CHEM. RES.

CHINESE UNIVERSITIES 2011, 27(1), 3844

*Corresponding author. E-mail: lyliurh@126.com
Received February 5, 2010; accepted March 10, 2010.
Supported by the National Natural Science Foundation of China(No.30725045), the Foundation of Eleventh Five-Year-Plan
of China(No.2008ZX09202-002), the Shanghai Leading Academic Discipline Project, China(No.B906) and the Scientific Foun-
dation of Shanghai City, China(No.07DZ19702).

Qualitative and Quantitative Analysis of Alkaloids in
Cortex Phellodendri by HPLC-ESI-MS/MS and HPLC-DAD
ZHU Shuang-lai
1,2
, DOU Sheng-shan
1
, LIU Xin-ru
1
, LIU Run-hui
1*
, ZHANG Wei-dong
1,3
,
HUANG Hong-lin
2
, ZHANG Yi
5
, HU Yao-hua
1,4
and WANG Shu-ping
1

1. School of Pharmacy, Second Military Medical University, Shanghai 200433, P. R. China;
2. School of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, P. R. China;
3. School of Pharmacy, Shanghai Jiaotong University, Shanghai 200240, P. R. China;
4. School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350108, P. R. China;
5. School of Pharmacy, Chengdu University of Traditional Chinese Medicine,
Chengdu 610075, P. R. China

Abstract A combined method of high performance liquid chromatograph-elecrtrospray-ionization mass spectro-
meter(HPLC-ESI-MS/MS) coupled with a photodiode array detector(HPLC-DAD) and principal component analy-
sis(PCA) was applied to the qualitative and quantitative analyses of alkaloids in Cortex Phellodendri(CP) samples,
and to the differentiation of two species of CP, Cortex Phellodendri Chinensis(CPC) and Cortex Phellodendri Amu-
rensis(CPA). Twenty-two peaks appeared in the HPLC-MS base peak chromatogram of CP detected by the
HPLC-ESI-MS/MS analysis, and the alkaloids were identified according to the MS
n
data, the known MS fragmenta-
tion rules and the literature data. Five alkaloids including berberine, palmatine, jatrorrhizine, phellodendrine and
magnoflorine were simultaneously determinated by the HPLC-DAD. Berberine was the primary component in all CP
samples, and the contents of berberine and palmatine were exploited to be two critical parameters for effective dis-
crimination between the two species of CP. The average content of berberine in CPC(58.75 mg/g) was higher than
that in CPA(9.16 mg/g), while the content of palmatine was less, only 0.25 mg/g in CPC and 4.19 mg/g in CPA. With
the use of PCA, samples datasets were separated successfully into two different clusters corresponding to the two
species, and berberine, palmatine, phellodendrine and magnoflorine contribute most to the above mentioned calssi-
fying. The proposed method proved to be a useful tool in the quality control of Chinese herbal medicines.
Keywords Cortex Phellodendri; Alkaloid; HPLC-ESI-MS/MS; Quantitative analysis; Qualitative analysis; Principal
component analysis
Article ID 1005-9040(2011)-01-038-07

1 Introduction
Alkaloids are a kind of cyclic compounds that contain
negative valent nitrogen atoms. Over 10000 alkaloids have
been isolated from nature so far, which are distributed widely
in plants
[1]
. Many studies demonstrate that alkaloids possessed
many kinds of biological activities
[24]
, such as anti-microbial,
anti-oxidants, anti-cancer, anti-inflammatory, and anti-virus
activities, etc.
Cortex Phellodendri(CP, Huangbo), the dry bark of Phel-
lodendri chinensis Schneid. or Phellodendri amurensis Rup., is
a commonly used traditional Chinese medicine herb with re-
moving damp heat, quenching fire, counteracting toxicity, and
the main active constituents are alkaloids. Modern pharmaco-
logical studies elucidate that CP has anti-microbial
[5]
, anti-
inflammatory
[6]
, anti-diarrhoea
[7]
, and anti-cancer properties
[6]
.
It has been officially listed in the Chinese Pharmacopoeia for a
long time. The Cortex Phellodendri Chinensis(CPC) and Cor-
tex Phellodendri Amurensis(CPA) are regarded as the two spe-
cies of CP. But CPC and CPA have been clarified into two
entries in the Chinese Pharmacopoeia of 2005 edition, in which
the quality assessment of the two medicines were based on one
mark compound berberine
[8]
. To our knowledge, the Pharma-
copoeia standard is lack of specificity, for berberine is a com-
mon ingredient in many herbal materials. Moreover, one mark
compound can not reflect the actual quality of herbs because
their therapeutic effect often results from multiple components.
Therefore, the effective method for the analysis of more alka-
loids in the quality control of CP is urgently needed.
Many analytical methods have been used for the study of
CP, such as thin layer chromatography(TLC)
[9,10]
, capillary
electrophoresis(CE)
[1113]
, high performance liquid chromato-
graphy(HPLC)
[1419]
, microcalorimetry
[2,20]
,
1
H NMR
[7]
and
NIRS
[21,22]
, most of methods are based on the analysis of
No.1 ZHU Shuang-lai et al. 39

berberine, palmatine and jatrorrhizine, and the HPLC is the
most preferred technique.
In the present study, a combination of high-performance
liquid chromatograph-electrospray-ionization mass spectrome-
ter(HPLC-ESI-MS/MS) coupled with a photodiode array de-
tector(HPLC-DAD) and principal component analysis(PCA)
was proposed for the quality control of CP. HPLC-ESI-MS/MS
with the advantage of rich structural information of analytes
acquired by MS
n
technique has been proved to be a modern
powerful tool for the identification of substances in herb ex-
tracts
[1,23,24]
. HPLC-DAD has high sensitivity, precision and
specificity. PCA can be employed to construct a linear discri-
mination model to classify samples and find the most important
principal components. Thus, the combined method can be used
to evaluate the quality of herbal medicines and classify the
samples from different habitats, different species or different
processing methods. The above method was successfully
applied to studying the alkaloids in CP for the quality control
purpose.
2 Experimental
2.1 Chemicals and Materials
HPLC-grade acetonitrile, formic acid and triethylamine
were purchased from Merck(Germany). Ultra pure water was
prepared by a Milli-Q50 SP Reagent Water System(Millipore
Corporation, USA). The reference standards of jatrorrhizine
hydrochloride, palmatine hydrochloride, berberine hydrochlo-
ride, magnoflorine hydrochloride and phellodendrine hydroch-
loride were purchased from the National Institute for the Con-
trol of Pharmaceutical and Biological Products(Beijing, China).
The purities of all the standards were not less than 98%, and
their chemical structures are shown in Fig.1. Twenty-two
batches of Cortex Phellodendri samples(Table 1) were
purchased from different places of China and identified
by Professor ZHANG Han-ming(Second Military Medical
University, Shanghai, China). The voucher specimens
(CPC09080922; CPA09010907) were deposited in the
Department of Phytochemistry, Second Military Medical
University(Shanghai, China).
2.2 Preparation of Samples
Each medicinal material of CP was dried at 60 C until
constant mass and ground to powder. Approximately 0.5 g of
the pulverized sample was accurately weighed and then ex-
tracted with 50 mL of acidified(1% HCl) methanol ultrasoni-
cally at room temperature for 40 min. The supernatant was
collected and filtered through a 0.45 m filter, and the filtrate
was used for the HPLC analysis.






Fig.1 Chemical structures of five reference standards
Table 1 Twenty-two batches of CP
Sample No. Species Origin Sample No. Species Origin
1 CPA Hebei Province 12 CPC Huaiyuan, Sichuan Province
2 CPA Hebei Province 13 CPC Dujiangyan, Sichuan Province
3 CPA Heilongjiang Province 14 CPC Dujiangyan, Sichuan Province
4 CPA Anhui Province 15 CPC Qingchengshan, Sichuan Province
5 CPA Liaoning Province 16 CPC Bazhong, Sichuan Province
6 CPA Jilin Province 17 CPC Beichuan, Sichuan Province
7 CPA Henan Province 18 CPC Beichuan, Sichuan Province
8 CPC Sichuan Province 19 CPC Beichuan, Sichuan Province
9 CPC Sichuan Province 20 CPC Yingbing, Sichuan Province
10 CPC Sichuan Province 21 CPC Yingbing, Sichuan Province
11 CPC Chengdu, Sichuan Province 22 CPC Yingbing, Sichuan Province

2.3 Preparation of Standard Solutions
Five reference standards were accurately weighed, then
dissolved in methanol and diluted to appropriate concentration,
respectively. Every standard stock solution was taken at the
correct volume to make a standard mixture.
All the solutions were stored in a refrigerator at 4 C for
analysis.
2.4 HPLC-ESI-MS/MS
Analysis was performed on an Agilent 1100 series LC/MS
ion trap mass spectrometer(Agilent, USA) connected to an
HPLC system with ESI ion source. Chromatography was car-
ried out on a C
18
XTerra column(4.6 mm250 mm, 5 m, Wa-
ters, USA) at room temperature via a gradient elution. The
mobile phase consisted of A(water, containing 0.3% formic
acid) and B(acetonitrile).
The gradient program was as follows: 025 min, 5%
25% B; 2535 min, 25%35% B; 3545 min, 35%45% B;
4560 min, 45%100% B. The flow rate was kept constantly
at 1 mL/min, and 0.2 mL/min of the eluant was introduced into
the MS system by means of split technique, and the sample
injection volume was 6 L. The conditions of MS analysis
40 CHEM. RES. CHINESE UNIVERSITIES Vol.27

were as follows: in the positive ion mode from m/z 100 to m/z
1000, drying gas, N
2
, flow rate, 9 L/min; gas temperature,
350 C; nebulizer, 2.7610
5
Pa; HV voltage, 3.5 kV.
2.5 HPLC-DAD Analysis
Analysis was performed on an Agilent series 1100 HPLC
system(Agilent Co., USA) consisting of a quaternary pump, a
column oven, an auto-sampler and diode array detector. The
mobile phase consisted of A(water, containing 0.3% formic
acid+0.3% triethylamine) and B(acetonitrile) in a gradient elu-
tion. The gradient program, column temperature and flow rate
were the same as those of the HPLC-ESI-MS/MS analysis. The
DAD recorded UV spectra in a range from 190 nm to 400 nm,
the investigated compounds were determined at a wavelength
of 265 nm. The sample injection volume was 8 L. Data acqui-
sition was performed by a ChemStation workstation.
2.6 Statistical Analysis
The peak area for each peak detected was put into the
SIMCA-P software Version 11.0(Umetrics, Umea, Sweden) for
multivariate analysis. The data set was unite varicance(UV)-
scaled in a column wise manner.
3 Results and Discussion
3.1 Method Development
Four different solvent systems: 50%(volume fraction)
methanol, 70% methanol, methanol and acidified methanol(1%
HCl)
[21]
were selected for extracting alkaloid components of
CP samples in order to obtain optimal extraction medium. The
ultrasonic extraction was selected, and the extraction time(20,
40 and 60 min) and suitable sample concentrations(0.02, 0.01,
0.004 g/mL) were investigated. The sum of peak area of five
reference standards was selected as criterion. The results sug-
gest that the acidified methanol is the best extracting solvent,
and the target compounds could be sufficiently extracted via
ultrasonic for 40 min.
The chromatographic conditions were optimized in order
to obtain chromatograms with a good resolution of the targeted
analyte peaks. Different mobile phases were investigated ac-
cording to the reported literature
[25]
. In the HPLC-DAD analy-
sis, the optimal mobile phase, consisting of water(containing
0.3% formic acid+0.3% triethylamine) and acetonitrile, was
finally employed. During the HPLC-MS/MS analysis, triethy-
lamine was not used due to its bad compatibility with the
HPLC-MS/MS ionisation conditions. The DAD was employed
in a wavelength range of 190400 nm to identify the target
compounds. It was found that 265 nm was the best wavelength
for the detection because almost all the investigated consti-
tuents have the maximum absorption at 265 nm.
3.2 Method Validation
The standard solutions respectively containing jatrorrhi-
zine hydrochloride, palmatine hydrochloride, berberine hy-
drochloride, magnoflorine hydrochloride or phellodendrine
hydrochloride were prepared and diluted to appropriate con-
centrations for the construction of calibration curves. Six con-
centrations of each analyte were injected in triplicate, and then
the calibration curves were constructed by plotting the peak
area versus the concentration of each analyte. All the calibra-
tion curves showed good linear regression(r
2
>0.9995) within
tested concentration ranges(Table 2). The limit of detection
(LOD) and limit of quantitation(LOQ) were measured on the
basis of the signal-to-noise(S/N) ratio of 3 and 10, respectively.
The values of LOD and LOQ for each compound are listed in
Table 2.
Table 2 Statistical results of linear regression equation analysis in the determination of alkaloids
*
Analyte Regression equation Linear range/(gmL
1
) r
2
LOD/ng LOQ/ng
Berberine hydrochloride y=3642.1x135.7 9.2612.0 0.9999 1.8 7.1
Palmatine hydrochloride y=2780.1x+16.5 3.1201.0 0.9997 1.5 6.7
Jatrorrhizine hydrochloride y=3028.1x+19.8 2.3152.0 0.9995 1.7 6.3
Magnoflorine hydrochloride y=1465.1x+3.6 1.2148.0 0.9995 2.5 9.2
Phellodendrine hydrochloride y=386.4x+0.5 2.6104.0 0.9997 1.3 7.3
* In the regression equation y=ax+b, y refers to the peak area(A), x the concentration of the reference standard substance(g/mL), r
2
the correlation coeffi-
cient of the equation, LOD the limit of detection(S/N=3) and LOQ the limit of quantitation(S/N=10).
The mixture standard solution was analyzed under the op-
timal conditions six times in 1 d for intra-day variation and in 3
successive days for inter-day variation to evaluate the precision.
The stability tests were performed by analyzing the same sam-
ple during periods of 0, 2, 4, 8, 16 and 24 h. Repeatability was
determined by analyzing the six different solutions made from
the same analyte and using the same method. The relative
standard deviation(RSD) was taken as a measure. Table 3 lists
the validation results of precision, repeatability and stability
tests.
Table 3 Precision, repeatability and stability data of five compounds
Compound
Precision Repeatability Stability
Intra-day(n=6) Inter-day(n=6) (n=6) (n=6)
Mean/(mgg
1
) RSD(%) Mean/(mgg
1
) RSD(%) Mean/(mgg
1
) RSD(%) RSD(%)
Berberine hydrochloride 19.06 1.78 19.22 1.86 19.08 2.16 1.56
Palmatine hydrochloride 0.41 2.03 0.42 2.48 0.42 3.05 1.83
Jatrorrhizine hydrochloride 0.21 1.97 0.22 1.86 0.21 3.72 1.59
Magnoflorine hydrochloride 5.23 2.25 5.18 2.57 5.14 2.88 1.47
Phellodendrine hydrochloride 1.98 2.85 2.04 2.07 1.92 1.98 1.77

No.1 ZHU Shuang-lai et al. 41


The recovery test was carried as follows: three different
quantities(about 0.8, 1.0 and 1.2 times the concentration of the
matrix) of authentic standards were added into the sample(S
8
)
which had been analyzed previously. The resultant sample
was extracted and analyzed as described above. The quantity of
each analyte was subsequently obtained from the corresponding
calibration curve. The recovery of the five standards ranged
from 95.1% to 104.8%, and most RSDs were less than 4.31%
(n=3). These results demonstrate that the method manifestes a
good accuracy.
3.3 Qualitative Analysis of Alkaloids in CP by
HPLC-ESI-MS/MS
The MS spectra of alkaloids from CP were acquired in
positive ion mode. The base peak chromatogram(BPC) of CP
in positive mode is shown in Fig.2. Totally 22 peaks(peak
No.122) appear in the HPLC-MS base peak chromatograms
of both CPC and CPA.












Fig.2 HPLC-MS base peak chromatograms(BPC) of Cortex Phellodendri in positive mode
(A) CPC; (B) CPA.
Table 4 lists their retention time(t
R
), MS and MS
n
frag-
mentation ions. The five authentic standards exhibited their
quasi-molecular ions [M]
+
. Fragment ions obtained, e.g., by the
loss of CH
3
[M15]
+
, C
2
H
7
N[M45]
+
, C
3
H
9
N[M59]
+
, CH
4
,
Table 4 Compounds detected in Cortex Phellodendri
by HPLC-MS
n

Peak
No.
t
R
/min MS, m/z MS
n
, m/z Identification
1 5.1 180.1[M]
+
121.1 Candicine
2 15.8 314.2[M]
+
298.1, 283.1, 268.1 Not identified
3 18.1 342.1[M]
+
192.1,177.1 Phellodendrine
4 19.2 344.1[M]
+
299.7, 267.1, 235.1 Not identified
5 20.2 342.2[M]
+
297.1, 265.1, 237.1 Magnoflorine
6 22.6 358.2[M]
+
313.2, 281.1, 189.2 Not identified
7 24.5 606.3[M]
+
286.1, 255.1, 178.1 Non-alkaloids
8 25.0 356.1[M]
+
311.1, 279.1, 265.1 Menisperine
9 26.1 328.1[M]
+
283.1, 225 Tetrahydroreticuline
10 28.5 356.2[M]
+
192.1, 177.1 Tetrahydropalmatine
11 28.9 324.1[M]
+
309.1, 280.1 Demethyleneberberine
12 29.4 352.1[M]
+
337.1, 308.1 Oxyberberine
13 31.3 383.1[M+H]
+
787.2, 177.1, 193.1 Non-alkaloids
14 32.1 338.2[M]
+
323.1, 294.1 Columbamine
15 32.5 338.2[M]
+
323.1, 294.1 Jatrorrhizine
16 34.1 322.1[M]
+
307.1, 279.1, 245.1 Berberrubine
17 34.9 352.1[M]
+
337.1, 308.1 Palmatine
18 35.3 336.2[M]
+
321.1, 292.2 Berberine
19 45.1 470.1[M+H]
+
493.2, 425.2, 409.2 Obaculactone(non-
alkaloids)
20 45.9 330.1[M+H]
+
284.2, 256.2, 238.2 Non-alkaloids
21 48.9 455.2[M+H]
+
477.2, 427.1, 409.9 Obacunone(non-
alkaloids)
22 52.8 511.1[M+H]
+
511.1, 421.2, 380.1 Non-alkaloids
CO, etc., could also be observed in the MS
n
spectra. Among the
five alkaloids, phellodendrine showed typical characteristic
Retro-Diels-Alder(RDA) cleavage fragmentation patterns as
previously reported
[26]
.
On the basis of the MS and the comparison of the chro-
matographic retention time with those of authentic standards,
peaks 3, 5, 15, 17 and 18 are unequivocally identified as phel-
lodendrine, magnoflorine, jatrorrhizine, palmatine and berbe-
rine, respectively, and the proposed mechanistic pathways for
the five alkaloids are shown in Scheme 1. Other components
were tentatively identified through the examination of the
fragmentation pathways of known alkaloids, published litera-
tures
[19,26,27]
, the mass spectra and product ion data.
3.4 Quantitative Analysis of Alkaloids in CP by
HPLC-DAD
Seven batches of CPA samples and fifteen batches of CPC
samples were investigated by the proposed HPLC-DAD
method, two typical chromatograms of CPA and CPC are
shown in Fig.3, and 12 common peaks in the whole chromato-
gram were aligned according to a typical spectrum and the
professional software Similarity Evaluation System for Chro-
matographic Fingerprint of Traditional Chinese Medicine
(Version 2004A).
Contents of the phellodendrine, magnoflorine, jatrorrhi-
zine, palmatine and berberine in each sample are listed in Table
5, and the data are expressed as meandeviation. As deduced
from the data, the content of each constituent differs greatly
among various batches of CP samples. Berberine is the primary
component in all the samples. The average contents of
42 CHEM. RES. CHINESE UNIVERSITIES Vol.27




























Scheme 1 Proposed mechanistic pathway for reference standards in Cortex Phellodendri
(A) Fragmentations of phellodendrine; (B) fragmentations of magnoflorine; (C) fragmentations of jatrorrhizine, palmatine and berberine.







Fig.3 HPLC-UV chromatography of CP
(A) CPC; (B) CPA. Twelve common peaks were denoted, respectively. Peaks: 1. phellodendrine; 2. magnoflorine;
8. jatrorrhizine; 9. palmatine; 10. berberine.
berberine and total alkaloids(amounts of five alkaloids) in CPC
(58.75 and 64.36 mg/g, respectively) are higher than those in
CPA(9.16 and 24.35 mg/g, respectively), while palmatine in
CPC(0.25 mg/g) is less than that in CPA(4.19 mg/g). The above
results reveal that berberine, palmatine and total alkaloids could
be used for the distinguishing of CPC and CPA. Since the alka-
loids are bioactive ingredients of CP, the further researches of
CP might be carried out to reveal the relationship between the
amount of alkaloids and pharmacological effect.
Contents of the phellodendrine, magnoflorine, jatrorrhi-
zine, palmatine and berberine in each sample are listed in Table
5, and the data are expressed as meandeviation. As deduced
from the data, the content of each constituent differs greatly
among various batches of CP samples. Berberine is the primary
component in all the samples. The average contents of berbe-
rine and total alkaloids(amounts of five alkaloids) in CPC
(58.75 and 64.36 mg/g, respectively) are higher than those in
CPA(9.16 and 24.35 mg/g, respectively), while palmatine in
CPC(0.25 mg/g) is less than that in CPA(4.19 mg/g). The above
results reveal that berberine, palmatine and total alkaloids could
be used for the distinguishing of CPC and CPA. Since the alka-
loids are bioactive ingredients of CP, the further researches of
CP might be carried out to reveal the relationship between the
amount of alkaloids and pharmacological effect.
No.1 ZHU Shuang-lai et al. 43


Table 5 Contents(mg/g) of five compounds in 22 samples(n=3)
Sample Species Phellodendrine Magnoflorine Jatrorrhizine Palmatine Berberine Total content of five alkaloids
1 CPA 1.300.16 6.190.43 0.490.08 5.090.16 8.820.42 21.90
2 CPA 3.180.46 2.540.31 0.700.05 1.780.12 5.150.51 14.40
3 CPA 1.690.25 8.190.56 0.570.08 5.350.21 9.050.74 24.90
4 CPA 0.840.14 3.580.33 0.250.04 1.980.16 5.330.35 12.00
5 CPA 1.810.21 8.710.71 0.580.04 5.180.28 10.220.80 26.50
6 CPA 1.850.19 6.970.53 0.670.03 5.150.34 12.860.82 27.50
7 CPA 1.660.23 9.470.62 0.600.01 4.830.25 12.701.07 29.30
8 CPC 1.980.21 5.230.42 0.210.03 0.430.02 19.061.50 26.90
9 CPC 2.320.16 1.920.31 0.480.09 0.350.01 40.501.80 45.60
10 CPC 2.490.29 1.730.25 0.390.06 0.400.03 53.733.10 58.70
11 CPC 2.690.30 2.300.16 0.550.02 0.330.04 70.924.60 76.80
12 CPC 1.730.16 3.210.27 0.430.03 0.190.03 66.602.80 72.20
13 CPC 2.030.22 1.660.19 0.390.05 0.130.04 67.103.70 71.30
14 CPC 1.920.18 2.540.21 0.360.03 0.160.01 62.722.60 67.70
15 CPC 1.710.16 3.200.34 0.410.05 0.210.01 68.924.30 74.40
16 CPC 1.930.20 2.380.29 0.450.03 0.260.02 59.362.10 64.40
17 CPC 2.160.23 1.810.34 0.310.02 0.220.01 49.893.00 54.40
18 CPC 1.760.18 2.240.18 0.390.01 0.140.01 66.952.50 71.50
19 CPC 1.530.17 6.680.68 0.500.01 0.310.02 67.852.60 76.90
20 CPC 2.100.16 2.150.36 0.600.02 0.200.01 74.253.50 79.30
21 CPC 2.560.51 3.040.26 0.500.03 0.270.01 63.543.40 69.90
22 CPC 2.020.26 2.890.33 0.460.01 0.180.01 49.852.90 55.40

3.5 Principal Component Analysis(PCA) of CP
The chemical profiles and quantitative analysis results
show that the PCA contents of PC in the samples from the dif-
ferent origins are dissimilar. To further confirm and explore the
components which are responsible to the classification of the
two species, principal component analysis(PCA), one of most
powerful tools in explorative data analysis, was utilized in this
study.
PCA is a statistical approach to facilitate an understanding
into the causes and effects indicated by the relationship of
multivariate dataset, the core of this method is to generate new
principal components(PCs) which are independent of the
original variables but shows linear combinations of them, each
PC consists of a set of values called score defining the posi-
tion of each sample in the new coordinate space, and the other
set of values called loading that gives the relative contribu-
tions of each variable in calculating the scores
[22,28]
. Peak
area-retention time pairs of 12 common compounds in 22 sam-
ples were inputted into the software by this method. After ap-
plying PCA, score and loading plots were generated, as shown
in Fig.4. The score plot shows the samples of CP are clearly
divided into different clusters corresponding to two species
[Fig.4(A)]; the loading plot demonstrates that four components
including berberine, phellodendrine, palmatine and magnoflo-
rine might contribute most to separating CPC from CPA
[Fig.4(B)].






Fig.4 Score plot(A) and loading plot(B) from PCA model
(A) Each point representing a particular sample, the 22 batches of samples were clearly divided into two groups; (B) berberine(peak 10),
palmatine(peak 9), phellodendrine(peak 1) and magnoflorine(peak 2).

4 Conclusions
A combined method using HPLC-ESI-MS/MS, HPLC-
DAD and PCA was performed for the qualitative and quantita-
tive analysis of alkaloids in Cortex Phellodendri, and for the
discrimination of Cortex Phellodendri Chinensis(CPC) and
Cortex Phellodendri Amurensis(CPA). The results suggest that
above method could be considered as routine screening in the
quality control of Chinese herbal medicines.

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