Supernatant was trans-

Y.-x. Chang et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 110

Fig. 1. Chemical structures of 11 components and 2 internal standards.

ferred and condensed to dryness by nitrogen gas. The dried residue

was reconstituted by 100 L methanol by vortex-mixing for 1 min
and centrifuged at 14,000 rpm for 10 min. Then, 5 L of the solution
was injected into the LCMS/MS system for analysis.
2.6. Method validation
A full validation according to the industrial guidelines for
bioanalytical method validation from the US Food and Drug Administration (FDA) was carried out for the assay in rat plasma (US Food
and Drug Administration, 2001) [16].
2.6.1. Selectivity
The selectivity was assessed by comparing the chromatograms
of six different batches of blank rat plasma samples with corresponding spiked plasma samples and obtained plasma after oral
administration of JJT tablet.
2.6.2. Linearity and lower limit of quantication (LLOQ)
For the calibration curves, the diluted standard working solutions were spiked into blank rat plasma to obtain the nal
concentrations in the ranges of 0.0415 ng mL1 for berberine
and palmatine; 0.0830 ng mL1 for epiberberine and berberubine;
0.1660 ng mL1 for jatrorrhizin; 0.275 ng mL1 for coptisine;
2750 ng mL1 for magnoorine; 0.4150 ng mL1 for chlorogenic
acid; 41200 ng mL1 for ferulic acid and 207500 ng mL1 for
cryptochlorogenic acid and neochlorogenic acid. All the standard
working solutions were kept at 4 C before use. The calibration

curve of each compound was constructed by plotting the ratio of

the peaks area of analytes and internal standard (IS) (y) against the
corresponding concentration (x, ng mL1 ) using a 1/X2 weighted
linear least-squares regression model.
The lower limit of quantication (LLOQ) is the lowest concentration of analyte in a sample which can be quantied reliably, with
an acceptable accuracy and precision. In addition, the analyte signal of the LLOQ sample should be at least 5 times the signal of a
blank sample. The relative standard deviation (RSD) of the LLOQ
sample within 20% and the accuracy was in the range of 80% to
120% according to the USFDA guidelines.

2.6.3. Precision and accuracy

The accuracy and precision were determined by using six replicates of QC samples at four levels on the same day (intra-day)
and between three different days (inter-day) and nally assessed
using the calibration curve constructed on the same testing day.
The precision of intra-day and inter-day were expressed as RSD
and accuracy was evaluated by comparing the calculated concentration with the spiked concentration. The RSD values should not
be more than 15% and accuracy should be in the range of 85% to
115%.

2.6.4. The recovery and matrix effect

The recovery and matrix effects at four QC levels were determined in sets of six replicates. The recovery was assayed by
comparing the peak areas obtained from plasma samples spiked

Table 1
Source and SRM parametersof 11 compounds and two internal standards (IS).
Compounds

CUR

CAD

IS

TEM

GS1

GS2

Q1

Q3

DP(V)

EP(V)

CE(V)

CXP(V)

Neochlorogenic acid
Chlorogenic acid
Cryptochlorogenic acid
Ferulic acid
Magnoorine
Coptisine
Epiberberine
Jatrorrhizin
Berberine
Palmatine
Berberubine
Tetrahydropalmatine (IS1)
Rosmarinic acid (IS2)

30
30
30
30
40
40
40
40
40
40
40
40
30

4
4
4
4
12
12
12
12
12
12
12
12
4

4500
4500
4500
4500
5000
5000
5000
5000
5000
5000
5000
5000
4500

500
500
500
500
600
600
600
600
600
600
600
600
500

40
40
40
40
40
40
40
40
40
40
40
40
40

30
30
30
30
20
20
20
20
20
20
20
20
30

353.0
353.0
353.1
192.9
343.3
320.1
337.2
339.3
337.3
353.1
323.2
356.2
359.1

191.1
191.0
190.7
133.9
266.3
292.1
320.1
323.2
321.1
337.2
308.1
192.2
161.1

33
33
33
35
45
45
58
55
45
55
52
45
35

5
5
5
5
5
6
4
5
4
5
7
5
5

33
25
22
22
30
40
42
39
40
33
37
40
23

2
2
2
2
3
3
3
3
4
3
3
2
5

Y.-x. Chang et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 110

Fig. 2. The chromatograms of the analytes in rat plasma: blank plasma (A), blank rat plasma spiked with standard compounds (B) and plasma samples taken from rats 30 min
after oral administration of traditional Chinese medicinal preparation Jinqi Jiangtang (C).

before extraction to those of analytes from solutions spiked in postextracted blank plasma at equivalent concentration.
The matrix effect is dened as the ion suppression/enhancement
on the ionization of analytes, which was measured via comparison
of the peak response from post extraction blank plasma samples
spiked with standard solutions at three levels in three replicates to
those of the corresponding concentration neat standard solutions.
The same procedure was applied for IS at a single concentration.
The matrix effect is not negligible if the ratio is less than 85% or
more than 115%..

2.6.5. Stability
The stability of analytes in plasma was investigated by analyzing
QC samples at four concentrations. The autosampler 24 h stability
was assayed after keeping the treated sample under autosampler conditions (room temperature) for 24 h. The QC samples were
subjected to three freeze (at about 20 C) to thaw (at room temperature) cycles for freeze and thaw stability. The stability was
expressed as RSD and accuracy. The long-term stability was examined by analyzing samples stored at about 80 C for 1 month.
For all stability testing of QC samples, the concentration was
compared with those of freshly prepared QC samples and the per-

Table 2
The calibration curves,linearity range,LLOQs of the assay(n = 6).
Compounds

Regression equation

Neochlorogenic acid
Chlorogenic acid
Cryptochlorogenic acid
Ferulic acid
Magnoorine
Coptisine
Epiberberine
Jatrorrhizin
Berberine
Palmatine
Berberubine

Y = 0.00566X + 0.236
Y = 0.173X + 0.492
Y = 0.0229X + 0.0414
Y = 0.0803X + 0.129
Y = 0.00706X 0.000419
Y = 0.0154X + 0.0154
Y = 0.0206X + 0.00792
Y = 0.019X + 0.000988
Y = 0.0201X + 0.0201
Y = 0.021X + 0.000668
Y = 0.0447X + 0.00429

0.9966
0.9954
0.9917
0.9944
0.9944
0.9917
0.9909
0.9900
0.9984
0.9963
0.9926

Linearity range
20-7500
0.4-150
20-7500
4-1500
2-250
0.2-75
0.08-30
0.16-60
0.04-15
0.04-15
0.09-30

LLOQ
(ng mL1 )
20
0.4
20
4
2
0.2
0.08
0.16
0.04
0.04
0.08

Y.-x. Chang et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 110

Table 3
Intra-day, inter-day accuracy and precision (n = 6).
Compounds

Concentration
(ng mL1 )

Neochlorogenic
acid

20
60
600
6000
0.4
1.2
12
120
20
60
600
6000
4
12
120
1200
2
6
60
600
0.2
0.6
6
60
0.08
0.24
2.4
24
0.16
0.48
4.8
48
0.04
0.12
1.2
12
0.04
0.12
1.2
12
0.08
0.24
2.4
24

Chlorogenic
acid

Cryptochlorogenic
acid

Ferulic
acid

Magnoorine

Coptisine

Epiberberine

Jatrorrhizin

Berberine

Palmatine

Berberubine

Intra-day

Inter-day

Accuracy (%)

RSD(%)

Accuracy()

RSD(%)

108
101
105
103
99.7
107
105
105
91.2
103
100
102
110
107
106
95.9
93.6
103
103
99.1
104
107
104
101
108
107
112
95.6
103
101
98.2
102
80.5
103
120
103
111
110
115
107
80.0
101
103
93.6

6.06
4.94
3.82
2.68
13.5
7.61
8.31
5.97
12.2
3.77
4.27
3.61
15.0
6.25
6.55
6.35
17.4
5.16
2.75
3.71
9.67
9.90
6.01
6.41
14.4
15.0
11.0
7.55
13.9
5.94
3.16
4.93
15.4
14.6
15.0
7.40
4.47
15.0
14.5
7.71
11.3
14.4
4.46
7.89

118
99.7
102
102
115
104
103
103
118
102
99.8
101
99.8
103
102
98.9
103
100
100
101
95.9
103
98.7
100
107
105
104
99.5
118
93.8
99.6
100
93.2
110
109
102
117
112
106
104
95.0
104
100
98.7

16.2
1.45
2.37
0.93
7.75
2.80
1.70
2.37
14.0
2.67
0.65
0.98
15.1
3.43
3.55
2.52
16.4
1.97
1.58
0.86
8.38
3.95
6.88
0.79
11.9
2.11
7.47
3.46
13.1
13.8
1.47
1.67
5.70
9.89
8.75
2.01
12.8
2.46
7.02
2.23
14.2
5.44
2.37
4.63

centage concentration deviations were calculated to evaluate the

stability.
2.6.6. Cross-talk
The cross-talk was evaluated by comparing the observed peak
area of the single analyte with that of the corresponding mix standards QC samples at high level and this was to make sure that each
analyte could not be inuenced by others during a simultaneous
determination.

dose in rat is 1.31 g/kg. Therefore, the JJT extract at 1.31 g kg1
(suspended in 0.5% carboxymethyl cellulose sodium salt aqueous solution) was administrated orally to the rats. Blood samples
(about 200 L) were collected before dosing and at 5, 10, 15, 30,
45 min, 1, 1.5, 2, 4, 6, 8, 12, 24 h after administration from the
fossa orbitalis of rats. After centrifugation at 7000 rpm for 10 min
at 4 C, the samples were immediately transferred into heparinized
tubes. The plasma was stored frozen at about 80 C until analysis.

2.7. Applications to pharmacokinetic studies

2.8. Data analysis
The PK study was carried out in accordance with the guidelines
for the care and use of laboratory animals and approved by the Animal Ethics Committee of Tianjin University of Traditional Chinese
Medicine. Seven male Sprague-Dawley rats weighing 180220 g
were kept at the animal center of Tianjin University of Traditional Chinese Medicine (Tianjin, China) with controlled conditions
(temperature at 25 2 C and relative humidity of 55 5%). They
were allowed free access to food and water and acclimatized for
several days until 12 h before the experiment. According to the
clinical dose used in human for each day which is 0.21 g/kg, the

All the pharmacokinetic parameters were calculated using the

DAS 1.0 software (Drug and Statistics 1.0, Medical College of
Wannan, China) [17]. The appropriate pharmacokinetic model
was chosen to t the concentrationtime curve in plasma. The
maximum drug concentration in plasma (Cmax ) and the time to
reach maximum drug concentration (Tmax ) were obtained directly
from concentrationtime data. Other pharmacokinetic parameters like the area under the plasma concentrationtime curve
(AUC), distribution half-life (t1/2 ), elimination half-life (t1/2 ), and

Y.-x. Chang et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 110

Table 4
Recoveries and matrix effects of 11compounds (n = 6).
Compounds

Concentration
(ng mL1 )

Neochlorogenic
acid

20
60
600
6000
0.4
1.2
12
120
20
60
600
6000
4
12
120
1200
2
6
60
600
0.2
0.6
6
60
0.08
0.24
2.4
24
0.16
0.48
4.8
48
0.04
0.12
1.2
12
0.04
0.12
1.2
12
0.08
0.24
2.4
24

Chlorogenic
acid

Cryptochlorogenic
acid

Ferulic
acid

Magnoorine

Coptisine

Epiberberine

Jatrorrhizin

Berberine

Palmatine

Berberubine

Recovery

Matrix effect

Mean (%)

RSD(%)

Mean(%)

RSD(%)

89.4
105
85.5
100
98.1
105
110
95.1
87.4
101
81.2
89.9
80.1
85.8
80.0
105
92.6
94.9
108
100
96.4
91.4
106
86.4
91.5
95.8
105
81.8
91.6
105
106
90.1
88.2
91.4
106
86.4
91.3
99.0
92.4
88.0
118
86.7
93.0
99.7

5.38
9.02
11.6
6.65
9.03
5.20
5.56
4.53
8.25
6.95
6.81
13.9
2.40
13.5
8.99
5.69
14.7
14.4
11.8
13.3
4.93
14.6
10.2
8.95
11.6
15.0
14.7
15.0
14.0
12.5
12.2
5.28
12.5
15.1
5.49
14.1
11.1
15.0
7.57
13.2
15.8
12.5
14.4
6.76

115
103
91.7
83.4
97.8
100
100
104
113
95
104
91.7
118
104
91.9
80.9
109
82.2
92.2
84.4
96.4
88.5
79.9
81.1
84.9
95.4
76.7
84.0
97.5
101
88.9
94.2
115
96.8
80.8
94.5
98.1
96.6
89.5
96.6
107
81.5
79.3
82.2

6.23
14.1
11.7
12.6
12.4
8.42
8.42
5.99
5.90
13.2
11.9
7.68
7.27
7.75
10.2
14.8
13.9
14.2
13.9
15.3
3.14
14.8
12.1
6.55
14.7
8.44
14.5
14.9
10.9
8.05
12.6
14.5
12.3
13.9
14.2
12.9
11.4
13.4
14.1
9.47
10.6
10.9
12.3
8.15

mean residence time (MRT) were also calculated. The values were
expressed as mean SD.

3. Results and discussion

were optimized to obtain the maximum sensitivity and response

of every component. The results are listed in Table 1.
Other parameters such as drying gas ow rate, drying gas
temperature, nebulizing gas pressure, and capillary voltage were
optimized to obtain the highest intensity of the charged molecules
of analytes.

3.1. Optimization of LCMS/MS conditions

3.2. Optimization of sample preparation
The HPLC conditions were optimized to obtain better separation in a short time. Different mobile phases (acetonitrile-water,
methanol-water), concentrations of additive (0.05%, 0.1%, and 0.2%
formic acid), column temperatures (20, 30 and 40 C) and ow rates
(0.2, 0.3 and 0.4 mL min1 ) were optimized. After continuous and
appropriate adjustment of the chromatographic separation conditions, the best results were obtained when the HPLC conditions
were set as follows: acetonitrile-0.1% formic acid as mobile phases,
ow rate at 0.3 mL min1 and column temperature at 30 C.
To optimize ESI conditions, the analytes and ISs were characterized by MS scan and MS/MS products in SRM mode. The ESIMS
data were obtained in both negative and positive mode. The parent ions and product ions of the analytes, source and parameters

Sample preparation is a critical step for developing an accurate and reliable LC-ESI MS/MS method to eliminate interference
from the sample matrix and achieve satisfactory recovery. In order
to extract the target analytes from biological uids more effectively, we used the liquidliquid extraction (LLE) with ethyl acetate
method. However, matrix effects of some analytes were lower
than 50% which could not meet the analysis requirements. Compared with above methods, protein precipitation with acetonitrile
(PPA) was found to offer satisfactory recovery and desired extract
efciency in determining the concentrations of the analytes. In
addition, PPA was much simpler and less time-consuming than LLE
with ethyl acetate. In order to improve the extraction efciency,

Y.-x. Chang et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 110

Table 5
Stability of the 11 analytes (n = 6).
Compounds

Concentration
(ng mL1 )

Neochlorogenic
acid

20
60
600
6000
0.4
1.2
12
120
20
60
600
6000
4
12
120
1200
2
6
60
600
0.2
0.6
6
60
0.08
0.24
2.4
24
0.16
0.48
4.8
48
0.04
0.12
1.2
12
0.04
0.12
1.2
12
0.08
0.24
2.4
24

Chlorogenic
acid

Cryptochlorogenic
acid

Ferulic
acid

Magnoorine

Coptisine

Epiberberine

Jatrorrhizin

Berberine

Palmatine

Berberubine

At 80 C for 1 month

Freeze-thaw cycles

Autosampler for 24 h

Accuracy(%)

RSD(%)

Accuracy(%)

RSD(%)

Accuracy(%)

RSD(%)

96
100
101
100
88.5
100
96.9
99.0
87.3
102
100
99.3
119
102
99.4
99.0
112
100
98.9
98.3
102
99.4
99.0
99.9
105
94.4
104
97.3
97.8
97.9
96.1
95.6
86.8
95.6
99.2
101
120
104
99.9
102
91.8
94.4
97.4
95.1

13.2
1.56
2.31
2.40
14.8
3.39
6.05
5.00
19.4
7.36
7.28
4.43
8.69
4.02
5.32
1.30
13.8
8.50
2.60
1.62
11.8
3.30
2.25
5.52
14.3
7.01
9.54
10.8
17.0
1.90
8.88
9.19
12.0
10.0
7.87
2.37
9.26
8.39
6.00
4.40
6.95
7.01
5.41
9.10

96.1
94.1
90.0
92.0
107
98.3
101
99.5
93.9
92.6
89.7
82.8
81.8
92.7
88.1
89.7
94.0
82.2
82.9
73.2
85.3
89.0
86.4
84.1
88.7
85.8
85.9
83.5
105
88.4
81.1
72.9
102
104
99.8
100
114
101
96.4
87.1
85.9
86.9
85.2
87.9

7.91
9.90
9.85
9.49
14.2
14.6
15.0
16.5
13.8
18.0
12.0
10.0
13.9
10.3
15.6
8.39
20.5
20.0
6.76
7.18
19.0
10.8
13.2
6.51
9.09
14.4
10.5
5.52
11.2
9.65
12.4
1.26
14.3
13.0
7.02
10.3
19.2
14.9
15.4
7.79
10.2
17.1
15.4
9.36

86.5
96.6
101
101
93.8
102
95.8
96.9
85.0
96.6
96.7
110
105
96.7
99.4
104
114
104
105
97.9
101
100
106
105
119
101
92.7
99.2
109
96.6
97.8
98.1
80
103
94.4
103
121
106
94.3
97.2
117
97.9
109
102

10.3
1.45
2.31
2.49
13.3
6.10
9.52
5.26
9.31
8.73
11.2
14.1
8.66
4.56
6.42
3.28
13.1
9.67
3.71
6.01
7.81
4.13
14.2
7.61
15.8
14.7
15.0
11.9
11.3
1.45
4.96
3.90
7.43
10.0
7.62
4.87
11.4
13.7
12.3
8.66
15.7
9.61
12.5
9.98

the volumes of formic acid (5, 10, and 20 L) and the precipitation
reagents (methanol and acetonitrile) were optimized. Finally, the
highest extraction recoveries of all analytes were obtained when
10 L formic acid (v/v) and 400 L acetonitrile were employed for
the sample pre-preparation.
3.3. Method validation
3.3.1. Specicity
Under the optimized LCMS/MS conditions, the specicity was
evaluated by analyzing six blank plasma samples, blank plasma
added to 11 analytes and 2 ISs, and the obtained plasma after oral
administration of JJT tablet The results demonstrated that there
were no interfering peaks in the peaks region of analytes (Fig. 2).
3.3.2. Linearity and LLOQ
The calibration curves were constructed with a series of diluted
concentrations in blank plasma by plotting the peak area ratio of
the analytes to IS against the concentration of each analyte. The
results are illustrated in Table 2. The results demonstrated that calibration curves showed good linearity in the linear range of the

analytes with the correlation coefcient r more than 0.99. The LLOQ
samples of six rat plasma samples independent of the calibration
curves were determined. The results are listed in Table 3. It was
observed that the LLOQs of seven alkaloids were less than 2 ng mL1
and that of four phenolic acids were less than 20 ng mL1 .Their
RSDs of inter-day and intra-day were less than 15% and the
accuracies ranged from 80% to 118%, respectively. These results
demonstrated that the limits were sufcient enough for the pharmacokinetic study of 11 analytes after oral administration of JJT
tablet.
3.3.3. Precision and accuracy
The intra-day and inter-day precision and accuracy were calculated by preparing and analyzing six replicates of the analytes at
spiked QC samples at LLOQ low, medium and high concentration
levels on the same day and continuously for 3 days, respectively.
The results are shown in Table 3. The RSD and accuracy at three levels QC samples was less than 15% and more than 93.6.The RSD at
LLOQ was less than 20% with accuracies in the range of 80118%. It
was concluded that the precision and accuracy of the method were
acceptable.

Y.-x. Chang et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 110

Table 6
The cross-talk of each compounds(n = 6).

3.4. Selection of internal standard

Compounds

Concentration
(ng mL-1)

Accuracy(%)

RSD(%)

Neochlorogenic acid
Chlorogenic acid
Cryptochlorogenic acid
Ferulic acid
Magnoorine
Coptisine
Epiberberin
Jatrorrhizin
Berberine
Palmatine
Berberubine

6000
120
6000
1200
600
60
24
48
12
12
24

100
98.7
97.2
94.3
90.1
103
111
98.7
96.5
102
111

2.64
7.54
4.24
5.74
4.40
4.42
3.13
5.26
5.48
3.24
3.14

In the study, the selection of the internal standard was investigated. Usually, the internal standard should have similar chemical
structures and physicochemical property and should not interfere
with the analytes. Based on this, two types of chemical components, tetrahydropalmatine (IS1) and rosmarinic acid (IS2) were
selected for the alkaloids and phenolic acids, respectively. The
results showed that the internal standards had no interference with
the endogenous matrix and suitable retention time for separation
was obtained for the analytes. As a result, the tetrahydropalmatine
(IS1) and rosmarinic acid (IS2) were chosen to evaluate the concentration of alkaloids and phenolic acids respectively in rat plasma
after orally administrated the JJT tablet.

3.3.4. Recovery and matrix effect

The results of recovery and matrix effect of the analytes are listed
in Table 4. The mean recoveries of the analytes of the QC samples
at four concentrations were more than 80.0% and the matrix effect
were higher than 76.7% and less than 115%, indicating that the
method was precise, consistent and reproducible and the assessment of the matrix effect showed no effect.

3.5. Pharmacokinetic study

The developed method was employed to determine the concentration of seven alkaloids and four phenolic acids in rat
plasma after oral administration of JJT tablet at a dose of
1.31 g kg1 (27.3 mg kg1 berberine, 7.22 mg kg1 epiberberine,
10.9 mg kg1 coptisine, 0.29 mg kg1 magnoorine, 0.13 mg kg1
berberubine, 4.57 mg kg1 palmatine, 11.4 mg kg1 jatrorrhizine,
36.0 mg kg1 chlorogenic acid, 1.96 mg kg1 cryptochlorogenic
acid, 1.54 mg kg1 neochlorogenic acid and 0.25 mg kg1 ferulic acid). The results showed that cryptochlorogenic acid and
neochlorogenic acid were not detected in rat plasma after orally
administrated JJT tablet. The possible reasons might be that the
concentration of these two compounds in plasma was below the
LLOQ, or they were not absorbed after oral administration, or
they were metabolized rapidly and their metabolites were not
detected in plasma. Further experiments are in progress to validate
the reason. The mean plasma concentrationtime curve proles
of berberine, epiberberine, coptisine, magnoorine, berberubine,
palmatine, jatrorrhizine, chlorogenic acid and ferulic acid are
shown in Fig. 3. The compartmental pharmacokinetic analysis of
concentrationtime data was performed by DAS 1.0 software. The
results showed that all the compounds except palmatine were
tted best to the one-compartment model and palmatine was consistent with two-compartment model. The mean pharmacokinetic
parameters are presented in Table 7.
As shown in Fig 3, multiple plasma concentration peaks were
observed in the mean plasma concentration curves proles of
berberine, epiberberine, coptisine, palmatine and jatrorrhizine,
which was consistent with results from previous studies [1820].
Previous studies reported multiple blood concentration peaks in
the alkaloid PK and attributed it to the distribution, reabsorption,
enterohepatic circulation or the administered drugs having been in
a complex prescription [2123]. Other studies have demonstrated
that TCMs and natural components from TCMs can be identied as
inhibitors, substrates, and/or inducers of a variety of CYP enzymes,
and TCMs-CYP interactions may occur and affect the pharmacoki-

3.3.5. Stability
The freezethaw stability, auto-sampler for 24 h stability and
long-term stability were evaluated by the mean concentrations of
the analytes of the QC samples at four levels. The results are listed
in Table 5, indicating that all the analytes were stable in rat plasma
for three freeze/thaw cycles, auto-sampler for 24 h at room temperature. The long term stability for 1 month was relatively stable
for all the target analytes with the accuracy for all the compounds
analysed passing FDA approved range.
3.3.6. Cross-talk validation
The accuracy of each compound is shown in Table 6. There
were two types of isomers including neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid isomer and berberine and
epiberberine isomer. The extracted ion pair results was obtained
simultaneously with others, however, it could not be infulenced
owing to retention time. For examples, the retention time of
neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid
were different, and the three peaks were separated well. The Q3
of jatrorrhizin was the same with the Q1 of the berberubine. There
are two peaks which were separated well (rententian times were
24.76 min and 25.12 min) in the chromatographic gures when the
ion pair of berberubine in the mix compound was extracted. When
integrated, the area of berberubine, the peak with retention time
25.12 min was calculated. The results showed that jatrorrhizin did
not inuence the accuracy of berberubine. Based on above results, it
was concluded that there was no cross-talk inuence in the newly
established LCMS/MS methods.

Table 7
Pharmacokinetic parameters of the nine components in JinqiJiangtang tablet (n = 7, mean SD).
Compounds

Dose
(mg kg1 )

Chlorogenic acid
Ferulic acid
Magnoorine
Coptisine
Epiberberine
Jatrorrhizin
Berberine
Palmatine
Berberubine

36.0
0.25
0.29
10.9
7.22
11.4
27.3
4.57
0.13

Tmax (h)
0.70
1.80
0.64
0.42
1.83
0.39
0.30
0.46
0.82

0.19
0.41
0.20
0.69
2.85
0.54
0.20
0.50
0.40

Cmax (ng mL1 )

161
40.1
13.4
15.8
10.8
6.58
15.4
13.6
1.37

55.2
34.3
0.69
2.39
2.01
1.54
3.62
12.6
0.45

AUC0-24
(ng mL1 h1 )
521
46.2
53.4
126
48.2
22.9
80.7
37.9
9.02

155
21.7
8.60
19.6
16.0
6.24
9.89
9.38
1.57

AUC0-
(ng mL1 h1 )
616
83.1
84.1
199
151
80.0
92.9
56.2
49.5

97.8
44.2
26.4
68.7
103
19.1
16.7
23.2
90.3

MRT0 24 (h)

T1/2 ka (h)

0.18
0.05
0.0763
0.0059
0.0097
0.0097
0.0186
0.10
0.15

5.08
2.90
8.18
8.16
10.5
8.68
7.35
7.73
9.40

0.89
1.10
0.64
1.00
2.07
2.04
1.19
0.57
0.67

T1/2 (h)
0.24
0.02
0.0758
0.0017
0.0048
0.0059
0.0105
0.15
0.11

4.41
4.42
2.67
12.4
8.17
3.97
4.41
0.28
4.44

2.56
4.90
0.97
4.52
9.82
4.22
1.78
0.40
2.77

Y.-x. Chang et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 110

Fig. 3. Mean plasma concentrationtime proles in rats after oral administration of traditional Chinese medicinal preparation Jinqi Jiangtang.

netics [23]. The results also indicated that the absorption of the
seven alkaloids and two phenolic acids might be rapid with the
Tmax values within 2 h.
However, the Cmax of seven alkaloids was less than 20 ng mL1 .
The AUC024 h values were 80.7 9.89 ng mL1 h1 for berberine, 126 19.6 ng mL1 h1 for coptisine, 48.2 16.0 ng mL1 h1
for epiberberine, 37.9 9.38 ng mL1 h1 for palmatine and
22.9 6.24 ng mL1 h1 for jatrorrhizine. These showed that these
alkaloids shared low plasma concentration. Previous studies concluded that the poor absorption and extensive metabolism might
result in low plasma concentration of berberine after oral administration [23]. Because of their similar structures with berberine and
lower contents for administration, the plasma concentration of the
other four protoberberine-type alkaloids was lower. The values of
T1/2 were in the range of 0.2812.4 h, which were greatly different
from previous study [23]. This was probably due to the different
proportions of the components in JJT tablet and the individual differences in rats.
As we can see from Fig 3, the mean plasma concentrationtime
curve proles of magnoorine and berberubine was remarkably
different from the above alkaloids. They only have a typical singlepeak in the concentrationtime curve. The Tmax value exceeded
epiberberine and jatrorrhizine and the AUC0 24 h value was higher
than epiberberine, jatrorrhizine and palmatine, though the content
of magnoorine was much lower than them. However, the Tmax and
T1/2 values were similar to them. The results might be presumably
due to the different physicochemical properties of compounds or
the PK interaction of the prescribed chemical constituents. The Tmax
and AUC0 24 h values of berberubine were low for its low concentration in JJT tablet. It is the rst time that pharmacokinetic study
of berberubine after oral administration has been reported.
It was found that values of Tmax and T1/2 of chlorogenic acid
and ferulic acid were much closer, demonstrating that they have
similar absorption and elimination rate. The Cmax values were
161 55.2 and 40.1 34.3 ng mL1 and values of AUC0 24 h were
521 155 and 46.2 21.7 ng mL1 h1 respectively. The results
were not correspondent with their dosages administered to the
rats (6.0 mg kg1 chlorogenic acid and 0.25 mg kg1 ferulic acid).

The possible reason may be that some of the ferulic acid detected
in rat plasma was the metabolites of the components in JJT tablet.
3.6. Method comparison with existing reports
There are several studies on evaluating the alkaloids in Rhizome Coptidis and its preparations [912]. In general, it is essential
to simultaneously determine different kinds of components from
TCMs and preparations in biological samples in clinical practice. But
no analytical methods for simultaneous determination of chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, ferulic
acid, magnoorine, coptisine, epiberberine, jatrorrhizin, berberine,
berberubine and palmatine in biological samples were available
in these studies. These studies mainly focused on the pharmacokinetics of alkaloids with high contents. There was a report on
the simultaneous determination of wogonin, coptisine, berberine, palmatine, jatrorrhizine, phellodendrine, magnoorine and
wogonoside from Huanglian Jiedu Decoction in rat plasma [24].
It was the rst study on the pharmacokinetics of magnoorine
(low content). However, the LLOQs for coptisine, berberine, jatrorrhizine, magnoorine and palmatine were 0.20, 0.48, 0.10, 0.32,
and 0.30 ng mL1 , respectively. The berberubine was not detected
in plasma. In our study, the LLOQs were 0.04 ng mL1 for berberine
and palmatine, 0.16 ng mL1 for jatrorrhizine, 0.2 ng mL1 for coptisine, 2 ng mL1 for magnoorine. Though the LLOQ of magnoorine
was a little higher than the above study, the method described in
our study is more suitable for clinical applications. To the best of our
knowledge, this is the rst time chlorogenic acid, cryptochlorogenic
acid, neochlorogenic acid, ferulic acid, magnoorine, coptisine,
epiberberine, jatrorrhizin, berberine, berberubine and palmatine
has been simultaneously determined using an LCMSMS method.
4. Conclusion
For the rst time, a rapid, sensitive, and convenient LCESIMS/MS method for the simultaneous determination of four
phenolic acids (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and ferulic acid) and seven alkaloids (berberine,

10

Y.-x. Chang et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 110

epiberberine, coptisine, magnoorine, berberubine, palmatine and

jatrorrhizine) in rat plasma has been developed and validated. The
method offers the advantages of high sensitivity and simple plasma
sample preparation. It was successfully applied to simultaneously
evaluate the PK properties of the bioactive components after oral
administration of JJT tablet. The PK parameters obtained from this
study and the validated method would be useful in clinical applications of JJT tablet and other related TCM preparations.
Acknowledgments
This research was supported National Natural Science Foundation of China (81001632 and 81374050), National Science and
Technology Support Program Projects (2014BA105B01), Program
for Innovative Research Team in Universities of Tianjin (TD125033), PCSIRT (IRT-14R41) and State the Science & Technology
Commission of MOST of China (2014ZX09304307001). The authors
have declared no conict of interest.
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