Analytica Chimica Acta 596 (2007) 273–280

Determination of four major saponins in the seeds of Aesculus

chinensis Bunge using accelerated solvent extraction followed
by high-performance liquid chromatography and
electrospray-time of flight mass spectrometry
Junhui Chen a,b , Wenlong Li a , Baijuan Yang a , Xiuchun Guo a ,
Frank Sen-Chun Lee a , Xiaoru Wang a,c,∗
a QingDao Key Lab on Analytical Technology Development and Standardization of Chinese Medicines,
First Institute Oceanography of SOA, Qingdao 266061, China
b College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266003, China
c Department of Chemistry and the Key Laboratory of Analytical Science of the MOE, College of Chemistry and

Chemical Engineering, Xiamen University, Xiamen 361005, China

Received 24 November 2006; received in revised form 4 June 2007; accepted 6 June 2007
Available online 12 June 2007

Abstract
A new method based on accelerated solvent extraction (ASE) followed by a reliable high-performance liquid chromatography-diode array detector
(HPLC-DAD) and positive ion electrospray-time of flight mass spectrometry (ESI-TOF/MS) analysis has been developed for the characterization
and quantification of four major saponins in extracts of the seeds of Aesculus chinensis Bunge (Semen Aesculi). The saponins escin Ia, escin
Ib, isoescin Ia and isoescin Ib were extracted from seeds of A. chinesis Bunge via ASE, and the operational parameters of ASE were optimized,
such as extraction solvent, extraction temperature, static extraction time and extraction cycles. The optimized procedure employed 70% MeOH as
extraction solvent, 120 ◦ C of extraction temperature, 7 min of static extraction time, 60% flush volume and the extraction recoveries of the four
compounds were nearly to 100% for two cycles. The HPLC conditions are as follows: SinoChrom ODS BP C18 (4.6 mm × 200 mm, 5 ␮m) column,
acetonitrile and 0.10% phosphoric acid solution as mobile phase, flow rate is 1.0 mL min−1 , detection length of UV is 203 nm, injection volume
is 10 ␮L. The results indicated that the developed HPLC method is simple, sensitive and reliable for the determination of four major saponins in
seeds of A. chinesis Bunge with a good linearity (r2 > 0.9994), precision (relative standard deviation (R.S.D.) <1.5%) and the recovery ranges of
95.2–97.3%. The limits of detection (LOD) of the four compounds were in the range of 0.40–0.75 ␮g mL−1 . This assay can be readily utilized as
a quality control method for Semen Aesculi and other related medicinal plants.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Semen Aesculi; Saponins; Accelerated solvent extraction; High-performance liquid chromatography; Electrospray-time of flight mass spectrometry

1. Introduction
Traditional Chinese medicine has gained popularity in many
countries. The seeds of Aesculus chinesis Bunge, Aesculus
chinensis Bunge. Var. chekiangensis (Hu et Fang) Fang and
Aesculus wilsonii Rehd are a resource of the Traditional Chi-
nese medicine Semen Aesculi [1]. The seeds and the bark of
young branches of Aesculus hippocastanum (the horse chest-
∗ Corresponding author.
E-mail address: mt2elp@fio.org.cn (X. Wang).
nut tree) congeneric plant of A. chinesis Bunge are also widely
used as medicines in Europe. A number of reports dating from
the early 18th century have indicated therapeutic properties for
horse chestnut. These have ranged from anti-fever to, at the end
of the 19th century, anti-haemorrhoidal properties. Escins, the
major active principle from A. hippocastanum the horse chestnut
tree is a natural mixture of triterpene saponins, has shown sat-
isfactory evidence for a clinically significant activity in chronic
venous insufficiency (CVI), haemorrhoids and post operative
oedema [2]. The chemistry of saponins in Aesculus plant have
been extensively studied in the literature, and so far about 30
individual compounds of saponins have been isolated and iden-

0003-2670/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2007.06.011
274 J. Chen et al. / Analytica Chimica Acta 596 (2007) 273–280
Fig. 1. Chemical structures of saponins used in the study.
tified. Among them the major ones include four escins of escin
Ia, escin Ib, isoescin Ia and isoescin Ib [1,3,4]. In the official Chi-
nese Pharmacopoeia of 2005 edition, only escin Ia is assigned as
the marker species for Semen Aesculi. Thus, an effective quality
control practice for Semen Aesculi can be realized by the assay
of the four saponins escin Ia, escin Ib, isoescin Ia and isoescin
Ib. In Fig. 1, the structures of the four compounds are illustrated
[5].
During the last decades, many analytical approaches includ-
ing colorimetry [6–7], thin-layer chromatography (TLC) [8–9]
and high-performance liquid chromatography [3,10–13] have
been developed to assay saponins in A. chinesis Bunge, A. chi-
nensis Bunge. Var. chekiangensis (Hu et Fang) Fang, A. wilsonii
Rehd and A. hippocastanum (the horse chestnut tree) extracts.
In the German Pharmacopoeia (DAB) the escin content of A.
hippocastanum and its extract are determined by means of a
colorimetric method. The colorimetric method determines only
the total saponins content and is non-specific towards individ-
ual escin. Several TLC-densitometric methods [8–9] have been
described for the quality control of escin and single Aesculus
preparations. However, the thin-layer chromatographic methods
are also developed to analyze the total saponins content. HPLC
is a popular method, and various detectors including ultraviolet
(UV), evaporative light scattering detection (ELSD) have been
used to determine escin [3,10]. Reports on mass spectrometric
analysis of escin has been scarce although one paper has been
published recently on the determination of escin Ia and escin Ib
in Symplocos chinensis by electrospray ionization tandem mass
spectrometry [14].
Accelerated solvent extraction (ASE) operates at high pres-
sures and temperature above point of the boiling point of the
organic solvent. The use of higher temperature increases the abil-
ity of solvent to solubilize the analyte, decreases the viscosity
of liquid solvents, allowing better penetration of the solvent into
the matrix. The use of higher pressure facilitates the extraction
of the analytes from samples by improving the solvent accessi-
bility to the analytes that is trapped in the matrix pores. The use
of ASE decreases significantly the total time of treatment and in
addition, this extraction method can be more effective and selec-
tive by changing some parameters like temperature, time, cycles
and solvent [15–17]. In recent years, ASE has been widely used
to extract the bioactive components in natural products [18–21].
In the present paper, a reliable HPLC method has been devel-
oped for the quantitative determination of four major saponins
escin Ia, escin Ib, isoescin Ia and isoescin Ib in the crude extract
of Semen Aesculi. ESI-TOF/MS coupled with HPLC has also
been applied to the analysis of Semen Aesculi extracts and the
identification of individual escin. In addition, a new extraction
process, accelerated solvent extraction, was optimized to max-
imize escins extraction yield. The validated method was also
applied to quantify the investigated components in Semen Aes-
culi material.
2. Experimental
2.1. Materials and reagents
Five samples of Semen Aesculi were purchased from dif-
ferent medical halls in Qingdao of China. Standards of escin
Ia, escin Ib, isoescin Ia and isoescin Ib were obtained from
National Institute for the Control of Pharmaceutical and Biolog-
ical Products of China. Acetonitrile was of HPLC grade from
Fisher Chemicals (USA). Other chemicals, such as methanol,
ethanol, etc. were all of analytical grade from Shanghai Chemi-
cal Factory. Water was purified using a Milli-Q water purification
system (Milipore, USA).
2.2. Sample preparation
The dried seeds of A. chinensis Bunge were grounded by
using a pestle and mortar. They were then sieved through a stain-
less steel sieve with pore size of 0.3 mm before extraction. No.
1 Semen Aesculi sample was used in all solvent and extraction
method studies.
2.2.1. Reflux extraction
Heated reflux extraction was performed using a cooled con-
denser and a round-bottom flask of 100 mL. 70% (v/v) aqueous
methanol was used as the solvent. Two grams of powdered sam-
ple and 35 mL of solvent were added into the round-bottom flask.
The suspension was extracted for 1 h at 90 ◦ C with a water bath
and filtered. The extraction was repeated two additional times
and the extracts were combined. The combined extract was fil-
tered, and the filtrate was evaporated to dryness using a rotary
evaporator at 50 ◦ C. The residue was then dissolved in 50 mL of
methanol and filtered through a 0.45 ␮m nylon filter membrane.
2.2.2. Ultrasonic extraction
Ultrasonic extraction was carried out by mixing 2 g of pow-
dered sample and 35 mL of 70% methanol in a flask, which was
then placed in an ultrasonic bath for 30 min. The extraction was
 J. Chen et al. / Analytica Chimica Acta 596 (2007) 273–280
repeated two additional times and the extracts were combined.
The combined extract was filtered, and the filtrate was evapo-
rated to dryness using a rotary evaporator at 50 ◦ C. The residue
was then dissolved in 50 mL of methanol and filtered through a
0.45 ␮m nylon filter membrane.
2.2.3. Accelerated solvent extraction
An ASE 100 System (Dionex, Sunnyvale, CA, USA) with
34-mL stainless steel ASE vessels was used for the pressurized
liquid extraction. About 2.0 g of Semen Aesculi powder was
mixed homogeneously with same weight of diatomaceous earth
and placed into an extraction cell. The extraction cells were
placed into the carousel and the samples were extracted under
some specified condition. The extract was evaporated to dry-
ness using a rotary evaporator at 50 ◦ C. The residue was then
dissolved in 50 mL of methanol and filtered through a 0.45 ␮m
nylon filter membrane prior to injection into the HPLC system.
2.3. Standards preparation and calibration curves
Standard stock solutions containing 1 mg mL−1 each of
saponins were prepared by dissolving approximately 10 mg
(range 9–10 mg, weighed accurately) of each pure compound in
10 mL methanol. Calibration standard working solutions were
prepared by mixing the above standard solutions in appropriate
proportions, and then diluted with methanol to the desired con-
centrations. Each calibration curve was constructed by running
samples at six different concentrations in triplicate.
2.4. HPLC and ESI-TOF/MS conditions
The HPLC analyses were performed with a HPLC instru-
ment (Agilent1100, USA) equipped with a quaternary solvent
delivery system, an autosampler, a column oven and a diode
array UV/Vis detector. The column (Sinochrom ODS-BP C18 ,
200 mm × 4.6 mm, 5 ␮m, Dalian Elite Analytical Instruments
Co. Ltd.) was eluted isocratically with a binary mixture of
acetonitrile and 0.10% phosphoric acid solution (volume ratio
42:58) at a total flow rate of 1.0 mL min−1 . Elution was moni-
tored at 203 nm on the diode array detector, the injection volume
was 10 ␮L, and separations were carried out isothermally at
30 ◦ C in a heated chamber.
A G1969A TOF mass spectrometer (Agilent, USA) with an
electrospray ion source coupled to the HPLC system described
above was used to analyze the escin Ia, escin Ib, isoescin Ia
and isoescin Ib in Semen Aesculi. Data were processed using
Analyst QS software equipped in Agilent TOF/MS. The chro-
matographic conditions were the same as those described above,
except an extra 0.20% acetic acid instead of 0.10% phospho-
ric acid was added into the mobile phase. The outlet of flow
cell was connected to a split valve in order to divert a flow of
0.4 mL min−1 to the electrospray ion source via a short length of
fused silica tubing. The mass spectrometer conditions were opti-
mized for saponins detection as follows. The lens voltages were
in positive ion mode by turning on the respective ions of interest.
A spray voltage of 4.0 kV was employed and the velocity of dry-
ing gas was 11.0 L min−1 . The MS data acquisition process was
275
composed of two scans, each with different collision induced
dissociation (CID) voltage of 100, 250 V, respectively. The tem-
perature of the heated transfer capillary was 340 ◦ C. The mass
spectrometer was scanned from m/z 100 to 1500 in full scan
mode. Reference solution was used and two m/z, 121.050873
and 922.009798 were selected for saponins detection.
2.5. HPLC method validation
The calibration curve was constructed by running six mixture
standards of different concentrations in triplicate. Consequently,
calibration curves were constructed. The correlation coefficient
was determined using a linear regression model. The limit of
detection was defined as three times the noise level obtained
from running matrix blank samples.
The precisions of escin Ia, escin Ib, isoescin Ia and isoescin
Ib and HPLC retention time (repeatability) measurements were
established by the observed relative standard deviations of the
respective measurements from six repeated runs. The repro-
ducibility of the method was also evaluated by running six
replicate samples (No. 1) prepared independently in a single
day. Sample stability was tested by periodically analyzing sam-
ple solution which was kept at room temperature at a frequency
of every 12 h within 3 days. The relative standard deviation was
taken as the indicator for stability.
The recoveries of the saponins were determined by standard
addition method. Four saponins in a mixed standard were spiked
into the samples (No. 1) and extracted and the results were
compared with an unspiked sample analyzed under the same
conditions.
3. Results and discussion
3.1. Optimization and validation of HPLC method
The chromatographic conditions were developed and opti-
mized using both saponin standards and real Semen Aesculi
sample. Reversed phase HPLC was used according the previous
literatures [10–11]. Varying the ratios of water and acetonitrile
in the mobile phase provided little improvement in separa-
tion. However, considerable improvement in peak resolution
and peak shape was observed upon the addition of acetic acid,
trifluoroacetic acid or phosphoric acid in the mobile phase.
Our phosphoric acid–acetonitrile system here is better than the
reported acetic acid or trifluoroacetic acid systems [10–11]. A
concentration 0.1% phosphoric acid in the mobile phase pro-
vided the best resolution and signal/noise ratio. Because the
absorptions of all the target compounds lie in the vacuum UV
range with λmax of 200 nm (Fig. 2), 203 nm was therefore chosen
as the detection wavelength. The use of such short wavelength
UV dictates the use of phosphoric acid instead of acetic acid
and trifluoroacetic acid in water as the mobile phase in order
to minimize detector baseline noise. Compared with results pre-
sented in previous reports [10,11] with the use of UV 210 nm and
evaporative light scattering detectors, our results show signifi-
cant improvement in detection sensitivity for all the four escins.
Fig. 2 compares the chromatograms of a mixed saponins stan-
276 J. Chen et al. / Analytica Chimica Acta 596 (2007) 273–280

Fig. 2. HPLC chromatograms of standard solution of four saponins (A); extracts of Semen Aesculi sample (B); (1) escin Ia, (2) escin Ib, (3) isoescin Ia, (4) isoescin
Ib.

Table 1
Calibration curve, linear range and detection limits of saponins escin Ia, escin Ib, isoescin Ia and isoescin Ib determined for the presented HPLC assay
Saponins Calibration curve Linear range (␮g) Correlation coefficient Detection limits (␮g mL−1 )

Escin Ia Y = 0.002X + 0.1793 0.151–60.4 0.9995 0.75

Escin Ib Y = 0.0019X + 0.1547 0.105–42.0 0.9997 0.50
Isoescin Ia Y = 0.0019X + 0.1262 0.084–16.8 0.9994 0.40
Isoescin Ib Y = 0.002X + 0.0162 0.056–5.6 0.9998 0.60

dard solution versus those of Semen Aesculi samples under the
same HPLC conditions. All the saponin peaks are symmetrical
and well resolved from each other. The sample chromatograms
contain more peaks than those of the standard, indicating the
presence of unknown components in Semen Aesculi extract
besides escin Ia, escin Ib, isoescin Ia and isoescin Ib.
3.1.1. Linearity and detection limit
Results from the calibration study and the limits of detection
for the four saponins escin Ia, escin Ib, isoescin Ia and isoescin
Ib are summarized in Table 1. The linearity of the calibration
curves have been verified by correlation study and the correlation
coefficients are all better than 0.9994. The linear dynamic ranges
of the calibration curves exceed two orders of magnitudes. The
detection limits are in the range of 0.40–0.75 ␮g mL−1 for the
four saponins, which are better than those reported for other
studies using ELSD or UV–vis detection in HPLC separation
[10,11].
3.1.2. Precision, reproducibility and stability
The precisions of both peak area and retention time mea-
surements were found to be better than 1.5% (R.S.D., n = 6)
for all the target saponins. The reproducibility (R.S.D.) of the
proposed method, on the basis of peak areas for six replicate
injections, was 1.62–3.4%. The variation in the retention times
of all the peaks was less than 0.52% for six replicate injections.
The stability (R.S.D.) of the measurements for the four saponins
is 2.09–3.87% (n = 6) for the peak areas and 0.49–0.86% (n = 6)
for the retention times.
3.1.3. Recovery
Recoveries for the four saponins were determined by stan-
dard addition method in which three repeated analyses of the
spiked samples were run within the same day. The results are
summarized in Table 2. The recoveries are within the range of
95.2–97.3%; and the R.S.D. values of all the four saponins from
three replicate injections are better than 5.0%, demonstrating the
good recovery and precision of the method.
3.2. Extraction method development
3.2.1. Optimization of the accelerated solvent extraction
Extraction conditions, namely, extraction solvent, tempera-
ture, static time, flush volume and extraction cycle are important
parameters for achieving a quantitative extraction [21,22]. Some
of these parameters were optimized with univariate approach,
based on the relative extraction ratio of escin Ia, escin Ib, isoescin
Ia and isoescin Ib.

Table 2
Recovery of saponins determined by standard addition method (n = 3)
Saponins Original amount (mg) Added amount (mg) Detected amount (mg) Recovery (%) R.S.D. (%)

Aescin Ia 18.4 14.6 14.2 97.3 2.54

Aescin Ib 12.0 10.4 9.9 95.2 3.09
Isoaescin Ia 8.9 8.0 7.7 96.3 3.41
Isoaescin Ib 5.3 4.8 4.6 95.8 4.01
 J. Chen et al. / Analytica Chimica Acta 596 (2007) 273–280 277

Table 3
Relative amount of four escins in Semen Aesculi extracted by accelerated solvent extraction with different solvents (mean ± S.D.; n = 3)
Solvents Escin Ia Escin Ib Isoescin Ia Isoescin Ib

n-Butanol 0.355 ± 0.012 0.524 ± 0.017 0.309 ± 0.012 0.386 ± 0.015

70% Methanol 1.000 ± 0.028 1.000 ± 0.024 1.000 ± 0.027 1.000 ± 0.030
Methanol 0.996 ± 0.031 0.975 ± 0.029 0.839 ± 0.022 0.985 ± 0.028
30% Ethanol 0.776 ± 0.028 0.766 ± 0.020 0.703 ± 0.021 0.971 ± 0.026
50% Ethanol 0.837 ± 0.032 0.836 ± 0.024 0.704 ± 0.023 0.865 ± 0.029
80% Ethanol with 0.5% acetic acid 0.345 ± 0.010 0.345 ± 0.012 0.267 ± 0.011 0.357 ± 0.015

The extraction solvent, which is critical for recovery of the
analytes [23], was firstly evaluated. n-Butanol, methanol, aque-
ous methanol and aqueous ethanol were investigated based on
the polarity of the target compounds and the literatures [1,10].
The ASE conditions used are as follows: 100 ◦ C of extraction
temperature, 10 min of static extraction time, 60% flush vol-
ume, with two cycles. Of the seven solvent systems compared in
Table 3, the relative quantities of escin Ia, escin Ib, isoescin
Ia and isoescin Ib extracted in 70% (v/v) aqueous methanol
are the highest, followed in decreasing order by pure methanol
and 50% (v/v) aqueous ethanol. Considering the extraction effi-
ciency, 70% (v/v) aqueous methanol was chosen as the extraction
solvent for further investigations.
Since the increase of temperature can improve the extrac-
tion of natural compounds, a series of experiments at different
temperatures (60–140 ◦ C) was performed to determine the best
extraction temperature by using 70% methanol as extraction sol-
vent, 10 min of static extraction time, 60% flush volume, with
two cycles. The results for the extractions at different temper-
atures are shown in Fig. 3. In the experiments, the extraction
efficiency of escin Ia and escin Ib was stable up to 120 ◦ C and
above this temperature it started to decrease. The decrease in the
extraction efficiency was possibly due to degradation of these
two compounds at temperatures higher than 120 ◦ C. The extrac-
tion efficiency of isoescin Ia and isoescin Ib increased up to
140 ◦ C, suggesting that isoescin Ia and isoescin Ib were more
stable than escin Ia and escin Ib at high temperature. Therefore,
120 ◦ C was chosen to be used as extraction temperature not only
for the extraction efficiency of isoescin Ia and isoescin Ib but also
for escin Ia and escin Ib.
To evaluate if static extraction time could influence extraction
efficiency of saponins in Semen Aesculi, different extractions (4,
7, 10, 13, 16 min) were performed with the following ASE condi-
tions: 70% methanol as extraction solvent, 100 ◦ C of extraction
temperature, 60% flush volume, with two cycles. The results for
the extractions at different static extraction time are shown in
Fig. 4. Increasing the static extraction time from 7 to 13 min did
not influence the extraction of target compounds. One important
point, when the static extraction time above 13 min, the extrac-
tion efficiency of escin Ia, escin Ib, isoescin Ia and isoescin Ib
decreased significantly. So, the static extraction time was set at
7 min for one cycle.
The flush volume (20, 40, 60, 80%) did not significantly affect
the extraction efficiencies of the target compounds in Semen
Aesculi. So, the flush volume was set at their default values,
60%.
At last, the extraction times of ASE was determined by per-
forming consecutive accelerated solvent extractions three times
on the same sample. As shown in Table 4, two extraction cycles
were sufficient to completely extract the target components from
Semen Aesculi.
To evaluate the repeatability of the extraction procedure, a
series of six replicated extractions were performed in the same
and different days. The results obtained for all target compounds
revealed a R.S.D. lower than 5%.
3.2.2. Comparison of ASE, reflux and sonication
The extraction efficiency of ASE for escin Ia, escin Ib,
isoescin Ia and isoescin Ib was compared with those of reflux and
sonication extractions. The optimal ASE conditions optimized
above were used and the relative yields of escins extracted from

Fig. 3. Effect of extraction temperature on extraction efficiency of escins for Fig. 4. Effect of static extraction time on extraction efficiency of escins for two
two cycles. cycles.
278 J. Chen et al. / Analytica Chimica Acta 596 (2007) 273–280

Table 4
Effect of extraction times on extraction efficiency of escins (mean ± S.D.; n = 3)
Extraction times Escin Ia (%) Escin Ib (%) Isoescin Ia (%) Isoescin Ib (%)

1 95.20 ± 2.00 95.41 ± 1.81 94.13 ± 2.26 94.72 ± 2.37

2 4.65 ± 0.13 4.56 ± 0.14 5.55 ± 0.15 5.11 ± 0.15
3 0.12 ± 0.01 0.07 ± 0.02 0.33 ± 0.01 0.15 ± 0.01

Table 5
Relative amount of four escins in Semen Aesculi extracted by different extraction methods (mean ± S.D.; n = 3)
Method Time Escin Ia Escin Ib Isoescin Ia Isoescin Ib

Reflux 1h×3 0.810 ± 0.023 0.834 ± 0.025 0.874 ± 0.030 0.868 ± 0.029
Sonication 30 min × 3 0.781 ± 0.032 0.765 ± 0.033 0.756 ± 0.028 0.707 ± 0.026
ASE 7 min × 2 1.000 ± 0.025 1.000 ± 0.027 1.000 ± 0.031 1.000 ± 0.29

Semen Aesculi samples were compared. As shown in Table 5, 3.3. Identification of saponins by LC–ESI-TOF/MS
the extraction efficiency of ASE was a little higher than those of
sonication and reflux extraction, and ASE has the advantage of A typical HPLC chromatogram of an extract sample and the
shorter extraction time than the other two. UV spectra of escin Ia peak are shown in Fig. 2B. With a diode-

Fig. 5. (A) MS spectra of escin Ia obtained using acetonitrile, water and acetic acid (volume ratio 58:42:0.2) as mobile phase in the positive mode; (B) escin Ib; (C)
isoescin Ia; (D) isoescin Ib.
 J. Chen et al. / Analytica Chimica Acta 596 (2007) 273–280 279

array detector and the corresponding computer software, the

evaluation of peak purity allows checking the singularity of the
peak component. Using this peak purity check routine, all the
four peaks in Fig. 2B have high peak purity according to the
normalized match factor value (>990) (with 1000 being 100%
pure).
The samples were also run by LC–ESI-TOF/MS to identify
unequivocally the target species. Our preliminary direct infusion
studies with escin Ia confirmed that positive ion mode was more
sensitive. The mass spectrometer conditions were optimized for
escin Ia standard in order to achieve maximum sensitivity. The
results showed that the spray voltage of 4.0 kV could give a best
sensitivity. The CID voltage of 100 V could produce the best
signals and the lowest limits. The use of 0.1–1.0% acetic acid in
mobile phase was found to give satisfactory sensitivity towards
escin Ia detection by ESI-TOF/MS.
The extracts of the Semen Aesculi sample was analyzed
by LC–ESI-TOF/MS with positive ion mode under the opti-
mized mass spectrometer conditions. All the four target saponins
unambiguously identified by TOF/MS from both exact mass
measurement and by comparing with authentic standard. For
escin Ia within the mass range scanned, series of fragment
ions were observed as shown in Fig. 5A. The exact masses
of these ions 1153.5420 ([M + Na]+ ), 1131.5571 ([M + H]+ ),
1113.5478 ([M − H2 O + H]+ , 969.5057 ([M − C6 H10 O5 + H]+ ),
807.4551 ([M − 2C6 H10 O5 + H]+ ), etc. were consistent with the
elemental compositions of C55 H86 O24 Na, C55 H87 O24 , C55
H85 O23 , C49 H77 O19 and C43 H67 O14 with mass errors under
5 ppm, respectively. The molecular mass of escin Ia was cal-
culated to be 1130 which was confirmed by the observation
of an [M + H]+ ion of 1131.5571 using positive ion ESI-TOF Fig. 6. (A) Observed mass ([M + H]+ ) of escin Ia and its isotopics compared
mass spectrometry. Fig. 6A–C shows that measured isotopics of with calculated isotopics; (B) observed mass ([M − C6 H10 O5 + H]+ ) of escin
the three formulas (C55 H87 O24 , C49 H77 O19 and C43 H67 O14 ) Ia and its isotopics compared with calculated isotopics; (C) observed mass
match perfectly with the theoretical values (including positions ([M − 2C6 H10 O5 + H]+ ) of escin Ia and its isotopics compared with calculated
isotopics.
and intensities). For escin Ib, isoescin Ia and isoescin Ib similar
fragment ions could be observed from the mass spectra shown in
Fig. 5B–D. with those of the actual examples for compound identification.
In our TOF-MS analysis, an isotope ratio matching technique In this study, the excellent agreement obtained between the pro-
was used in the compound identification process using the Ana- posed and the actual isotopic ratios of the peaks associated with
lyst QS software available in the instrument. In the exercise, once escin Ia can be seen in Fig. 6A–C.
the exact mass of the peak was decided, one or several possi- Yang et al. [24] have identified saponins in seeds of A. wilsonii
ble elemental compositions will then be proposed. The software by ES-MS in positive mode and NMR. The main characteristic
can then give the theoretical isotopic ratios (shown with broken fragment ions of escin Ia, escin Ib, isoescin Ia and isoescin Ib
lines) of all the possible structures. The exact masses and the for MS analysis also consisted of m/z 1131, 969, 807, 613, 513
intensities of these theoretical isotopic peaks are then compared and 453 ions consistent with our results.

Table 6
Contents of saponins in different Semen Aesculi samples (mean ± S.D.; n = 3)
No. Escin Ia (mg g−1 ) Escin Ib (mg g−1 ) Isoescin Ia (mg g−1 ) Isoescin Ib (mg g−1 ) Total (mg g−1 )

1 18.4 ± 0.4 12.0 ± 0.3 8.9 ± 0.3 5.3 ± 0.2 44.6 ± 1.2
2 17.1 ± 0.4 8.3 ± 0.2 13.8 ± 0.4 6.9 ± 0.2 46.1 ± 1.2
3 15.6 ± 0.5 9.2 ± 0.4 7.7 ± 0.3 5.0 ± 0.1 37.5 ± 1.3
4 19.1 ± 0.3 11.8 ± 0.3 8.3 ± 0.2 6.4 ± 0.2 45.6 ± 1.0
5 16.3 ± 0.4 10.6 ± 0.3 7.9 ± 0.3 5.8 ± 0.2 40.6 ± 1.2
Average 17.3 ± 1.4 10.4 ± 1.6 9.3 ± 2.5 5.9 ± 0.8 42.8 ± 3.7
280 J. Chen et al. / Analytica Chimica Acta 596 (2007) 273–280

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