Chinese Medicine, 2011, 2, 20-28
doi:10.4236/cm.2011.21004 Published Online March 2011 (http://www.SciRP.org/journal/cm)
Development and Validation of an HPLC Method for
Simultaneous Determination of Nine Active Components
in ‘Da-Chai-Hu-Tang’
Lingli Zheng, Deshi Dong*
The First Affiliated Hospital, Dalian Medical University, Dalian, China
Received December 21, 201 0; revised January 25, 2011; accepted February 2, 2011
Abstract
In this study, a simple, reliable and accurate method for the simultaneous separation and determination of
naringin, hesperidin, neohesperidin, baicalin, wogonoside, baicalein, wogonin, emodin and chrysophanol in
‘Da-Chai-Hu-Tang’ was developed by reverse-phase high-performance liquid chromatography (RP-HPLC).
The chromatographic separation was performed on an Agilent ZORBAX C18 column (250 mm × 4.6 mm
i.d., 5.0 μm), and the mobile phase composed of methanol and water containing 1% (v/v) acetic acid was
used to elute the targets in a gradient elution mode. The flow rate and detection wavelength were set at 0.8
ml/min and 280 nm, respectively. All calibration curves of the nine components expressed good linearities
(r2 ≥ 0.9992) within the tested ranges. The RSD values demonstrated the intra- and inter-day precisions were
less than 2.89%, and the recoveries of the investigated compounds were between 96.22% and 105.28%. The
proposed method is simple, precise, specific, sensitive, and successfully applied to determine the nine marker
compounds in ‘Da-Chai-Hu-Tang’ for quality control.
Keywords: High-Performance Liquid Chromatography, ‘Da-Chai-Hu-Tang’, Tradi tional Ch ine se Med icin e,
Multiple Compounds Determination
1. Introduction
Traditional Chinese medicines (TCMs), especially in
China, have played an important role in clinical therapy,
and been widely used for the prevention and treatment
various diseases because of its high effectiveness and
low toxicit y for thousands of years [1,2].
Generally, herbal medicines are used in combinations
to afford a formula composed of several single herbs.
Combining the herbs together and boiled in solvent can
make different preparations, and multiple constituents
are usually responsible for the therapeutic effects by
synergistic or antagonistic interactions. Each herb has its
own bioactivities, but when many herbs are combined,
there may be changes of active components, especially in
their contents. Moreover, some TCMs have been widely
administrated directly after boiling with water without
any quality assessment in some areas of China, which
may produce some side effects and influence the activi-
ties of herbal products because of different herbs from
different regions with different contents of active com-
pounds. That is why the quality of TCMs is very critical
important for affording the efficiency and avoiding the
toxicity. Thus, sensitive and reliable holistic analytical
approach is necessary.
Mostly, single marker compound is analyzed to eva-
luate the quality of T CMs [3] , which is simple but cannot
totally demonstrate the quality of herbal prescriptions.
Then, multiple components analysis (MCA) method has
been developed, which can simultaneously evaluate
many active compounds from different herbs and has
been widely used for the quality control of TCMs [4-6].
In the process of component determination, analytical
methods and technologies are essential. Up to date, two
kinds of chromatographic techniques, high-performance
liquid chro matography (HPLC) and HPLC-mass spectro-
metry (HPLC-MS), have been used more and more fre-
quentl y for the quality control of various kinds of herbal
medicines [7-9]. The former, has been universally used
as a convenient and sensitive method because of its con-
venience, precision, cheapness, sensitivity and reprodu-
cibility [10]. The later, can screen the chemical constitu-

L. L. ZHENG ET AL.
21
ents high-throughput in TCMs, especially those trace
components which are difficult for analysis by conven-
tional methods. Hence, HPLC-MS is a powerful tool for
its high level of sensiti vity a nd selectiv it y, but t he exp en-
sive running cost violates its application in routine anal-
ysis. Thus, in this paper, HPLC method was established
to achieve quality control.
The Chinese for mular ‘Da-Chai-Hu-Tang’ ( DCHT), is
a botanical drug and composed of Radix bupleuri, Fruc-
tus aurantii immatur us, Rhizo ma zingiber is rece ns, Radi x
scutellariae, Radix paeoniae alba, Rhizoma pinelli and
Fructus jujubae. Because of its therapeutic effectiveness
and few side effects, DCHT has been widely used to treat
acute cholecystitis, cholelithiasis, pancreatitis and ap-
pendicitis in China [11]. By now, pharmacological re-
search has demonstrated that it also shows a good effect
in inhibition atherosclerosi s and fatty liver [12,13].
In this decoction, there are several compounds with
significant pharmacological effects, such as flavonoids
including naringin and hesperidin from F. aurantii im-
maturus [14-18], baicalin and wogonoside from R. scut-
ellariae [19-24], and anthraquinones including emodin
and chrysophanol from R. paeoniae alba [25-29], etc.
Thus, selection of these marker compounds for totally
quality research of DH CT is critical important. But there
have no papers reported for simultaneous determination
of the nine marker compounds i n DHCT for q ualit y con-
trol through a literature search as far as we known.
The aim of the present paper was to establish a simple,
efficient and sensitive method for simultaneous analysis
of nine marker compounds i ncluding naringin, hesperi din,
neohesperidin, b aicalin, wogono side, b aicale in, wogon in,
emodin and chrysophanol (shown in Figure 1) in DHCT
for quality co ntrol by HPLC.
2. Experimental
2.1. Materials and Reagents
Nine standard compounds of naringin, hesperidin, neo-
hesperidin, baicalin, wogonoside, baicalein, wogonin,
emodin and chrysophanol were purchased from the Na-
tional Institute for Control of Pharmaceuticals and Bio-
logical Products (Beijing, China). Medicinal plants, Ra-
dix bupleuri, Fructus aurantii immaturus, Rhizoma zin-
giberis recens, Radix scutellariae, Radix paeoniae alba,
Rhizoma pinelli, Fructus jujubae and Radix et Rhizoma
Rhei were purchased from a local drug store (Dalian,
China) and identified by Dr. Yun-Peng Diao (Dalian
Medical University, Dalian, China). Voucher specimens
were deposited in College of Pharmacy, Dalian Medical
University (Dalian, China). Methanol was HPLC grade
(TEDIA, USA), and water for HPLC analysis was pre-
pared using a Millipore (Millipore, USA). Acetic acid
and other reagents were analytical grade purchased fro m
ShenLian Chemical Factory (Shenyang, China). All the
solvents and solutions were filtered through a Millipore
filter (0.45 μm) before use.
2.2. Standard Solution Preparation
A mixed stock standard solution containing naringin,
hesperidin, neohesperidin, baicalin, wogonoside, baical-
ein, wogonin, emodin and chrysophanol was prepared by
Figure 1. The chemical structures of the nine components: (1) naringin; (2) hesperidin; (3) neohesperidin; (4) baicalin; (5)
wogonoside; (6) baicalein; (7) wogoni n; (8) emodin and (9 ) chrysophano l.

L. L. ZHENG ET AL.
22
accurately weighing appropriate amounts of the nine
reference compounds and dissolving in methanol. All the
standard stock and working solutions were prepared in
dark brown calibrated flasks and stored at 4˚C.
2.3. Preparation of Sample Solutions and
Negative Control Solutions
Ten medical plants were triturated into powders in the
particle size of 40-60 mesh, and then weighed according
to DCHT formula and blended. The mixed powder (0.70
g) was extracted by 20 ml methanol for 20 min in an
ultrasonic bath. In order to keep the repeatability of the
extraction procedure, lost volume of methanol was com-
pensated after extraction. After filtration, 2 ml filtrate
was transferred into a 10 ml volumetric flask with MeOH
and 10 μl of the resultant solution was injected into the
LC system for analysis after through a 0.45 μm Millipore
filter.
According to the prescription and preparation protocol
of DCHT formula, three kinds of negative control sam-
ples in which the formula without F. aurantii Imma t-
urus, R. scutella riae, or R. et Rhizoma Rhei, respectively,
were prepared to validate the specificity of the method.
The negative samples were prepared according to the
method mentioned above.
2.4. Apparatus and Chromatographic
Conditi ons
Chromatography was performed with an Agilent Tech-
nologies 1200 series HPLC system consisting of a qua-
ternary delivery system, an auto-sampler and a DAD
detector. All the separations were carried out on a
ZORBAX SB C18 column (250 mm × 4.6 mm I.D., 5
μm). The mobile phase consisted of methanol (A) and
water containing 1% acetic acid (B) at a flow rate of 0.8
ml/min with a gradient elution mode was carried out as
follows: 0-20 min, linear gradient from 15% A to 35% A;
20-40 min, the mobile phase was held on 35% A; 40-60
min, linear gradient to 50% A; 60-110 min, the linear
gradient to 80% A; 110-120 min, the linear gradient to
95% A. Each run was followed by equilibration time of
15 min. Ultraviolet (UV) spectra were monitored at 280
nm. All the data were collected and analyzed with
Chemsta tion software.
3. Results and Discussions
3.1. Optimization of Chromatographic
Conditi ons
To develop an accurate, valid and optimal chromatogra m,
some HPLC analytical parameters including separation
column, mobile phase and its elution mode were all in-
vestigated in this study. Four kinds of reversed-phase
columns, Lichrosorb C18 column (150 mm × 4.6 mm
I.D., 5 μm), Johnsson ODS2 C18 column (250 mm × 4.6
mm I.D., 5 μm), Agilent XDB C18 column (150 mm ×
4.6 mm I.D., 5 μm) and Agilent ZORBAX C18 column
(250 mm × 4.6 mm I.D., 5 μm) were tested under differ-
ent elution modes of using methanol–water or acetoni-
trile–water containing different concentrations of acetic
acid as the mobile p hase (listed in Table 1). After a seri-
al of experiments, we found that the separation was per-
formed on an Agilent ZORBAX C18 column (250 mm ×
4.6 mm I.D., 5 μm) using the solvent system composed
of methanol (A)–water containing 1% acetic acid (B) as
the mobile phase with gradient elution mode as follows:
0-20 min, linear gradient from 15% A to 35% A; 20-40
min, the mobile phase was held on 35% A; 40-60 min,
linear gradien t to 50% A; 60 -11 0 min, t he l inea r gra die nt
to 80% A; 110-120 min, the linear gradient to 95% A.
The flow rate was 0.8 ml/min. The detection wavelength
was set at 280 nm on the basis of the UV spectra with
three dimension chromatograms of DAD detection,
where all the compounds could be detected and had
adequate adsorption. Selectivity was assessed by com-
paring chromatograms obtained from the blank sample
and from the corresponding spiked sample. Typical
chromatograms are shown in Figure 2, in which chro-
matograms of A, B and C correspond to blank mobile
phase, mixed standards, DCHT, and the peaks 1, 2, 3, 4,
5, 6, 7, 8 and 9 re present na ringin, he speri din, neohespe-
ridin, baicalin, wogonoside, baicalein, wogonin, emodin
and c hrysophanol, r espectively.
3.2. Optimization Sample Extraction Protoco l
The extra ctio n co nditi ons, for e xample ext rac tion s ol vent ,
method and time, can easily influence the efficiency of
the extraction. As a result, it is necessary to estimate and
optimize the factors affecting extraction recovery. Two
methods, boiling and ultrasonic are often used to extract
the ta rgets fr om matrix. T he d isadva ntages of the boiling
procedure are the loss of the compounds due to ioniza-
tion, hydrolysis and oxidation during extraction, the
consumption of a large amount of solvent, low extraction
efficiency, and time-consuming. These shortcomings
have led to the consideration of ultrasound-assisted ex-
traction (UAE) method, which has been widely used in
quality control of TCMs. In UAE process, extraction
solvent, sample-solvent ratio and extraction time are
critical i mporta nt for high extraction efficie ncy.
Methanol is often used as the extraction solvent be-
cause of its high efficiency and directly application for

L. L. ZHENG ET AL.
23
Table 1. The tried column a nd mobile pha se in optimization of HP LC co nditions.
Colu mn Solvent system Elution mode
Lichrosorb C18 (4.6 mm × 150 mm I.D., 5 μm)
(Zhonghuida, Dalian, China) Acetonitrile (A)
and water (B) 0~20 min , 1 5% A; 20~30 min, 15%~40 % A; 30~60 min, 40% A
ODS2 C18 (4.6 mm × 250 mm I.D., 5 μm)
(Johnsson, Dalian, China) Acetonitrile (A) and water (B) 0~30 min , 1 0~40% A; 30~ 60 min, 40% A
ODS2 C18 (4.6 mm × 250 mm I.D., 5 μm)
(Johnsson, Dalian, China) Methan ol (A) And wat er (B) 0~10 min, 10% A; 10~40 min, 10~40% A; 40~80 min, 40% A; 80~120 m in, 40~60% A
XDB C18 (4.6 mm × 150 mm I.D., 5 μm)
(Agilent, USA) Methanol (A) and water (B) 0~15 min, 10~30% A; 15~30 min, 30~40% A; 30~40 min , 40% A; 40~60 m in, 40~60% A; 60~80 min, 60~ 80% A
ZORBAX C18 (4.6 mm × 250 mm I.D., 5μm)
(Agilent, USA) Metha nol (A) and 1% acetic acid water (B) 0~15 min, 20~40% A; 15~35 min, 40% A; 35~40 min, 40~45% A; 40~60 min, 45~ 60% A; 60~100 min, 60~ 95% A
ZORBAX C18 (4.6 mm × 250 mm I.D., 5 μm)
(Agilent, USA) Methanol (A) and 1% acetic acid water (B) 0~10 min, 22~35%
A; 10~30 min, 35~38% A; 30~40 min,
38~45% A ; 40~6 0 m in , 45~ 6 0% A; 60~100 min, 60~95% A
ZORBAX C18 (4.6 mm × 250 mm I.D., 5 μm)
(Agilent, USA) Methanol (A) and 1% acetic acid water (B) 0~20 min, 15~35% A; 20~40 min, 35% A; 40~60 min, 35~50% A; 60~110 min, 50~ 80% A; 110~12 0 m in, 80~95% A
(a)
(b)
(c)
Figure 2 . Represent ative HPLC chromatograms of: (a) mo-
bile phas e; (b) mixed standard so lutions; (c) DCHT sample .
HPLC analysis without any more preparation. In the
present paper, pure and aqueous methanol (20%, 40%,
60% and 80%) were tried and examined as theextraction
solvent for DCHT by UAE for 30 min. The results
sho wn in Figure 3(a ) showed that the extraction rates of
all targets were gradually increased along with the in-
crease of methanol concentrations, and pure methanol
was selected as the extraction solvent. Second, three le-
vels of the use of methanol (10, 20 and 30 ml) were in-
vestigated, and the results are shown in Figure 3(b). It
was evident that 20 ml methanol was the best for the
extraction. Furthermore, the extract time, including 10,
20, 30 and 45 min were also optimized and the result
shown in Figure 3(c) indicated that the extraction time
contr olle d at 20 min was e nough. In the end, the sui tabl e
extraction conditions were as follows: the samples were
extracted by UAE using 20 ml methanol as the extraction
solvent, and the pro cess was lasted for 20 min.
3.3. Specificity of the Method
In order to investigate the specificity of the method, dif-
ferent negative control samples of DHCT were prepared
and analyzed by HPLC, and the chromatograms are
shown in Figure 4. It was obvious that there were no
interferences for determination of the nine compounds by
comparing the retention times with the standards. Fur-
thermore, the purities of the investigated peaks were all
confirmed to be pure through DAD purity studies .
3.4. Calibration Curves, the Limit of Detection
(LOD) and Quantification (LOQ)
The external standard method was used to obtain regression

L. L. ZHENG ET AL.
24
(a)
(b)
(c)
Figure 3. Efficiencies of the extraction for the nine com-
pou nds in DCHT us ing diff erent: (a) e xtr action solve nt; ( b)
the use of methanol; (c) extraction time.
(a)
(b)
(c)
Figure 4. Representative HPLC chromatograms of: (a) the
negat ive s ample w ithout F. a uran tii im maturu s; ( b) the neg-
ative s ampl e w ithout R. scutellariae; (c) the neg ative sa mple
without R.et Rhi zoma Rhei.
equations. The calculated results are shown in Table 2.
In the r e gr e ss io n eq ua t io n y = ax + b, y refers to the peak
area, x is the concentration of the standard compounds
(µg/ml), while a is the slope rate of the line and b is the
intercept of the straight line with y-axis. All the standard
compounds showed good linearity (r2 ≥ 0.9992) in the
tested concentratio n ranges. The limit of detec tion (LOD)
and quantification (LOQ) were also measured. The stan-
dard so lution was d iluted with MEOH to the appropriate
concentrations. The detection limit was defined as the
lowest concentration level resulting in a peak area of
three times the baseline noise. LOD was in the range of
0.07-0.30 µg/ml. The LOQ was obtained as amount to
give a signal-to-noise ratio (S/N) of 10 in the range of
0.35-0.87 µg/ml (listed in Table 2).

L. L. ZHENG ET AL.
25
Table 2. Regres sion data, linear range and the LOD a nd LOQ of the develope d method.
Analytes Calibration curvea r2 Linear r ange (μg/ml) LODb (μg/ml) LOQc (μg/ml)
Nari ngin y = 17.40x + 20.12 1.0000 1.53-49.00 0.25 0.75
Hesperidin y = 21.75x + 12.72 0.9999 2.70-43.20 0.20 0.48
Neohesperidin y = 23.38x + 28.20 0.9999 9.75-312.00 0.08 0.40
Baicalin y = 41.80x – 80.41 0.9998 6.25-200.00 0.07 0.35
Wogonoside y = 45.22x + 62.24 0.9998 3.25-120.00 0.18 0.50
Baicalein y = 68.27x – 36.53 0.9994 1.25-40.00 0.30 0.55
Wogonin y = 64.31x – 105.86 0.9996 3.12-50.00 0.30 0.74
Emod in y = 25.72x + 30.86 0.9993 4.00-64.00 0.25 0.87
Chrysophanol y = 33.47x + 23.22 0.9992 2.38-76.00 0.28 0.74
ay is the pea k area in HPLC analysis m onitored at 280 nm, x is the concentration of compound (μg/ml); bLOD refers to the limi t of
detection, S/N＝3; cLOQ refers to the limit of quantification, S/N＝10.
3.5. Assay Precision, Repeatability, Stability and
Recovery
The precision of the method was validated by both intra-
and inter-day precisions. The assays were carried out on
the same mixed standard solutions at low, medium and
high concentration levels during one day and one assay
each day for three consecutive days, respectively. Rela-
tive standard deviation (RSD) of the mean content for
each compound was calculated and ranged from 0.46%
to 2.89% for intra- and inter-day precisions, which is
sho wn in Ta b le 3. T he results indicated that the accuracy
and precision of the proposed method were sufficient for
determinatio n of the nine compounds in the sample of
DCHT.
The analysis repeatability of the nine components
(Table 4) was determined by analysis of six samples
which were prepared with the same preparation proce-
dure and processed in parallel as described above. The
RSD wa s ca lculated as a measurement for the repeatabil-
ity of the method. The results indicated that the RSD
values of each compound detected were all less than
1.97%, which showed good reproducibility of the devel-
oped method.
For the stab ility test, a sample of DCHT was analyzed
with the interval of 6 h (0, 6, 12 and 18 h) at room tem-
perature, and the sample solution was found to be stable
(RSD values of the mean content were lower than
2.15%). The results are listed in Table 4.
The recovery assays were carried out by adding
known contents of the standard samples to the known
amounts of samples of DCHT and comparing the deter-
mined amount of these standards with the amount origi-
nally added. Table 4 shows these results of recovery
tests. The mean recovery of the met hod was in the range
of 96.22-105.28%, with RSD of less than 2.33%. Consi-
dering the results of the recovery assays, the method was
thus acceptable.
Table 3. Intra- and inter-day variability of the 9 analytes.
Analytes Concentration
(μg/ml)
Int r a -day (n = 6) Int e r-day (n = 3)
RSD
a
(%) Accuracy
b
(%) RSD (%)
Accuracy
(%)
Nari ngin
3.06 1.87 98.37 2.89 96.08
6.12 2.06 102.61 2.04 103.43
24.50 2.69 98.78 2.49 99.22
Hesperidin
5.40 1.97 98.70 1.27 96.11
10.80 1.83 102.13 0.89 99.63
21.60 1.64 103.70 1.64 101.99
Neohesperidin
19.50 0.86 97.23 1.97 98.56
78.00 1.94 102.32 2.46 98.96
312.00 2.06 100.98 1.48 96.51
Baicalin
12.50 2.29 101.20 1.83 101.52
50.00 1.64 96.70 0.94 97.52
100.00 0.73 97.62 0.81 98.19
Wogonoside
7.50 0.94 98.13 1.94 98.80
30.00 0.46 103.60 2.19 96.90
60.00 1.33 96.40 1.45 97.88
Baicalein
2.50 1.81 96.40 1.70 102.80
10.00 1.90 98.50 1.54 98.10
20.00 2.05 103.85 0.96 96.20
Wogonin
6.25 1.05 98.08 1.57 97.12
12.50 0.46 99.52 2.06 98.88
25.00 1.07 98.76 2.16 101.40
Emod in
8.00 1.41 99.75 1.62 99.25
16.00 0.99 97.94 1.97 102.63
32.00 0.71 98.50 2.05 102.84
Chrysophanol
4.75 1.45 98.11 0.87 100.42
9.50 1.70 101.16 1.67 98.74
38.00 1.11 99.05 1.56 98.66

L. L. ZHENG ET AL.
26
aRSD(%) = (SD/mean) × 100; baccuracy(%) = (mean of measured concen- tration/spiked concentration) × 100.
Table 4. Rep eatbility, stability and recove ry results for the assay of the 9 analytes.
Analyte Repeatabi l ity (n = 6) Stability (18 h, n = 3) Recoverya (n = 3)
Mea n (mg/g) RSD (%) Mean (mg/g) RSD (%) Reco very (% ) RSD (%)
Nari ngin 13.92 0.94 13.87 1.68 98.65 1.40
Hesperidin 1.06 1.37 1.02 0.67 99.03 2.33
Neohesperidin 11.51 0.48 11.46 1.27 96.22 1.00
Baicalin 3.49 1.32 3.52 0.80 101.04 0.81
Wogonoside 0.98 0.87 0.94 0.94 98.33 0.48
Baicalein 0.92 0.94 0.95 0.73 105.28 0.92
Wogonin 0.84 1.38 0.86 2.15 97.17 1.05
Emod in 0.70 1.97 0.73 1.67 98.73 1.23
Chrysophanol 0.78 0.86 0.77 1.38 97.54 1.44
aRecovery(%) = (detected amount − original a mount)/spiked amount × 100.
4. Conclusions
An HPLC method for simultaneous determination of
nine active compounds including rhaponticin, naringin,
hesperidin, neohesperidin, baicalin, wogonoside, bai-
calein, wogoni n, emod in and chryso phano l in DHC T has
not been reported. The presented method in addition to
its novelty for determination of nine ingre dients wa s suf-
ficiently rapid, simple and sensitive as well as precise
and accurate, and it was not interfered with other chemi-
cal constituents in DCHT. The linearity, accuracy, preci-
sion, LOD and LOQ, specificity-selectivity of the method
and sample stability were all established. Although nine
compounds were quantitated, there are many other com-
ponents in DCHT. More researches can be practiced for
further investigation. But the method has several advan-
tages, including rapid analysis, simple mobile phase, and
simple sample preparation. It was success- fully used for
the analysis of compatibility study of a formulation pre-
pared in our laboratory and suitable for routine analysis
in quality-control la boratories.
5. Acknowledgemen ts
This research was partially supported by the excellent
young scientists funds (No.2006 J23JH024) of the
Science and Technology Foundation of Dalian, China.
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