Analysis of compositional carbohydrates in polysaccharides and foods by capillary zone electrophoresis

Article abstract of DOI:10.1016/j.carbpol.2012.01.039

A simple, sensitive and specific analytical method has been established for high efficient separation and simultaneously high sensitive determination of thirteen reducing carbohydrates, including aldohexose and aldopentoses as well as maltose and lactose. Reducing carbohydrates were derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP), separated by capillary zone electrophoresis (CZE) with use of methanol modifier in 175 mM borate buffer (pH 11.0), and detected by UV at 245 nm. The optimized CZE method was found to be well suited to examine the compositional reducing monosaccharides of the isolated polysaccharides from Lycopus lucidus and Jujube, and free mono- and disaccharides in beer and milk. Quantitative recoveries of the compositional carbohydrates in the samples were in the range of 93.2-104.0%, and RSD values ranged from 2.9% to 4.9%. The developed CZE method proves to be precise and practical for quality control of reducing carbohydrates, and will provide more highly efficient separation in food analysis in the future.

Full text of DOI:10.1016/j.carbpol.2012.01.039

Carbohydrate Polymers 88 (2012) 754–762
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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Analysis of compositional carbohydrates in polysaccharides and foods
by capillary zone electrophoresis
Tong Wanga, Xingbin Yangb, Dongying Wangb, Yadong Jiaob, Yu Wanga, Yan Zhaoa,∗
a School of Pharmacy, Fourth Military Medical University, Xi’an 710032, China
b Key Laboratory of Ministry of Education for Medicinal Resource and Natural Pharmaceutical Chemistry, College of Food Engineering and Nutritional Science, Shaanxi Normal
University, Xi’an 710062, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple, sensitive and specific analytical method has been established for high efficient separation
and simultaneously high sensitive determination of thirteen reducing carbohydrates, including aldo-
hexose and aldopentoses as well as maltose and lactose. Reducing carbohydrates were derivatized with
1-phenyl-3-methyl-5-pyrazolone (PMP), separated by capillary zone electrophoresis (CZE) with use of
methanol modifier in 175 mM borate buffer (pH 11.0), and detected by UV at 245 nm. The optimized
CZE method was found to be well suited to examine the compositional reducing monosaccharides of
the isolated polysaccharides from Lycopus lucidus and Jujube, and free mono- and disaccharides in beer
and milk. Quantitative recoveries of the compositional carbohydrates in the samples were in the range
of 93.2–104.0%, and RSD values ranged from 2.9% to 4.9%. The developed CZE method proves to be pre-
cise and practical for quality control of reducing carbohydrates, and will provide more highly efficient
separation in food analysis in the future.
Received 8 November 2011
Received in revised form 10 January 2012
Accepted 14 January 2012
Available online 21 January 2012
Keywords:
Monosaccharides
Disaccharides
Polysaccharides
Beer and milk
Capillary zone electrophoresis
© 2012 Elsevier Ltd. All rights reserved.
1. Introduction
natural oligo- or polysaccharide preparations can serve as incen-
tive for the greater consumption of functional mono-, oligo-
Carbohydrates are the major source of energy in the human diet
with a consumption ranging from 40 to 80% of total energy require-
ments. In addition to the provision of energy, it is becoming clear
that carbohydrates play a essential role in the process of life, and are
found to have a wide range of effects on human physiology includ-
ing effects on satiety and gastric emptying, control of blood glucose,
insulin metabolism, and serum cholesterol and influencing colonic
microflora and gastrointestinal processes such as laxation and fer-
mentation (Muir et al., 2009). Carbohydrates as the major classes of
importance to human nutrition are a diverse and complex family of
compounds, including monosaccharides, oligosaccharides and the
polysaccharides (starch and nonstarch polysaccharides) (Molnár-
Perl, 2000; Muir et al., 2009). Clearly, a greater understanding of the
physiological effects of carbohydrates will only be possible with the
separation, identification, and quantification of the different classes
of carbohydrates present in foods.
and polysaccharides with beneficial effects on health (Lachman,
Havrland, Fernández, & Dudjak, 2004; Parr & Bolwell, 2000; Wang
et al., 2002). At the same time, lactose and maltose as common
reducing disaccharide sweeteners are widely applied to many foods
(Paulus & Klockow, 1996). Therefore, the monitoring of reducing
monosaccharides and oligosaccharides which frequently occur in
food samples is very important for in the fields of nutrition, biology
and food science.
The separation of complex mono- and oligosaccharide mixtures
is a challenging analytical task for numerous reasons. At first, the
analytes show differences only in the configuration of the hydroxyl
groups and possess therefore very similar separation properties
(Frügel, Carle, & Schieber, 2005; Yang, Zhao, Wang, Wang, & Mei,
2005). Furthermore, they generally lack UV-absorbing or fluores-
cent functions as well as readily ionizable charged groups. For
these reasons, many different analytical methods (Currie & Perry,
2006; Rumpel & Dignac, 2006; Sanz, Martínez-Castro, & Moreno-
Arribas, 2008; Tetsuo, Zhang, Matsumoto, & Matsumoto, 1999;
Yang & Ding, 2000), especially those based on high-performance
liquid chromatographic (HPLC) techniques using various different
detectors, have been assayed (Blanco, Muro, & Gutierrez, 2004;
Lopes & Gaspar, 2008; Nogueira, Silva, Ferreira, & Trugo, 2005;
Panagiotopoulos, Sempere, Lafont, & Kerherve, 2001). Although
HPLC coupled with refractive index detector (RID) has been
accepted as one of the major techniques for the direct analysis
Monosaccharides are the most basic units of biologically impor-
tant carbohydrates, which mainly include the reducing hexoses
(glucose, glucosamine, galactose, mannose, etc.) and pentoses
(ribose, arabinose, etc.). In this regards, a understanding of free
or conjunct monosaccharides frequently occur in foodstaffs or
∗
Corresponding author. Tel.: +86 29 84774473; fax: +86 29 84776945.
E-mail address: yanzhao@fmmu.edu.cn (Y. Zhao).
0144-8617/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2012.01.039

T. Wang et al. / Carbohydrate Polymers 88 (2012) 754–762
755
of carbohydrates, it has many disadvantages, such as lack of sen-
2.2. Extraction of the plant polysaccharides
sitivity and incompatibility with gradient elution (Yang & Ding,
2000). To overcome the high detection limit of carbohydrates,
most of the methods described in the literatures rely on some
sort of derivatization technique since the introduction of a chro-
mophoric moiety can substantially improve the sensitivity, and a
suitable tagging of reducing saccharides usually results in enhanced
resolution and less matrix disturbances (Honda, Isawe, Makino,
& Fujiwara, 1989). The reagent 1-phenyl-3-methyl-5-pyrazolone
(PMP) with strong UV absorbance at 245 nm is one of the pop-
ular labels for the HPLC method that can react with reducing
carbohydrates under mild conditions, requiring no acids cata-
lyst and causing no desialylation and isomerization (Fu & O’Neill,
1995; Kakehi, Ueda, Suzuki, & Honda, 1993; Yang, Zhao, & Lv,
2007).
As an alternative to HPLC, capillary zone electrophoresis (CZE)
is shown to be a powerful separation technique which pro-
vides high-resolution results and is becoming a standard tool
for the analysis of many compounds (Cortacero-Ramírez, Segura-
Carretero, Cruces-Blanco, & Fernández-Gutiérrez, 2003; Morales,
2002; You et al., 2008). Recently, we have also described a CZE
method for separation of 8 monosaccharides, where the intro-
duction of a chromophic PMP into the reducing monosaccharide
molecule can be easily achieved by chelation of the monosac-
charides with a suitable ion of borate (Yang, Zhao, Zhou, et al.,
2007; Yang, Zhao, Yang, & Ruan, 2008). Although methods for
the separation of the sugars in samples have been examined by
a great quantity of researchers, only 7–8 monosaccharides were
usually baseline separated, which significantly affected the ana-
lytical accuracy (Gao, Araiab, Lecka, & Emmerb, 2010; Ijiri et al.,
2010; Rovio, Simolin, Koljonen, & Siren, 2008; Santos, Duarte, &
Esteves, 2007; Yang, Zhao, Zhou, et al., 2007). Therefore, the poten-
tial CZE method needs to be further challenged for the accurate
identification of complex mono- and oligosaccharide ingredi-
ents.
The plant polysaccharide extracts were isolated by hot-water
extraction and ethanol precipitation as previously described (Tian,
Zhao, Guo, & Yang, 2010). In brief, the dried powder of L. lucidus
and Chinese jujube (150 g) were defatted with 95% alcohol and
then extracted with distilled water (w/v, 1:10) at 80 ◦C for three
times, 1 h each time. After extraction, the combined extracts were
pooled, concentrated to 30% of the original volume under a reduced
pressure and then centrifuged at 3000 rpm for 15 min. The super-
natant was collected and 3 volume of 95% alcohol was added slowly
by stirring to precipitate the polysaccharide, and then kept at 4 ◦C
overnight and finally the polysaccharide pellets were obtained
by centrifugation at 4000 rpm for 15 min and repeatedly washed
sequentially with possibly less amounts of ethanol, acetone and
ether, respectively. The refined polysaccharide pellets were com-
pletely dissolved in appropriate volume of distilled water and
intensively dialyzed for 3 days against distilled water (cut-off Mw
8000 Da), and then the retentate portion was deproteinized by a
freeze–thaw process (FD-1, Henan Yuhua Instrument Co., China)
for repeating 10 times followed by filtration. Finally, the filtrate
was lyophilized to yield brownish water-soluble polysaccharides.
2.3. Hydrolysis of the polysaccharides
22.4 mg of the L. lucidus polysaccharide or 19.8 mg of Jujube
polysaccharide sample was placed in 2 mL of 2 M TFA in an ampoule
(10 mL). The ampoule was sealed under a nitrogen atmosphere and
then kept in an oil bath at 110 ◦C for 8 h. The resulting reaction solu-
tion was cooled to room temperature, and was further centrifuged
at 1000 rpm for 5 min. The supernatant was collected and dried to
remove the excess TFA under a stream of nitrogen gas. The sample
solution was added with 1.0 mL distilled water and then ready for
the further experiments.
The aim in this work was to present an optimized, powerful
and robust CZE analytical technique capable of simultaneously
separating thirteen saccharides, including a set of stereoiso-
mers of aldopentose and aldohexose as well as reducing lactose
and maltose which frequently occur in natural plants or food
industry. Furthermore, the developed CZE method has been
successfully applied to the analysis of the compositional monosac-
charides released from the polysaccharides from herbal Lycopus
lucidus Turcz. and Jujube as well as the analysis of free
monosaccharides and disaccharides in food such as beer and
milk.
2.4. Preparation of buffer and standard solution
The stock solution of 400 mM boric acid was prepared in water.
The running buffers were prepared by diluting the stock solution
to the appropriate concentrations and were adjusted to the desired
pH values with sodium hydroxide. The stock solution of standard
monosaccharides and disaccharides (10 mM) were prepared and
diluted with ultrapure water. The sample solutions were filtered
through a 0.22 ␮m syringe filter and were degassed using an ultra-
sonic bath for 2 min prior to use. All the solutions prepared were
stored in the dark at 4 ◦C until being used.
2.5. PMP derivatization procedures for reducing monosaccharides
and disaccharides
2. Materials and methods
2.1. Materials and chemicals
The PMP derivatization procedure was carried out as our
described previously (Lv et al., 2009; Zhang, Xu, Zhang, Zhang, &
Zhang, 2003). Briefly, 20 ␮L aqueous of standard monosaccharides
or disaccharides or the hydrolyzed samples of the polysaccharides
was spiked with 400 ␮L of 0.3 M aqueous NaOH and 400 ␮L 0.5 M
PMP–methanol solution. It was showed no glucosamine existed in
all the tested samples, and thus glucosamine as an internal stan-
dard was added to each sample before the derivatization. After
the solution was shaken for 10 second, the mixture was allowed
to react for 30 min at 70 ◦C, then cooled to room temperature
and neutralized with 400 ␮L of 0.3 M HCl. The resulting solution
was extracted with chloroform, and the organic phase was dis-
carded after shaking, and the extraction process was repeated for
3 times. Finally, the aqueous layer was filtered through a 0.22 ␮m
membrane for CZE analysis. The reagent solution was freshly pre-
pared before derivatization, and for the beer and milk analysis, the
The aerial parts (leaves and stems) of L. lucidus Turcz., and Chi-
nese jujube (Zizyphus jujuba) were harvested from the Northern
countryside region of Shaanxi province, China. The samples were
thoroughly washed with water, air-dried and finely pulverized into
a powder. The milk and beer samples were purchased at local mar-
kets. d-Mannose, d-ribose, d-glucose, l-rhamnose, d-glucuronic
acid, d-galacturonic acid, glucosamine, d-xylose, d-galactose, l-
arabinose, d-fucose, maltose, and lactose were obtained from
Merck (Darmstadt, Germany). 1-Phenyl-3-methyl-5-pyrazolone
(PMP) and trifluoroacetic acid (TFA) were the products of Beijing
Reagent Plant (Beijing, China). HPLC grade methanol was purchased
from Honeywell (USA). Water was purified on a Milli-Q system
(Millipore, Bedford, MA), and all of the other chemicals were of
analytical grade.

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T. Wang et al. / Carbohydrate Polymers 88 (2012) 754–762
samples were degassed before being derivatized and diluted by
1:20 with water and passed through a 0.22 ␮m membrane filter
prior to injection.
arabinose, glucose, ribose, rhamnose, fucose, galactose, mannose,
glucuronic acid and galacturonic acid (Fig. 1E). As a result, pH 11.0
was chosen as the preliminary optimized pH value for further inves-
tigated.
Borate concentration is another important parameter in our sep-
aration procedure, and therefore, it must also be optimized. Herein,
the borate was not only used as a background electrolyte but also
exerted complexation with the tested carbohydrates, which can
influence both electro-osmotic flow and electrophoretic mobility,
and may also affect the symmetry of the peaks in CZE (Zhao, Yang,
Sun, Jiang, & Zhang, 2006). As expected, different borate concen-
trations (125, 150, 175, 200 and 250 mM) at pH 11.0 were shown
to have a greater impact on the resolution and tm of the reducing
carbohydrates. As shown in Fig. 2A–E, the best resolution versus
the lowest tm was obtained at 175 mM borate buffer at pH 11.0. It
was also noted that when the excessive concentrations of borate
high than 200 mM, it was troublesome to obtain a stable base-
line, and the electric field in the capillary may become distorted
because of Joule heating (Cross & Cao, 1998), leading to an even
worse separation and irregular peak shapes, and the reproducibil-
ity of the separation was poor (Frazier, Inns, Dossi, Ames, & Nursten,
2000). As a result, 175 mM borate buffer at pH 11.0 was chose to
accomplish a good compromise.
In this study, it was difficult to achieve a fully simultaneous
CZE baseline separation for a large number of the PMP-labeled
nine aldohexose and two aldopentoses as well as maltose and
lactose only via optimizing borate buffer concentrations and pH
values as described above. Therefore the separation condition
needed to be further optimized to achieve a robust CZE approach
for improving the accuracy for sugar composition analysis. It is
well known that the addition of an organic modifier can either
improve or deteriorate the CZE separation (Egeberg & Bergli, 2002;
Koike, Kitagawa, & Otsuka, 2007). Interestingly, a steady improve-
ment in the resolution of the tested reducing carbohydrates was
observed with increased proportions of methanol in the increasing
range of 0.5–4% (v/v), where the mobility of the PMP–carbohydrate
derivatives increased concomitantly (Fig. 3A–E). The best separa-
tion was achieved at 4% methanol under the condition of 175 mM
borate buffer at pH 11.0, and thirteen PMP-labeled carbohydrate
derivatives were baseline separated as respect. Fig. 3E shows a
simultaneous separation of the tested thirteen standard carbo-
hydrates as PMP derivatives, and the peaks were identified in
the order of glucosamine, maltose, lactose, xylose, arabinose, glu-
cose, ribose, rhamnose, fucose, galactose, mannose, glucuronic
acid, and galacturonic acid. Taking all effects into account, pH
11.0, 175 mM borate buffer containing 4% methanol at 25 ◦C
and 15 kV are generally recommended as the optimum condi-
tion.
2.6. Apparatus and electrophoretic procedures
Separation and analysis were carried out with a P/ACE MDQ cap-
illary electrophoresis instrument (Beckman-Coulter, Fullerton, CA,
USA). Detection was done with direct UV monitoring. Data acqui-
sition and processing were carried out with Beckman System Gold
software. All capillaries (fused silica) had an internal diameter of
50 ␮m and were 58.5 cm in total length (effective length 48.5 cm).
The new capillaries (from Yongnian Optical Fiber Factory, Hebei),
were conditioned by rinsing with 1 M sodium hydroxide for 20 min,
0.1 M hydrochloric acid (20 min), and deionized water (30 min). At
the start of each working day, the capillary was washed with water
for 5 min, 0.1 M HCl for 5 min, 0.1 M NaOH for 10 min and water
for 5 min. Between analyses the capillaries were rinsed with 0.1 M
NaOH for 3 min followed by water for 2 min and then equilibrated
with running buffer at a pressure of 20 psi for 3 min. Samples were
introduced by pressure injection mode at 0.5 psi for 5 s into the cap-
illary. At the end of the working day, the capillary was washed with
water for 5 min and dipping the capillary end in a vial containing
water. Absorbance was detected with a photodiode array detector
set at 245 nm.
3. Results and discussion
3.1. CZE separation profile of the PMP-labeled reducing
carbohydrates
For quantification of reducing saccharides by selective, sensi-
tive, and robust CZE methods, pre-column derivatization is among
the most popular approaches. In the present case, reductive 1-
phenyl-3-methyl-5-pyrazolone (PMP) as the strong UV-active tag
was chosen and an excess of PMP label was used to guaran-
tee optimal conversion into the labeled saccharide derivatives,
which works in a simplified aqueous methanol system, and affords
stable PMP derivative as our previously described (Yang et al.,
2005). In our study, the CZE separation of the standard PMP-
carbohydrate derivatives was performed in alkaline borate buffers,
where analytes can be transformed into negatively charged com-
plexes with borate (Gro˝ ßl, Harrison, Kamla, & Kenndler, 2005;
Wang & Fang, 2004; Zhao et al., 2007), and detection was carried
out at 245 nm and identified by spiking samples with stan-
dards.
To guarantee the ionization of the analytes, we made a detailed
study of the pH of the running borate buffer over a range between
pH 9 and 11.2 under a constant condition of 175 mM borate buffer,
applied voltage 15 kV and capillary temperature 25 ◦C, and the
effects are shown in Fig. 1. It was found that with the buffer pH
increased, the migration time (tm) of the tested nine aldohexose
and two aldopentoses as well as maltose and lactose tagged with
PMP were prolonged and the resolution was obviously increased
initially, and when the pH was higher than 11.0, the resolution
decreased and a further deteriorated peak shape was observed
(Fig. 1F). As illustrated in Fig. 1A–D, at the pH 9.0, 9.5, 10.0 and
10.5, all the tested thirteen reducing monosaccharides and disac-
charides can not reach baseline separation. As the pH increased to
11.0, the resolutions of all the analytes were obviously improved,
and it can be identified thirteen significant peaks in spite of ribose
and glucose, fucose, mannose and galactose can not still reach com-
pletely baseline separation. It seems to show that the order of the
peaks remained changeless, and the elution of the PMP-labeled sac-
charides was in a order of glucosamine, maltose, lactose, xylose,
3.2. Validation of the CZE analysis
The CZE method was validated by determining linearity, sensi-
tivity and repeatability. The linearity was verified by the analysis of
six points in the range of 3.0–137.0 ␮M of standard sugars (maltose,
lactose, xylose, arabinose, glucose, ribose, rhamnose, galactose,
mannose, fucose, glucuronic acid and galacturonic acid), each point
of the calibration plot was repeated three times in an independent
solution, and the linear regression parameters of the calibration
curves are shown in Table 1. As a consequence, the CZE assay had
excellent linearity between Y (peak area ratio of the analytes with
internal standard) and X (concentration of the standards) from
3 ␮M to 137 ␮M with the correlation coefficients (r) in the range of
0.9902–0.9985. Furthermore, limit of detection (LOD) of each tested
analytes at the signal-noise ratio (S/N) of 3 were determined to be
ranged from 1.3 to 1.9 ␮mol by successive dilutions of PMP-labeled

T. Wang et al. / Carbohydrate Polymers 88 (2012) 754–762
757
Fig. 1. Effects of pH values on the separation of thirteen PMP-labeled standard reducing carbohydrates by CZE. Analytical conditions: fused-capillary 58.5 cm (48.5 cm to
the detector) × 50 ␮m i.d., 175 mM borate buffer, applied voltage 15 kV, capillary temperature 25 ◦C, UV detection at 245 nm. The pH of borate buffer: (A) pH 9.0, (B) pH 9.5,
(C) pH 10.0, (D) pH 10.5, (E) pH 11.0, and (F) pH 11.2. Peaks: 1. glucosamine, 2. maltose, 3. lactose, 4. xylose, 5. arabinose, 6. glucose, 7. ribose, 8. rhamnose, 9. fucose, 10.
galactose, 11. mannose, 12. glucuronic acid, and 13. galacturonic acid.
carbohydrate mixture (Table 1), indicating that the sensitivity of the
method was satisfactory.
As shown in Table 2, the method repeatability was also deter-
mined by estimating the method precision expressed as coefficient
of variation (CV%). CV% was obtained by the analysis of both
intra-day variability and inter-day variability of tm and corrected
peak areas (A/t), which was estimated by making five repetitive
injections of a standard mixture solution (10 ␮M for each analyte)
under the same CZE conditions. The results showed that the CV val-
ues in intra-day were less than 3.7% for tm and 4.1% for A/t, and the

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T. Wang et al. / Carbohydrate Polymers 88 (2012) 754–762
Table 1
Migration time (tm), regression analysis and limit of detection (LOD) of the proposed CZE method.
Carbohydrates
tm (min)a
Equation, Y = aX + bb
Correlation coefficient
LOD (␮M)c
a
b
Maltose
Lactose
Xylose
Arabinose
Glucose
Ribose
Rhamnose
Fucose
Galactose
Mannose
Glucuronic acid
Galacturonic acid
18.856
22.387
23.207
24.617
26.104
27.790
29.725
31.304
33.108
34.687
49.007
63.899
118.64
121.31
177.65
227.01
181.29
211.42
173.73
190.50
244.06
251.29
218.89
270.28
−0.0175
−0.0959
−0.0285
−0.0639
−0.0952
−0.0531
−0.0801
−0.0202
−0.1542
−0.0885
−0.1304
−0.2463
0.9948
0.9985
0.9902
0.9908
0.9915
0.9936
0.9965
0.9943
0.9918
0.9941
0.9913
0.9974
1.5
1.5
1.5
1.4
1.3
1.3
1.6
1.6
1.3
1.3
1.9
1.9
a
tm: migration time.
b
The Y and X are peak area ratio of the analytes to internal standard (glucosamine) and concentration of the analytes (3–137 ␮M), respectively.
LOD correspond to concentrations giving a signal-to-noise ratio of 3.
c
Fig. 2. Effects of borate concentrations at pH 11.0 on the separation of thirteen PMP–carbohydrates. Borate concentration: (A) 125 mM, (B) 150 mM, (C) 175 mM, (D) 200 mM,
and (E) 250 mM. CZE conditions and peak assignments as described in Fig. 1.

T. Wang et al. / Carbohydrate Polymers 88 (2012) 754–762
759
Table 2
Precision of the migration time (tm) and corrected peak area (A) of the tested carbohydrates by the developed CZE method.
Carbohydrates
Intra-day precision (CV%, n = 5)
Inter-day precision (CV%, n = 5)
tm
A
tm
A
Maltose
Lactose
Xylose
Arabinose
Glucose
Ribose
Rhamnose
Fuccose
Galactose
Mannose
Glucuronic acid
Galacturonic acid
1.0
1.0
2.0
1.9
3.3
3.5
2.2
2.0
2.6
2.4
3.7
3.1
1.0
1.2
1.8
1.4
4.1
3.7
1.4
2.7
1.5
2.9
2.8
2.3
1.5
1.3
1.8
1.7
2.8
3.0
2.0
2.1
2.4
3.1
4.0
3.5
1.3
2.4
3.2
2.8
4.4
4.2
2.3
3.2
3.6
5.1
4.6
5.4
Fig. 3. Influence of methanol modifier on the separation of thirteen PMP-labeled standard carbohydrates. Methanol concentrations in borate buffer: (A) 0.5%, (B) 1%, (C) 2%,
(D) 3%, and (E) 4%. CZE conditions and peak assignment as mentioned in Fig. 1.

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T. Wang et al. / Carbohydrate Polymers 88 (2012) 754–762
inter-day CV values were less than 5.4% for all variables, respec-
tively. The validated results indicated that this method was
accurate and reliable.
3.3. Application to real polysaccharide samples derived from L.
lucidus and Jujube
The present study was designed to develop a repeatable and
accurate CZE analytical method for the quantification of most
component reducing monosaccharides and disaccharides. To eval-
uate the applicability of the proposed CZE method, the two
isolated polysaccharides from L. lucidus (LPs) and Jujube (JPs) were
hydrolyzed with TFA, dried and PMP-labeled as described in Sec-
tion 2 and finally the released monosaccharide as PMP derivatives
were analyzed by the described CZE method under the optimized
conditions using glucosamine as internal standard. The typical elec-
tropherograms of the hydrolyzed monosaccharides from LPs and
JPs are shown in Fig. 4B and C, respectively. Peaks were identi-
fied by comparing their tm with those of the standards spiked in
the samples (Fig. 4A). As can be seen in Fig. 4B and Table 3, LPs
were composed of xylose, arabinose, glucose, ribose, rhamnose,
galactose, mannose, glucuronic acid, and galacturonic acid in the
molar contents of 31.5, 27.5, 22.5, 17.5, 6.0, 71.5, 13.5, 5.5 and
25.0 ␮M, respectively, and their corresponding mole percentages
were 14.3%, 12.5%, 10.2%, 7.9%, 2.7%, 32.4%, 6.1%, 2.5% and 11.3%,
respectively. JPs consisted of arabinose, glucose, ribose, rhamnose,
and galactose in the molar contents of 8.4, 103.2, 44.1, 5.7, and
19.8 ␮M, and their corresponding mole percentages were 4.6%,
57.1%, 24.3%, 3.1% and 10.9%, respectively (Fig. 4C and Table 3). The
results showed that LPs and JPs were the typically acidic and neutral
heteropolysaccharide with significantly differentiable properties in
monosaccharide composition, respectively.
3.4. Analysis of reducing mono- and oligosaccharides in beer and
milk
Fig. 4D shows a typical electropherogram of the compositional
reducing monosaccharides and disaccharides tagged with PMP
derived from beer sample. Maltose and glucose were found in
beer in the molar content of 16.3 and 6.6 ␮M, respectively, indicat-
ing that the predominantly composition reducing carbohydrates in
beer was maltose. As can be seen in Fig. 4E, milk included maltose,
lactose and glucose in the molar content of 56.6, 21.2 and 6.1 ␮M.
Furthermore, recovery experiments were performed in order to
investigate the accuracy of the CZE method. Known amounts of
each standard monosaccharides or disaccharides with the samples
were added to the hydrolyzed polysaccharide samples or food sam-
ples, and the resulting spiked sample was subjected to the entire
analytical sequence. Each solute was spiked at a close concentration
with the sample and recoveries were calculated based on the differ-
ence between the total amount determined in the spiked samples
and the amount observed in the non-spiked samples. All analyses
were carried out in triplicate. The results show that the recoveries of
all the nine monosaccharides in the polysaccharide samples ranged
between 93.2 and 104.0% and the RSD values fell within 3.0–4.9%
(Table 3). In the analysis of beer and milk, the recovery was between
94.0% and 102.5% (Table 3), and the RSD values fell within 2.9–3.9%.
These results also showed that the proposed CZE method was not
only suitable for the determination of the component reducing
monosaccharides in polysaccharides, but also practical for the anal-
ysis of the reducing mono- and oligosaccharides in beer and milk.
The major aim of this study was to develop an analytical tech-
nique, based on CZE with PMP-tagged UV detection, which could be
used to quantify the major reducing carbohydrates frequently occur
in foods. Although there is some published information in this area
(Honda, Suzuki, & Taga, 2003; Sjo˝ berg, Adorjan, Rosenau, & Kosma,
Fig. 4. Typical electropherograms of thirteen standard reducing carbohydrates (A),
and the compositional mono- and di-saccharides of L. lucidus polysaccharides (LPs,
B), Jujube polysaccharides (JPs, C), beer (D), and milk (E) under the optimum con-
ditions of pH 11, 175 mM borate buffer containing 4% methanol at applied voltage
15 kV and capillary temperature 25 ◦C and detection wavelength at 245 nm. The
polysaccharide samples were hydrolyzed with TFA and then were labeled with PMP,
and food samples were diluted 1:20 with distilled water, and then was labeled with
PMP as described in Section 2. CZE conditions and peak assignment as described in
Fig. 1.
2004), no previous studies have attempted to quantify all of these
carbohydrates. To our knowledge, this is the first report showing a
CZE separation method for the thirteen aldohexose, aldopentoses,
maltose and lactose. It is well known that the separation and iden-
tification of a large number of carbohydrates is a complex and
challenging area of research. One of the major problems is the risk
of coelution of the different sugars. Here, a combined optimiza-
tion techniques of borate buffer with methanol modifier provided
a good separation of most reducing monosaccharides and disac-
charides of interest in foods. The developed CZE method has been
tested with quality control samples of polysaccharides, beer and
milk samples and has been found to provide accurate and precise
results.

T. Wang et al. / Carbohydrate Polymers 88 (2012) 754–762
761
Table 3
CZE determination of the component carbohydrates in the sample of polysaccharide and food and their recovery analysis (n = 3).
Samples
LPs
Components
Content (␮M)
Spiked (␮M)
Found (␮M)
Recovery (%)
RSD (%)
Xylose
Arabinose
Glucose
31.5
27.5
22.5
17.5
6.0
71.5
13.5
5.5
20.0
20.0
20.0
20.0
5.0
50.0
20.0
5.0
52.0
47.1
43.2
36.9
11.2
119.3
33.8
10.2
44.1
102.5
98.0
103.5
97.0
104.0
95.6
101.5
94.0
4.9
4.0
4.1
3.6
3.0
4.1
3.6
4.4
4.6
Ribose
Rhamnose
Galactose
Mannose
Glucuronic acid
Galacturonic acid
25.0
20.0
95.5
JPs
Arabinose
Glucose
Ribose
Rhamnose
Galactose
8.4
103.2
44.1
5.7
5.0
50.0
50.0
5.0
13.2
149.8
94.9
10.5
40.2
96.0
93.2
101.6
96.0
3.7
4.7
3.7
4.0
4.2
19.8
20.0
102.0
Beer
Milk
Maltose
Glucose
16.3
6.6
20.0
5.0
36.8
11.3
102.5
94.0
3.9
3.0
Maltose
Lactose
Glucose
56.6
21.2
6.1
50.0
20.0
5.0
105.9
40.7
11.2
98.6
97.5
102.0
3.6
2.9
3.2
4. Conclusions
Egeberg, P. K., & Bergli, S. O. (2002). Fingerprinting of natural organic matter by
capillary zone electrophoresis using organic modifiers and pattern recognition
analysis. Journal of Chromatography A, 950, 221–231.
Reducing carbohydrates is considered to be one of the most
abundant ingredients in nature. To investigate their natural distri-
bution and biological roles, a robust analytical system must be used
to isolate, identify, and quantify them. Herein, we report the devel-
opment of such a system that can chromatographically separate
and specifically quantify nine aldohexoses, and two aldopentoses,
and two disaccharides tagged with PMP. Furthermore, the CZE
method with indirect UV detection was optimized and validated
for quantification of reducing carbohydrates in different types of
polysaccharides, beer and milk. The proposed CZE method provides
a repeatable, accurate and economic alternative for the separation
of the natural mono- and disaccharides. The proposed method is
particularly suitable for determining the component monosaccha-
rides in natural polysaccharide and can also be applied to routine
analysis of monosaccharides or disaccharides in the other real-life
samples, such as other plant polysaccharides, fruits, vegetables,
juices, wines, and grain. Besides, the analytical cost is lower and
the CZE analysis can be carried out in common laboratory. This find-
ing is of significance in the discovery of new functional foods, and
expanding our understanding of food composition, and will assist
in the refinement of design of dietary strategies to improve health
outcomes.
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purées, fruit preparations and jams—A review. Trends in Food Science & Technol-
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Acknowledgments
Koike, R., Kitagawa, F., & Otsuka, K. (2007). Simultaneous determination of ampho-
teric surfactants in detergents by capillary electrophoresis with indirect UV
detection. Journal of Chromatography A, 1139, 136–142.
Lachman, J., Havrland, B., Fernández, E. C., & Dudjak, J. (2004). Saccharides of yacon
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This study was supported by a grant from the National Nat-
ural Science Foundation of China (C30972054, C20802091, and
C31171678).
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