Development and application of a rapid and efficient CZE method coupled with correction factors for determination of monosaccharide composition of acidic hetero-polysaccharides from Ephedra sinica

Article abstract of DOI:10.1002/pca.1235

Introduction - Ephedrine alkaloids cannot account for all the effects of Ephedra sinica and the polysaccharides are also demonstrated to be one of the main bioactive constituents of E. sinica. However, no work has been reported on the analysis of monosacc

Full text of DOI:10.1002/pca.1235

Research Article
Received: 2 January 2010;
Revised: 20 February 2010;
Accepted: 29 March 2010
Published online in Wiley Online Library: 30 December 2010
(wileyonlinelibrary.com) DOI 10.1002/pca.1235
Development and Application of a Rapid and
Efficient CZE Method Coupled with Correction
Factors for Determination of Monosaccharide
Composition of Acidic Hetero-polysaccharides
from Ephedra sinica
Yong-gang Xia,a Qiu-hong Wang,a Jun Liang,a Bing-you Yang,a Guo-yu Lib
and Hai-xue Kuanga*
ABSTRACT:
Introduction – Ephedrine alkaloids cannot account for all the effects of Ephedra sinica and the polysaccharides are also
demonstrated to be one of the main bioactive constituents of E. sinica. However, no work has been reported on the analysis of
monosaccharide composition of purified polysaccharides isolated from the stem of E. sinica.
Objective – To develop a rapid and efficient capillary zone electrophoresis (CZE) method based on pre-column derivatisation
with 1-phenyl-3-methyl-5-pyrazolone for the simultaneous determination of neutral and acidic sugars of purified polysaccha-
rides from E. sinica.
Methodology – Three polysaccharides (ESP-A3, ESP-A4 and ESP-B4) were isolated and purified by ion exchange and gel-
filtration chromatography from the stem of E. sinica. The effects of background electrolyte pH and concentration, applied
voltage and temperature on the separation were investigated. Meanwhile, factors affecting the hydrolysis of ESP-B4 with
sulphuric acid were investigated by changing the hydrolysis time, acid concentration and hydrolytic temperature to achieve
complete hydrolysis. The standard curves coupled with correction factors were used to calculate molar ratios.
Results – The optimal CZE method coupled with correction factors was successfully applied to the determination of molar ratios
of three purified polysaccharides and their corresponding partial acid hydrolysis products. ESP-A3, ESP-A4 and ESP-B4 were all
typical acidic hetero-polysaccharides and consisted of xylose, arabinose, glucose, rhamnose, mannose, galactose, glucuronic
acid and galacturonic acid, and their corresponding molar ratios were 6.8:7.5:1.0:14.0:13.7:22.3:10.2:3.8 for ESP-A3,
1.2:4.1:1.0:5.1:1.6:17.3:3.1:2.2 for ESP-A4, and 1.0:4.5:1.0:2.0:1.0:5.5:1.5:50.0 for ESP-B4.
Conclusion – The results provided scientific evidence for the further study of the structure and bioactivity of complex acidic E.
sinica polysaccharides. Copyright © 2010 John Wiley & Sons, Ltd.
Keywords: capillary zone electrophoresis; polysaccharides; monosaccharide composition; molar ratios; correction factors; Ephedra
sinica
antitussive, central nervous system stimulant, anti-inflammatory
and colds (Trujillo and Sorenson, 2003; Roman et al., 2004;
Introduction
Botanical polysaccharides with multiple pharmacological activi-
Dunnick et al., 2007). A series of ephedrine alkaloids have for a
ties have recently stimulated the interest of academia and the
long time been considered as the pharmacologically active ingre-
pharmaceutical industries (Hurtley et al., 2001). In fact, the roles of
dients of E. sinica for the treatment of various diseases and symp-
water-soluble polysaccharides from traditional Chinese medi-
toms (Konno et al., 1985; Meng et al., 2007), but they cannot
cines in biological processes have been studied with increasing
account for all the effects mentioned above and the water-
attention over the past recent years because of their broad
soluble polysaccharides have also been demonstrated to be one
spectrum of therapeutic properties and relatively low toxicity
(Schepetkin and Quinn, 2006; Chen et al., 2007; Fang and Ding,
2007). Indeed, immunomodulation, anti-tumour, antivirus,
anticoagulant, hypoglycaemic, anti-complementary, anti-
University of Chinese Medicine, 24 Heping Road, Harbin 150040, People’s
* Correspondence to: Hai-xue Kuang, Pharmaceutical College, Heilongjiang
inflammatory and antioxidation bioactivities have been pre-
sented by many polysaccharides extracted from medicinal fungi
and plants (Schepetkin and Quinn, 2006; Chen et al., 2007; Fang
and Ding, 2007; Liu et al., 2007; Yu et al., 2007; Hsieh et al., 2008).
Ephedra sinica is a known traditional Chinese medicine that has
been used for thousands of years to treat asthma, bronchitis,
Republic of China. E-mail: hxkuang@hotmail.com
a
Pharmaceutical College, Heilongjiang University of Chinese Medicine, 24
Heping Road, Harbin 150040, People’s Republic of China
b
Pharmaceutical College, Harbin Medical University, Harbin 150081, 157
Baojian Road, People’s Republic of China
Phytochem. Anal. 2011, 22, 103–111
Copyright © 2010 John Wiley & Sons, Ltd.

Y. Xia et al.
of the main bioactive constituents of E. sinica (Konno et al., 1985;
Cheng et al., 2001; Ding et al., 2006).
to the best of our knowledge rather limited monosaccharides
were usually baseline or fast separated, which significantly
affected the analytical accuracy or efficiency (Wang and Fang,
2004; Größl et al., 2005). Therefore, the improved separation of a
series of monosaccharides needs to be further investigated in the
presence of borate complexation. In this paper, a simple and
rapid CZE method based on PMP derivatisation has been devel-
oped for simultaneous separation and determination of the
neutral and acidic sugars. The effects of background electrolyte
pH and concentration, applied voltage and temperature on the
separation were investigated. Meanwhile, the factors affecting
hydrolysis of E. sinica polysaccharide with sulphuric acid were
also investigated by changing the hydrolysis time, acid concen-
tration and hydrolytic temperature to achieve complete hydroly-
sis of the polysaccharide polymer. Finally, the developed
optimised CZE method coupled with correction factors was suc-
cessfully applied to the determination of monosaccharide com-
position of three purified polysaccharides from E. sinica and their
corresponding partial acid hydrolysis products.
Accordingly, an understanding of the basic composition prop-
erties of polysaccharides from E. sinica could contribute to our
understanding of their practical applications. Many international
researches have suggested that polysaccharides of medicinal
plants composed of glucan boost the immune system and have
perfect anti-tumour activities (Yoon et al., 2008). In another
report, researchers found that sulphation of the neutral (1 →
3)-linked D-galactan from lac tree afforded derivatives with good
antioxidant capacities (Aguirre et al., 2009). In addition, more het-
eropolysaccharides have been isolated from herb plants with
obvious bioactivities. For example, Physalis alkekengi polysaccha-
ride consisting of Ara, Gal, Glc and GalA in the ratio of 2.6:3.6:2:1
can significantly reduce blood glucose levels and water intake,
and increase the body weight in alloxan-induced diabetic mice
(Tong et al., 2008). Therefore, investigation of the basic sugar
composition of the polysaccharides is particularly necessary to
better discover their functional properties for wide application in
the pharmaceutical industries.
There are several methods currently used for the separation of
carbohydrates, including thin-layer chromatography (TLC), gas
chromatography (GC), high-performance liquid chromatography
(HPLC) (Lee and Lin, 1996) and high-performance capillary elec-
trophoresis (HPCE) (El Rassi, 1996). Although TLC method is
simple and convenient, it is adequate only for qualitative analysis
due to its poor separation efficiency and poor precision for quan-
titative purposes (Lee and Lin, 1996; Zhang et al., 2003). The GC
method has good sensitivity and separation efficiency as a
popular tool, however; the sample preparation procedure before
the analysis is very labourious (Lee and Lin, 1996; Lv et al., 2009).
Because of the inherent hydrophilic nature of carbohydrates,
aqueous-based separation methods including HPLC and HPCE
are very suitable for their analysis (El Rassi, 1996). Presently, HPLC
is one of the most widely used and important separation
methods for the analysis of carbohydrates. Various kinds of
bonded phase and ion-exchange columns have been developed
for the HPLC method (Honda, 1984). A major drawback of HPLC is
its lack of separation efficiency. In addition, HPLC often needs a
longer time for column equilibration and the cost of a specialised
column is substantial. In this regard, HPCE seems to possess
several advantages over HPLC by offering higher separation effi-
ciencies, yielding shorter analysis time, requiring smaller sample
amounts and consuming lower amounts of expensive reagents
and solvents (Honda, 1996; Paulus and Klockow, 1996; El Rassi,
1999). However, a major difficulty that has to be surmounted for
HPCE is the separation of complex carbohydrate samples. The
lack of chromophores or fluorophores in the structure of
monosaccharides limits their sensitive detection in HPCE (Plocek
and Novotny, 1997; Boulis et al., 1998; Altria, 1999; Soga and
Serwe, 2000; Yang et al., 2007; Rovio et al., 2007). Therefore, car-
bohydrates are generally tagged with a suitable chromophore or
fluorophore to obtain highly sensitive detection. The reagent
1-phenyl-3-methyl-5-pyrazolone (PMP) is one of the popular
labels and reacts with reducing carbohydrate under mild condi-
tions, requiring no acid catalyst and causing no isomerisation
(Honda et al., 1989, 2003; Daotian and Roger, 1995; Andersen
et al., 2003; Zhang et al., 2003; Lv et al., 2009).
Experimental
Plant material
The dry stems of E. sinica were collected in March 2007 from Datong of
Shanxi Province, China and identified by Professor Zhenyue Wang of
Heilongjiang University of Chinese Medicine. A voucher specimen
(20070016) was deposited at Herbarium of Heilongjiang University of
Chinese Medicine, Harbin, P. R. China.
Reagents and standards
D-mannose (Man), L-rhamnose (Rha), D-glucose (Glc), D-galactose (Gal),
L-arabinose (Ara), D-xylose (Xyl), D-glucuronic acid (GlcUA), D-galacturonic
acid (GalUA) and sulphuric acid (H2SO4) were purchased from Sigma (St.
Louis, MO, USA). Trifluoroacetic acid (TFA) was obtained from Merck
(Darmstadt, Germany). 1-Phenyl-3-methyl-5-pyrazolone, purchased from
Beijing Reagent Plant (Beijing, China), was recrystallised twice from
chromatographic-grade methanol before use. Throughout the study
deionised water was used, prepared by a Milli-Q water system (Millipore,
MA, USA). The pH value of the electrolyte solution was measured with a
Sartorius PB-20 pH meter with Sartorius pH/ATC electrode (Sartorius, CO,
Germany) calibrated with commercial buffers of pH 7.00, 10.00 and 12.0
(Titrisol, Merck kGaA, Germany). All other chemicals were of the highest
grade available.
CZE equipment and conditions
The analysis of PMP-labeled monosaccharides was carried out on a P/ACE
MDQ capillary electrophoresis instrument (Beckman Coulter, Fullerton,
CA, USA). An integrated P/ACE 32 Karat Station (software version 4.0) was
used to perform the data collection and to control the operational vari-
ables of the system. Separation was carried out in an unmodified fused
silica capillary (48.5 cm ¥ 50 mm i.d., effective length 40 cm). Both the
capillary and samples were thermostatted to 25°C. The samples were
injected with a pressure of 0.5 psi for 5 s. The separation voltage was
raised linearly within 0.2 min from 0 to 20 kV. Detection was done with
direct UV monitoring using a photodiode array detector at wavelength
254 nm.
A new capillary from Yongnian Optical Fiber Factory (Hebei Province,
China) was activated by washing consecutively with each of 0.1 M phos-
phoric acid (15 min), water (10 min), 0.1 M sodium hydroxide (15 min), and
water (10 min). At the beginning of each working day, the capillary was
prewashed with 0.1 M phosphoric acid (2 min), water (2 min), 0.1 M
sodium hydroxide (2 min), water (2 min) and running buffer (2 min),
Although the principle of the complexation between borate
and vicinal hydroxy groups in sugars to form negative complexes
was widely used for the separation of carbohydrate compounds
(Hoffstetter-Kuhn et al., 1991; Guttman, 1997; David et al., 2005),
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Phytochem. Anal. 2011, 22, 103–111

Determination of Monosaccharide Composition of Acidic Hetero-Polysaccharides
respectively. Between analyses the capillaries were consecutively rinsed
with 0.1 M sodium hydroxide (1 min), water (1 min) and running buffer
(1 min).
and acid was removed in vacuum by addition of methanol repeatedly.
The hydrolysis sample was dialysed with distilled water for 48 h in a dialy-
sis sack (cut-off Mw 3500 Da), and then diluted the solution in the sack
with ethanol. After hydrolysis, the precipitate (a) and supernatant in the
sack (b) and the fraction out of sack (c) were dried, and then CZE analysis
was carried out.
Isolation, purification and acid hydrolysis
Isolation and purification. The dry stems of E. sinica were ground to
powder, and submitted to sequential extractions as follows: dry powders
(1.0 kg) were extracted three times with 10 vols of 95% EtOH under reflux
for 3 h each time to remove lipids. The residue was dried in air and then
extracted three times with 10 vols of distilled water for 24 h (each time) at
4°C. The combined aqueous extracts were filtered, concentrated 10-fold,
and 95% EtOH added to final concentration of 80%. The precipitate was
dissolved in 600 mL of water and deproteinated 15 times with 200 mL of
5:1 chloroform-n-butanol as described by Staub (1965). The resulting
aqueous fraction was extensively dialysed (cut-off Mw 3500 Da) against
tap water for 48 h and distilled water for 48 h and precipitated again by
adding a 5-fold volume of ethanol. After centrifugation, the precipitate
was washed with anhydrous ethanol and then dissolved in water and
lyophilised to yield the crude polysaccharide A (8.5 g) collected by cen-
trifugation (3000 rpm, 10 min, 20°C). A similar procedure was used with
10 vols of hot water (100°C, 3 h, three times) for above the residue after
cold water extraction. The fraction obtained was labeled crude polysac-
charide B (35.7 g).
Crude polysaccharide A (3.0 g) was dissolved in distilled water and
passed through two series connected resin columns [Amberlite FPA90-Cl
(Cl- form) and Amberlite IRC-84 (H+ form)] eluting with distilled water and
1.0 M NaCl to yield fractions Fr. A1 (1.2 g) and Fr. A2 (0.9 g), respectively. Fr.
A1 (1.0 g) was chromatographed over DEAE-Sepharose F. F eluting with
distilled water and 0.2 M NaCl to yield subfractions Fr. A1-1 (400.0 mg) and
Fr. A1-2 (380.0 mg), respectively. Fr. A1-2 (380.0 mg) was further purified
by gel-permeation chromatography on a high-resolution Sephacryl S-300
eluting with distilled water to afford ESP-A3 (310.0 mg). Fr. A2 (550.0 mg)
was chromatographed over DEAE-Sepharose F.F eluting with 1.0 M NaCl
to afford ESP-A4 (430.0 mg). Crude polysaccharide B (6.0 g) was dissolved
in distilled water and passed through two series-connected resin
columns [Amberlite FPA90-Cl (Cl- form) and Amberlite IRC-84 (H+ form)]
eluting with distilled water and 1.0 M NaCl to yield fractions Fr. B1 (2.3 g)
and Fr. B2 (2.2 g), respectively. Fr. B2 (1.0 g) was applied to a DEAE-
cellulose 52 column with 1.0 M NaCl to afford ESP-B4 (880.0 mg). Polysac-
charides in the eluted fractions were detected using the phenol–
sulphuric acid method and all NaCl elute fractions in column
chromatography were dialysed against distilled water for 48 h (cut-off Mw
3500 Da) and then freeze-dried.
Derivatisation procedure
Preparation of standard solution. The individual standard
monosaccharide (1.0 mM, each), and mix monosaccharide solution
(1.0 mM) were prepared with deionised water.Working standard solutions
were further obtained by appropriate dilution of the stock mix monosac-
charide sample solutions with deionised water. All sample solutions were
filtered through a 0.45 mm syringe filter and were degassed using an
ultrasonic bath for 2 min prior to use. All the solutions prepared were
stored in a refrigerator at +4°C.
Derivatisation procedure. PMP derivatisation of monosaccharides
was carried out as described previously with proper modification (Honda
et al., 1989, 2003; Daotian and Roger, 1995; Andersen et al., 2003; Zhang
et al., 2003; Lv et al., 2009). Aliquots of 200 mL of individual standard
monosaccharide, or mixed standard monosaccharide solutions, or the
hydrolysed polysaccharide samples were placed in the 2.0 mL centrifuge
tubes, then 0.5 M methanol solution (100 mL) of PMP and 0.3 M aqueous
NaOH (100 mL) were added to each. Each mixture was allowed to react for
30 min in a 70°C water bath, then cooled to room temperature and neu-
tralised with 100 mL of 0.3 M HCl. The resulting solution was performed on
liquid–liquid extraction with same volume of isoamyl acetate (twice) and
chloroform (one time), respectively. After being shaken vigorously and
centrifuged, the organic phase was carefully discarded to remove the
excess reagents. Then the aqueous layer was filtered through a 0.45 mm
membrane and diluted with water before HPCE analysis.
Method validation
The regression equations were calculated in the form of y = ax + b, where
y was the peak areas and x was the concentration of analytes. The signal-
to-noise ratios of 3:1 and 10:1 were used to establish LOD and LOQ,
respectively. The measurement of intra- and inter-day variability was uti-
lised to determine the precision of this newly developed method. The
intraday variation was determined by analyzing the same mixed standard
water solution five times within 1 day, while for inter-day variability test,
the solution was examined in triplicate for a consecutive three days. Sta-
bility of sample solution was tested at 0, 4, 8, 12, 24 and 48 h within 2 days.
All solutions were kept at 4°C before analysis. The analytes showed very
stable in water solution (RSD 3.45%) at 4°C during the tested period.
Identification of purity of polysaccharides. Each polysaccharide
(ESP-A3, ESP-A4 and ESP-B4) was dissolved in distilled water at a concen-
tration of 5 mg/mL and analysed on aWaters 2695 HPLC system equipped
with a quaternary solvent delivery system, a Waters 600 controller, two
Waters 600 pumps, a 2695 autosampler, a Waters 2695 column oven and
an Alltech ELSD 2000 detector. The separation was carried out on a
Shodex sugar KS-805 column (8.0 ¥ 300 mm, 17 mm) coupled with a
Shodex KS-G guard column (6 ¥ 50 mm, 7 mm). The isocratic elution was
employed using water with 0.5 mL/min at 30°C and the injection volume
was 10 mL. The drift tube temperature for ELSD was set at 116°C and the
nitrogen flow rate was 3.3 L/min for the determination of polysaccha-
rides. Their purities were over 98% by HPLC analysis.
Results and Discussion
CZE method development
We can only label carbohydrates as shown in Fig. 1, which usually
yields neutral sugar derivatives which become negatively
charged in aqueous basic solutions due to the partial dissociation
of the enolic hydroxyl group of the PMP tag. In this study, the
method development was achieved by optimising background
electrolyte pH and concentration, applied voltage and capillary
temperature.
Complete acid hydrolysis of polysaccharides. Each polysaccha-
ride sample (20 mg) was dissolved in 2 mL of 2.0 M H2SO4 in an ampoule
(5 mL). The ampoule was sealed under a nitrogen atmosphere and kept at
110°C to hydrolyse the polysaccharide into component monosaccharides
for 6 h, then cooled to room temperature and neutralised with 2 mL of
4.0 M NaOH. The reaction mixture was diluted to 5 mL with deionised
water and was centrifugalised at 1000 rpm for 5 min. Then the superna-
tant was ready for the following experiments.
Effect of pH
The pH of background electrolyte is a very important parameter
in capillary electrophoresis. It can affect the EOF rate and the
degree of ionisation of analytes. Hence, the effect of running
buffer pH (9.54, 10.02, 10.26) in 35 mM borate on the separation
of monosaccharides was investigated. For GlcUA and GalUA, a
Partial acid hydrolysis of polysaccharides. Each polysaccharide
sample (100 mg) was hydrolysed with 0.1 M TFA (3 mL) for 6 h at 100°C,
Phytochem. Anal. 2011, 22, 103–111
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Y. Xia et al.
H3C
O
N
N
CHO
OH
N
CH3
N
H
HO
H
CH
H3C
O
OH
OH
N
H
OH
O
N
H
HO
H
H
H
OH
OH
OH
CH2OH
H
PMP
Reducing sugar
CH2OH
Bis-PMP Derivative
Figure 1. Illustration of condensation reaction with PMP.
Figure 2. Electropherograms of PMP derivatives of eight standard monosaccharides at the optimal conditions including 35 mM borate at pH 10.02,
capillary temperature 25°C and applied voltage 20 kV, 0.2 mM each. Peak identities: 1, Xyl; 2, Ara; 3, Glc; 4, Rha; 5, Man; 6, Gal; 7, GlcUA; 8, GalUA.
Detection, 254 nm direct mode; injection pressure, 0.5 psi for 5 s; capillary, fused-silica 40.0/48.5 cm (Ldet/Ltot); separation temperature, 25°C.
steady baseline resolution could always be achieved at the inves-
tigated pH range whereas the separation of the neutral sugars
showed relatively strong dependence on the pH as compared
with the uronic acids. In particular, for Ara and Glc, assuming an
appropriate pH (9.54) had been chosen, Ara and Glc co-eluted.
If a higher pH (10.02) was adopted, good symmetry and resolu-
tion for each analyte were achieved. At pH 10.26, there was a
longer analysis time for GalUA. Thus pH 10.02 was chosen as the
optimal pH.
Selection of the capillary temperature and voltage
The temperature of the analysis may sometimes be important in
CZE, as fluctuations in the temperature may affect the viscosity of
the running buffer, leading to higher analyte electrophoretic
mobilities and shorter analysis time. The temperature changes
can also affect the pH of the buffer. In this case, 25°C is most
optimal. In addition, the effects of three voltage values (18, 20,
22 kV) on separation of the analytes were also studied.The results
showed that good resolution and acceptable migration time
were achieved at 20 kV.
Under the proposed conditions including 35 mM borate at pH
10.02, capillary temperature 25°C and applied voltage 20 kV, a
complete baseline resolution for carbohydrate derivatives was
achieved within 16.5 min (Fig. 2).
Effect of buffer concentration
The effect of different concentrations of borate (20, 35 and
50 mM) at pH 10.02 on the separation of analytes was studied. It
was found that Xyl, Ara and Glc were not well separated at 20 mM
of borate with poor resolution, but the best resolution and
highest theory plates were achieved at 35 mM of borate. Increas-
ing borate concentration, however, remarkably increased the
migration time of all analytes. The result was in agreement with
previous studies (Honda et al., 1989; Zhang et al., 2003). With a
further increase in buffer concentration, a large amount of Joule
heating affected separation efficiency. Taking both shorter run-
time and separation efficiency into consideration, the borate
buffer concentration was selected as 35 mM.
Method validation
As shown in Table 1, all calibration curves showed good linear
regression (r Ն 0.9779) within the test ranges. The LOD (S/N = 3)
and the LOQ (S/N = 10) were less than 1.95 and 5.85 mM for all
analytes, respectively. The measurement of intra- and inter-day
variability was utilised to determine the precision of this newly
developed method, which was investigated by the relative stan-
dard deviations (RSDs) of the migration time and peak area
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Determination of Monosaccharide Composition of Acidic Hetero-Polysaccharides
Table 1. Linearity of CZE method of different carbohydrate
Carbohydratea
Regression equationb
r
Linear range (mM)
LOD (mM)
LOQ (mM)
Xyl
Ara
Glc
Rha
Man
Gal
GlcUA
GalUA
y = 267.68 x - 709.79
y = 321.64 x + 332.25
y = 238.74 x - 251.21
y = 175.04 x - 1437.3
y = 342.59 x + 316.33
y = 387.90 x + 1352
y = 335.28 x + 1614.6
y = 347.73 x + 1597.5
0.9966
0.9988
0.9793
0.9971
0.9779
0.9840
0.9980
0.9953
5.00–400.00
5.00–400.00
5.00–400.00
5.00–400.00
5.00–400.00
5.00–400.00
5.00–400.00
5.00–400.00
1.40
1.09
1.63
1.95
1.22
0.98
1.47
1.12
4.20
3.27
4.89
5.85
3.66
2.94
4.41
3.36
a Quantitated with a calibration curve at = 254 nm.
b y and x stand for the peak area and the contentration (mM) of compounds, respectively.
Table 2. Precision of the retention time and peak area of analytes in the present method
Sugars
Intra-day precision (RSD%, n = 5)
Retention timea Peak areab
Inter-day precision (RSD%, n = 3)
Retention timea Peak areab
Xyl
Ara
Glc
Rha
Man
Gal
1.25
1.03
1.39
2.07
1.09
2.05
1.89
3.68
2.84
2.82
2.93
1.66
3.74
2.89
1.75
2.08
1.29
2.61
1.56
2.72
2.55
4.17
3.46
4.45
4.79
4.23
4.96
4.01
GlcUA
a Retention time ration between analytes and GalUA.
b Peak area ration between analytes and GalUA.
rations between analytes and GalUA. In order to overcome injec-
tion volume errors between injections, GalUA had to be chosen
as the reference peak for the precision experiments. The overall
intra- and inter-day variabilities of migration time rations were
less than 2.72%, while the overall intra- and inter-day variabilities
of peak area rations were less than 4.96% (Table 2).
dard curves. Thus the standard curves have to play an important
role in providing the linearity range for the tested sample
solutions.
In the process of structural analysis of polysaccharides, the
molar ratio of each monosaccharide in polysaccharides is one of
the most important parameters (Roy et al., 2008; Tong et al.,
2008). It is unnecessary to obtain the specific amount of each
monosaccharide because researchers just want to know the
molar ratios of each one (Dong et al., 2007; Yu et al., 2007; Hsieh
et al., 2008; Xu et al., 2009). Therefore, the molar ratio can be
calculated by the equation [Ri/n = fi/n ¥ (A’i/A’n)] rather than specific
amount of each sugar obtained. We believe that the former is
more favourable in the elimination of systematic errors.
Correction factors and molar ratios
The injection volumes in HPCE are typically small, 5–50 nL, and
maintaining precision between injections is difficult. Hence, an
internal standard is always used to overcome this problem.
However, there are no less than eight monosaccharides in
complex acidic hetero-polysaccharides, so it is not easy to find a
suitable internal standard to overcome this problem. Here we
used the standard curves coupled with relative correction
factors for determination of monosaccharide composition
(molar ratios) of acidic hetero-polysaccharides from E. sinica as
follows. The correction factor is shown in the equation: fi/n = (mi/
Ai)/(mn/An), where Ai and An are the values of the peak areas in
the standard monosaccharide, respectively; mi and mn are the
values of quantities (nmol) in the standard monosaccharide,
respectively. The molar ratio value is shown in the equation:
Ri/n = fi/n ¥ (A’i/A’n), where A’i/A’n is the ratio value of the corre-
sponding peak areas in the tested samples and fi/n is the relative
correction factor. However, fi/n should be a constant in both the
standard monosaccharides and the tested samples for the
monosaccharides i and n in the linearity range from the stan-
Optimisation of acid hydrolysis procedures
It is very difficult to achieve complete acidic hydrolysis of
the polysaccharide polymers that contain uronic acid groups
(Smidsrod et al., 1966; Ucar and Balaban, 2003). If they have more
uronic acid groups, it will be more difficult to achieve complete
hydrolysis. Therefore, optimisation of acidic hydrolysis conditions
is necessary for the accurate analysis of each polysaccharide. In
this study, sulphuric acid, an effective acid in degrading polysac-
charides, was chosen to hydrolyse polysaccharides into their
component monosaccharides (Kekos and Koukios, 1985; Yani-
shevskaya et al., 1987; Ucar and Balaban, 2003), and the factors
affecting hydrolysis of ESP-B4 with sulphuric acid were investi-
gated by changing the hydrolysis time, acid concentration, and
Phytochem. Anal. 2011, 22, 103–111
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Y. Xia et al.
Table 3. CZE analysis results of three purified acidic hetero-
polysaccharides and their corresponding partial acid hydroly-
sis products
Fractions
Molar ratios
Xyl Ara Glc Rha Man Gal GlcUA GalUA
ESP-A3
ESP-A4
ESP-B4
ESP-A3-a 4.3 3.6
ESP-A3-b
ESP-A3-c
6.8 7.5 1.0 14.0 13.7 22.3 10.2
3.8
2.2
50.0
1.0
—
5.1
1.2 4.1 1.0 5.1 1.6 17.3
1.0 4.5 1.0 2.0 1.0 5.5
3.1
1.5
4.2
—
—
4.8 8.9 7.7
—
—
1.8 1.1 1.9 1.0 3.7
12.6 2.0 7.1 1.0 15.7
—
ESP-A4-a 1.5 3.8
—
3.2 1.1 8.8
1.0
3.5
1.5
1.9
1.0
—
4.6
1.1
2.4
71.1
23.6
17.2
ESP-A4-b 1.2 5.5 1.0 4.3 3.7 32.4
ESP-A4-c 1.0 5.2 3.8 5.2 3.3 14.1
ESP-B4-a 3.1 7.3 1.0 3.7 1.4 11.6
ESP-B4-b 1.8 5.3
ESP-B4-c 1.9
—
—
—
—
1.8 6.4
— 1.0
—
a Precipitation in the sack.
b Supernatant in the sack.
c Fraction out of the sack.
ESP-B4, and such destruction was markedly accelerated at 3.0 M
H2SO4. The results indicated that uronic acid groups in polysac-
charide cannot achieve complete hydrolysis with 1.0 M H2SO4,
while most sugar moieties are destroyed with 3.0 M H2SO4. Thus
2.0 M H2SO4 was chosen as the optimal acid hydrolysis concentra-
tion. When we fixed acid concentration as 2.0 M H2SO4; the other
six neutral sugars and GlcUA also obtained better recoveries at
the hydrolysis time of 6 h (Fig. 3B). In addition, as shown in
Fig. 3(C), the release of most neutral sugars and GlcUA was sig-
nificantly dependent on the hydrolysis temperature and went
through a peak (110°C) and then decreased. Thus the optimal
hydrolysis temperature and time were chosen as 110°C and 6 h.
Analysis of purified polysaccharides from E. sinica
ESP-A3, ESP-A4 and ESP-B4 were isolated and purified by ion
exchange and gel-filtration chromatography. Determination of
their chemical structures and biological activities is in progress.
Here, in order to evaluate the applicability of the proposed
method, the isolated polysaccharide was hydrolysed with 2 M
H2SO4 for 6 h at 110°C, and PMP-labeled as described in the
Experimental section. The released monosaccharide derivatives
were analysed by the described CZE method under the optimised
conditions. Figure 4 shows typical chromatograms of E. sinica
polysaccharide samples and the detected contents are listed in
Table 3. As can be seen, the PMP derivatives of the component
monosaccharides released from ESP-A3, ESP-A4 and ESP-B4
could be still perfectly baseline separated from each other and
the component monosaccharides could be identified by compar-
ing with the chromatogram of the mixture of standard monosac-
charides (Fig. 2). The results showed that ESP-A3, ESP-A4 and
ESP-B4 were all typical acidic hetero-polysaccharides and were
composed of Xyl, Ara, Glc, Rha, Man, Gal, GlcUA and GalUA
in the molar ratios of 6.8:7.5:1.0:14.0:13.7:22.3:10.2:3.8
for ESP-A3, 1.2:4.1:1.0:5.1:1.6:17.3:3.1:2.2 for ESP-A4 and
1.0:4.5:1.0:2.0:1.0:5.5:1.5:50.0 for ESP-B4. Their corresponding
molar percentages (mol%) were 8.6, 9.4, 1.3, 17.7, 17.3, 28.1, 12.9
and 4.9% for ESP-A3, 3.4, 11.5, 2.8, 14.3, 4.6, 48.6, 8.7 and 6.2% for
Figure 3. (A) Molar ratios of GalUA (mol%) released from ESP-B4 sub-
jected to treatment with 1.0, 2.0, and 3.0 M H2SO4 at 110°C vs. different
hydrolysis times; (B) molar ratios of seven sugar (mol%) released from
ESP-B4 subjected to treatment with 2.0 M H2SO4 at 110°C vs. different
hydrolysis times; (C) molar ratios of seven sugar (mol%) released from
ESP-B4 subjected to treatment with 2.0 M H2SO4 at 6 h vs. different tem-
perature. Hydrolysis procedure and CZE conditions are as described in
Experimental section.
hydrolytic temperature to achieve complete hydrolysis of the
polysaccharide polymer.
To further confirm the influence of acid hydrolysis, the hydroly-
sis results of ESP-B4 with different molarities of acid (1.0, 2.0, and
3.0 M H2SO4) at 110°C for different times were characterised by
CZE coupled with correction factors for the determination of the
liberation rate of the GalUA. As depicted in Fig. 3(A), although the
release of GalUA from ESP-B4 was considerably accelerated with
the increase in H2SO4 concentration, the hydrolysis with 2.0 M
H2SO4 for 6 h gave the highest recovery of free GalUA, followed by
3.0 M H2SO4 for 4 h, and with 1.0 M H2SO4 for 8 h in decreasing
order. When the hydrolysis time was further prolonged, the
destruction rate of GalUA was greater than the release rate from
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Phytochem. Anal. 2011, 22, 103–111

Determination of Monosaccharide Composition of Acidic Hetero-Polysaccharides
Figure 4. Electropherograms of PMP derivatives of monosaccharides in ESP-A3 (A), ESP-A4 (B) and ESP-B4 (C). Hydrolysis conditions as 2.0 M H2SO4 at
110°C for 6 h. CZE condition and peak identities were described in Fig. 2.
ESP-A4, and 1.5, 6.8, 1.5, 3.0, 1.5, 8.3, 2.3 and 75.2% for ESP-B4,
respectively. It is clear that the predominant composition
monosaccharide in ESP-B4 was GalUA, with up to 75.2% (mol%)
of total identified carbohydrates, and 22.5% of total identified
carbohydrates were neutral sugars.
Partial acid hydrolysis is a very useful method in the structure
analysis of polysaccharide so that some detailed information on
structures can be provided (Dong et al., 2007; Tong et al., 2008).
The results from CZE analysis are shown inTable 3. Taking ESP-A3,
for example (Fig. 5), all the fractions including ESP-A3-a, ESP-A3-b
and ESP-A3-c recovered after partial acid hydrolysis of ESP-A3
and CZE analysis with pre-column derivatisation. The material
retained in the sack after dialysis of the partial acid hydrolysate
was named as ESP-A3-a, and it had the highest molecular
size of the three fractions. Its structure was proposed to
be closest to ESP-A3. The component of ESP-A3-a, in which
the ratio value of Xyl : Ara : Rha : Man : Gal : GlcUA : GalUA was
4.3:3.6:4.8:8.9:7.7:4.2:1.0, indicated that Man and Gal may be the
backbone of the structure of ESP-A3, and Xyl, Ara, Rha, GlcUA and
GalUA may be close to this backbone. The analysis results for
ESP-A3-b and ESP-A3-c indicated that the branched structure of
ESP-A3 was composed of Ara, Glc, Rha, Man, Gal and GalUA
because these sugar residues could be obtained easily after
hydrolysis.
Phytochem. Anal. 2011, 22, 103–111
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Y. Xia et al.
Figure 5. Electropherograms of PMP derivatives of partial acid hydrolysis products for ESP-A3. (A) Precipitation in the sack (ESP-A3a); (B) supernatant
in the sack (ESP-A3b); (C) fraction out of sack (ESP-A3c). Hydrolysis conditions as 2.0 M H2SO4 at 110°C for 6 h. CZE condition and peak identities were
described in Fig. 2.
other complex plant polysaccharides and some real-life samples,
such as fruit juices, wines and brandies.
Conclusion
The results reported in this paper demonstrate that the proposed
CZE method coupled with correction factors provides an excel-
lent example for the determination of molar ratios among
Acknowledgements
Our work was supported by the Major State Basic Research Devel-
opment Program of China (973 Program 2006CB504708) and the
National Natural Science Foundation of China (no.30973870).
monosaccharides of complex acidic hetero-polysaccharide from
E. sinica. Such information can provide a scientific evidence for
further exploitation of the chemical structure and bioactivity of
complex acidic E. sinica polysaccharides. In addition, it would
facilitate the use of E. sinica polysaccharide in pharmaceutical and
other technical applications, which would contribute to the sus-
tainable use of E. sinica plant resources. Meanwhile, it can also be
applied to routine analysis of monosaccharide composition in
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