Article

A simple and sensitive HPLC method for the simultaneous determination of eight bioactive components and fingerprint analysis of Schisandra sphenanthera

Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
Analytica chimica acta (Impact Factor: 4.51). 03/2010; 662(1):97-104. DOI: 10.1016/j.aca.2009.12.039
Source: PubMed

ABSTRACT

A simple and sensitive high performance liquid chromatography method with photodiode array detection (HPLC-DAD) was developed for simultaneous determination of eight bioactive constituents (schisandrin, schisandrol B, schisantherin A, schisanhenol, anwulignan, deoxyshisandrin, schisandrin B and schisandrin C) in the ripe fruit of Schisandra sphenanthera and its traditional Chinese herbal preparations Wuzhi-capsule by optimizing the extraction, separation and analytical conditions of HPLC-DAD. The chemical fingerprint of S. sphenanthera was established using raw materials of 15 different origins in China. The chromatographic separations were obtained by an Agilent Eclipse XDB-C18 reserved-phase column (250 mm x 4.6 mm i.d., 5 microm) using gradient elution with water-formic acid (100:0.1, v/v) and acetonitrile, at a flow rate of 1.0 mL min(-1), an operating temperature of 35 degrees C, and a wavelength of 230 nm. The constituents were confirmed by (+) electrospray ionization LC-MS. The new method was validated and was successfully applied to simultaneous determination of components in 13 batches of Wuzhi-capsule. The results indicate that this multi-component determination method in combination with chromatographic fingerprint analysis is suitable for quantitative analysis and quality control of S. sphenanthera.

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Available from: Wan-Sheng Chen, Nov 06, 2014
Analytica Chimica Acta 662 (2010) 97–104
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A simple and sensitive HPLC method for the simultaneous determination of eight
bioactive components and fingerprint analysis of Schisandra sphenanthera
Hua Wei
a,b
, Lianna Sun
b
, Zongguang Tai
a
, Shouhong Gao
a
, Wen Xu
a
, Wansheng Chen
a,
a
Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China
b
Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai 200433, China
article info
Article history:
Received 18 August 2009
Received in revised form
19 December 2009
Accepted 31 December 2009
Available online 11 January 2010
Keywords:
Schisandra sphenanthera
Chromatographic fingerprint
Quality evaluation
High performance liquid chromatography
method with photodiode array detection
Liquid chromatography-mass spectrometry
abstract
A simple and sensitive high performance liquid chromatography method with photodiode array detection
(HPLC-DAD) was developed for simultaneous determination of eight bioactive constituents (schisandrin,
schisandrol B, schisantherin A, schisanhenol, anwulignan, deoxyshisandrin, schisandrin B and schisandrin
C) in the ripe fruit of Schisandra sphenanthera and its traditional Chinese herbal preparations Wuzhi-
capsule by optimizing the extraction, separation and analytical conditions of HPLC-DAD. The chemical
fingerprint of S. sphenanthera was established using raw materials of 15 different origins in China. The
chromatographic separations were obtained by an Agilent Eclipse XDB-C18 reserved-phase column
(250 mm × 4.6 mm i.d., 5 m) using gradient elution with water–formic acid (100:0.1, v/v) and acetoni-
trile, at a flow rate of 1.0 mL min
1
, an operating temperature of 35
C, and a wavelength of 230 nm. The
constituents were confirmed by (+) electrospray ionization LC–MS. The new method was validated and
was successfully applied to simultaneous determination of components in 13 batches of Wuzhi-capsule.
The results indicate that this multi-component determination method in combination with chromato-
graphic fingerprint analysis is suitable for quantitative analysis and quality control of S. sphenanthera.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Schisandra sphenanthera Rehd. et Wils (Schisandraceae), known
as Nan-wuweizi”, and Schisandra chinensis (Turcz.), known as Bei-
wuweizi”, have been widely used as a traditional Chinese medicine
(TCM) for thousands of years in China. Since 2000, the fruits of these
two plants have been officially listed in the Chinese Pharmacopoeia
(State Pharmacopoeia Committee, 2000) as two different crude
drugs [1]. Nan-wuweizi is widely used for the treatment of hepatitis,
hepatic renal insufficiency, menstrual disorders, and neuroasthe-
nia [2] owing to its hepatoprotective, anti-oxidant, anti-tumor,
detoxificant, anti-HIV and platelet-activating factor antagonistic
activities [3–5]. It has become a component of various traditional
Chinese medicine preparations (TCMPs), of which Wuzhi-capsule
(WZC) is widely used as a hepar-protecting and enzyme-decreasing
drug in clinical practice. Nan-wuweizi was found to have many
bioactive schisandra lignans with a dibenzocyclooctadiene skele-
ton, including deoxyshisandrin, schisantherin A, and schisanhenol
[2].AsNan-wuweizi is distributed in different parts of China [6],
the contents of the bioactive components vary greatly depending
Corresponding author. Fax: +86 21 81886181.
E-mail addresses: chenws@vent.citiz.net, chenwanshengchzh@yahoo.com.cn
(W. Chen).
on the geographical locations, climate, and other factors [2]. The
fact that many other species of genus Schisandra (Schisandraceae)
are also found in the same location of Nan-wuweizi makes it even
more difficult to identify and collect it.
Considering the above description, there is an urgent need
to develop a satisfactory method for controlling the quality of
Nan-wuweizi. Although there have been many reports about the
quality control of Wuweizi, most of them are limited to qualita-
tive and quantitative analysis of several main components rather
than chemical fingerprinting study of Nan-wuweizi [2,7,8]. Analy-
sis of chemical fingerprint has been accepted by the WHO [9], FDA
(2000) [10], and other authorities for quality assessment of herbal
medicines [11]. Determination of particular lignan constituents
alone is not enough due to the fact that common compounds
exist in both Nan-wuweizi and Bei-wuweizi. For example, schisan-
therin A has been used for quality evaluation of Nan-wuweizi owing
to its hepatoprotective effect and its high content in the herb
[1], but it also exists in Bei-wuweizi [12–14]. Moreover, a herbal
medicine is often a complex mixture containing dozens or even
hundreds of chemical components. Clinically some low-content
compounds had very strong activities [15]. A single component
such as schisantherin A alone cannot be responsible for the overall
pharmacological activities of Nan-wuweizi; other important lig-
nans, such as deoxyschizandrin, schisandrin, schisandrin C, and
schisandrin B [16–18], should also be considered for quality con-
0003-2670/$ see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2009.12.039
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98 H. Wei et al. / Analytica Chimica Acta 662 (2010) 97–104
Fig. 1. The structures of eight bioactive components in Schisandra sphenanthera.
trol of Nan-wuweizi. Although some compounds may not be active
themselves, they can affect the potency of other bioactive con-
stituents [15]. Some researchers suggest using as many as possible
components simultaneously to evaluate the quality of the herbal
medicines, but their suggestion seems unfeasible because most
reference components are not commercially available. In addi-
tion, most herbal medicines are so complex that the procedures
of separating, identifying and determining several components at
the same time are rather tedious for pharmaceutical quality eval-
uation [19]. Although there are reports in the literature [2,8,13]
about simultaneous determination of the main components in
Nan-wuweizi, few studies have been reported about some very
important components, such as schisanhenol and anwulignan.
In the present study, we developed a high performance liq-
uid chromatography method with photodiode array detection
(HPLC-DAD) by optimizing the extraction, separation and analyti-
cal conditions for characteristic fingerprint analysis in combination
with simultaneous determination of eight bioactive components,
including schisandrin (1), schisandrol B (2), schisantherin A (3),
schisanhenol (4), anwulignan (5), deoxyshisandrin (6), schisandrin
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H. Wei et al. / Analytica Chimica Acta 662 (2010) 97–104 99
B (7) and schisandrin C (8) (Fig. 1)inNan-Wuweizi and Nan-
Wuweizi-based TCMPs such as WZC, where chromatographic peaks
were confirmed by LC–MS analysis. The combination of chromato-
graphic fingerprint analysis and simultaneous determination of the
eight bioactive components offers a more comprehensive strategy
for quality evaluation of Nan-wuweizi.
2. Materials and methods
2.1. Materials and reagents
Fifteen raw material samples of Nan-wuweizi were collected
from different provinces in China. All the voucher specimens,
which were authenticated by Prof. Han-Ming Zhang, were kept
at our department for future reference. The air-dried samples
were smashed into powder and passed through a 40-mesh sieve
before analysis and stored in a desiccator. Thirteen batches of
WZC were purchased from Hezheng Pharmaceutical Company
(Chengdu, China), each capsule containing 11.25 mg deoxyschizan-
drin.
Reference compounds, schisandrin, deoxyshisandrin, schisan-
drin B, schisantherin A, schisandrin C and schisanhenol were
purchased from Shanghai R&D Center for Standardization of Tra-
ditional Chinese Medicines (Shanghai, China). Purity of these
compounds was higher than 98% as determined by HPLC. Schisan-
drol B and anwulignan (99% purity) were isolated and purified from
the ripe fruits of Nan-wuweizi and Bei-wuweizi. The chemical struc-
tures of the two compounds were confirmed by UV, IR, ESI-MS,
1
H NMR and
13
C NMR. A chromatogram of the mixture of stan-
dard compounds is shown in Fig. 2. The reference compounds were
accurately weighed and dissolved in methanol and then diluted
to appropriate concentrations for establishing calibration curves.
All stock and working standard solutions were stored at 4
C until
use.
HPLC-grade acetonitrile was purchased from Caledon Laborato-
ries Ltd. (Ontario, Canada). Analytical grade methanol and ethanol
were purchased from China Medicine (Group) Shanghai Chemical
Reagent Corporation (Shanghai, China). Water for HPLC analysis
was purified by a Milli-Q water purification system (Millipore,
USA).
2.2. Instrumentation and chromatographic condition
2.2.1. HPLC instrumentation and optimization of
chromatographic condition
Chromatographic analysis was performed on an Agilent 1200
series high performance liquid chromatography system equipped
with a diode array detector (190–400 nm), a quaternary solvent
delivery system, a column temperature controller and an autosam-
pler. The chromatographic data were processed with Agilent
Chromatographic Work Station software. Different HPLC param-
eters were examined and compared, including various columns,
mobile phases, column temperatures, and mobile phase flow rates.
Finally, analysis was carried out at 35
C on an Eclipse XDB-C18
column (250 mm × 4.6 mm i.d., 5 m). A linear gradient elution
of eluents A (water–formic acid, 100:0.1, v/v) and B (acetonitrile)
was used for separation. The elution program was optimized and
conducted as follows: a linear gradient of 45–52% B (0–12 min),
a linear gradient of 52–53% B (12–15 min), a linear gradient of
53–58% B (15–16 min), a linear gradient of 58–54%B (16–24 min),
a linear gradient of 54–75% B (24–26 min), a linear gradient of
75–76% B (26–40 min), a linear gradient of 76–100% B (40–41 min)
and 100% B (41–55 min). After a 10 min equilibration period, the
samples were used for injection. The peaks were recorded using
DAD absorbance at 230 nm and the solvent flow rate was kept
at 1.0 mL min
1
.
Fig. 2. Typical chromatograms for determination of eight bioactive com-
pounds in Nan-wuweizi and WZ-capsules. (A) Mixed standards; (B) Nan-wuweizi
(batch no. 070915); (C) the negative sample of WZ-capsule; (D) WZ-capsule
(batch no. 070601). Peak 1 = schisandrin, 2 = schisandrol B, 3 = schisantherin
A, 4 = schisanhenol, 5 = anwulignan, 6 = deoxyshisandrin, 7 = schisandrin B,
8 = schisandrin C, respectively. The separation conditions are described in Section
2.2.1.
2.2.2. LC–MS instrumentation and chromatographic conditions
All the chromatographic peaks were confirmed by an LC–MS
experiment using the Agilent 6410 triple-quadrupole mass spec-
trometer under a positive electrospray ionization (ESI) mode with
the spray voltage set at 4000 V. Desolvation gas (nitrogen) was
heated to 325
C and delivered at a flow rate of 10 L min
1
. The
nitrogen nebulizing gas was set at 40 psi with a source temper-
ature of 105
C. The HPLC condition was the same as described
above.
2.3. Preparation of standard solutions
Standard stock solutions of the 8 reference standards (schisan-
drin, schisandrol B, schisantherin A, schisanhenol, anwulignan,
deoxyshisandrin, schisandrin B and schisandrin C) were prepared
by dissolving them in methanol. They were then diluted to seven
concentrations for construction of calibration plots in the ranges
of 2.65–530, 2.53–506, 2.74–548, 2.60–520, 2.65–530, 2.53–506,
2.54–508, and 2.66–532 gmL
1
, respectively. Further dilution
with the lowest concentrations in the calibration curves were car-
ried out to provide a series of standard solutions for evaluating the
limits of detection (LOD) and the limits of quantification (LOQ) of
the compounds. The stock and working solutions were stored at
4
C.
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100 H. Wei et al. / Analytica Chimica Acta 662 (2010) 97–104
2.4. Optimization of sample extraction methods and preparation
of sample and negative control solutions
All the samples were oven-dried at 50
C until the weight
remained constant. Different extraction factors, including con-
centrations of ethanol, sample–solvent ratios, and extraction
times were tested. Finally, each dried sample (1.0 g powder) was
extracted with 40 mL ethanol aqueous solution (90%, v/v) in an
ultrasonic water bath for 45 min. The extracted solution was cen-
trifuged at 1735 × g for 10 min. The supernatant was collected and
the solutions (2 mL) were filtered through a 0.45 m membrane fil-
ter prior to HPLC analysis. An aliquot of 20 L solution was injected
for HPLC analysis.
According to the prescription and preparation protocol of WZC
formula provided by the Drug Standard of the Ministry of Public
Health of China, negative control samples of WZC, which contain
no Nan-wuweizi, were prepared to validate the specificity of the
method.
2.5. Method validation
The method was validated for linearity, limits of detection and
quantification (LODs and LOQs), precision (inter-day and intra-
day precision), reproducibility, stability and accuracy following the
International Conference on Harmonization (ICH) guideline [20]
and some reports on determination analysis [6,8,13].
2.6. Similarity calculation of HPLC fingerprint analysis
Data analysis was performed by the Similarity Evaluation
System for Chromatographic Fingerprint of Traditional Chinese
Medicine (Version 2004A), which was recommended by State Food
and Drug Administration (SFDA). The software was employed for
synchronization and quantitative comparison between different
samples. The correlation coefficients of entire chromatographic
profiles of the samples were calculated, and the simulative mean
chromatogram was also calculated and generated. The similarities
of different chromatographic patterns were compared with mean
chromatogram between the samples tested. In addition, the rel-
ative retention time (RRT) and relative peak area (RPA) of each
characteristic peak related to the reference peak were also calcu-
lated for quantitative expression of the chemical properties in the
chromatographic pattern of Nan-wuweizi.
3. Results and discussion
3.1. Optimization of HPLC conditions
To obtain accurate, valid and optimal chromatographic condi-
tions, different HPLC parameters were examined and compared,
including various columns (Diamonsil 250 mm × 4.6 mm i.d.,
5 m; ZORBAX SB-C18 250 mm × 4.6 mm i.d., 5 m; Eclipse
XDB-C18 250 mm × 4.6 mm i.d., 5 m; CALESIL ODS-100 C18
250 mm × 4.6 mm i.d., 5 m; Eclipse XDB-C18 150 mm × 4.6 mm
i.d., 5 m), mobile phases (methanol–water and acetonitrile–water
with different modifiers, including acetic acid, formic acid, and
phosphoric acid), column temperatures (20, 30, 35, or 40
C), and
mobile phase flow rates (0.8, 1.0, 1.2, or 1.5 mL min
1
). Based on the
maximum absorption of the marker compounds in the UV spectra
of the three-dimensional chromatograms obtained by DAD detec-
tion, the detection wavelength was set at 230 nm, where all the
marker compounds could be detected and had adequate absorp-
tion. Finally, the optimized HPLC condition was established by
comparing the resolution, baseline, elution time, and the number of
characteristic peaks in each chromatogram after repeated testing.
In Fig. 2 shows chromatograms A, B, C, and D corresponding to the
Fig. 3. Simultaneous LC–DAD (A) and LC–MS/MS (B) analysis of Nan-wuweizi (batch
no. 070915).
mixture of the standards, Nan-wuweizi, negative control sample of
WZC, and WZC.
3.2. Optimization of the extraction methods
To obtain satisfactory extraction efficiency, ultrasonic, reflux-
ing, and soxhlet extraction were compared. It was found that the
ultrasonic extraction was simpler and more effective for lignan
extraction, and therefore used in further experiments. Pure and
aqueous ethanol solutions were tested as the extraction solvents.
In the present study, different concentrations (20%, 40%, 60%, 80%,
90%, and 100%) of ethanol solutions were used for extraction pro-
cedure of Nan-wuweizi (batch no. 070915). It was found that the
extraction values of all targets increased gradually with the ethanol
concentration increasing when the concentration of ethanol was
<90%. A high ethanol concentration (100%) did not benefit efficient
extraction. Thus, 90% aqueous ethanol was selected as the extrac-
tion solvent. Four sample–solvent ratios (1:20, 1:40, 1:60, and 1:80,
w/v) were tested and compared, and it was found that the ratio of
1:40 was the best. Different extraction times (10, 30,45, and 60 min)
were also tested and 45 min was selected. The suitable extraction
condition was established as follows: samples were extracted by
ultrasonic extraction using 40-time of 90% aqueous ethanol as the
extraction solvent, and the process lasted for 45 min.
3.3. LC–MS identity confirmation
LC–MS was used to identify the chemical constituents of the 8
schisandra lignans (1–8). ESI in both negative and positive modes
was used to analyze Nan-wuweizi (batch no. 070915) under the
conditions mentioned above. Positive mode ESI was found to be
sensitive for schisandra lignans. The chromatograms from the
sequential DAD and MS (in ESI positive mode) are presented in
Fig. 3. Table 1 lists the retention times (tR) and MS data. The mass
spectra matched with those obtained for the pure standards for
Table 1
Identification of the eight compounds by HPLC-ESI–MS.
Peak Compound tR (min) MS data in positive ion mode (m/z)
1 Schisandrin 12.1 455 [M+Na]
+
2 Schisandrol B 15.2 439 [M+Na]
+
3 Schisantherin A 25.1 559 [M+Na]
+
4 Schisanhenol 28.8 425 [M+Na]
+
5 Anwulignan 30.7 329 [M+H]
+
6 Deoxyshisandrin 33.5 417 [M+H]
+
7 Schisandrin B 35.5 423 [M+Na]
+
8 Schisandrin C 37.8 385 [M+H]
+
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H. Wei et al. / Analytica Chimica Acta 662 (2010) 97–104 101
Table 2
The regression data, LODs and LOQs for eight bioactive compounds analyzed by HPLC.
Compound Regression equation (Y = aX + b) R
2
Linear range (gmL
1
) LOD
a
(gmL
1
) LOQ
b
(gmL
1
)
Schisandrin Y = 41,397X + 139.76 0.9989 2.51–502.00 0.56 1.97
Schisandrol B Y = 43,953X + 129.52 0.9989 2.53–506.00. 0.59 2.12
Schisantherin A Y = 48,035X + 127.48 0.9989 2.65–530.00 0.84 2.17
Schisanhenol Y = 47,992X + 241.951 0.9986 2.54–508.00 0.32 1.25
Anwulignan Y = 18,328X + 20.841 0.9989 2.66–532.00 0.99 3.50
Deoxyshisandrin Y = 47,978X + 214.68 0.9988 2.74–548.00 0.40 1.53
Schisandrin B Y = 18,428X + 20.841 0.9989 2.60–520.00 0.52 1.88
Schisandrin C Y = 49,542X + 151.31 0.9991 2.53–506.00 0.56 1.99
a
LOD refers to the limits of detection, S/N = 3.
b
LOQ refers to the limits of quantity, S/N = 10.
each of the components of interest 1–8, thus confirming their iden-
tity. As listed in Table 1, the eight components exhibited their
quasi-molecular ions [M+Na]
+
and [M+H]
+
. Their fragmentation
patterns well matched with their chemical structures. According to
the m/z values and retention features, the 8 components were iden-
tified from 90% ethanol extract of 15 batches of Nan-wuweizi. The
results further revealed that the 8 investigated compounds were
the main constituents of Nan-wuweizi, which is of great importance
to establish a better determination method for its quality control.
3.4. Method validation of quantitative analysis
The method was validated in terms of linearity, LOD and LOQ,
precision, reproducibility and recovery test.
3.4.1. Calibration curves, LOD and LOQ
Methanol stock solutions containing the 8 analytes were pre-
pared and diluted to appropriate concentrations for construction of
the calibration curves. The 8 analytes solutions at 6 different con-
centrations were injected in triplicate, and the calibration curves
were constructed by plotting the peak areas versus the concentra-
tions of each analyte. Satisfactory calibration curves of the eight
bioactive components were obtained. LOD and LOQ, which were
expressed by 3- and 10-fold of the ratio of the signal-to-noise (S/N),
were also acquired. Detailed information regarding the calibration
curves, linear ranges, LOD and LOQ is listed in Table 2.
3.4.2. Precision
Intra- and inter-day variations were chosen to determine the
precision of the developed assay. Intra-day precision was validated
with three concentrations of mixed standard solutions under the
optimized conditions for five times within 1 day. Inter-day preci-
sion was validated with the mixed standard solutions used above
once a day for 3 consecutive days. Inter- and intra-day precisions for
all investigated components were expressed as relative standard
deviation (RSD) (Table 3).
3.4.3. Reproducibility and stability
To test the reproducibility of our assay, six independently pre-
pared samples of Nan-wuweizi (batch no. 070915) in parallel were
prepared and analyzed. Variations were expressed as RSD. The
results of reproducibility and stability tests are shown in Table 4.
Stability was tested at room temperature and samples were ana-
lyzed in triplicate every 8 h within 24 h. Stability was expressed
as the percentage decrease of sample solution: (content in sam-
ple solution at 0 content in sample solution at 24 h)/content in
sample solution at 0 h. RSD values were lower than 3.00% for all
components.
3.4.4. Recovery test
The recovery test was done by the standard addition method.
Low, medium and large amounts of the 8 standards were added
to the known sample (Nan-wuweizi, batch no. 070915) and then
extraction and analysis were done as described in Section 2.4.
The mean recovery was counted according to the following for-
mula: recovery (%) = (amount found original amount)/amount
spiked × 100%, and RSD (%) = (SD/mean) × 100%. The mean recov-
ery of the 8 lignan compounds was 96.32–103.9% and their RSD
value was less than 3.13% (Table 5). It was found that the HPLC-DAD
method was precise, accurate and sensitive enough for simul-
taneous quantitative evaluation of the 8 lignan compounds in
Nan-wuweizi.
3.5. Sample analysis
The newly established analytical method was subsequently
applied to simultaneous determination of 8 active compounds in
15 commercial samples of Nan-wuweizi and WZC (13 batches)
from the same manufacturer. All samples were analyzed using
the optimized extraction method under optimized HPLC con-
ditions. Each sample was analyzed in triplicate to determine
the mean content (mg g
1
). Table 6 showed that deoxyshisan-
drin (1.54–6.63 mg g
1
), schisantherin A (0.80–5.66 mg g
1
), and
Table 3
Intra-day and inter-day precision of eight bioactive compounds.
Compound Concentration
(gmL
1
)
Intra-day
a
(n =5)
Inter-day
b
(n =3)
RSD (%) RSD (%)
Schisandrin 25.10 0.26 0.58
100.40 0.49 0.75
301.20 0.40 1.60
Schisandrol B 25.30 0.15 0.97
101.20 0.49 0.84
303.60 0.41 0.73
Schisantherin A 26.50 0.21 1.31
106.00 0.49 1.79
318.00 0.47 0.93
Schisanhenol 25.40 0.20 2.65
101.60 0.50 1.16
304.80 0.38 1.04
Anwulignan 26.60 0.26 1.84
106.40 0.74 1.21
319.20 1.72 0.96
Deoxyshisandrin 27.40 0.19 1.55
109.60 0.48 0.82
328.80 0.44 1.73
Schisandrin B 26.00 0.20 3.35
104.00 0.48 4.86
312.00 0.45 2.21
Schisandrin C 25.30 0.19 0.46
101.20 0.57 0.79
303.60 0.48 1.18
a
Intra-day precision on 1 day for tested five times.
b
Inter-day precision on 3 different days.
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102 H. Wei et al. / Analytica Chimica Acta 662 (2010) 97–104
Table 4
Reproducibility and stability of eight bioactive compounds.
Compound Reproducibility (n = 6) Stability (24 h, n =3)
Mean (mg g
1
)
a
RSD (%)
b
Percentage decrease
b
(%) RSD (%)
Schisandrin 0.881 ± 0.014 1.57 1.011 ± 0.012 1.19
Schisandrol B 0.270 ± 0.002 0.70 0.812 ± 0.020 2.46
Schisantherin A 0.846 ± 0.014 1.61 0.591 ± 0.009 1.52
Schisanhenol 0.047 ± 0.001 2.57 0.678 ± 0.011 1.62
Anwulignan 3.090 ± 0.053 1.71 1.036 ± 0.021 2.03
Deoxyshisandrin 1.424 ± 0.026 1.80 0.843 ± 0.013 1.54
Schisandrin B 0.551 ± 0.010 1.87 0.736 ± 0.008 1.09
Schisandrin C 0.080 ± 0.002 2.70 2.113 ± 0.015 0.71
a
Content = mean ± S.D.
b
Percentage decrease (%): [the mean content (mg g
1
)of prepared sample of Nan-wuweizi (batch no. 070915) at 0 h those at 24 h/those at 0 h] × 100.
Table 5
Recovery of the eight bioactive compounds.
Compound Original (mg g
1
) Spiked (mg g
1
) Found (mg g
1
) Recovery
a
(%) Average recovery (%) RSD (%) (n =3)
Schisandrin 1.040 0.470 1.522 102.17 100.11 2.16
0.970 1.991 97.85
1.380 2.427 100.32
Schisandrol B 0.383 0.140 0.519 96.74 100.12 3.13
0.310 0.702 102.94
0.500 0.887 100.69
Schisantherin A 1.056 0.490 1.532 98.86 98.22 0.89
1.010 2.051 98.58
1.530 2.533 97.21
Schisanhenol 0.190 0.090 0.276 96.53 96.85 1.51
0.170 0.352 95.57
0.320 0.505 98.46
Anwulignan 3.447 0.990 4.456 101.85 102.85 0.94
1.870 5.391 103.78
3.020 6.558 102.93
Deoxyshisandrin 1.776 0.810 2.555 96.05 96.32 1.29
1.600 3.306 95.23
2.410 4.130 97.68
Schisandrin B 0.671 0.290 0.976 105.24 103.90 1.28
0.620 1.314 103.90
0.980 1.675 102.57
Schisandrin C 0.179 0.090 0.272 101.20 99.13 2.07
0.150 0.328 99.12
0.270 0.439 97.08
a
Recovery (%) = [(found amount original amount)/spiked amount] × 100.
anwulignan (1.57–3.82 mg g
1
) were the main components of
Nan-wuweizi commercial samples, followed by schisanhenol
(0.01–1.48 mg g
1
) and schisandrin C (0.03–0.89 mg g
1
), partic-
ularly in those purchased from Hunan province. The targets
with the lowest contents included schisandrin (<0.76 mg g
1
),
schisandrol B (<0.51 mg g
1
), and schisandrin B (<0.21 mg g
1
),
and they could not be detected in some samples. The com-
mercial samples from Anhui province (A) had a low con-
Table 6
The contents (mg g
1
) of the 8 targets in 15 Nan-wuweizi commercial samples purchased from different places.
No. Batch number Sample source 1 2 345678Total
A 070915 Anhui 0.88 0.34 0.84 0.02 1.08 1.49 0.67 0.04 5.36
B 070721 Hunan Tr
a
N.D.
b
3.64 1.48 2.14 3.93 Tr
a
0.33 11.53
C 071001 Henan Tr
a
N.D. 1.30 0.09 3.08 1.78 Tr
a
0.06 6.31
D 070829 Shanxi Tr
a
N.D. 4.34 0.31 2.53 4.83 N.D. 0.37 12.38
E 080216 Henan Tr
a
N.D. 3.53 0.53 2.41 4.58 Tr
a
0.40 11.45
F 070926 Jiangsu Tr
a
N.D. 4.07 0.34 2.49 4.56 N.D. 0.48 11.94
G 071204 Shanxi Tr
a
N.D. 2.54 0.13 1.85 2.36 N.D. 0.23 7.11
H 071012 Yunnan Tr
a
N.D. 3.31 0.30 3.82 3.70 Tr
a
0.48 11.61
I 070705 Shanxi Tr
a
N.D. 3.60 0.36 3.18 4.21 Tr
a
0.42 11.77
J 070816 Hubei 0.02 N.D. 2.17 0.17 3.79 2.09 Tr
a
0.24 8.48
K 071225 Shanxi Tr
a
N.D. 2.89 0.37 2.55 4.14 N.D. 0.22 10.18
L 050829 Hebei 0.04 N.D. 3.63 0.36 3.04 4.82 Tr
a
0.80 12.70
M 071018 Hebei Tr
a
N.D. 2.45 0.19 3.21 2.62 Tr
a
0.14 8.61
N 061107 Shanxi Tr
a
N.D. 5.66 0.65 2.91 6.63 Tr
a
0.64 16.49
O 071006 Hubei Tr
a
N.D. 3.05 0.48 2.30 4.42 N.D. 0.89 11.15
a
Trace: below LOQ.
b
N.D.: below LOD.
Page 6
H. Wei et al. / Analytica Chimica Acta 662 (2010) 97–104 103
Fig. 4. Representative HPLC chromatograms for fingerprint analysis.
tent of total lignans (5.54%), although it had all the 8
components; the samples from Shanxi province (D, G, I,
and N) had a higher content of total lignans (16.49%). The
results of the WZC shows that the content of deoxyshisandrin
(5.20–6.54 mg capsule
1
), schisantherin A (3.4–6.05 mg capsule
1
),
and anwulignan (2.78–5.39 mg capsule
1
) were also the highest
targets in the WZC, but the content of schisandrin, schisandrin C,
and schisandrol B were extremely low. Overall, the content of the
eight bioactive components in the 13 batches of WZC was similar.
3.6. Establishment of chromatographic fingerprint of
Nan-wuweizi and similarity evaluation
To obtain a standard fingerprint, standard samples of good
quality are needed to establish the mean chromatogram. Fifteen
Nan-wuweizi samples (see Table 6) that met the requirement of the
Pharmacopoeia of the People’s Republic of China (2005 Edition)
were selected as the standard samples [21]. The average chro-
matogram of the 15 batches of commercial samples was taken
as the standard characteristic fingerprint of Nan-wuweizi. Peaks
existing in all chromatograms of the samples were assigned as the
“common peak”. The chromatograms of Nan-wuweizi containing
13 distinct common peaks within 55 min are shown in Fig. 4. Peak
6 (schisantherin A) was chosen to calculate the relative retention
time (RRT) and relative peak area (RPA). RRT and RPA of character-
istic peaks in 15 samples are shown in Table 7. SFDA suggests that
all herbal chromatograms should be evaluated in terms of similar-
ity by calculation of the correlation coefficient and/or angle cosine
value of original data [22–24]. Similarity analysis was therefore
Table 7
The relative retention time (RRT ± SD) and the relative peak area (RPA ± SD) of 13
characteristic peaks in chromatograms of 15 samples.
Peak no. RRT
a
± SD RPA
b
± SD
1 0.25 ± 0.0002 0.03 ± 0.0103
2 0.46 ± 0.0015 0.08 ± 0.2142
3 0.48 ± 0.0006 0.02 ± 0.0031
4 0.53 ± 0.0009 0.03 ± 0.0128
5 0.96 ± 0.0005 0.37 ± 0.0841
6
c
11
7 1.04 ± 0.0010 0.22 ± 0.0400
8 1.07 ± 0.0019 0.15 ± 0.0635
9 1.12 ± 0.0019 0.18 ± 0.0806
10 1.19 ± 0.0028 0.03 ± 0.0074
11 1.22 ± 0.0028 0.39 ± 0.1982
12 1.28 ± 0.0026 1.25 ± 0.2342
13 1.40 ± 0.0025 0.05 ± 0.1772
a
RRT: the ratio between peak retention time of target and internal reference
substance.
b
RPA: the ratio between peak retention time of target and internal reference
substance.
c
The internal reference substance.
Table 8
The similarities of chromatograms of 15
samples.
No. Similarities
a
A 0.836
B 0.979
C 0.913
D 0.996
E 0.994
F 0.997
G 0.984
H 0.983
I 0.983
J 0.989
K 0.947
L 0.955
M 0.993
N 0.926
O 0.950
a
The reference fingerprint was developed
with the median of all chromatograms.
conducted based on the standard fingerprints, and the results are
shown in Table 8. The closer the cosine values are to 1, the more
similar the two chromatograms are. The similarity values of all the
15 samples was more than 0.80. Samples with the smallest similar-
ity value or below a certain value, 0.8 for example, were regarded
as nonqualified. Therefore, if 0.80 is set as an appropriate thresh-
old, it is easy to identify acceptable based on the chromatographic
fingerprint. The fingerprint patterns of the different samples are
different, and by comparing the each fingerprint pattern with the
mean chromatogram, we can obtain the similarity value of each
fingerprint pattern, which can help us to evaluate the quality of
different samples.
4. Conclusions
Based on HPLC-DAD, we developed a simple quantitative
and qualitative method for simultaneous analysis of 8 active
compounds in the Chinese herbal drug Nan-wuweizi and Nan-
wuweizi-based TCMPs such as WZC. The method proved to have
good linearity, precision, repeatability, stability and recovery. For
the first time, we have also successfully established the HPLC
fingerprint of Nan-wuweizi. Fifteen batches of Nan-wuweizi from
different sources were assessed and distinguished by chromato-
graphic fingerprint analysis in combination with similarity analysis.
It is demonstrated that HPLC coupled with multiple compound
determination and fingerprint analysis is a powerful, practical tool
for comprehensive quality control of traditional Chinese medicines.
Acknowledgements
The authors thank Shanghai R&D Center for Standardization of
Traditional Chinese Medicines (Shanghai) and Prof. Dao-Feng Chen
from the Department of Pharmacognosy of Fudan University School
of Pharmacy for their excellent assistance.
References
[1] China Pharmacopoeia Committee, Pharmacopoeia of the People’s Republic of
China, China Chemical Industry Press, Beijing, 2000.
[2] J.P. Gao, Y.H. Wang, Y.Q. Yu, D.F. Chen, Zhongguo Tian Ran Yao Wu 1 (2003) 89.
[3] S.J. Jing, Y.H. Wang, D.F. Chen, Zhongguo Tian Ran Yao Wu 3 (2005) 78.
[4] W.L. Xiao, S.X. Huang, R.R. Wang, Phytochemistry 69 (2008) 2862.
[5] M. Xu, G. Wang, H. Xie, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 828
(2005) 55.
[6] Y.H. Liu, X.R. Luo, R.F. Wu, B.N. Zhang, Flora of China, Science Press, 1996, p.
252.
[7] X. Huang, F.R. Song, Zh.Q. Liu, Sh.Y. Liu, Huaxue Xuebao 66 (2008) 1059.
[8] B. Wang, J. Hu, W. Tan, J. Chromatogr. B 865 (2008) 114.
Page 7
104 H. Wei et al. / Analytica Chimica Acta 662 (2010) 97–104
[9] Z. Xiang, H. Li, L. Zhang, Chin. J. Chromatogr. 21 (2003) 568.
[10] World Health Organization, Guidelines for the Assessment of Herbal Medicines,
WHO, Munich, Geneva, 1991.
[11] State Food Drug Administration of China, Technical Requirements for the
Development of Fingerprints of TCM Injections, SFDA, Beijing, 2000.
[12] X.R. Yang, Q.X. Xiang, S.R. Xiong, J.H. Song, Yao Wu Fen Xi Za Zhi 26 (2006) 1558.
[13] S.P. Fu, F. Zhang, H. Zhang, Q. Xu, X.M. Liang, Xian Dai Zhong Yao Yan Jiu Yu Shi
Jian 18 (2004) 32.
[14] C.W. Halstead, S. Lee, C.S. Khoo, J. Pharm. Biomed. Anal. 45 (2007) 30.
[15] X. Deng, X. Chen, R. Yin, J. Pharm. Biomed. Anal. 46 (2008) 121.
[16] P.Y. Chiu, K.F. Luk, H.Y. Leung, K. Ng, K.Mi. Ko, Phytother. Res. 1002 (2009) 2826.
[17] L. Opletal, H. Sovov, M. Brtlov, J. Chromatogr. B 812 (2004) 357.
[18] X. Deng, X. Chen, W. Cheng, Chromatographia 67 (2008) 559.
[19] K. Yu, Y.F. Gong, Z.Y. Lin, J. Pharm. Biomed. Anal. 43 (2007) 540.
[20] ICH, Guidance for Industry, Q2B Validation of Analytical Procedures: Method-
ology, 1996.
[21] China Pharmacopoeia Committee, Pharmacopoeia of the People’s Republic of
China, China Chemical Industry Press, Beijing, 2005, p. 527.
[22] X. Wang, W.Y. Wang, K.R. Zhang, K.S. Bi, J. Shenyang Pharm. Univ. 20 (2003)
36.
[23] L.X. Wang, H.B. Xiao, X.M. Liang, K.S. Bi, Acta Pharm. Sin. 37 (2002) 713.
[24] F. Gong, Y.Z. Liang, P.S. Xie, F.T. Chau, J. Chromatogr. A 1002 (2003) 25.
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