Journal of Chromatography A, 1216 (2009) 2063–2070

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Qualitative evaluation and quantitative determination of 10 major active

components in Carthamus tinctorius L. by high-performance liquid
chromatography coupled with diode array detector
Li Fan, Hai-Yu Zhao, Man Xu, Lei Zhou, Hui Guo, Jian Han, Bao-Rong Wang, De-An Guo ∗
The State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center,
No. 38 Xueyuan Road, Beijing 10083, China

a r t i c l e i n f o a b s t r a c t

Article history: Flavonoids in the water extract of Carthamus tinctorius L. exhibit potent biological activities such
Available online 21 March 2008 as anti-coagulant, vasodilation, anti-oxidant, neuroprotection and immunosuppressant. A high-
performance liquid chromatographic method was established to evaluate the quality of Carthamus
Keywords: tinctorius through a simultaneous quantitation of eight flavonoids, hydroxysafflor yellow A (2),
HPLC-DAD 6-hydroxykaempferol 3,6-di-O-ˇ-glucoside-7-O-ˇ-glucuronide (3), 6-hydroxykaempferol 3,6,7-tri-O-
Carthamus tinctorius L.
ˇ-glucoside (4), 6-hydroxykaempferol 3-O-ˇ-rutinoside-6-O-ˇ-glucoside (6), 6-hydroxykaempferol
Determination
3,6-di-O-ˇ-glucoside (7), 6-hydroxyapigenin 6-O-glucoside-7-O-glucuronide (8), anhydrosafflor yellow
Quality control
6-Hydroxyapigenin
B (9), and kaempferol 3-O-ˇ-rutinoside (10), together with two other compounds named guanosine (1)
6-O-glucoside-7-O-glucuronide and syringin (5). Among them, compound 8 was identified as a new compound. The compounds were
separated on an Alltech Alltima-C18 column with gradient elution of acetonitrile and 0.01% trifluoroacetic
acid. The detection wavelength was 280 nm. All the compounds showed good linearity (r2 ≥ 0.9989). The
recoveries, measured at three concentration levels, varied from 94.9% to 105.2%. This method was also
validated with respect to precision, repeatability and accuracy, and was successfully applied to quan-
tify the 10 components in 46 batches of C. tinctorius samples from different areas. Significant variations
were found in the contents of these compounds in these samples. Compared with the reported analytical
methods of C. tinctorius, this simple and reliable method provided a new basis for overall assessment on
quality of C. tinctorius and should be considered as a suitable quality control method.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction melanin production [15], immunosuppressant [16], anti-tumor [17]

Carthamus L. is a genus belonging to the Compositae family.
Carthamus tinctorius L. is the only species of this genus in China,
which has been used as a food additive, a natural pigment and a
famous traditional Chinese medicine (TCM) having the function of
promoting blood circulation by removing blood stasis. Saffron is
used for the same purpose, but it is more expensive than C. tincto-
rius. Therefore, it is commonly used for the replacement of Saffron
in traditional medicine.
The florets of Carthamus tinctorius L. are popularly used for
the treatment of cardiovascular, cerebrovascular and gynecologi-
cal diseases. Modern pharmacological studies demonstrated that C.
tinctorius extracts had a number of effects, such as anti-coagulant
[1–4], vasodilation [2–4], anti-hypertension [5,6], anti-oxidant
[7–12], neuroprotection [13], hepatic protection [14], inhibiting the
∗ Corresponding author. Tel.: +86 10 82802024; fax: +86 10 82802700.
E-mail address: gda@bjmu.edu.cn (D.-A. Guo).
and treating dysmenorrhea [18]. Most of the biological activities
are produced by its water extract. In China, the water extract of
C. tinctorius has been developed as an intravenous injection, and
has been extensively applied in hospitals to treat cardiovascular
diseases. More than 50 Chinese patent drugs containing C. tincto-
rius were listed in Chinese Pharmacopoeia. Many of them such as
Zhenghonghua oil, Dieda pill and Qili powder [19], are very popular
in oriental medicines.
More than 200 compounds were isolated from C. tinctorius
including flavonoids, phenylethanoid glycosides, coumarins, fatty
acids, steriods and safflower polysaccharides [20–27]. Flavonoids
are considered as the active components of many medicinal herbs
with health-related properties. Chalcone flavonoids are the main
compounds in the water extract of C. tinctorius such as hydroxysaf-
flor yellow A, which is responsible for the main curative effects of
safflower. It was also reported that 6-hydroxykaempferol glycosides
also showed anti-platelet aggregative effects [28]. Consequently,
quantification of these active compounds in C. tinctorius would be
of great significance for quality evaluation of this herb. In previous

0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2008.03.046
2064 L. Fan et al. / J. Chromatogr. A 1216 (2009) 2063–2070

Fig. 1. The structures of 10 major components in C. tinctorius.

reports, bioactive compounds in C. tinctorius have been analyzed by
TLC [19], HPLC [29–34] and CE [38]. Unfortunately, these methods
only determined single or few bioactive components as chemical
markers to evaluate the quality of crude drugs. HPLC fingerprint-
ing methods have also been established to control the quality of
safflower [34–37], but little information of the major bioactive com-
ponents was mentioned except hydroxysafflor yellow A. Therefore,
those methods were not suitable for comprehensive quality eval-
uation of C. tinctorius. In this paper, a simple and accurate HPLC
method for the simultaneous quantification of major components
in the water extract of C. tinctorius was successfully established for
the quality control of this important TCM. Up to now, this is the
first report on the simultaneous identification and determination
of multi-components in C. tinctorius.
2. Experiment
2.1. Reagents and materials
Acetonitrile is of HPLC grade (Fisher Scientific, Fairlawn, NJ).
Trifluoroacetic acid (TFA) is of reagent grade from Nankai Univer-
sity’s chemical plant. The deionized water was prepared by using
Millipore purification system (Millipore, Milford, MA, USA) and
filtered with 0.45 ␮m membranes. Sephadex LH-20 (Amersham
 L. Fan et al. / J. Chromatogr. A 1216 (2009) 2063–2070 2065

Fig. 2. Extraction efficiency of different extraction temperature: (1) guanosine;
(2) hydroxysafflor yellow A; (3) 6-hydroxykaempferol 3,6-di-O-ˇ-d-glucoside-
7-O-ˇ-d-glucuronide; (4) 6-hydroxykaempferol 3,6,7-tri-O-ˇ-d-glucoside; (5)
syringin; (6) 6-hydroxykaempferol 3-O-ˇ-rutinoside-6-O-ˇ-d-glucoside; (7) 6-
hydroxykaempferol 3,6-di-O-ˇ-d-glucoside; (8) 6-hydroxyapigenin 6-O-glucoside-
7-O-glucuronide; (9) anhydrosafflor yellow B; (10) kaempferol 3-O-ˇ-rutinoside.
(The right Y-axis is correlated with point 2 while the left Y-axis is correlated with
the other points.)
Fig. 3. Extraction efficiency of different extraction time: (1) guanosine; (2) hydrox-
ysafflor yellow A; (3) 6-hydroxykaempferol 3,6-di-O-ˇ-d-glucoside-7-O-ˇ-d-
glucuronide; (4) 6-hydroxykaempferol 3,6,7-tri-O-ˇ-d-glucoside; (5) syringin; (6) 6-
hydroxykaempferol 3-O-ˇ-rutinoside-6-O-ˇ-d-glucoside; (7) 6-hydroxykaempferol
3,6-di-O-ˇ-d-glucoside; (8) 6-hydroxyapigenin 6-O-glucoside-7-O-glucuronide; (9)
anhydrosafflor yellow B; (10) kaempferol 3-O-ˇ-rutinoside. (The right Y-axis is cor-
related with point 2 while the left Y-axis is correlated with the other points.)

Biosciences) and C18 reversed-phase (RP) silica gel (50 ␮m, Merck)
were used in the separation process of the crude drug.
The crude drug samples were purchased from local phar-
macies or drug markets in different areas in China. The 10
standard compounds were isolated by the author from the florets
of C. tinctorius L. They were guanosine (1), hydroxysafflor yel-
low A (2), 6-hydroxykaempferol 3,6-di-O-ˇ-d-glucoside-7-O-ˇ-d-
glucuronide (3), 6-hydroxykaempferol-3,6,7-tri-O-ˇ-d-glucoside
(4), syringin (5), 6-hydroxykaempferol 3-O-ˇ-rutinoside-6-O-
ˇ-d-glucoside (6), 6-hydroxykaempferol 3,6-di-O-ˇ-d-glucoside
(7), 6-hydroxyapigenin 6-O-glucoside-7-O-glucuronide (8), anhy-
drosafflor yellow B (9) and kaempferol 3-O-ˇ-rutinoside (10).
Structures of these compounds were identified by 1 H NMR, 13 C
NMR, HMQC, HMBC, ESI–MS and other spectroscopic methods. On
the basis of UV, NMR, MS and HPLC all reference compounds are
considered to have a purity of 95% or more. Compound 8 is a new
compound, and was named 6-hydroxyapigenin 6-O-glucoside-7-
O-glucuronide. The structures of the 10 compounds are shown in
Fig. 1.
2.2. Apparatus and chromatographic conditions
gradient to A–B (10:90, v/v); 30–60 min, linear gradient to A–B
(20:80, v/v), which was held for 5 min. Each run was followed
by equilibration time of 5 min. The flow rate was 1.0 ml/min. The
column temperature was set at 30 ◦ C. Ultraviolet (UV) spectra
were monitored at 280 nm. The injection volume was 10 ␮l. The
data were collected and analyzed with Chemstation 10.02 soft-
ware.
Preparative HPLC: Spectra-P-100 pump (Thermo Separation
Products), connected to a Spectra-UV-100 detector (Thermo Sep-
aration Products), Zorbax Extend C18 column (150 mm × 20 mm,
10 ␮m), flow rate at 1.8 ml/min and wavelength detection at
280 nm. IR: Nicolet Avatar-FT-IR spectrometer; KBr pellets. UV: TU-
1901-UV–vis spectrophotometer. Optical rotation: AA10R digital
polarimeter; in MeOH at 25 ◦ C. Melting point (m.p.): X-6 melting-
point apparatus (Beijing TECH Instrument Co., Ltd.); uncorrected.
ESI–MSn : Finnigan LCQ-Advantage ion-trap mass spectrometer
(Thermo Finnigan, San Jose, CA, USA). HR-ESI–MS: Bruker APEX
IV FT-MS. NMR spectrometer: Bruker ARX-400 and DRX-500
spectrometers; in d6 -DMSO. CD Spectra: JASCO J-810 spectropo-
larimeter.

An Agilent 1100 HPLC system equipped with a quaternary sol-
vent delivery system, an autosampler and a DAD detector was
used. Separation was achieved on a Alltech Alltima-C18 column
(250 mm × 4.6 mm, 5 ␮m) coupled with a Alltech Alltima-C18
guard column (12.5 mm × 4.6 mm, 5 ␮m). The mobile phase con-
sisted of acetonitrile (A) and 0.05% aqueous TFA (B), which were
applied in the gradient elution as follows: 0–20 min, linear gra-
dient from A–B (2:98, v/v) to A–B (5:95, v/v); 20–30 min, linear
2.3. Preparation of standard solutions and calibration curve
A 20% acetonitrile stock solution containing all the reference
standards was prepared and diluted to appropriate concentra-
tion range to establish calibration curves. Each calibration curve
was analyzed in triplicate. All calibration curves were con-
structed from peak areas of the reference standards versus their
concentrations.

Table 1
Calibration curves of 10 components

Marker compound Regression equation r2 Linear range (␮g/ml) LOQ (␮g/ml) LOD (␮g/ml)

1 y = 941.2x − 6.89 0.9995 5–80 0.30 0.10

2 y = 289.1x − 10.20 0.9999 80–960 2.00 0.40
3 y = 816.3x + 14.29 0.9992 8–300 0.38 0.10
4 y = 612.1x − 8.66 0.9989 10–120 0.50 0.13
5 y = 1241.5x − 4.92 0.9996 4–48 0.27 0.07
6 y = 535.0x − 6.46 0.9998 10–160 0.67 0.20
7 y = 1030.9x − 7.06 0.9998 6–96 0.40 0.12
8 y = 628.1x − 1.95 0.9990 5–100 0.50 0.20
9 y = 350.5x − 19.81 0.9998 31–628 1.05 0.31
10 y = 574.8x − 10.11 0.9990 4–80 0.40 0.16

y, peak area; x, concentration of compound (␮g/ml); limit of detection (LOD), S/N = 3; limit of quantification (LOQ), S/N = 10.
2066 L. Fan et al. / J. Chromatogr. A 1216 (2009) 2063–2070

Table 2
Intra- and inter-day variability for the assay of 10 components

Compound Concentration (␮g/ml) Intra-day (n = 6) Inter-day (n = 3)

a b
Found RSD (%) Accuracy (%) Found RSD (%) Accuracy (%)

1 10.0 10.4 ± 0.1 0.57 103.7 10.5 ± 0.2 1.62 105.3

40.0 39.4 ± 0.1 0.36 98.5 40.3 ± 0.8 2.02 100.8
60.0 59.8 ± 0.4 0.70 99.7 60.3 ± 0.4 0.63 100.4

2 160.0 162.0 ± 2.0 1.23 101.3 164.6 ± 2.7 1.61 102.9

640.0 632.3 ± 1.0 0.16 98.8 647.7 ± 14.5 2.23 101.2
960.0 964.6 ± 1.5 0.16 100.5 978.2 ± 21.6 2.21 101.9

3 14.3 13. 7 ± 0.5 3.79 95.6 14.4 ± 0.7 4.65 100.9

61.0 61.6 ± 0.2 0.36 101.0 64.2 ± 2.4 3.74 105.3
95.6 99.1 ± 0.2 0.22 103.7 100.6 ± 1.3 1.31 105.3

4 20.0 20.6 ± 0.1 0.69 102.9 20.8 ± 0.2 0.93 103.9

80.0 78.3 ± 0.2 0.23 97.9 79.6 ± 1.3 1.65 99.5
120.0 122.3 ± 0.1 0.12 101.9 126.5 ± 3.7 2.93 105.4

5 8.0 8.1 ± 0.1 1.14 101.3 8.4 ± 0.3 3.02 104.7

32.0 32.0 ± 0.3 0.81 100.0 33.2 ± 1.2 3.63 103.9
48.0 47.8 ± 0.1 0.28 99.6 48.8 ± 0.9 1.75 101.6

6 20.0 20.4 ± 0.0 0.21 101.9 20.7 ± 0.4 1.68 103.6

80.0 79.1 ± 0.2 0.23 98.9 80.7 ± 1.6 2.00 100.9
120.0 119.7 ± 0.6 0.49 99.8 122.1 ± 3.0 2.43 101.8

7 12.0 12.3 ± 0.0 0.26 102.3 12.6 ± 0.3 2.55 105.2

48.0 47.5 ± 0.2 0.35 99.0 49.2 ± 1.5 3.12 102.6
72.0 71.9 ± 0.1 0.17 99.8 74.1 ± 1.9 2.60 102.9

8 10.0 9.8 ± 0.0 0.32 98.1 9.9 ± 0.1 1.29 99.1

40.0 39.4 ± 0.3 0.63 98.4 40.49 ± 1.1 2.71 101.2
60.0 61.2 ± 0.6 0.93 101.9 62.4 ± 1.8 2.83 104.0

9 62.8 65.0 ± 0.1 0.10 103.5 65.2 ± 0.3 0.42 103.9

251.2 246.4 ± 0. 7 0.27 98.1 247.6 ± 2.3 0.93 98.6
376.8 374.5 ± 0.4 0.10 99.4 375.2 ± 2.6 0.70 99.6

10 8.2 8.6 ± 0.0 0.28 104.7 8.3 ± 0.3 3.13 102.0

30.3 29.37 ± 0.10 0.35 96.9 28.7 ± 0.6 2.17 94.8
45.3 44.64 ± 0.58 1.30 98.5 43.2 ± 1.4 3.25 95.4
a
RSD (%) = (SD/mean) × 100.
b
Accuracy (%) = (mean of measured concentration/spiked concentration) × 100.

2.4. Preparation of sample solutions
The dried florets of C. tinctorius were comminuted (0.28 mm).
Each sample (0.2 g) was accurately weighed and extracted by reflux-
ing at 60 ◦ C with 6 ml water for 45 min. Then the extraction solution
was adjusted to the original weight, filtered through 0.45 ␮m mem-
branes and injected into HPLC system. In a second filtrate obtained
by flushing the filter with an additional volume of solvent, no sig-
nificant quantities of analyte could be detected. Thus the recovery
of the filtration step is 100% or near 100%.
2.5. Method validation
The method was validated for linearity, precision (inter-day,
intra-day precision and intermediate precision), accuracy, stability,
specificity and selectivity following the International Conference
on Harmonization (ICH) guideline [39] and some reports on deter-
mination analysis [40–45].
3. Results and discussion
3.1. Identification of compound 8
6-Hydroxyapigenin 6-O-glucoside-7-O-glucuronide (com-
pound 8): yellow amorphous powder; C27 H28 O17 ; m.p. 254–256 ◦ C;
[˛]25 ◦
D −96 (c 1.200, MeOH); UV max (MeOH) (nm): 215, 275,
333; IR max (KBr) (cm−1 ): 3404, 2924, 1657, 1607, 1455, 1360,
1285, 1249, 1072, 836, 591; ESI–MSn (negative ion) m/z: 623
[M − H]− , 447 [M-GluA-H]− , 285 [M-GluA-Glc-H]− ; HR-ESI–MS
m/z: 625.1399 [M + H]+ , calculated for C27 H28 O17 , found 625.1407;
CD spectrum of the compound showed negative Cotton effect at
213 nm (ε = −3.49) and 282 nm (ε = −1.04); 1 H NMR (500 MHz,
DMSO-d6 ) ı: 6.85 (1H, s, 3-H), 7.03 (1H, s, 8-H), 7.94 (2H, d,
J = 8.5 Hz, 2 - and 6 -H), 6.94 (2H, d, J = 8.5 Hz, 3 - and 5 -H), 10.46
(1H, s, 4 -OH), 13.08 (1H, s, 5-OH); 6-O-Glc, 4.88(1H, d, J = 7.0 Hz,
HGlc -1 ); 7-O-GluA, 5.23(1H, d, J = 7.5 Hz, HGlu -1 ), 3.98(1H, d,
J = 9.5 Hz, HGlu -5 ); 13 C NMR (125 MHz, DMSO-d6 ) ı: 164.3 (C-2),
102.7 (C-3), 182.2 (C-4), 152.7 (C-5), 129.2 (C-6), 155.7 (C-7), 94.2
(C-8), 152.3 (C-9), 105.9 (C-10), 121.0 (C-1 ), 128.5 (C-2 ), 116.0
(C-3 ), 161.3 (C-4 ), 116.0 (C-5 ), 128.5 (C-6 ); 6-O-Glc, 103.3 (C-1 ),
74.1 (C-2 ), 76.3 (C-3 ), 69.7 (C-4 ), 77.0 (C-5 ), 60.7 (C-6 ); 7-O-GluA,
100.2 (C-1 ), 73.0 (C-2 ), 75.3 (C-3 ), 71.3 (C-4 ), 75.3 (C-5 ),
170.1 (C-6 ). The 1 H NMR and 13 C NMR data were compared with
6-hydroxyapigenin 6,7-di-O-ˇ-d-glucoside previously reported
[21]. In the HMBC spectrum, cross-peak between ı 5.23 (HGluA -1 )
and ı 155.7 (C-7), ı 3.98 (HGluA -5 ) and ı 170.1 (C-6 ) disclosed the
possible linking site of 7-O-GluA. However, the relative bonding
site of 6-O-Glc was not observed in the HMBC. Consequently,
TOCSY was carried out to prove their relationship. In the TOCSY
spectrum, the cross-peak between ı 5.23 (HGluA -1 ) and 3.98
(HGluA -5 ) confirmed that glucuronide was attached to aromatic
ring A at C-7 position and glucoside should be linked to aromatic
ring A at C-6 position. From the above deduction, compound 8 was
identified as 6-hydroxyapigenin 6-O-glucoside-7-O-glucuronide.
 L. Fan et al. / J. Chromatogr. A 1216 (2009) 2063–2070 2067

Table 3
Repeatability, stability and intermediate precision of 10 components in C. tinctorius

Compound Repeatability (n = 6) Stability (18 h, n = 3) Intermediate precision (n = 3)

a b
Content (mg/g) RSD (%) Content (mg/g) RSD (%) Content (mg/g) RSD (%)

1 0.97 ± 0.01 1.05 0.99 ± 0.00 0.09 0.97 ± 0.01 1.21

2 16.85 ± 0.30 1.77 16.80 ± 0.30 0.50 16.88 ± 0.21 1.24
3 1.72 ± 0.02 0.95 1.72 ± 0.03 1.51 1.71 ± 0.03 1.95
4 1.79 ± 0.04 2.22 1.79 ± 0.05 2.55 1.80 ± 0.05 2.73
5 0.57 ± 0.02 3.37 0.56 ± 0.01 1.90 0.57 ± 0.03 4.65
6 1.81 ± 0.04 2.08 1.81 ± 0.02 0.97 1.82 ± 0.03 1.72
7 1.46 ± 0.04 3.04 1.47 ± 0.03 1.92 1.45 ± 0.07 4.79
8 1.11 ± 0.04 3.19 1.10 ± 0.02 1.92 1.10 ± 0.03 2.60
9 7.86 ± 0.12 1.52 7.89 ± 0.07 0.90 7.87 ± 0.08 0.93
10 1.50 ± 0.02 1.43 1.48 ± 0.02 1.34 1.51 ± 0.03 1.72
a
Content = mean ± SD (n = 3).
b
RSD (%) = (SD/mean) × 100.

3.2. Optimization of chromatographic conditions
According to the maximum absorption of all the marker com-
pounds on the UV spectra with three-dimension chromatograms
of DAD detection, 280 nm was selected as detection wavelength,
where all the marker compounds could be detected and had ade-
quate absorption. Different types of chromatographic columns
were tested to optimize the separation, such as Intersil ODS-3
column, Waters Spnerisorb ODS2 column, Zobax Extend-C18 col-
umn, BDS Hypersil-C18 column, Phenomenes Luna-C18 column,
Alltech Alltima-C18 column and several other types of Alltech
columns. Finally, Alltech Alltima-C18 column (250 mm × 4.6 mm,
5 ␮m) was proved to be the best in this application. Different
ratios of water and acetonitrile were compared, but no satisfac-
tory separation was obtained. According to the literatures [31–37],
formic acid, acetic acid, phosphoric acid and TFA were added to
the mobile phase to enhance the resolution and eliminate the
peak tailing of the target compounds. As a result, acetonitrile and
water containing 0.01% TFA were chosen as the eluting solvent
and a gradient elution programs was performed to ensure that
each compound could be well separated. Besides, it was found
that the separation was better when the column temperature
was kept at 30 than 20, 25 or 35 ◦ C. The flow rate was set at
1.0 ml/min to maintain the satisfactory separation and reasonable
analytical time. Fig. 4 showed the typical separation of a stan-
dard mixture (A) and C. tinctorius extracts of different origins
(B, C and D) obtained under the above optimized HPLC condi-
tions.

Fig. 4. Representative HPLC chromatograms of: (A) mixed standard solution; (B) C. tinctorius. (Chongqing, China); (C) C. tinctorius. (Hefei, Anhui, China); (D) C. tinctorius.
(Xinjiang, China). (1) guanosine; (2) hydroxysafflor yellow A; (3) 6-hydroxykaempferol 3,6-di-O-ˇ-d-glucoside-7-O-ˇ-d-glucuronide; (4) 6-hydroxykaempferol 3,6,7-tri-O-
ˇ-d-glucoside; (5) syringin; (6) 6-hydroxykaempferol 3-O-ˇ-rutinoside-6-O-ˇ-d-glucoside; (7) 6-hydroxykaempferol 3,6-di-O-ˇ-d-glucoside; (8) 6-hydroxyapigenin 6-O-
glucoside-7-O-glucuronide; (9) anhydrosafflor yellow B; (10) kaempferol 3-O-ˇ-rutinoside.
2068 L. Fan et al. / J. Chromatogr. A 1216 (2009) 2063–2070

3.3. Sample extraction optimization Table 4

Accuracy of 10 components in C. tinctorius (n = 3)

In order to obtain satisfactory extraction efficiency, variables Compound Spiked amount (␮g/ml) Found (␮g/ml) Recoverya (%) RSDb (%)
involved in the procedure such as solvent, extraction method, 1 10.0 9.8 ± 0.2 98.1 2.00
extraction temperature and extraction time were optimized. Water, 20.0 19.4 ± 0.3 97.1 1.62
50% methanol, 95% ethanol and methanol were tested as extraction 30.0 29.1 ± 0.5 97.1 1.74
solvents. As main components in safflower had good water- 2 160.0 161.2 ± 2.2 100.7 1.34
solubility, the results showed that the 10 components could be 320.0 327.7 ± 3.7 102.4 1.12
adequately extracted by water. Then reflux, ultrasonic extraction, 480.0 491.3 ± 6.7 102.4 1.36
soak at room temperature were investigated as extraction meth- 3 50.0 51.4 ± 0.6 102.8 1.20
ods. The results suggested that reflux was better than ultrasonic 100.0 101.3 ± 1.4 101.3 1.36
extraction and soak, thus reflux was used in further experiments. An 150.0 153.8 ± 1.5 102.6 0.98
aliquot of 0.2 g sample was extracted with 6 ml water by refluxing at 4 20.0 19.8 ± 0.1 98.9 0.69
40, 60, 80 and 100 ◦ C, respectively. As depicted in Fig. 2, the results 40.0 41.1 ± 0.5 102.7 1.22
indicated that the best extraction temperature was 60 ◦ C. The opti- 60.0 61.1 ± 0.7 101.8 1.06

mal extraction time was performed afterwards. The peak areas of 5 8.0 8.1 ± 0.1 100.9 1.39
the marker compounds obtained in 10, 30, 45, 60 and 120 min were 16.0 15.8 ± 0.2 98.8 1.41
24.0 24.0 ± 0.5 100.0 2.05
shown in Fig. 3. The marker compounds were completely extracted
within 45 min. Therefore, the most suitable extraction conditions 6 20.0 20.1 ± 0.3 100.5 1.55
were refluxing at 60 ◦ C by water for 45 min. 40.0 40.2 ± 0.9 100.4 2.26
60.0 60.5 ± 0.8 100.9 1.36

3.4. Calibration curves, the limit of detection and quantification 7 12.0 12.0 ± 0.5 99.9 3.89
24.0 23.7 ± 0.6 98.8 2.59
36.0 35.8 ± 1.2 99.4 3.32
Linear regression analysis for each of the 10 compounds was
performed by external standard method. All the calibration curves 8 10.0 10.1 ± 0.1 101.1 1.14
20.0 20.5 ± 0.4 102.7 2.16
showed good linearity (r2 ≥ 0.9989). The standard solutions were
30.0 30.8 ± 0.4 102.5 1.36
diluted with 20% acetonitrile to provide a series of standard solu-
tions with the appropriate concentrations. The limit of detection 9 62.8 64.7 ± 1.5 103.1 2.32
125.6 129.1 ± 1.9 102.8 1.45
and quantification under the chromatographic conditions were
188.4 192.1 ± 0.9 102.0 0.44
determined by injecting a series of standard solutions until the
signal-to-noise (S/N) ratio for each compound was 3 for LOD and 10 8.0 8.1 ± 0.1 101.1 1.64
16.0 16.2 ± 0.2 101.3 1.22
10 for LOQ. The results are given in Table 1. 24.0 24.3 ± 0.4 101.4 1.77
a
Recovery (%) = (detected amount − original amount)/spiked amount × 100.
3.5. Precision b
RSD (%) = (SD/mean) × 100.

Precision of the method was evaluated by analyzing the stan-

dard solutions containing 10 standard compounds at three different
concentration levels (high, middle and low). The experiment was
repeated six times on the same day and additionally on three
consecutive days to determine intra- and inter-day precision,
respectively. Relative standard deviation (RSD) of peak area for each
of the marker compounds was calculated respectively, and was no
more than 4.65%. The results are given in Table 2.
3.6. Repeatability, stability, intermediate precision, specificity and
selectivity
Six samples from the same origin were extracted and analyzed
with the proposed method. The RSD value was calculated as a
measurement of method repeatability. RSD values of the 10 com-
pounds were less than 3.37%, which showed high repeatability of
the method.
The same sample was stored at 25 ◦ C, and analyzed at 0, 12, 18,
24, and 48 h, respectively. The RSD of peak areas was taken as a
measure of stability. The results in Table 3 showed that the sample
solution was found to be stable within 18 h (RSD <2.55%).
The intermediate precision of the method was evaluated by
applying the proposed method to analyze the same sample using
different instruments by different analysts. With RSD less than
4.79%, the method could be considered reliable. All the results were
shown in Table 3.
Furthermore, specificity of the method was assessed by study
of the resolution factor of the marker compounds peaks from the
nearest resolving peaks. The selectivity was checked by analyzing
peak purity of all the marker compounds. As shown in Fig. 4, the
proposed method had sufficient specificity and selectivity since the
10 components were well separated from each other and all the
peaks were confirmed to be pure through DAD purity studies.
3.7. Accuracy
Accuracy was determined by adding the mixed standard solu-
tions with three different concentration levels (high, middle and
low) to the known amounts of C. tinctorius samples. Then the
resultant samples were extracted and analyzed with the pro-
posed method and triplicate experiments were performed at each
level. The percentage recoveries were calculated according to the
following equation: (detected amount − original amount)/spiked
amount × 100. As shown in Table 4, the developed analytical
method was reproducible with good accuracy in the range of
97.06–103.08% (RSD <3.89%).
3.8. Sample analysis
The newly established analytical method was subsequently
applied to simultaneously determine the 10 compounds in 46 C.
tinctorius commercial samples purchased from different places.
Representative chromatograms of the extracts of C. tinctorius sam-
ples are shown in Fig. 4. All the contents are summarized in Table 5.
The results showed that the total amounts of the 10 components
determined in the samples varied from 4.88 to 37.55 mg/g, with 7-
fold variation. The results showed that 42.8% of the samples with
the total amount above 25 mg/g originated in Xinjiang, the dom-
inant producing area in China. As far as a single component was
concerned, hydroxysafflor yellow A was the major compound, the
contents of which varied from 2.35 to 20.74 mg/g, with 8.8-fold
Table 5
Contents of 10 components in 46 C. tinctorius samples

No. Collection province Contenta (mg/g) (n = 3)

1 2 3 4 5 6 7 8 9 10

1 Sichuan 0.39 ± 0.00 7.43 ± 0.03 0.75 ± 0.00 0.79 ± 0.01 0.21 ± 0.01 0.79 ± 0.01 0.69 ± 0.00 0.53 ± 0.00 2.51 ± 0.01 0.67 ± 0.01
2 Sichuan 0.85 ± 0.00 16.34 ± 0.06 1.33 ± 0.01 1.36 ± 0.01 0.37 ± 0.01 1.17 ± 0.01 0.61 ± 0.00 1.11 ± 0.01 6.84 ± 0.07 1.82 ± 0.01
3 Sichuan 0.78 ± 0.01 13.33 ± 0.04 1.12 ± 0.01 1.23 ± 0.02 0.35 ± 0.00 1.18 ± 0.01 0.71 ± 0.01 0.82 ± 0.01 6.63 ± 0.03 1.33 ± 0.01
4 Chongqing 0.33 ± 0.01 7.12 ± 0.03 0.62 ± 0.01 0.75 ± 0.01 0.21 ± 0.01 0.67 ± 0.01 0.61 ± 0.01 0.41 ± 0.01 2.87 ± 0.01 0.70 ± 0.01
5 Anhui 0.38 ± 0.00 7.58 ± 0.02 1.20 ± 0.01 1.21 ± 0.01 0.30 ± 0.00 0.49 ± 0.01 0.74 ± 0.01 0.86 ± 0.01 3.37 ± 0.01 0.37 ± 0.00
6 Anhui 0.22 ± 0.00 2.35 ± 0.00 0.18 ± 0.00 0.24 ± 0.00 0.05 ± 0.00 0.12 ± 0.00 0.07 ± 0.00 0.15 ± 0.00 1.29 ± 0.01 0.21 ± 0.00
7 Anhui 0.43 ± 0.01 8.09 ± 0.01 0.55 ± 0.01 0.67 ± 0.00 0.18 ± 0.00 0.83 ± 0.02 0.77 ± 0.02 0.54 ± 0.01 3.72 ± 0.01 0.69 ± 0.02
8 Anhui 0.44 ± 0.00 8.42 ± 0.01 0.86 ± 0.01 1.20 ± 0.01 0.25 ± 0.00 1.19 ± 0.02 0.88 ± 0.01 0.68 ± 0.01 2.93 ± 0.01 0.81 ± 0.00
9 Anhui 0.53 ± 0.00 7.88 ± 0.02 0.99 ± 0.00 1.28 ± 0.01 0.28 ± 0.00 1.08 ± 0.02 0.53 ± 0.01 0.85 ± 0.01 3.32 ± 0.01 0.77 ± 0.01
10 Guangdong 0.79 ± 0.01 11.70 ± 0.07 1.92 ± 0.03 1.86 ± 0.01 0.65 ± 0.00 2.57 ± 0.02 1.46 ± 0.01 1.70 ± 0.01 8.03 ± 0.05 1.89 ± 0.02
11 Xinjiang 0.46 ± 0.01 8.37 ± 0.04 0.62 ± 0.00 0.86 ± 0.00 0.21 ± 0.00 0.81 ± 0.00 0.64 ± 0.00 0.50 ± 0.00 2.78 ± 0.01 0.78 ± 0.01
12 Guizhou 0.47 ± 0.01 7.53 ± 0.01 0.95 ± 0.01 1.15 ± 0.01 0.24 ± 0.00 0.97 ± 0.01 0.59 ± 0.01 0.70 ± 0.00 3.34 ± 0.01 0.88 ± 0.01
13 Shanxi 0.29 ± 0.00 6.12 ± 0.06 0.59 ± 0.01 0.78 ± 0.01 0.18 ± 0.00 0.27 ± 0.01 0.51 ± 0.00 0.36 ± 0.00 2.15 ± 0.01 0.37 ± 0.01
14 Liaoning 1.07 ± 0.00 19.33 ± 0.21 1.11 ± 0.02 1.50 ± 0.01 0.47 ± 0.01 1.69 ± 0.01 1.29 ± 0.01 0.85 ± 0.01 7.77 ± 0.02 1.71 ± 0.01

L. Fan et al. / J. Chromatogr. A 1216 (2009) 2063–2070

15 Anhui 1.09 ± 0.00 20.74 ± 0.15 1.38 ± 0.01 1.63 ± 0.01 0.45 ± 0.01 1.42 ± 0.01 1.16 ± 0.01 0.83 ± 0.01 7.12 ± 0.02 1.51 ± 0.01
16 Zhejiang 0.93 ± 0.00 16.89 ± 0.14 1.19 ± 0.01 1.51 ± 0.00 0.43 ± 0.00 1.34 ± 0.04 1.17 ± 0.01 0.78 ± 0.01 6.10 ± 0.03 1.42 ± 0.01
17 Liaoning 0.83 ± 0.00 15.03 ± 0.19 1.41 ± 0.01 1.67 ± 0.01 0.45 ± 0.00 1.49 ± 0.01 0.93 ± 0.01 0.99 ± 0.01 7.03 ± 0.03 1.61 ± 0.04
18 Henan 0.56 ± 0.01 5.22 ± 0.01 0.91 ± 0.01 0.95 ± 0.01 0.18 ± 0.00 0.58 ± 0.02 0.22 ± 0.01 0.92 ± 0.01 3.12 ± 0.03 0.64 ± 0.01
19 Liaoning 0.79 ± 0.00 13.83 ± 0.10 1.23 ± 0.02 1.64 ± 0.03 0.40 ± 0.01 1.21 ± 0.01 0.95 ± 0.01 0.80 ± 0.01 5.15 ± 0.01 1.35 ± 0.01
20 Xinjiang 1.17 ± 0.00 20.23 ± 0.08 1.64 ± 0.00 1.63 ± 0.01 0.48 ± 0.00 0.91 ± 0.02 0.39 ± 0.01 1.00 ± 0.01 8.97 ± 0.05 1.13 ± 0.01
21 Chongqing 1.01 ± 0.00 15.65 ± 0.08 1.62 ± 0.01 1.94 ± 0.02 0.49 ± 0.01 2.21 ± 0.01 1.56 ± 0.01 1.21 ± 0.02 8.26 ± 0.01 1.88 ± 0.01
22 Heilongjiang 0.37 ± 0.00 6.02 ± 0.10 0.42 ± 0.01 0.52 ± 0.01 0.14 ± 0.01 0.41 ± 0.00 0.33 ± 0.00 0.32 ± 0.01 2.39 ± 0.02 0.49 ± 0.01
23 Guizhou 0.36 ± 0.00 6.75 ± 0.03 0.50 ± 0.00 0.50 ± 0.00 0.12 ± 0.00 0.62 ± 0.01 0.77 ± 0.00 0.35 ± 0.00 4.56 ± 0.06 1.12 ± 0.00
24 Anhui 0.62 ± 0.00 11.73 ± 0.01 0.86 ± 0.01 1.02 ± 0.01 0.30 ± 0.00 0.94 ± 0.01 0.64 ± 0.01 0.63 ± 0.01 5.20 ± 0.01 0.79 ± 0.01
25 Gansu 0.84 ± 0.00 17.06 ± 0.01 1.66 ± 0.00 1.98 ± 0.02 0.48 ± 0.01 0.74 ± 0.01 1.02 ± 0.01 1.08 ± 0.01 6.08 ± 0.02 0.80 ± 0.01
26 Zhejiang 0.58 ± 0.01 9.96 ± 0.05 0.88 ± 0.01 1.23 ± 0.01 0.29 ± 0.01 0.60 ± 0.01 0.53 ± 0.01 0.54 ± 0.00 3.65 ± 0.01 0.96 ± 0.01
27 Xinjiang 0.97 ± 0.01 16.85 ± 0.30 1.72 ± 0.02 1.79 ± 0.04 0.57 ± 0.02 1.81 ± 0.04 1.46 ± 0.04 1.11 ± 0.04 7.86 ± 0.12 1.50 ± 0.02
28 Liaoning 0.94 ± 0.00 7.39 ± 0.04 1.44 ± 0.01 1.39 ± 0.01 0.37 ± 0.01 1.61 ± 0.05 0.71 ± 0.01 1.54 ± 0.01 6.78 ± 0.09 1.15 ± 0.02
29 Liaoning 1.75 ± 0.01 10.28 ± 0.03 1.02 ± 0.01 0.89 ± 0.02 0.17 ± 0.01 0.54 ± 0.01 N.D.b 1.21 ± 0.02 5.07 ± 0.04 1.11 ± 0.02
30 Liaoning 0.93 ± 0.01 4.68 ± 0.02 1.34 ± 0.00 1.40 ± 0.00 0.36 ± 0.01 1.59 ± 0.01 1.08 ± 0.01 1.27 ± 0.01 5.45 ± 0.02 1.18 ± 0.02
31 Xinjiang 0.81 ± 0.01 12.49 ± 0.04 1.49 ± 0.01 1.94 ± 0.02 0.52 ± 0.01 1.69 ± 0.02 1.19 ± 0.01 1.05 ± 0.01 7.16 ± 0.08 1.96 ± 0.03
32 Xinjiang 0.96 ± 0.01 16.41 ± 0.06 1.73 ± 0.01 1.97 ± 0.01 0.52 ± 0.01 2.05 ± 0.03 1.34 ± 0.01 1.12 ± 0.01 7.79 ± 0.01 2.03 ± 0.01
33 Heilongjiang 0.79 ± 0.01 14.04 ± 0.01 1.83 ± 0.01 1.99 ± 0.01 0.59 ± 0.01 2.42 ± 0.03 1.47 ± 0.01 1.67 ± 0.01 10.43 ± 0.04 1.90 ± 0.01
34 Xinjiang 0.45 ± 0.01 6.22 ± 0.01 1.35 ± 0.01 1.60 ± 0.01 0.30 ± 0.01 1.16 ± 0.01 1.13 ± 0.01 0.96 ± 0.01 3.84 ± 0.01 0.81 ± 0.01
35 Gansu 0.79 ± 0.01 11.15 ± 0.13 1.34 ± 0.02 1.51 ± 0.02 0.37 ± 0.01 1.18 ± 0.01 0.59 ± 0.01 0.87 ± 0.01 4.78 ± 0.05 1.15 ± 0.01
36 Xinjiang 0.73 ± 0.02 12.39 ± 0.04 1.83 ± 0.03 2.04 ± 0.02 0.49 ± 0.01 2.58 ± 0.02 1.84 ± 0.01 1.41 ± 0.01 8.29 ± 0.05 1.79 ± 0.01
37 Xinjiang 1.06 ± 0.01 12.22 ± 0.10 1.35 ± 0.04 1.40 ± 0.01 0.38 ± 0.01 0.78 ± 0.01 0.23 ± 0.01 0.97 ± 0.01 6.34 ± 0.02 0.98 ± 0.02
38 Heilongjiang 1.39 ± 0.01 11.50 ± 0.04 1.48 ± 0.01 1.78 ± 0.02 0.38 ± 0.01 0.94 ± 0.01 0.37 ± 0.01 1.23 ± 0.01 5.47 ± 0.08 1.22 ± 0.02
39 Shanxi 0.60 ± 0.01 9.60 ± 0.10 1.27 ± 0.02 1.43 ± 0.02 0.29 ± 0.01 1.08 ± 0.01 0.55 ± 0.01 1.05 ± 0.01 5.48 ± 0.08 1.41 ± 0.02
40 Liaoning 0.62 ± 0.01 9.27 ± 0.03 2.19 ± 0.03 2.64 ± 0.02 0.59 ± 0.01 2.67 ± 0.01 1.89 ± 0.01 1.84 ± 0.01 7.50 ± 0.04 2.05 ± 0.02
41 Sichuan 0.55 ± 0.01 7.99 ± 0.03 1.80 ± 0.02 2.21 ± 0.02 0.50 ± 0.01 2.21 ± 0.03 1.25 ± 0.01 1.45 ± 0.04 5.28 ± 0.07 1.46 ± 0.02
42 Jiangxi 0.43 ± 0.01 5.48 ± 0.01 0.97 ± 0.01 1.20 ± 0.01 0.26 ± 0.00 0.95 ± 0.01 0.59 ± 0.00 0.69 ± 0.01 3.56 ± 0.02 0.61 ± 0.01
43 Shanxi 0.72 ± 0.01 10.69 ± 0.04 1.83 ± 0.03 2.00 ± 0.01 0.52 ± 0.01 0.98 ± 0.01 1.35 ± 0.01 1.06 ± 0.01 6.63 ± 0.05 1.00 ± 0.02
44 Hebei 0.69 ± 0.02 8.40 ± 0.05 1.72 ± 0.06 2.00 ± 0.01 0.50 ± 0.01 1.76 ± 0.01 0.94 ± 0.01 1.33 ± 0.01 5.63 ± 0.05 1.65 ± 0.02
45 Anhui 0.58 ± 0.01 5.82 ± 0.03 1.70 ± 0.02 2.18 ± 0.02 0.41 ± 0.01 1.96 ± 0.01 1.36 ± 0.01 1.28 ± 0.01 5.25 ± 0.04 1.74 ± 0.02
46 Sichuan 0.73 ± 0.01 8.58 ± 0.03 1.59 ± 0.02 2.08 ± 0.01 0.48 ± 0.01 1.94 ± 0.01 1.27 ± 0.01 1.25 ± 0.01 5.60 ± 0.04 1.59 ± 0.02

Rangec 0.22–1.75 2.35–20.74 0.18–2.19 0.24–2.64 0.05–0.65 0.12–2.67 0.07–1.89 0.15–1.84 1.29–10.43 0.21–2.05
a
Content = mean ± SD (n = 3).
b
N.D.: not detected.
c
Range: the range of contents of each compound in all the collected samples.

2069
2070 L. Fan et al. / J. Chromatogr. A 1216 (2009) 2063–2070
variation in all the samples. With respect to other compounds, obvi-
ous variation could also be found with at least 2.5-fold variation. A
number of factors may contribute to the variation of contents, such
as plant origin, genetic variation, growth circumstance, process-
ing, storage conditions and so on. The variation in contents of the
“marker compounds” could certainly lead to the variation of thera-
peutic effects. That is why each aspect involved procedure needed
to be standardized.
Compared with the reported analytical methods of C. tinctorius,
this newly established method provided much higher specificity,
precision and accuracy. By simultaneous determination of 10 major
bioactive components, the quality of C. tinctorius could be con-
trolled effectively.
4. Conclusion
A simple and accurate HPLC-DAD method is firstly established
for the simultaneous determination of 10 major components in dif-
ferent origins of C. tinctorius. The fine validation results showed
that the proposed method could be considered as a reliable and
sensitive quality control supplement for the safflower’s specifica-
tion in Chinese Pharmacopoeia. The characteristic compounds with
the structure of 6-hydroxykaempferol such as compounds 3 and 4
are suggested as the reasonable markers for the quality control due
to their strong bioactivities. The present study laid solid foundation
for the establishment of comprehensive quality control method of C.
tinctorius and its related herbal products in Chinese Pharmacopoeia.
Acknowledgement
We thank the program for Changjiang Scholar and Innovative
Research Team in University (Grant number: 985-2-063-112) and
the Cultivation Fund of the Key Scientific and Technical Innovation
Project (No. 104218) the Ministry of Education of China for their
generous financial support for this work.
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