Journal of Chromatography A, 1109 (2006) 279–284
Separation and on-line concentration of saponins from Panax notoginseng
by micellar electrokinetic chromatography
Shufang Wang, Shu Ye, Yiyu Cheng ∗
Department of Chinese Medicine Science and Engineering, College of Pharmaceutical Sciences,
Zhejiang University, Hangzhou 310027, China
Received 13 October 2005; received in revised form 19 December 2005; accepted 9 January 2006
Available online 15 February 2006
Abstract
A simple, reproducible and sensitive micellar electrokinetic chromatography (MEKC) method was developed for the separation and determination
of 10 saponins from Panax notoginseng. Field-enhanced sample injection with reverse migrating micelles (FESI–RMM) was used for on-line
concentration of the saponins. The effects of concentrations of sodium dodecyl sulfate (SDS) and organic modifier, the sample matrix, the injection
time of water plug, the injection voltage and injection time of sample on the separation and stacking efficiency were investigated. The optimum
buffer contained 10 mM H3 PO4 , 140 mM SDS, 20% acetonitrile and 15% 2-propanol and the pH of buffer was 2.4. The sample solution was
diluted with 15 mM SDS and injected for 15 s with −8 kV after injection of 2 s water plug. Under the optimum conditions, the analytes were well
separated with very high plate number (8.0 × 105 –1.2 × 106 N/m); the limits of detection of the analytes were 1.7–6.3 ␮g/mL. The high sensitivity
permitted the determination of two minor saponins Rh1 (1.01 mg/g) and Rg2 (0.62 mg/g) in P. notoginseng, which were not determined in the
literature.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Ginsenosides; Notoginsenosides; On-line concentration; Micellar electrokinetic chromatography; Field-enhanced sample injection with reverse migrating
micelles
1. Introduction
Panax notoginseng (Araliaceae), named as Sanqi or Tianqi in
Chinese, is precious medicinal material of China. Modern pharmacological research has proved that P. notoginseng saponins
(PNSs), including ginsenosides and notoginsenosides, have the
effects of improving heart muscle ischemia, resisting arrhythmia, decreasing blood fat, decreasing blood pressure, decreasing blood sugar, resisting thrombus, increasing coronary flow,
anti-inflammatory, analgesia, conscious-sedation, anti-aging,
restraining tumor cell of liver, resisting hepatic fibrosis and
protecting liver and renal [1]. P. notoginseng has been extensively used to treat cardiovascular disease, hepatitis, headache
and surgical diseases in clinic [2]. PNSs including ginsenosides
and notoginsenosides are its major effective components. Ginsenosides are also the main effective components of the two
∗
Corresponding author. Tel.: +86 571 87951138; fax: +86 571 87951138.
E-mail address: [email protected] (Y. Cheng).
0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2006.01.023
well-known ginseng genuses, i.e. Panax ginseng and Panax
quinquefolium. PNSs are often used as the markers of quality control of P. notoginseng and its preparations. Considering its extensive application in clinic, developing a rapid and
effective method to analyze PNSs in P. notoginseng is very
important.
Recently, Fuzzati reviewed the analysis methods of ginsenosides [3]. Ginsenosides were mainly analyzed by highperformance liquid chromatography (HPLC) [4–7]. Besides
HPLC technique, thin-layer chromatography (TLC) [8,9],
gas chromatography (GC) [10,11], near-infrared spectroscopy
(NIR) [12] and enzyme immunoassay (EIA) [13] were also
used to analyze ginsenosides. Among the five techniques, the
separation efficiency, precision and accuracy of HPLC was the
best, but it has the drawbacks of time-consuming sample preparations, requiring large amounts of organic solvent and long
analysis times (usually more than 60 min) when more analytes
were simultaneously separated. Some of the structures of PNSs
are very similar, such as the structures of ginsenoside Rg1 and
notoginsenoside Re, so the separation of Rg1 and Re is often
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S. Wang et al. / J. Chromatogr. A 1109 (2006) 279–284
the capital problem that should be resolved when analyzing
ginsenosides by HPLC. And, baseline drift was serious when
the detection wavelength was lower than 203 nm under gradient
elution condition, which limited its sensitivity because the UV
absorption of ginsenosides and notoginsenosides were the end
absorption.
Capillary electrophoresis (CE) has many advantages over
other techniques, including short analysis time, high efficiency,
technical simplicity and applicability to most analytes, small
sample and reagent requirements. Up to now, there have been
only two reports on the analysis of ginsenosides by CE [14,15].
Glöckl et al. separated ginsenosides Rb1 , Rb2, Re, Rc, Rf, Rd
and Rg1 in P. ginseng extract using 100 mM borate containing
80 mM cholate (pH 10) [14]. In their study, the lower limit of
quantitation of Rb1 was higher (100 ␮g/mL) and the extract of
real sample had to be cleaned by solid-phase extraction (SPE)
before analysis. Because the UV absorption of PNSs is weak
and the contents of some saponins (such as Rh1, Rg2, Rc,
Rb2, and Rf) are rather low in P. notoginseng, it is necessary
to develop a simple and sensitive MEKC for the analysis of
PNSs.
The concentration sensitivity of CE is poor due to short optimal path and small volume of sample that can be injected.
In order to improve the concentration sensitivity of MEKC,
Quirino and Terabe developed an on-line concentration technique, field-enhanced sample injection with reverse migrating
micelles (FESI–RMM) [16]. Low-pH buffer was used to reduce
the electroosmotic flow. The sample is dissolved with the aid of
micelles in solution and injected into the capillary using voltage. This concentration technique has been shown to provide
more than 100-fold increase in UV detector response with very
high plate numbers. The FESI–RMM technique was especially
useful for the compounds with high retention factor. The higher
is the retention factor of the analyte, the better is the stacking
enhancement factor.
In this work, FESI–RMM MEKC was used to separate and
on-line concentrate the saponins (the structures are shown in
Fig. 1) in P. notoginseng. The optimum separation and stacking conditions were achieved by systematically optimizing the
concentrations of sodium dodecyl sulfate (SDS) and organic
modifier, the sample matrix, the injection time of water plug
and the injection voltage and injection time of sample.
Fig. 1. The structures of analytes and internal standard. (Glu: glucose, Ara(f): arabinose in furanose form, Rha: rhamnose, Xyl: xylose).
S. Wang et al. / J. Chromatogr. A 1109 (2006) 279–284
2. Experimental
2.1. Chemicals and materials
Ginsenosides Rb1 , Rg1 and Re and notoginsenoside R1 were
purchased from National Institute for the Control of Pharmaceutical and Biological Products of China. Ginsenosides Rd,
Rc, Rb3 , Rh1 , Rg2 , and Rf were purchased from Jilin University (Jilin Province, China). Naringenin purchased from
Sigma–Aldrich was used as internal standard. Phosphoric acid
(H3 PO4 ) and sodium dodecyl sulfate were analytical grade. Acetonitrile, methanol (Merck, Germany) and 2-propanol (Shanghai
Ludu chemicals, Shanghai, China) were chromatographic pure.
Water was purified by a Milli-Q water purification system (Millipore, USA). P. notoginseng was purchased from Tianjin Tasly
Pharmaceutical Co. (Tianjin, China).
2.2. Standards mixture, internal standard and buffer
solution preparation
Stock solution of standards containing 2520 ␮g/mL Rd,
1520 ␮g/mL Rc, 1515 ␮g/mL Rb3, 1540 ␮g/mL Rb1, 1505 ␮g/
mL Rh1, 1130 ␮g/mL Rg2, 2795 ␮g/mL Rf, 1500 ␮g/mL Rg1,
1590 ␮g/mL Re and 1575 ␮g/mL R1 was prepared in methanol.
Solutions of lower concentration were obtained by diluting the
stock solution with appropriate amount of methanol. 102 ␮g/mL
naringenin (internal standard) was prepared in 5% methanol
(methanol:water = 5:95). The solution of standards was mixed
with internal standard and 15 mM SDS solutions according to
the volume ratio of 2:1:7 before being injected into the electrophoresis system.
The running buffer solution was prepared by mixing 0.5 mL
200 mM H3 PO4 , 3.5 mL 400 mM SDS in water, 2.0 mL acetonitrile and 1.5 mL 2-propanol in a 10 mL volumetric flask and
diluting to graduation line with water. All solutions for CE were
filtered through a 0.45 ␮m microfilter (Shanghai Yadong Nulear
Grade Resin Co., Shanghai, China).
281
lent Technologies). Capillary electrophoresis was performed
on a 70.0 cm (61.5 cm to the detector) × 75 ␮m I.D. fused silica capillary (Yongnian Photoconductive Fibre Factory, Hebei
Province, China). The detection wavelength was 195 nm. The
temperature was controlled at 25 ◦ C. The separation voltage was
−20 kV. The capillary was conditioned prior to its first use by
consecutively flushing with acetonitrile for 10 min, 0.1 M HCl
for 10 min, H2 O for 5 min, 1 M NaOH for 10 min, H2 O for 5 min
and the electrophoresis buffer for 15 min. After each run the capillary was rinsed with water for 1 min, followed by 0.1 M NaOH
for 2 min, water for 2 min and then the electrophoresis buffer for
3 min. The buffer was renewed after every three runs for good
reproducibility.
2.5. Operation procedure
A water plug was injected into the capillary by 30 mbar for
2 s from the cathode end. Voltage was applied at negative polarity with the sample solution in the cathodic vial and the buffer
solution in the anodic vial. During sample injection, the current
was −28 ␮A. After the sample was injected, the voltage was
shut off and a buffer vial was placed at the cathodic end, then
the voltage was applied again at negative polarity until all the
peaks was detected.
2.6. Calculation of the stacking enhancement factor
The stacking enhancement factors are calculated using the
equations [16] SEheight = Hstack /H and SEarea = Astack /A, where
the numerator is the peak height or peak area obtained with
stacking and the denominator is the peak height or peak area
obtained from 12 s hydrodynamic injection.
The hydrodynamic injection time was optimized. When the
injection time increased from 2 to 12 s, the peak areas and the
peak heights of the analytes increased. When the injection time
was further increased, the peak areas and the peak heights of
some analytes began to decrease. So the hydrodynamic injection
time was selected as 12 s.
2.3. Sample preparation
3. Results and discussion
P. notoginseng was pulverized and then sieved (60 mesh).
One gram of powdered P. notoginseng was extracted with 50 mL
methanol for 2 h in an ultrasonic bath. The original extract solution (SA) was used for the determination of Rd, Rb1, Rg1, Re and
R1. 20 mL of the SA was evaporated to dryness and redissolved
with 2 mL methanol, i.e. SA was condensed 10-fold. The condensed solution (SB) was used for the determination of Rh1 and
Rg2. All the sample solutions were passed through a 0.22 ␮m
microfilter (Shanghai Yadong Nulear Grade Resin Co.) before
being injected into the capillary electrophoresis system.
2.4. Apparatus
An HP3D capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany) equipped with diode-array
detector (190–600 nm) was used in this study. Data acquisition
and analysis were carried out with ChemStation software (Agi-
3.1. Optimizing the separation conditions
Initially, we used the separation conditions of Ref. [14], but
some of the analytes could not be separated well and the concentration sensitivity detected with our electrophoresis system was
poor (about 100 ␮g/mL). Because sensitivity could be improved
and the column efficiency was very high under the FESI–RMM
MEKC condition, we attempted to analyze the PNSs using
FESI–RMM MEKC method. After preliminary experiments, an
electrolyte containing 10 mM H3 PO4 , 140 mM SDS, 20% acetonitrile and 15% 2-propanol was chosen as the initial buffer.
The optimum separation was achieved by systematically optimizing the concentrations of SDS, organic modifier and buffer.
In order to reduce the electroosmotic flow, the pH of buffer (pH
2.4) was not adjusted. Because the effect of buffer concentration
on the separation was not significant, it was not discussed below.
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S. Wang et al. / J. Chromatogr. A 1109 (2006) 279–284
3.1.1. Effect of SDS concentration
In this work, the effect of SDS concentration on the separation
and stacking of analytes was important. Different SDS concentrations (80, 100, 120, 140 and 160 mM) in the electrolytes were
used to study the effect of SDS concentration. With the SDS
concentration increasing, the peak areas and peak heights of
analytes increased and the migration times decreased. But the
resolution of the consecutive peaks gradually decreased with
the SDS concentration increasing. When the SDS concentration
increased to 160 mM, the baseline separation of Rc and Rb3
was not achieved. Finally, 140 mM SDS was chosen for higher
stacking efficiency and shorter analysis time.
3.1.2. Effect of organic modifier
The effect of acetonitrile on the separation was studied by
adding different concentrations (0, 5, 10, 15, 20 and 25%) of acetonitrile to the electrolyte. The result indicated that the migration
times of the analytes and the resolution between the consecutive
peaks increased with the increasing of acetonitrile concentration. When the acetonitrile concentration increased to 15%, all
the analytes could be well separated from each other, but the
peak of Rd was partly overlapped with the peak of interference
in the real sample. Finally, 20% was decided as the optimum
acetonitrile concentration, at which the analytes were well separated in the real sample and the analysis time was shorter than
that at 25% acetonitrile.
The 2-propanol concentration was also optimized by adding
different concentrations (0, 5, 10, 15 and 20%) of 2-propanol to
the electrolyte. The result of experiments proved that most of the
peaks of analytes were partly or completely overlapped without
addition of 2-propanol as another organic modifier. The effect of
2-propanol was similar to that of acetonitrile, i.e. the migration
times of analytes and the resolution between the consecutive
peaks increased with the increasing of 2-propanol concentration.
The baseline separations of 10 analytes in the standards mixture
and real sample were achieved at 15% 2-propanol.
3.2. Optimizing the stacking conditions
3.2.1. Effect of sample matrix on the peak area
In order to investigate the effect of sample matrix on the
peak area, the solution of standards mixture was diluted with
different concentration (0, 10, 15 and 20 mM) of SDS solution,
and the result was shown in Fig. 2. Because the analytes were
not stable under acid condition, H3 PO4 was not added to the
sample solution. As shown in Fig. 2, the peak areas of Rd, Rc,
Rb3 and Rb1 were the highest at 10 mM SDS, while the peak
areas of the other six analytes increased with the increase of SDS
concentrations. The peaks of Re and R1 badly broadened when
the SDS concentration was 20 mM. Therefore, 15 mM SDS was
selected.
3.2.2. Effect of the injection time of water plug on the peak
areas and repeatability
The peak heights increased with the injection time of water
plug increasing from 0 to 2 s. The reason is that the water plug
provides a higher electric field at the tip of the capillary, which
Fig. 2. The effect of SDS concentration in sample solution on the peak areas
of analytes. The solution standards were mixed with the internal standard solution and 0–20 mM SDS solution according to the volume ratio of 2:1:7 and
then injected for 15 s with −8 kV after injection of 2 s water plug. Buffer:
10 mM H3 PO4 —140 mM SDS—20% acetonitrile—15% isopropanol (pH 2.4).
Capillary: 70.0 cm (61.5 cm to the detector) × 75 ␮m I.D. fused silica capillary. Detection wavelength: 195 nm. Temperature: 25 ◦ C. Separation voltage:
−20 kV.
will eventually improve the sample stacking procedure [17].
When the injection time was longer than 2 s, the peak heights
began to decrease. This may be because when the water plug was
too long, a strong laminar flow generated as a result of the mismatch of EOF velocity in the sample and buffer zones [18,19].
The introduction of water plug could also greatly improve the
repeatability of the peak areas [20]. Finally, the injection time
of water plug was selected as 2 s.
3.2.3. Effect of the injection time and the injection voltage
of sample on the peak area and peak height
With the injection time increasing from 9 to 18 s, the
peak areas of analytes increased. But when the injection time
increased to 18 s, the peak height of R1 began to decrease and
the peak badly broadened, which may be due to sample overloading. So the injection time was selected as 15 s.
The peak areas of the analytes increased with the injection
voltage increasing from −4 to −14 kV. However, when the injection voltage was higher than −8 kV, the peak heights of some
peaks began to decrease, and the peaks began to broaden. This
may be because higher voltage makes the analytes get through
the boundary between the water plug and the buffer and disperse
into the buffer during injection procedure, which results in the
decrease of the stacking efficiency [20]. Therefore, −8 kV was
chosen.
3.3. The optimum separation and stacking conditions
According to the experiments mentioned above, the optimum
buffer contained 10 mM H3 PO4 , 140 mM SDS, 20% acetonitrile
and 15% 2-propanol and the pH of buffer was 2.4. The sample solution was diluted with 15 mM SDS and injected for 15 s
with −8 kV after injection of 2 s water plug. The typical electro-
S. Wang et al. / J. Chromatogr. A 1109 (2006) 279–284
283
Fig. 3. The typical electropherograms of standard mixture under normal condition (A) and stacking condition. (A) The solution standards were mixed with the
internal standard solution and water according to the volume ratio of 2:1:7 and
then injected for 12 s with 30 mbar pressure. (B) The solution standards were
mixed with the internal standard solution and 15 mM SDS solution according to
the volume ratio of 2:1:7 and then injected for 15 s with −8 kV after injection
of 2 s water plug. The final concentrations of analytes were 45.2–100.8 ␮g/mL.
Buffer and other conditions were the same as those in Fig. 2.
pherograms of standards mixture under the normal and stacking
conditions were shown in Fig. 3. The 10 saponins were separated within 32 min with very high plate numbers. The stacking
enhancement factors and the plate numbers of the analytes were
listed in Table 1.
3.4. Linearity, repeatability and limits of detection
Under the optimum conditions, regression equations revealed
linear relationships between the ratios of peak area of the
analytes to that of internal standard and the corresponding
concentrations. The correlation coefficients of the calibration
curves were 0.9992–0.9998 for the analytes. The linear ranges
were 25.2–504.0 ␮g/mL for 1, 15.2–304.0 ␮g/mL for 2, 15.2–
303.0 ␮g/mL for 3, 7.7–308.0 ␮g/mL for 4, 15.0–301.0 ␮g/mL
for 5, 11.3–226.0 ␮g/mL for 6, 14.0–559.0 ␮g/mL for 7,
15.0–300.0 ␮g/mL for 8, 15.9–318.0 ␮g/mL for 9, and 15.8–
315.0 ␮g/mL for 10. The limits of detection were determined
when the ratio of signal to noise was 3. The limits of detection
of analytes were 1.7–6.3 ␮g/mL, which is comparable to that
obtained by HPLC [4]. The repeatability of the proposed method
was determined by repeated (n = 5) injection of a standard mixture solution containing 90.4–201.6 ␮g/mL of the 10 analytes
Fig. 4. The typical electropherograms of standard mixture (A), SA (B) and SB
(C). Buffer and other conditions were the same as those in Fig. 3B.
under the optimum conditions. The relative standard deviations
(RSDs) of the relative migration time and relative peak area
of each peak were 0.2–0.9% and 0.6–3.8% for intra-day, and
0.2–1.0% and 1.1–5.9% for inter-day, respectively. The recoveries experiments were performed by adding accurate amounts
of the analytes to the real samples. The results were 97.8–103.3%
for the analytes.
3.5. Application
The optimum conditions were applied to the separation and
determination of the 10 saponins in the extract of P. notoginseng.
Fig. 4A–C were the typical electropherograms of the standards
mixture, the original extract solution and the condensed extract
solution, respectively. The peaks of analytes were identified by
comparing the migration times and the UV spectra of the analytes in real sample with those of standards and spiking the
standards to the real sample solution. The response at about
Table 1
The stacking enhancement factors and the plate numbers under the optimum conditions
Analytes
Rd
Rc
Rb3
Rb1
Rh1
Rg2
Rf
Rg1
Re
R1
SEarea
SEheight
Plate number
(×106 N/m)
12
8
1.1
13
8
1.1
12
8
1.0
12
7
0.8
9
7
1.0
9
6
1.0
9
6
1.0
7
6
1.2
6
7
1.2
4
3
1.0
284
S. Wang et al. / J. Chromatogr. A 1109 (2006) 279–284
29.8 min of Fig. 4B was the fluctuation of baseline. As shown
in Fig. 4B and C, seven saponins, except Rc, Rb3 and Rf, were
detected in the extraction of P. notoginseng. The contents of
Rc, Rb3 and Rf were lower than the LOD. The contents of
the other analytes were 18.0 ± 0.7 mg/g for Rd, 26.4 ± 0.9 mg/g
for Rb1, 1.01 ± 0.02 mg/g for Rh1, 0.62 ± 0.01 mg/g for
Rg2, 24.4 ± 0.4 mg/g for Rg1, 5.31 ± 0.08 mg/g for Re and
10.0 ± 0.1 mg/g for R1.
4. Conclusion
The PNSs could be well separated with very high column
efficiency and high sensitivity using the MEKC method developed. By using FESI–RMM on-line concentration technique, the
concentration sensitivity could be enhanced up more than one
magnitude than that in the literature [14]. The result demonstrated that the method was effective to the pharmaceutical
quality control of P. notoginseng. The high sensitivity permitted the determination of two minor saponins Rh1 and Rg2 in P.
notoginseng, which were not generally determined with other
methods in the literatures. The method could also be a promising alternative for the preliminary investigation of PNSs in the
preparations containing P. notoginseng and other Panax plants.
Acknowledgements
This work was financially supported by the Key Program
of National Natural Science Foundation of China (Grant No.
90209005) and the National Basic Research Program of China
(Grant No. 2005CB523402).
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