全 文 ：• 303 •中华中医药杂志(原中国医药学报)2012年2月第 27 卷第2期 CJTCMP , February 2012, Vol . 27, No. 2
·论著·
Validated methods for simultaneous analysis of the active
components alignment in Huangjinju Tablets
HUANG Rui1, ZHANG Gui-jun1, WANG Meng1, WANG Jing-juan1, ZHAO Yue2
( 1School of Traditional Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, China;
2School of Traditional Chinese Materia Medica, Guangdong Pharmaceutical University, Guangzhou 510006, China)
Abstract: Objective: To establish thin-layer chromatography (TLC) and reversed-phase high-performance liquid
chromatography (RP-HPLC) methods for simultaneous qualitative and quantitative analysis of the active components alignment
(ACA) (baicalin-chlorogenic acid-orientin-vitexin) in Huangjinju Tablets. To lay a foundation for the establishment of the active
components alignment quality evaluation system of traditional Chinese medicine based on clinic curative effects. Methods: TLC
chromatogram of the active components alignment was obtained on a polyamide sheet with a system consisting of chloroform-
ethyl acetate-methanol-formic acid, 5:5:4:0.6 (v/v/v/v). Visualization was accomplished with UV light (365nm) after spraying the
sheets with 1% aluminium chloride in ethanol. RP-HPLC method involves a gradient elution on an Agela Venusil column using
solvent A, 0.2% phosphoric acid in water and solvent B, acetonitrile as mobile phase at a fl ow rate of 1mL/min. A linear gradient
from 15% to 30% of solvent B in 30min. UV detection was monitored at 340nm. The HPLC method was validated for specifi city,
linearity, accuracy and precision. Results: Four compounds in Huangjinju Tablets were successfully separated from each other.
The HPLC standards showed a good linearity for the four analytes with determination coeffi cients higher than 0.9995. The average
recoveries were ranged from 98.99% to 100.33% with RSD less than 1.79% for these components. Conclusion: Both methods were
proved to be simple, accurate and sensitive and are applicable to the quality control of Huangjinju Tablets.
Key words: Huangjinju Tablets; Active components alignment; TLC; RP-HPLC; Baicalin-chlorogenic acid-orientin-
vitexin
Fund assistiance: Enterprise-university-institute Integrated Project of Ministry of Education of Guangdong Province
(No.2007B090400085)
同时分析黄金菊片剂中药效组分的方法
黄睿1，张贵君1，汪萌1，王晶娟1，赵越2
（1北京中医药大学中药学院，北京 100102；2广东药学院中药学院，广州 510006）
摘要：目的：建立用薄层色谱法（TLC）同时定性分析和反相高效液相色谱法（RP-HPLC）同时定量分析黄
金菊片剂中药效组分黄芩苷-绿原酸-荭草苷-牡荆苷的方法，为建立中药与临床疗效对应的药效组分质量评价体
系奠定基础。方法：TLC鉴别是以聚酰胺薄膜为固定相，以氯仿-乙酸乙酯-甲醇-甲酸（5:5:4:0.6）为展开剂，喷
以1%三氯化铝乙醇溶液，置紫外光灯365nm下检视。RP-HPLC法以Agela Venusil为色谱柱，以0.2%磷酸水（A）
-乙腈（B）（0-30min，B 15%-30%线性洗脱）为流动相，流速1mL/min，检测波长为340nm，并对专属性、线
性、准确度和精密度等进行了方法学验证。结果：4种成分TLC法检测分离度较好，RP-HPLC法标准曲线线性关系
良好，R2均大于0.9995，平均回收率为98.99%-100.33%，相对标准偏差RSD均小于1.79%。结论：两种方法均操作
简便、结果准确、灵敏度高，可用于黄金菊片剂的质量控制。
关键词：黄金菊片剂；药效组分；薄层色谱法；反相高效液相色法；黄芩苷-绿原酸-荭草苷-牡荆苷
基金资助：广东省教育部产学研结合项目（No.2007B090400085）
通讯作者：张贵君，北京市朝阳区望京中环南路6号北京中医药大学中药学院，邮编：100102，电话：010-84738624
E-mail：guijunzhang@163.com
Huangjinju Tablets is a refined oral formulation of
Huangjinju decoction. Huangjinju decoction consists
of Radix Scutellariae, Flos Trollii Chinensis and
Flos Chrysanthemi Indici and is a traditional clinical
prescription in China used in the treatment of acute and
chronic pharyngitis, tonsillitis and upper respiratory
• 304 • 中华中医药杂志(原中国医药学报)2012年2月第27卷第2期 CJTCMP , February 2012, Vol . 27, No. 2
tract infections[1]. This research was under the instruction
of the active components alignment (ACA) theory[2]
which indicates that the effects of traditional Chinese
medicine (TCM) are not decided by a single component,
but by many components, i.e. the active components.
These active components form the ACA with an exact
relationship of quality and proportion. The TCM will
be discarded the dross, be selected the essential and
be refined if the ACA were used for medicine. Baicalin
(BAI), chlorogenic acid (CHA), orientin (ORI) and
vitexin (VIT) are the ACA of Huangjinju which was
obtained by a lot of pharmacology experiments and the
results indicated that Huangjinju ACA and Huangjinju
decoction have bioequivalence[3]. The ACA can be
prepared to make many preparations such as granules,
capsules and injection besides tablets.
The chemical structures of BAI (5,6-Dihydroxy-
7-O-β-D-glucopyranosyl-2-penyl-4H 1-benzopyran-
4 - one) , CH A ( [1S - (1α , 3β , 4α , 5α )] -3 - [ [3 - (3 ,4 -
dihydroxyphenyl) -1-oxy-2-propenyl]oxy] -1,4,5 -
t r ihyd roxy-1) , OR I (5 ,7-Dihydroxy-8 -beta -D -
g lucopyranosyl-2 - (3,4 -dihydroxyphenyl) - 4H-1-
benzopyran-4-one) and VIT (5,7-Dihydroxy-8-beta-D-
glucopyranosyl-2-(4-hydroxyphenyl)-4H-1-benzopyran-
4-one) are presented in Fig. 1.
There have been many methods to analyze
single compound of BAI, CHA, ORI and VIT. Some
new methods for simultaneous analysis of the four
components by HPLC and TLC is needed. Our research
group has been studing the ACA and preparations of
Huangjinju for many years[4-7]. The aim of this study
was to develop and to validate simple and rapid TLC
and HPLC methods for the quantitative and qualitative
analysis of Huangjinju Tablets.
Materials
The quantitative determinations were carried out
on an Agilent 1100 series liquid chromatograph (Agilent
Technologies Inc., USA) equipped with a G1315B diode
array detector (DAD), a G1311A quaternary pump, a
G1379A degasser, a G1316A column thermostat, and
a G1313A auto-sampler. Agilent Chemstation LC 3D
systems software was used for data acquisition.
TP-250 Ultrasonic Cleaner (Tian Peng Electricity
New Technology Co., Ltd., Beijing). BT25S Electronic
Analytical Balance (Sartorius Scientific Instruments Co.,
Ltd., Beijing). UV-Ⅲ Ultraviolet Analysis Instrument
(BEONY Science & Technology Co., Ltd., Beijing).
Reference standards of Baicalin (No.110715-200815),
Chlorogenic acid (No.110753-200413), Orientin
(No.111777-200801) and Vitexin (No.111687-200501)
were all purchased from the National Institute for the
Control of Pharmaceutical and Biological Products
(Beijing, China). Huangjinju Tablets (Batch No.100601)
and negative controls (Batch No. were all 100701) were
supplied by Hongtaikangda Co., Ltd. (Beijing, China).
Polyamide sheets (8cm×8cm) were purchased from
Zhejiang Taizhou Luqiao Sijia Biochemical Plastics
Factory (Taizhou, Zhejiang, China). Acetonitrile was
HPLC-grade quality purchased from Fisher Scientific
(Fair Lawn, NJ, USA). Water was HPLC grade (Watson
Group HK Ltd.). Phosphoric acid, chloroform, ethyl
acetate, methanol, and formic acid were of analytical
grade (Beijing, China).
Methods
1. Chromatographic conditions
1.1 TLC system Solutions of all analytes were
loaded on a polyamide sheet and developed with a
solvent system consisting of chloroform-ethyl acetate-
methanol-formic acid, 5:5:4:0.6 (v/v/v/v). The volumes
applied were all 1μL. Visualization was accomplished
with UV light (365nm) after spraying the sheets with 1%
aluminium chloride in ethanol.
1.2 RP-HPLC system Chromatographic separation
was performed at 25℃ on a reversed-phase Venusil
ASB-C18 column (5μm, 150Å, 4.6mm×250mm, Agela
Technologies Inc., USA). The mobile phase consisted
of 0.2% phosphoric acid in water (solvent A) and
acetonitrile (solvent B). The elution program was followed
O
OH O
HO
O
OOH
OH
OH
COOH
O
O
OH
OH
OH
OH
HO
O
HO

Baicalin Chlorogenic acid
OHO
OH O
O
HO
CH2OH
HO OH
OH
OH
OHO
OH O
O
HO
CH2OH
HO OH
OH
Orientin Vitexin
Fig.1 Chemical structures of the four analytes
• 305 •中华中医药杂志(原中国医药学报)2012年2月第 27 卷第2期 CJTCMP , February 2012, Vol . 27, No. 2
by a linear gradient from 15% to 30% of solvent B in
30min. The detection was performed at a wavelength
of 340nm. The flow rate was set at 1.0mL/min and the
sample injection volume was 10μL. The mobile phase
was filtered through a 0.45μm nylon membrane.
2. Sample Preparation Huangjinju Tablets
and negative controls of each analyte were powdered
respectively. 50mg of each powder was weighed
accurately into a 50mL conical f lask with stopper
respectively and 50mL methanol was added. The flask
was sealed with stopper and weighed, then sonicated
(ultrasonic power 200W, frequency 30kHz) for 30min.
After cooling to room temperature, methanol was added
to make up the initial weight. After shaking, the solution
was filtered through a 0.45μm nylon membrane to get
test solution for TLC and RP-HPLC.
3. Standard Preparation
3.1 TLC system Baicalin, chlorogenic acid, orientin
and vitexin were dissolved in methanol, respectively, to
obtain standard solutions of 0.89mg/mL, 0.057mg/mL,
0.046mg/mL and 0.012mg/mL, respectively.
3.2 RP-HPLC system Stock standard solutions
of baicalin (1.78mg/mL), chlorogenic acid (0.114mg/mL),
orientin (0.092mg/mL) and vitexin (0.024mg/mL)
were prepared by dissolving the accurately weighed
reference standards in methanol, respectively, and stored
away from light at 4℃. Calibration standard solutions
were prepared in a concentration range of 0.356-1.424,
0.0228-0.0912, 0.0184-0.0736, 0.0048-0.0192mg/mL
for baicalin, chlorogenic acid, orientin and vitexin,
respectively, to generate 6-point calibration curves for
each by diluting the stock solution of standards with
methanol, corresponding to 40%-160% of expected
sample values.
Working standard solutions were prepared at 0.89,
0.057, 0.046 and 0.012mg/mL of baicalin, chlorogenic
acid, orientin and vitexin, respectively, corresponding to
100% of expected sample value.
4. System suitability System suitability test
is an important part of method validation to ensure
performance of the HPLC system. Our research group
checked the parameters retention time (Rt), resolution (Rs),
asymmetry factor (Af), repeatability and theoretical plate
number (N) for the peak of each compound by injecting
a mixed standard solution containing 0.89mg/mL BAI,
0.057mg/mL CHA, 0.046mg/mL ORI and 0.012mg/mL
VIT. All data were generated automatically by the
system.
5. Method validation RP-HPLC method was
validated for its linearity (determination coefficient),
accuracy (% of recovery, %RSD), precision (%RSD),
sensitivity and specificity according to International
Conference on Harmonization guidelines[8].
5.1 Linearity Six different concentration level
solutions of BAI, CHA, ORI and VIT, in the range
of 40%-160% of the expected sample values were
prepared and injected to show that there is a direct
proportional relationship between analyte response and
concentration. Six points standard calibration curves
were obtained by the method of least square regression
and evaluated by their determination coefficients (R2).
5.2 Accuracy Accuracy of an analytical method
expresses the closeness between the determination
results and the true value of the sample and was
determined by calculating recoveries by the standard
addition method. Recoveries of the method were studied
by adding known amount of standards to that of sample
at 3 concentration levels 80%, 100% and 120% of the
expected content for Huangjinju Tablets. Three series of
samples for each concentration levels were prepared and
injected in duplicate. Recoveries were calculated with
respect to the standard solutions. Good recoveries prove
good accuracy of the proposed method.
5.3 Specificity Specificity was studied to measure
the interference from components that may be expected
in Huangjinju Tablets and was verified by negative
controls of each analyte, and standards of BAI, CHA,
ORI and VIT. Solutions were analyzed in accordance
with the proposed method to make sure no extraneous
peak was observed.
5.4 Precision
5.4.1 System precision: System precision was
determined by six replicate injections of the system
suitability standard solutions. The relative standard
deviations (RSDs) should be less than 2.0%.
5.4.2 Repeatability (intraday): Repeatability was
assessed by analyzing 6 samples of the same lot, on the
same day, within the same laboratory.
• 306 • 中华中医药杂志(原中国医药学报)2012年2月第27卷第2期 CJTCMP , February 2012, Vol . 27, No. 2
5.4.3 Intermediate Precision (interday): The
intermediate precision was evaluated by another analyst
on a different day with 6 samples of the same lot.
5.5 Solution Stability The stability of solution for
sample was evaluated. The solution was stored at room
temperature and tested at initial, 2h, 4h, 6h and 8h. The
solutions were tested using a freshly prepared standard.
5.6 Ruggedness The test was performed on a
different column (Diamonsil, C18, 5μm, 4.6mm×250mm,
Dikma Technologies, Beijing, China).
Results
1. TLC analysis Four compounds in huangjinju
tablets were successfully separated from each other using
a mobile phase of chloroform-ethyl acetate-methanol-
formic acid (5:5:4:0.6, v/v/v/v) and polyamide sheets as
a stationary phase, with a retention factor (Rf) value of
0.45, 0.36, 0.17 and 0.28 for BAI, CHA, ORI and VIT,
respectively. As shown in Fig. 2, spots of BAI, CHA,
ORI and VIT in Huangjinju Tablets were corresponded
in position and color to the spots in the chromatogram
obtained with the standard solution. It was observed that
no spots were visible in the chromatogram of negative
controls. The BAI produced black spot, the CHA
appeared as a blue fluorescent spot, the VIT and ORI
produced glassy yellow-green fluorescent spots. The four
components were successfully distinguished by TLC.
Resolutions were higher than 1.5.
2. HPLC analysis
2.1 System suitability test Repeated injections
showed that the Rt of every analyte was stable and the
RSDs of repeatability were all less than 2%. Resolutions
were all higher than 1.5. Asymmetry factors were
observed to be very close to 1. Theoretical plates were all
higher than 2 000. Results are summarized in Table 1.Fig.3
shows the HPLC chromatograms of standard mixture
and sample. All the compounds could be well separated
with high efficiency of the symmetrical peaks.
NB B NC C 1 2 3 NO O NV V
Fig.2 TLC chromatograms of Huangjinju Tablets
Map Key: NB represents the negative control of Baicalin; B
represents Baicalin; NC represents the negative control of Chlorogenic
acid; C represents Chlorogenic acid; #1, #2, #3 represent the samples
of Huangjinju Tablets; NO represents the negative control of Orientin;
O represents Orientin; NV represents the negative control of Vitexin; V
represents Vitexin.
2.2 Specificity Specificity was evaluated by a
qualitative comparison between chromatograms obtained
from sample, 4 standards and 4 negative controls. It was
found that there were no peaks appeared at the same
retention time of BAI, CHA, ORI and VIT. As a result,
it was concluded that the method has a high degree of
specificity.
2.3 Linearity The standards showed a good
linearity for the four analytes with determination
coefficients higher than 0.9995. The linear equations
were Y=1941.6X+64.64 (R2=0.9996) for BAI, Y=
Fig.3 HPLC chromatograms of standard (A) and
sample (B) solutions
Map Key: 1. Chlorogenic acid; 2. Orientin; 3. Vitexin; 4. Baicalin.
A
B
2
3
1
4
2
3
4
1
时间/
时间/
• 307 •中华中医药杂志(原中国医药学报)2012年2月第 27 卷第2期 CJTCMP , February 2012, Vol . 27, No. 2
such as acetic acid, ethyl acetate-methanol-water-formic
acid, 10:4:0.5:1 (v/v/v/v) and n-butanol-formic acid-water,
7:2:2 (v/v/v). But the separation of the ACA was not
ideal at all. The mobile phase chloroform-ethyl acetate-
methanol-formic acid, 5:5:4:0.6 (v/v/v/v) was proved to
provide a better separation. The spots obtained on silica
gel G appeared big and indistinctly. So the polyamide
sheets were used as a good stationary phase to separate
the four components.
2. HPLC analysis
2.1 Selectivity of detection wavelength RP-HPLC
chromatographic conditions have been developed for
quantitative determination of huangjinju tablets. The
difficulty of this determination is due to the fact that
the concentration of BAI in huangjinju ACA is about
fifteen, twenty and eighty times higher than that of
CHA, ORI and VIT, respectively. Although the λmax
of BAI, CHA, ORI and VIT is 278nm, 330nm, 350nm
and 338nm, respectively, 340nm was selected as the
detection wavelength for the simultaneous determination
of the four analytes. In this way, CHA, ORI and VIT can
be detected at low concentrations while BAI is at high
concentration.
2.2 Selectivity of mobile phase To optimize
the RP-HPLC parameters, several mobile phase
compositions were used such as methanol-glacial acetic
acid-water, acetonitrile-water systems with different
proportions. The peak shapes and separations were very
poor. The acetonitrile-phosphoric acid-water system
was choosed in the end. The retention time of BAI
peak was very long and the peak shape was imperfect
when an isocratic elution adopted was suitable for other
components. So the method of gradient elution was
studied. The acidity of the mobile phase is an important
influence factor for the determination. But if the acidity
were too high, it would do harm to the column. A
satisfactory separation and good peak shapes for ACA
were obtained with a mobile phase consisting of 0.2%
phosphoric acid in water (solvent A) and acetonitrile
(solvent B). A linear gradient from 15% to 30% of solvent
B in 30min. Clear baseline was achieved with DAD
detection at 340nm.
2.3 Study on the sample preparation When the
concentration of sample solution was low, 0.1mg/mL,
Table 1 Method validation results for Huangjinju Tablets
System suitability test
Theoretical plates
Asymetry factor
Resolution
Repeatability (n=6)
Validation
Linearity (R2)
Accuracy (% recover)
(%RSD) (n=9)
System precision (%RSD) (n=6)
Intraday precision (%RSD) (n=6)
Interday precision (%RSD) (n=6)
Solution stability (%RSD)
Determination of sample
BAI

105034
0.97
31.65
0.27

0.9996
98.99
1.18
0.27
0.14
0.03
0.35
98.25
CHA

13777
1
-
0.58

0.9997
99.87
0.71
0.58
1.06
0.37
1.14
99.19
ORI

32878
0.99
17.80
1.12

0.9998
99.76
1.11
1.12
1.01
1.09
1.29
100.42
VIT

44306
0.99
7.90
1.88

0.9995
100.33
1.79
1.88
1.82
1.87
1.84
98.39
Limits

N>1500
1±0.05
R>1.5
RSD<2%

R2>0.99
(100±5)%
RSD<3%
RSD<2%
RSD<3%
RSD<3%
RSD<3%
(100±5)%
11837X+15.36 (R2=0.9997) for CHA, Y=8791X+37.24
(R2=0.9998) for ORI and Y=11152X+8.11 (R2=0.9995)
for VIT.The linearity ranges were in the range of
0.356-1.424, 0.0228-0.0912 , 0.0184-0.0736,
0.0048-0.0192mg/mL for BAI, CHA, ORI and VIT,
respectively.
2.4 Accuracy The average recoveries of all
components in three different concentrations were
98.99%, 99.87%, 99.76% and 100.33% for BAI, CHA,
ORI and VIT, respectively, and were found within the
range of 98.99% to 100.33%. The RSDs of accuracies
for all components were less than 1.79%. Such high
accuracies indicate that the proposed method is reliable
for the determination of huangjinju tablets.
2.5 Precision All the assay results about RSD
value were within the acceptance limit of 3%, and
expressed the good precision of the proposed method.
2.6 Solution Stability The RSDs of solution
stability experiments were found well within 2%. No
significant changes were found during the test. The
solution stability data confirmed that the sample
solutions were stable at least for 8 hours.
2.7 Ruggedness The resolutions, peak shapes,
peak areas and retention times obtained on the
Diamonsil column made no big difference as that on the
Agela Venusil column.
Discussion
1. TLC analysis A lot of mobile phases were tried
• 308 • 中华中医药杂志(原中国医药学报)2012年2月第27卷第2期 CJTCMP , February 2012, Vol . 27, No. 2
the peaks were deformed obtained by the above HPLC
method with high injection volume 100μL for the peak
areas of VIT and ORI were very small. The concentration
of 1mg/mL was proved ideal for the determination with
the injection volume 10μL. To optimize the method of
sample extraction, several solvent of 50% methanol, 80%
methanol, methanol, ethanol, different ultrasonic powers
of 180W, 200W, 250W, and different times of 20min,
30min, 40min were tested. Considering all factors of
high extraction rate and time, methanol was selected as
the solvent. Ultrasonic power was 200W. Ultrasonic time
was 30min.
Conclusion
A new HPLC analytical method has been developed
and makes a major contribution to the quantity control
of huangjinju tablets. In addition, TLC represents as a
quick, easy and reliable method for the identification
of huangjinju ACA and do not require expensive
instrument. Both methods are accurate, simple and
reproducible, and can be used for routine quality control
analysis. This research will open doors for further
research of Huangjinju and is hoped to provide a new
idea for TCM development.
REFERENCES
[1] 张春晖,张贵君,王晶娟,等.药效组分中药“黄金菊”对甲
型流感病毒的抑制作用.世界科学技术-中医药现代化 ,
2009,11(6):856-859
ZHANG Chun-hui,ZHANG Gui-jun,WANG Jing-juan,et al.Anti-
influenza effect of Huangjinju,a Chinese medicine with active
components alignment.Journal of World Science and Technology/
Modernization of Traditional Chinese Medicine and Materia
Medica,2009,11(6):856-859
[2] 张贵君,罗容,王奕洁.中药药效组分理论与中药组分学.中
药材,2007,30(2):125-126
ZHANG Gui-jun,LUO Rong,WANG Yi-jie.The active components
alignment theory and components subject of traditional Chinese
medicine.Journal of Chinese Medicinal Materials,2007,30(2):
125-126
[3] 张春晖.“黄金菊”红外光谱三级鉴定与药效组分生物效应
评价方法研究.北京:北京中医药大学,2010
ZHANG Chun-hui.Study on the identification of Huangjinju by
three-steps infrared spectra and biological evaluation method of
active components alignment.Beijing:Beijing university of Chinese
medicine,2010
[4] 韩茹,张贵君,张春晖,等.注射用黄金菊冻干粉针质量标准
研究.世界科学技术-中医药现代化,2010,12(2):237-240
HAN Ru,ZHANG Gui-jun,ZHANG Chun-hui,et al.The quality
standard for Huangjinju Injections.Journal of World Science and
Technology/Modernization of Traditional Chinese Medicine and
Materia Medica,2010,12(2):237-240
[5] 郑璐璐,张贵君,张春晖,等.黄金菊药效组分解热的生物
效应.中国实验方剂学杂志,2011,17(2):196-198
ZHENG Lu-lu,ZHANG Gui-jun,ZHANG Chun-hui,et al.Biological
effects on active antipyretic components alignment of huangjinju.
China Journal of Experimental Traditional Medical Formulae,
2011,17(2):196-198
[6] 尹秀丽.黄金菊制剂质量评价及生物鉴定方法探讨.北京: 北
京中医药大学,2006
YIN Xiu-li.Study on the quality evaluation and biological
identification methods of huangjinju formulations.Beijing:
Beijing university of Chinese medicine,2006
[7] 潘艳丽,张贵君,孙素琴.黄金菊粉针药效组分红外指纹表征
分析.中成药,2006,28(2):172-175
PAN Yan-li,ZHANG Gui-jun,SUN Su-qin.IR characteristic
analysis of the effective constituents in Huangjinju Powder
Injection.Chinese Traditional Patent Medicine,2006,28(2):
172-175
[8] International conference on harmonization of technical
requirements for registration of pharmaceuticals for human use,
validation of analytical procedures:methodology,current step 4
version.Available on www.ich.org
（收稿日期：2011年6月25日）