Journal of Chromatography B, 951952 (2014) 7888

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/chromb

Determination of six components of Andrographis paniculata extract

and one major metabolite of andrographolide in rat plasma by liquid
chromatographytandem mass spectrometry
Jian Wang, Wenqing Yang, Guanglin Wang, Pingming Tang, Yang Sai
Department of Drug Metabolism and Pharmacokinetics, Hutchison Medipharma Ltd., Shanghai 201203, PR China

a r t i c l e

i n f o

Article history:
Received 4 November 2013
Received in revised form 17 January 2014
Accepted 20 January 2014
Available online 28 January 2014
Keywords:
Andrographis paniculata
HMPL-004
Chemical components
LCMS/MS
Pharmacokinetic study

a b s t r a c t
Andrographis paniculata (AP) has been widely used in Asian countries to treat many kinds of diseases for several decades. Hutchison Medipharma Ltd. developed an aqueous ethanol extract of
A. paniculata (APE) named as HMPL-004 to treat inammatory bowel diseases. The representative
chemical components of HMPL-004 include andrographolide (AND), neoandrographolide (NAND),
14-deoxyandrographolide (DAND), 14-deoxy-11,12-didehydro-andrographolide (DDAND), apigenin-7O--d-glucuronopyranoside (AODG) and chlorogenic acid (CLA). HM5013620 is the major circulating
metabolite of AND. The purpose of this study was to develop a bioanalytical method to determine all seven
compounds in rat plasma using liquid chromatography coupled to electrospray tandem mass spectrometry (LCMS/MS). The assay was fully validated according to FDA guidelines. The LCMS/MS detection
was operated in the negative mode, and the multiple reaction monitoring (MRM) mode was used for
the quantication. The analyte extraction was performed by protein precipitation with acetonitrile after
adding a small volume (2% of the total volume) of 10% formic acid into plasma to stabilize AND under
bench-top condition (ice-bath). The linear ranges of the analytes were 82000 ng/mL for DDAND and
42000 ng/mL for others. Validation results demonstrate that AND, NAND, DAND, DDAND, CLA, AODG
and HM5013620 can be rapidly, accurately, precisely and robustly quantied in rat plasma. Furthermore,
the method was successfully applied to characterize the pharmacokinetic proles of all seven compounds
in Sprague-Dawley rats after a single oral administration of 750 mg of HMPL-004.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Traditional Chinese medicines (TCMs) are used throughout the
world but owing to their complex composition and lack of characterization of active components, it is difcult for developers to
perform quality control and ensure clinical efcacy. In addition, it is
also difcult to characterize the pharmacokinetic properties of the
many structure-diverse components when they are administered
simultaneously.
Andrographis paniculata (AP) is a member of the plant family
Acanthaceae. It has been widely used in Asian countries including
China, India, and Thailand to treat upper respiratory tract infection, HIV infection, ulcerative colitis, hepatitis, rhinitis, sinusitis,

Corresponding author at: Department of Drug Metabolism and Pharmacokinetics, Hutchison Medipharma Ltd., Building 4, No. 720 Cai Lun Road, Zhangjiang
Hi-Tech Park, Shanghai 201203, PR China. Tel.: +86 21 50790088;
fax: +86 21 50793595.
E-mail addresses: Yangs@hmplglobal.com, saiyang2006@gmail.com (Y. Sai).
1570-0232/$ see front matter 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jchromb.2014.01.028

bacillary dysentery, enteritis, leprosy and other inammatory and

infectious diseases [14]. AP extract (APE) are available in China
as oral preparations or injections for therapeutic use. The Chinese
Pharmacopoeia recommends a daily dose of APE oral preparations
of 0.631.26 g with a dehydroandrographolide (DDAND) content
greater than 3.8%.
The aqueous ethanol extract of AP leaves has been developed by Hutchison Medipharma Ltd. (HMP) named as HMPL-004
to treat inammatory bowel diseases including ulcerative colitis
(UC) and Crohns disease (CD). HMPL-004 includes many kinds of
active constituents. Andrographolide (AND), neoandrographolide
(NAND), 14-deoxyandrographolide (DAND), and 14-deoxy-11,12didehydroandro-grapholide (DDAND) have already been shown
to be the major diterpenoid components in AP [5]. In addition to these compounds, apigenin-7-O--d-glucuronopyranoside
(AODG) and chlorogenic acid (CLA) were identied to be the major
representative avonoid and polyphenol components in HMPL004, respectively. Among these six components, AND was the
most important one, showing anti-inammatory and antimicrobial activity [6,7]. Its pharmacokinetics has also been investigated

J. Wang et al. / J. Chromatogr. B 951952 (2014) 7888

79

Fig. 1. Structure information of AND, NAND, DAND, DDAND, CLA, AODG, HM5013620 and cortisone.

extensively. The previous reports of AND metabolism were mainly

focused on the phase II conjugated metabolites found in excreta
[810]. A study of metabolites in post-dose human plasma samples
showed that the major circulating metabolite of AND was generated via hydrolysis (In house data, not shown here). The structure
of this hydrolysis metabolite has been conrmed through chemically synthesizing the corresponding standard compound named
HM5013620 by the Medicinal Chemistry group of HMP. Besides
AND, DDAND also can be hydrolyzed to form a major circulating metabolite but it is not accessible through chemical synthesis.
Our previous study also found that AND was unstable in biological
matrices, such as blood and plasma, that over 20% of AND can be
metabolized to form dehydrated metabolite or GSH adducts after
storage on ice-water for 1 h and over 50% of AND can be gone after
storage at room temperature for 1 h. Although a number of studies
of AND have been reported, the stability of AND was not recognized
[1114] and is likely to have resulted in an underestimation of AND
in biological samples.
The aim of this study is to develop a simple, sensitive, selective
and robust method to quantitatively determine all seven compounds in rat plasma and then apply it to in vivo pharmacokinetic
studies.

The analysis was performed on a triple quadrupole mass spectrometer equipped with turbo ionspray interface (API4000, AB
Sciex, Foster City, CA, USA). An Agilent 1200 series liquid chromatography system was used consisting of a binary pump, an
on-line vacuum degasser and a thermostated column compartment
(Agilent Technologies, Palo Alto, CA, USA) and a CTC PAL autosampler (CTC Analytics AG, Zwingen, Switzerland). Data acquisition and
processing were performed using Analyst version 1.4.2 software
(AB Sciex).

2. Materials and methods

2.3. LCMS/MS conditions

2.1. Chemicals and reagents

Analytes were separated on a Waters Xbridge Shield RP C18

column (50 mm 2.1 mm ID, particle size 5 m; Waters Corp.,
Mildford, MA, USA) protected with a Supelco lter (Particle size
0.5 m; SigmaAldrich, St Louis, MO, USA). The column temperature was maintained at 30 C. Chromatography was performed
using a gradient system consisting of mobile phase solution
A (0.1% acetic acid in water) and solution B (50% acetonitrile in methanol). The gradient elution method was as follows:
0.002.00 min: 5% B55% B; 2.003.70 min: 55% B; 3.703.71 min:
55% B100% B; 3.715.00 min: 100% B; 5.005.01 min: 100% B5%
B; 5.016.50 min: 5% B. The ow rate was 0.4 mL/min and the
autosampler temperature was set to 8 C. During the rst 0.60 min
and after 5.10 min, the eluents were directed to waste. The injection

HMPL-004 was provided by the Pharmaceutical Science group

of HMP (Shanghai, China), containing 6.5% of AND, 3.1% of
NAND, 3.4% of DDAND, 0.4% of DAND, 1.9% of CLA and 1.0%
of AODG. AND (Purity 97.3%) was purchased from Guilin Sanleng Biotech Co., Ltd. (Guangxi, China). NAND (Purity 93.4%) was
purchased from Zhongxin Innova Laboratories (Tianjin, China).
DAND (Purity 97.0%), DDAND (Purity 97.4%), and AODG (Purity
89.6%) were all puried and characterized from HMPL-004 by
the Pharmaceutical Science group of HMP (Shanghai, China). CLA
(Purity 96.4%) was purchased from Sichuan Weikeqi Biotech Co.,
Ltd. (Sichuan, China). HM5013620 (Purity 98.0%) was chemically

synthesized by the Medicinal Chemistry group of HMP (Shanghai,

China). Cortisone, as the internal standard (Purity 98.0%), was purchased from SigmaAldrich (St Louis, MO, USA). The structures
of all seven compounds and the internal standard are shown in
Fig. 1. Demethylsulfoxide (DMSO), formic acid and acetic acid were
all HPLC grade and purchased from Tedia (Faireld, OH, USA).
Methanol, acetonitrile, and isopropanol were all HPLC grade and
purchased from Thermo-Fisher Scientic (Pittsburgh, PA, USA).
Deionized water (18.2 m) was prepared by a Millipore Milli-Q
gradient water purication system (Billerica, MA, USA).
2.2. Instruments

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J. Wang et al. / J. Chromatogr. B 951952 (2014) 7888

Table 1
The MS parameters of each analyte.
Analyte

Q1 (m/z)

Q3 (m/z)

DP

EP

CE

CXP

AND
NAND
DAND
DDAND
CLA
AODG
HM5013620
IS (Cortisone)

409
539
393
331
353
445
367
359

331
59
59
303
191
269
305
329

40
55
35
80
45
60
80
78

10
10
10
10
10
10
10
10

18
56
35
34
24
36
30
14

5
9
6
9
3
7
9
9

volume was 10 L. To avoid carryover, the syringe and injection

valve of the autosampler were set to be washed for 3 and 5 times
before and after injection with wash solution 1 (90% isopropanol
containing 0.1% NH4 OH) and wash solution 2 (0.1% acetic acid in
methanol/acetonitrile/water (1:1:2, v/v/v)), respectively.
The mass spectrometer was operated in the negative mode. The
ionspray voltage and the source temperature were set to 4500 V
and 500 C, respectively. The nebulizer gas, heater gas, curtain gas
and collision gas were set to 60, 50, 20 and 6 psi, respectively. The
multiple reaction monitoring (MRM) mode was used for analyte
quantication, and the MS parameters of each analyte are listed in
Table 1.

2.7. Validation of the method

The method was validated for specicity, selectivity, linearity,
LLOQ, accuracy and precision, extraction recovery, matrix effect,
hemolyzed plasma effect and stability according to US FDA guidelines [15].
2.7.1. Specicity and selectivity
Six different batches of analyte-free heparinized rat plasma
were used to investigate whether the endogenous matrix constituents interfered with the assay. The peak area at the retention
time for each analyte should be no more than 20% of the peak area
of the LLOQ standards.

2.4. Preparation of stock and working solutions

2.8. Linearity and LLOQ
For each of the seven compounds, two stock solutions
(1.0 mg/mL) were separately prepared in DMSO: one for the calibration standards and one for the quality control (QC) samples.
An appropriate volume of stock solution of each compound for the
calibration standards was spiked and mixed together rstly, then
diluted in acetonitrile to obtain eight calibration standard working solutions containing AND, NAND, DAND, DDAND, CLA, AODG
and HM5013620, each at concentration levels of 40,000, 20,000,
10,000, 4000, 1000, 400, 160 and 80 ng/mL. The QC working solutions containing the seven compounds were similarly prepared at
concentration levels of 32,000, 3000, 400, 160 and 80 ng/mL. For the
internal standard (IS), the stock solution was prepared at 1.0 mg/mL
in acetonitrile and then diluted in acetonitrile to obtain the working
solution at 500 ng/mL. All solutions were stored at 4 C.
2.5. Preparation of calibration standard and QC samples
Calibration standards were prepared by adding small volumes
(5% of the total volume) of working solutions into blank heparinized
rat plasma to give nal concentrations of 2000, 1000, 500, 200, 50,
20, 8 and 4 ng/mL for each analyte. QC samples were similarly prepared at concentration levels of 1600 (high QC), 150 (medium QC),
20 (low QC for DDAND), 8 (lower limit of quantication (LLOQ)
for DDAND and low QC for others) and 4 (LLOQ for all except
DDAND) ng/mL. A preliminary stability study showed that AND was
unstable in rat plasma, while formic acid can help to stabilize it. For
this reason, the blank plasma was rstly acidied with a small volume (2% of the total volume) of 10% formic acid before the addition
of working solution into blank plasma.
2.6. Sample preparation
A protein precipitation method was used for sample preparation. Simply, 150 L of IS working solution was added into 50 L of
plasma sample. After being vortexed for at least 1 min, the mixture
was centrifuged at 20,000 g for 10 min at 4 C. 100 L of the supernatant was then transferred into another polythene tube containing
100 L of water, and vortexed well before injection into LCMS/MS
system for analysis.

The linearity of the method was derived from three separate

analytical runs. In each analytical run, calibration curves containing double blank sample (no analyte or IS), blank sample (only IS)
and eight (seven for DDAND) non-zero concentration levels were
prepared and analyzed in duplicate. Calibration curves in each analytical run were tted by the peak area ratio (Analyte/IS) versus
analyte concentrations using a 1/x2 weighted linear least-squares
regression model. LLOQ was dened as the lowest drug concentration on the calibration curve. The bias between the nominal
concentration and the back-calculated concentration should be less
than 15%, except at LLOQ where the bias can be less than 20%.
2.9. Accuracy and precision
The intra-day accuracy and precision were evaluated at ve
(four for DDAND) concentration levels by the determination of six
replicate QC samples on the same day. The inter-day accuracy and
precision were assessed at ve (four for DDAND) concentration levels by analyzing six replicate QC samples over three consecutive
days. Precision was calculated as relative standard deviation (RSD),
which should not exceed 20% for the LLOQ samples and 15% for
other QC samples. The deviation of the mean values between the
nominal concentration and the back-calculated concentration (RE)
was used to evaluate the accuracy, which should be within 20%
for the LLOQ samples and 15% for other QC samples.
2.9.1. Extraction recovery and matrix effect
The extraction recovery of each analyte was determined at three
concentration levels by comparing the mean peak areas of the regularly pre-treated QC samples with those of the samples prepared
by directly adding QC working solutions into the post-precipitated
blank plasma.
The matrix effect was also assessed at three concentration levels by comparing the mean peak areas of the samples prepared
by directly adding QC working solutions into the post-precipitated
blank plasma with those of the samples prepared by directly
adding QC working solution into neat nal injected solution
(acetonitrile/H2 O = 1:1, v/v).

J. Wang et al. / J. Chromatogr. B 951952 (2014) 7888

Fig. 2. Product ion scans and proposed fragmentation patterns of AND (A), NAND (B), DAND (C), DDAND (D), CLA (E), AODG (F), HM5013620 (G) and cortisone (H).

81

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J. Wang et al. / J. Chromatogr. B 951952 (2014) 7888

2.10. Stability
The stability of each analyte was checked by analyzing three
replicates of plasma samples at low QC and high QC, which were
exposed to different storage conditions including bench-top stability (2.5 h on ice-water), three freeze/thaw cycle stability (80 C)
and long-term storage stability (80 C for 32 days). In addition, the
stability of each analyte was evaluated in plasma extracts exposed
to the storage condition of autosampler (8 C for 72 h). All analytes
were considered stable when the deviation between the nominal

concentration and the mean value of the back-calculated concentration (RE) was within 15%.
2.11. Hemolyzed plasma effect
The hemolyzed plasma was prepared by adding 10% (v/v) of
rat whole blood to rat plasma and vigorously vortexing to ensure
the blood hemolyzed and mixed well with the plasma. The effects
of hemolysis on AND, NAND, DAND, DDAND, CLA, AODG and
HM5013620 were evaluated by spiking the working solution to

Fig. 3. Representative LCMS/MS chromatograms from blank rat plasma, spiked plasma samples at the LLOQ of AND, NAND, DAND, DDAND, CLA, AODG, HM5013620 and IS
(cortisone).

J. Wang et al. / J. Chromatogr. B 951952 (2014) 7888

83

Fig. 3. (Continued ).

hemolyzed plasma matrices with six replicates at three concentration levels. The spiked QC samples were extracted and analyzed
with a set of regular plasma standards. The measured concentration values were compared with the nominal values. If the RSD
values did not exceed 15% and RE values were within 15%, it was
considered that the effect of hemolysis could be neglected.
2.12. Pharmacokinetic study
The validated LCMS/MS method was applied to investigate
the pharmacokinetic proles of each component of HMPL-004 in

Sprague-Dawley (SD) rats (n = 6, male/female = 1:1) following a

single oral administration of 750 mg/kg of HMPL-004. The study
protocol was evaluated and approved by Institutional Animal
Care and Use Committee (IACUC) of HMP. Venous blood samples
(<0.15 mL) were collected in heparinized tubes containing 3 L of
10% formic acid at 0 (pre-dose), 15, 30 min and 1, 1.5, 2, 4, 6, 8, 12
and 24 h post dosing from the retro-orbital sinus after the rat was
anesthetized by isourane. After inverting the tubes gently at least
5 times, blood samples were centrifuged at 3000 g for 10 min to
separate supernatant plasma, which was then stored at 80 C until
analysis.

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J. Wang et al. / J. Chromatogr. B 951952 (2014) 7888

Table 2
The regression equations, linear range and LLOQ for the determination of all seven analytes.
Analyte

Analytical run

Regression equation

Coefcient (R)

Linear range (ng/mL)

LLOQ (ng/mL)

y = 0.000857x + 0.000677
y = 0.000971x + 0.00072
y = 0.00101x + 0.000277

0.9977
0.9970
0.9965

42000

AND

1
2
3
1
2
3

y = 0.00117x + 0.000721
y = 0.00139x + 0.000973
y = 0.00140x + 0.000353

0.9974
0.9972
0.9964

42000

NAND

1
2
3

y = 0.000925x + 0.000109
y = 0.00110x + 0.000118
y = 0.00106x + 0.000796

0.9989
0.9989
0.9982

42000

AODG

1
2
3

y = 0.000923x + 0.000138
y = 0.000982x + 0.000518
y = 0.000982x + 0.000712

0.9983
0.9987
0.9979

42000

CLA

1
2
3

y = 0.000653x + 0.000254
y = 0.000790x + 0.000242
y = 0.000732x + 8.91e005

0.9980
0.9981
0.9977

42000

HM5013620

1
2
3

y = 0.000375x + 0.000338
y = 0.000428x + 0.000307
y = 0.000455x + 0.000149

0.9968
0.9950
0.9974

42000

DAND

1
2
3

y = 9.14e005x + 4.44e005
y = 0.000101x + 0.00019
y = 0.000106x + 9.51e006

0.9966
0.9979
0.9971

82000

DDAND

Table 3
The intra-day and inter-day accuracies and precisions for the analysis of all analytes (n = 3 days, 6 replicates per day).
Analyte

Nominal conc. (ng/mL)

Measured conc. (ng/mL)

Accuracy (%)

Precision (%)

Measured conc. (ng/mL)

AND

4
8
20
150
1600

3.58 0.44
7.56 0.32
19.00 0.59
140.67 5.61
1528.33 45.35

10.46
5.54
5.00
6.22
4.88

12.37
4.29
3.12
3.99
2.97

3.81 0.41
7.72 0.45
19.50 0.97
138.67 4.56
1505.56 46.81

4.65
3.45
2.50
7.56
5.90

10.81
5.83
4.95
3.29
3.11

NAND

4
8
20
150
1600

3.56 0.19
7.60 0.32
18.63 0.61
141.17 6.94
1506.67 39.33

11.08
5.04
6.83
5.89
5.83

5.20
4.18
3.25
4.92
2.61

3.70 0.26
7.63 0.35
19.05 0.71
138.22 5.87
1479.44 48.69

7.43
4.69
4.75
7.85
7.53

7.13
4.60
3.71
4.24
3.29

DAND

4
8
20
150
1600

3.45 0.36
7.34 0.70
18.23 0.92
136.50 8.53
1513.33 69.47

13.88
8.27
8.83
9.00
5.42

10.38
9.58
5.02
6.25
4.59

3.89 0.48
7.84 0.70
19.34 1.35
138.17 7.60
1476.11 77.09

2.76
1.98
3.28
7.89
7.74

12.25
8.89
7.00
5.50
5.22

DDAND

8
20
150
1600

7.93 0.64
20.25 1.12
145.50 5.96
1595.00 61.24

0.83
1.25
3.00
0.31

8.02
5.54
4.09
3.84

7.90 0.85
19.43 1.37
141.33 5.83
1551.67 70.15

1.26
2.86
5.78
3.02

10.79
7.08
4.13
4.52

CLA

4
8
20
150
1600

3.79 0.18
7.74 0.27
18.40 0.72
138.33 8.07
1615.00 161.59

5.29
3.23
8.00
7.78
0.94

4.85
3.43
3.89
5.83
10.01

3.83 0.24
7.70 0.43
18.46 0.69
133.78 8.71
1617.78 131.17

4.31
3.81
7.69
10.81
1.11

6.32
5.58
3.74
6.51
8.11

AODG

4
8
20
150
1600

3.94 0.34
7.92 0.39
18.48 0.78
138.00 5.62
1596.67 111.12

1.58
1.02
7.58
8.00
0.21

8.75
4.98
4.20
4.07
6.96

3.97 0.30
7.72 0.46
18.58 0.92
133.06 7.94
1552.22 94.72

0.76
3.55
7.08
11.3
2.99

7.46
5.95
4.95
5.97
6.10

HM5013620

4
8
20
150
1600

3.54 0.20
7.87 0.28
18.87 1.07
141.50 4.14
1528.33 84.00

11.63
1.67
5.67
5.67
4.48

5.79
3.54
5.66
2.92
5.50

3.71 0.26
7.67 0.30
18.96 0.85
136.39 7.37
1500.56 70.50

7.32
4.10
5.19
9.07
6.22

7.13
3.96
4.46
5.40
4.70

Intra-day

Inter-day
Accuracy (%)

Precision (%)

J. Wang et al. / J. Chromatogr. B 951952 (2014) 7888

85

Table 4
Matrix effect and extraction recovery for the analytes in rat plasma (n = 6).
Analyte

Nominal conc. (ng/mL)

Extraction recovery
Mean

Matrix effect
SD

SD

RSD (%)

AND

8
150
1600

119.86
103.62
98.68

6.38
3.21
3.76

5.33
3.10
3.81

85.66
90.43
88.41

2.18
3.08
0.60

2.55
3.40
0.67

NAND

8
150
1600

119.69
105.01
99.27

6.20
2.19
4.17

5.18
2.09
4.20

89.35
91.40
89.90

3.76
3.82
1.04

4.21
4.18
1.16

DAND

8
150
1600

114.91
103.75
101.03

12.81
4.16
5.52

11.15
4.01
5.47

93.60
95.32
91.82

7.56
3.04
3.97

8.07
3.19
4.33

DDAND

20
150
1600

126.16
104.82
103.23

8.82
3.04
5.78

6.99
2.90
5.60

98.00
96.55
93.62

7.67
5.83
2.75

7.83
6.04
2.94

CLA

8
150
1600

94.60
63.44
63.11

9.36
4.13
1.25

9.89
6.52
1.98

107.20
111.32
112.13

5.80
3.20
1.01

5.41
2.88
0.90

AODG

8
150
1600

113.83
81.83
78.98

11.01
4.20
2.42

9.67
5.14
3.07

103.04
105.00
104.83

7.92
2.91
1.14

7.69
2.77
1.09

HM5013620

8
150
1600

112.60
94.12
91.12

6.52
1.91
3.17

5.79
2.03
3.48

96.92
97.07
98.82

3.30
4.12
0.91

3.40
4.24
0.92

3. Results and discussion

3.1. Mass spectrometry
Q1 full scans were conducted by direct infusion of all the analytes in a solution of acetonitrile-water (1:1, v/v) containing 0.1%
formic acid under positive and negative electrospray ionization
(ESI) modes. Better responses were observed in negative mode than
those in positive mode for all compounds with the exception of
DDAND which showed a little higher signal intensity in positive
mode than that in negative mode. This phenomenon is predictable
as all the analytes are weakly acidic with DDAND the closest to a
neutral compound. A number of studies have reported that acetic
acid is an ideal negative-ion ESI modier which can signicantly
increase the negative-ion ESI response of the analytes due to the
high gas-phase proton afnity of its anion, its small anion volume
and its high acidity [1618]. In this study, after 0.1% formic acid
was replaced with 0.1% acetic acid, the negative ion responses of
all analytes were increased signicantly. Especially for AND, NAND
and DAND, the acetic acid adducts were formed so that the negative signal intensities were enhanced more signicantly. Fig. 2
depicts the MS/MS product ion scans and proposed fragmentation patterns of all analytes. Clear responses were shown for AND,
NAND and DAND at m/z of 409, 539 and 393 respectively, corresponding to the deprotonated molecular ions of acetic acid adducts
([M+HOACH] ), while for DDAND, CLA, AODG, HM5013620 and
cortisone, no acetic acid adducts were formed and only high abundant peaks of deprotonated molecular ions ([MH] ) at m/z of 331,
353, 445, 367 and 359 were found respectively. The most abundant product ions of AND, NAND and DAND were all at m/z of 59
corresponding to the deprotonated ion of acetic acid and the product ions generated by loss of acetic acid also showed high signal
intensities at m/z of 349, 479 and 333, respectively. For AND, an
abundant product ion at m/z of 331 was generated additionally by
further loss of water from m/z of 349. The molecular ion of DDAND
was more difcult to fragment usefully with low product ion intensities. Noteworthy amongst these from a mechanistic perspective

RSD (%)

Mean

is m/z of 303 which is generated by loss of the ketone moiety. The

most abundant fragment of CLA was the ion at m/z of 191, corresponding to the cyclohexanecarboxylic acid. The highest intensity
in the MS/MS spectrum of AODG was the peak at m/z of 269, which
was generated by loss of the sugar moiety. Low intensity ions were
also found for the product ions of HM5013620, among which the
most abundant was m/z of 305 which corresponds to loss of the
ethylene glycol. For the IS, the highest intensity product ion was
observed at m/z of 329, which was formed by loss of formaldehyde.
Table 1 shows the optimized MS/MS transitions and corresponding
MRM parameters of each analyte.
3.2. Chromatography
Different mobile phase compositions (methanolwater acidied with 0.1% acetic acid (v/v), acetonitrilewater acidied with
0.1% acetic acid (v/v) and methanol/acetonitrilewater acidied
with 0.1% acetic acid (v/v)) with different gradient elution methods
were explored to optimize chromatographic separation of analytes.
Compared to methanol or acetonitrile as mobile phase B alone, a 1:1
mixture (v/v) of methanol and acetonitrile was found to improve
the chromatographic separation of the analytes. In addition, in
order to minimize the carryover, different wash solutions and
washing times on the syringe and injection valve of autosampler
were examined and it was found that isopropanol in basic solution (0.1%NH4 OH) removed the analytes remaining in the sampling
system after injection of the highest calibration standard, and the
carryover was less than 20% of the area of LLOQ.
3.3. Sample preparation
A pilot study showed that AND could be stabilized in acidic condition by adding a small volume (2% of the total volume) of 10%
formic acid into the biological matrix. Considering the structure
diversity of the seven compounds, a simple protein precipitation approach was used as the sample preparation procedure and
no further concentration procedures were needed to meet the

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J. Wang et al. / J. Chromatogr. B 951952 (2014) 7888

Table 5
Stability data for each analyte (n = 3).
Analyte

Nominal conc. (ng/mL)

Bench-top stability in
plasma
(2.5 h on ice-water)

3 cycles freeze/thaw
stability
(80 C)

Long-term stability
(32 days at 80 C)

Autosampler stability
(72 h at 8 C)

RE (%)

RSD (%)

RE (%)

RSD (%)

RE (%)

RSD (%)

RE (%)

RSD (%)

AND

8.00
1600

7.08
1.67

3.14
3.50

3.71
1.46

0.70
4.54

0.42
11.46

4.06
0.41

5.13
2.92

2.31
2.26

NAND

8.00
1600

3.54
1.25

2.60
2.23

0.83
1.88

6.35
2.67

3.21
10.42

2.52
1.07

5.13
2.50

1.27
2.79

DAND

8.00
1600

7.46
3.54

4.16
3.64

0.00
8.13

5.20
3.61

5.92
10.21

4.85
0.80

5.21
1.25

4.65
1.90

DDAND

20.0
1600

5.50
9.79

2.31
5.53

5.50
10.83

5.72
1.17

0.33
5.63

3.04
2.89

4.00
1.04

1.38
4.44

CLA

8.00
1600

11.42
3.54

2.74
7.30

5.12
12.92

7.79
2.50

7.50
13.54

3.17
2.21

3.29
10.00

4.76
5.42

AODG

8.00
1600

6.29
2.29

5.15
2.54

1.63
9.79

10.43
2.92

5.46
9.79

3.32
3.27

3.54
3.96

3.73
0.35

HM5013620

8.00
1600

8.67
3.54

3.67
2.98

6.00
4.79

6.33
3.39

7.96
11.04

7.20
1.07

5.42
4.17

2.72
1.99

Table 6
Hemolyzed plasma effect for each analyte (n = 6).
Analyte

Nominal conc. (ng/mL)

Measured conc. (ng/mL)

RE (%)

RSD (%)

AND

8
150
1600

9.18 0.54
158.17 2.93
1518.33 38.17

14.71
5.44
5.10

5.93
1.85
2.51

NAND

8
150
1600

8.52 0.20
156.17 2.14
1548.33 44.46

6.48
4.11
3.23

2.37
1.37
2.87

DAND

8
150
1600

8.64 0.45
153.50 4.23
1691.67 51.15

7.98
2.33
5.73

5.20
2.76
3.02

DDAND

20
150
1600

20.82 1.03
163.17 5.42
1668.33 52.69

4.08
8.78
4.27

4.97
3.32
3.16

CLA

8
150
1600

7.50 0.21
147.33 2.42
1563.33 59.22

6.21
1.78
2.29

2.82
1.64
3.79

AODG

8
150
1600

7.81 0.51
155.33 2.50
1633.33 57.15

2.38
3.56
2.08

6.56
1.61
3.50

HM5013620

8
150
1600

8.60 0.28
159.50 2.81
1603.33 41.79

7.48
6.33
0.21

3.22
1.76
2.61

Table 7
Pharmacokinetic parameters of all components in SD rats after oral administration of 750 mg/kg HMPL-004 (mean SD, n = 6).
Parameters

Unit

AND

NAND

DAND

DDAND

CLA

AODG

HM5013620

t1/2
MRT
AUClast
AUCtotal
Cmax
Tmax

h
h
h ng/mL
h ng/mL
ng/mL
h

3.10 1.92
4.53 1.49
467.73 102.32
523.86 134.30
180.98 71.16
0.29 0.10

7.99 7.65
10.62 10.90
358.48 139.62
591.79 364.24
156.73 95.29
0.29 0.10

3.15 2.20
7.21 2.54
232.41 144.07
291.81 201.35
62.10 29.00
0.29 0.10

2.61 2.43
3.77 3.13
677.31 261.04
695.04 187.67
386.17 100.64
0.29 0.10

2.88 3.17
3.39 1.32
977.88 326.59
1049.80 283.68
299.67 100.13
1.71 1.35

5.96 9.69
11.62 13.06
613.44 629.80
810.21 669.78
98.87 93.19
6.00 2.19

3.37 1.65
5.66 2.17
1378.26 531.08
1575.12 635.54
366.33 86.63
0.67 0.49

J. Wang et al. / J. Chromatogr. B 951952 (2014) 7888

87

Fig. 4. The average plasma concentrationtime curves of AND, NAND, DAND, DDAND, CLA, AODG and HM5013620 after a single oral dose of 750 mg/kg of HMPL-004.

sensitivity requirements of the method. Cortisone was selected as

IS because it showed low interference in the biological matrix, has
good stability under the analysis conditions and showed consistent
extraction efciency.

3.4. Validation
3.4.1. Specicity and selectivity
Fig. 3 depicts the typical chromatograms for AND, NAND, DAND,
DDAND, CLA, AODG, HM5013620 and IS obtained from the analysis
of pre-treated calibration standard samples at the LLOQ and a blank
plasma sample. Except for AND and DDAND, no obvious interference peaks were observed at the retention times of the analytes
in the blank plasma sample. For AND and DDAND, the areas of the
interference peaks in the blank plasma samples were not exceeding
6% and 16% of the areas at the LLOQ levels, respectively.

3.5. Linearity and LLOQ

Table 2 lists the regression equations of calibration curves and
linearity ranges for all seven analytes. The calibration curves were
linear over the concentration range of 82000 ng/mL for DDAND
and 42000 ng/mL for others. The LLOQ of each analyte was 4 ng/mL
with the exception that the LLOQ of DDAND was 8 ng/mL.

3.6. Accuracy and precision

The intra- and inter-day precision and accuracy values for the
QC samples are summarized in Table 3. The intra- and inter-day
precision values of AND, NAND, DAND, DDAND, CLA, AODG and
HM5013620 were all less than 12.37%, whilst the accuracy values
were all within the range of 13.88% to 1.25%. These results indicate
that the method is both accurate and precise.

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J. Wang et al. / J. Chromatogr. B 951952 (2014) 7888

3.6.1. Extraction recovery and matrix effect

The extraction recoveries and matrix effects of all analytes are
summarized in Table 4. At three concentration levels, the mean
extraction recoveries were all higher than 90% with RSD less than
12% among six replicates, except for AODG and CLA. For AODG, the
mean extraction recoveries at medium and high concentration levels (150 and 1600 ng/mL) were around 80% with a RSD less than
6% and for CLA, the mean extraction recoveries at medium and
high concentration levels (150 and 1600 ng/mL) were around 63%
with a RSD less than 7%. All these data indicate that the extraction
method is efcient and reproducible. The matrix effects of each analyte determined at three concentration levels were all within the
range of 85115%, indicating that no signicant inuences on the
ionization of each analyte exist by co-eluted compounds.

In the past decades, a few papers have reported methods to

quantitatively determine AND in animals and humans [1114].
We nd it surprising that no one has ever realized the instability of AND in plasma, which led to the possible underestimation
of the exposure to AND. In this study, AND was found to be stable
in plasma under acidic condition on ice-water for at least about
2.5 h, which was long enough for the whole period of sample
pre-treatment. Using the current method, the exposure of AND following dose normalization was much higher than the previously
reported data (523.86 h ng/mL at 43.75 mg/kg versus reference data
of 490.83 h ng/mL at 120 mg/kg [14]).
AND, NAND, DAND and DDAND have already been shown to be
the major diterpenoid components in A. paniculata, but until now,
we have been unaware of methods to simultaneously determine
these four components.

3.7. Stability
4. Conclusion
The analytes were stable in plasma stored on ice-water for at
least 2.5 h after acidication with 10% formic acid (For each analyte:
RE in the range of 5.50% to 11.42% with RSD 7.30%), in plasma
after three freeze-thaw cycles at 80 C (RE in the range of 5.50%
to 12.92% with RSD 10.43% for each analyte), and in plasma stored
at 80 C for 32 days (RE in the range of 13.54% to 5.92% with
RSD 7.20% for each analyte). In addition, the analytes were also
demonstrated to be stable in the pre-treated samples at 8 C for up
to 72 h (RE in the range of 5.42% to 10.00% with RSD 5.42% for
each analyte). All these results are summarized in Table 5.
3.8. Hemolyzed plasma effect
The effects of hemolysis on AND, NAND, DAND, DDAND, CLA,
AODG and HM5013620 are summarized in Table 6. The RSD of
all analytes was no more than 6.56%, whilst RE values were all
within the range of 6.21% to 14.71%, indicating that hemolysis
has minimal effect on the quantication of all these seven analytes.
3.9. Method application
The validated method was successfully applied to simultaneously determine the plasma concentrations of AND, NAND,
DAND, DDAND, CLA, AODG and HM5013620 (the major circulating metabolite of AND) in SD rats after a single oral administration
of 750 mg/kg of HMPL-004, which is equivalent to 48.75 mg/kg of
AND, 23.25 mg/kg of NAND, 3.00 mg/kg of DAND, 25.50 mg/kg of
DDAND, 14.25 mg/kg of CLA and 7.50 mg/kg of AODG. The mean
plasma concentration-time curve of each analyte is shown in
Fig. 4 and the pharmacokinetic parameters were calculated by
non-compartmental analysis (NCA) using Thermo KineticaTM 4.4.1
(Thermo Electron Corporation) and listed in Table 7.
As the representative chemical components of HMPL-004, all
analytes were detectable until 12 h post dose except for AND, which
was detectable up to 24 h post dose. HM5013620, the hydrolysis
metabolite of AND, showed the highest plasma exposure (AUC,
the area under the curve) among all seven analytes. HM5013620
has therefore been shown to be one of the major metabolites of
AND with about 3-fold higher AUC than its parent compound in
the circulating system. Several studies have reported that phase
II conjugated metabolites were the major metabolites of AND in
the excreta [810]. Until now, no reports of circulating metabolites of AND have been found. In this study, a hydrolysis metabolite
of AND was found in the circulating system and quantitatively
analyzed in plasma samples. Our previous studies have shown
that few of HM5013620 was generated after incubation with liver
microsomes or S9s, indicating that it was possibly formed via nonhepatic enzymes such as the esterase existing in the blood (Data
not shown).

A simple LCMS/MS method for the simultaneous quantication of six chemical components of HMPL-004 (AND, NAND,
DAND, DDAND, CLA and AODG) and one major metabolite of AND
(HM5013620) in rat plasma was developed and validated in the
current study and proven to be highly robust, accurate, sensitive
and specic. In this study, AND was found to be unstable in plasma,
but could be stabilized via acidication. In addition, the hydrolysis
metabolite of AND, named HM5013620, was found to be the major
circulating metabolite with 3-fold higher exposure than AND in
rat plasma. The developed method has been applied successfully
in the rat pharmacokinetic study and will be favorable for the further studies of HMPL-004 and other pharmacokinetic studies of A.
paniculata related TCMs.
Acknowledgements
The author would like to thank Dr. Weihan Zhang, the senior
director of the Medicinal Chemistry group of HMP, and Dr. Zhenping
Wu, the senior vice president and group leader of the Pharmaceutical Science group of HMP, for providing the standard materials of
all analytes and also thank all other colleagues in HMPL-004 project
groups of HMP for the support on this study.
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