Determination of carbohydrates as their p-sulfophenylhydrazones by capillary zone electrophoresis

Article abstract of DOI:10.1016/S0008-6215(01)00090-8

p-Hydrazinobenzenesulfonic acid was explored as an ultraviolet labeling reagent for capillary electrophoresis of mono-, di- and trisaccharides. The labeling reaction that produces p-sulfophenylhydrazines took less than 8 min, and introduced both chromphore and charged groups into the carbohydrate molecules. The derivatives of nine mono- and disaccharides were completely separated in 9 min using a 100 mM borate buffer at pH 10.24. On-column UV detection at 200 nm allowed the detection of glucose with a mass detection limit of 17.6 fmol or a concentration limit of 3.6 μM. Reproducible quantification of carbohydrates was achieved in the concentration range of 0.1-9.1 mM in reaction solution. The method was applied successfully to determine the monosaccharide composition of laminaran.

Full text of DOI:10.1016/S0008-6215(01)00090-8

www.elsevier.nl/locate/carres
Carbohydrate Research 332 (2001) 191–196
Determination of carbohydrates as their
p-sulfophenylhydrazones by capillary zone
ꢀ
electrophoresis
Xiaoyan Wang, Yi Chen*
Center for Molecular Science, Institute of Chemistry, Chinese Academy of Sciences, Group 205, PO Box 2709,
Beijing 100080, People’s Republic of China
Received 18 January 2001; accepted 22 March 2001
Abstract
p-Hydrazinobenzenesulfonic acid was explored as an ultraviolet labeling reagent for capillary electrophoresis of
mono-, di- and trisaccharides. The labeling reaction that produces p-sulfophenylhydrazines took less than 8 min, and
introduced both chromphore and charged groups into the carbohydrate molecules. The derivatives of nine mono-
and disaccharides were completely separated in 9 min using a 100 mM borate buffer at pH 10.24. On-column UV
detection at 200 nm allowed the detection of glucose with a mass detection limit of 17.6 fmol or a concentration limit
of 3.6 mM. Reproducible quantification of carbohydrates was achieved in the concentration range of 0.1–9.1 mM in
reaction solution. The method was applied successfully to determine the monosaccharide composition of laminaran.
©
2001 Elsevier Science Ltd. All rights reserved.
Keywords: p-Hydrazinobenzenesulfonic acid; Carbohydrate; Capillary electrophoresis; Precolumn derivatization
are high efficiency, high speed, and the re-
quirement of only a minute amount of sample.
However, carbohydrates are in general
difficult to separate and detect due to their
lack of readily ionizable functional groups and
1
. Introduction
Carbohydrates are among the key biologi-
cal substances involved in many life processes
such as cell recognition and the immune re-
sponse. Since the function of carbohydrates
depends on their unique structures, their anal-
ysis and identification have become increas-
ingly important.
Capillary zone electrophoresis (CZE) has
been successfully applied to carbohydrate
analysis. The main features that CZE offers
2
chromophores. Various approaches have
been worked out to accommodate CZE analy-
sis, and these have been reviewed in detail by
3
–5
El Rassi.
Precolumn derivatization is the
most widely used means, and most of the
methods are based on reductive amination.
6
–9
Although this method gives excellent results, it
is generally time-consuming (requiring 1–4 h)
and may result in the cleavage of some impor-
tant sugar residues, such as sialic acid.
Abbre6iations: BHZ, p-hydrazinobenzenesulfonic acid;
Man, mannose; Gal, galactose; GlcNAc, N-acetylglu-
cosamine; Glc, glucose; Fuc, fucose; Ara, arabinose.
Hydrazine-containing reagents such as dab-
sylhydrazine, FMOC-hydrazine, and dansyl-
ꢀ
For a preliminary communication, see Ref. 1.
1
0,11
hydrazine have been applied to TLC
and
*
Corresponding author. Fax: +86-10-62559373.
1
1–13
E-mail address: chenyi@public.east.cn.net (Y. Chen).
HPLC procedures
with success. P e´ rez and
0008-6215/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(01)00090-8

192
X. Wang, Y. Chen / Carbohydrate Research 332 (2001) 191–196
1
4
Col o´ n reported the CZE of dansylhydrazine-
labeled sugars using laser-induced fluorescence
the osazone and hydrazone coexisting. There-
fore the products were the mixtures of the
isomeric compounds, perhaps either acyclic
and ring (pyranosyl or furanosyl) forms of the
(
LIF) detection. The most appealing feature
of it was that it took only 15 min for deriva-
tization. The detection limits of the deriva-
tized mono- and disaccharides reached 100
attomoles. Moreover, such reagents react spe-
cifically with ketone and aldehyde groups and
can be used directly on biological samples.
FMOC-hydrazine and dansylhydrazine have
been employed for the determination of sugars
1
8
hydrazones such as those of acetohydrazine
or a pair of syn and trans isomers of an
1
open-chain hydrazone. We conducted
H
NMR spectroscopy in an effort to identify the
derivatives, but further experiments are still
needed to definitely ascertain the structure of
the isomers.
1
5
14
in plasma and tear fluid, respectively. How-
ever, these reagents introduced no charge to
the sugar molecules, which is necessary for
CZE.
According to experimental results, isomer B
of the GlcNAc derivative seems to be thermo-
dynamically preferred over isomer A in this
case. The ratio of isomer B can thus increased
through increasing the reaction time, tempera-
ture and/or the ratio of BHZ to sugar (Fig. 1).
Isomer B of the GlcNAc derivative is the
predominant component under the optimum
reaction conditions. Moreover, the same trend
was not observed for some of the di- and
trisaccharides, e.g., maltose and melitose,
which were found yield two reaction products.
For maltose and melitose, the ratio of the two
isomers remained almost constant as the reac-
tion time varied.
It would be most convenient for the CZE of
carbohydrates if the derivatized sugars carried
charges over a wide pH range. This is espe-
cially desirable in oligosaccharide separa-
1
6
tions.
(
p-Hydrazinobenzenesulfonic acid
BHZ) was thus selected, and the results
showed that the labeling process could be
1
carried out within 10 min. In this work the
derivatization and CZE separation of sugars
were studied in detail.
pH of reaction solution.—The pH of the
reaction solution plays an important role in
derivatization. As shown in Fig. 1(a), the opti-
mum pH ranges for GlcNAc-b, Fuc, Gal were
pHꢀ3–4, ꢀ3–5 and ꢀ2–4, respectively.
Outside these ranges, the yields decreased.
This is apparently caused by the dual effects
2
. Results and discussion
Deri6atization. deri6ati6e configuration.—
The possible products of the hydrazine-con-
taining reagent labeled sugar are imide,
hydrazone and osazone. Our results showed
that some sugars such as GlcNAc and some
di- and trisaccharides yield two elec-
trophoretic peaks. This phenomenon also oc-
curred with some other hydrazine-containing
+
+
of H on the derivatization: H will proto-
nate both the aldehyde group of sugars and
the amino group of BHZ. The former reaction
increases the reaction activity, while the latter
reduces the nucleophilic properties of BHZ.
Moreover, the pH of the reaction mixture may
not be maintained if strong acids such as HCl
are used as catalysts. The situation becomes
worse if the BHZ concentration is increased,
because its sodium salt is a mild base. A
1
4,17,18
17
14
reagents.
Mopper and P e´ rez postu-
lated that some equilibration existed between
the hydrazone and osazone derivatives. How-
ever, Bendiak has demonstrated that both the
acyclic and cyclic hydrazones are produced
from an acetohydrazide-derivatized sugar by
1
18
H NMR spectroscopy. To confirm the
NH Ac–HCl buffer was finally chosen to con-
4
structure of the products of the derivatization,
in this paper, FABMS (negative-ion detection)
was performed with labeled GlcNAc. The MS
data showed a peak at m/z 390 that corre-
trol the pH of the reaction mixture. To suit
most of the sugars, it is suggested that the
solution pH be maintained in the range of
pHꢀ3–4.
The molar ratio of BHZ to sugar.—Fig.
1(b) shows that the peak heights of monosac-
charides increase sharply with the concentra-
−
sponds to the GlcNAc hydrazone [M−H] .
No imide or osazone peak was detected at m/z
4
08 or 519, which precludes the possibility of

X. Wang, Y. Chen / Carbohydrate Research 332 (2001) 191–196
193
tion of BHZ at the beginning of the reaction
and level off over the ratio 10:1. Excessive
reagent caused precipitation and interfered
with the subsequent CZE separations. The
best ratio was ꢀ10:1–20:1.
Temperature and time.—Fig. 1(c) shows
that the optimum reaction temperatures were
about 70, 60 and 50 °C, for GlcNAc-b, Fuc
and Gal, respectively. Interestingly, GlcNAc-b
was sensitive to temperature variations while
the other two sugars were not. For fast label-
ing, higher temperatures were preferred. It is
suggested that the derivatization be routinely
carried out at 70 °C. At this temperature the
reaction will be completed in about 10 min
behavior has been studied as detailed in the
following paragraphs.
Buffer pH and concentration.—In order to
be reproducible and to minimize errors from
1
9
temperature variation and other factors, rel-
ative mobility, v /v was adopted as the
ep eo
criterion in this study:
t0
v /v =1−
ep eo
tR
where t is the rentention time of dimethyl
0
sulfoxide and t the migration time of the test
R
substance. Fig. 2 shows the effects of pH and
concentration of borate buffer on the relative
mobility of the BHZ derivatives. It is evident
that with increasing pH or borate concentra-
tion the relative mobilities of the derivatives
became greater due to the facilitation of the
formation of borate complexes. According to
Fig. 2(a), optimum borate concentration range
(
see Fig. 1(d)).
Separation of BHZ hydrazones.—In an al-
kaline borate buffer, the BHZ hydrazones are
negatively charged, due to both the sulfonate
and the borate complex. Their electrophoretic
Fig. 1. Effects of various factors on precolumn derivatization of selecting sugar to BHZ. (a) pH value of reaction solution. The
concentration of BHZ was 0.2 M. Reaction temperature, 70 °C; reaction time, 20 min. (b) The molar ratio of BHZ to sugar. The
pH value of reaction solution was fixed at pH 3.0. Reaction temperature, 70 °C; reaction time 20 min. (c) Reaction temperature.
The pH value of reaction solution was pH 3.0. The concentration of BHZ was 0.2 M. Reaction time 20 min. (d) Time course of
derivatization. The pH value was 3.0. The concentration of BHZ was 0.2 M. The reaction temperature was 70 °C. In all
experiments the reaction products were analyzed by using 100 mM borate buffer, pH 10.4, 300 V/cm field intensity. On-column
detection was carried out at 206 nm. The concentration of carbohydrates in the sample solution was 5 mM.

194
X. Wang, Y. Chen / Carbohydrate Research 332 (2001) 191–196
Fig. 2. Effects of pH and borate concentration of buffer on the separation of BHZ-sugars. The borate concentration in (a) and
pH in (b) were fixed at pH 10.5 and 100 mM, respectively. The CZE conditions followed those in Fig. 1.
was 100–150 mM. In order to avoid Joule
heating and to shorten the separation time, a
lower concentration of borate was found to be
beneficial. Fig. 2(b) shows the relationship
between pH and the relative mobility at a
constant borate concentration of 100 mM.
Between pH 10.2 and 10.8, most monosacchar-
ides showed the best resolution. From the
results sketched in Fig. 2(a and b), a pHꢀ
obtained with five repeated runs. The mass
detection limit of glucose was 17.6 fmol or 3.6
mM in terms of concentration, at a signal-to-
noise ratio of 3. This indicated that the
1
0.2–10.8 and a borate concentration of 100
mM were chosen for the subsequent analyses.
Capillary zone electrophoresis (CZE).—Fig.
depicts the electrophorogram of the deriva-
3
tized mixture of nine reducing mono- and
disaccharides together with Me SO as the in-
2
ternal standard. The reagent migrated slower
than sugar derivatives, and the separation was
completed in 9 min. The efficiencies obtained
were between 110,000 and 290,000 theoretical
plates per meter for all the nine sugars. Both
maltose and melitose had two peaks, and the
minor peaks could not be suppressed signifi-
cantly by varying the reaction conditions.
Quantitati6e analysis and sensiti6ity.—
Sugar mixtures with differing concentrations
were derivatived and separated by CZE, with
UV detection at 200 nm. Although the yields
of the BHZ derivatives were not uniform, they
were reproducible enough to allow reliable
quantification. The calibration curves for the
test sugars showed quite acceptable linearity
over a concentration range of 0.1–9.1 mM.
The respective equations of the line and the
regression analysis results are listed in Table 1.
A coefficient of variation below 4.2% was
Fig. 3. Separation of nine sugar-BHZ derivatives: *,Me SO; 1,
cellobiose; 2, maltose (a); 3, N-acetylglucosamine; 4, melitose
2
(
a); 5, glucose; 6, fucose; 7, mannose; 8, galactose; 9, ara-
binose; 10, maltose (b); 11, melitose (b). Concentration of
each sugar was 0.2 mM. Separation conditions: Beckman
P/ACE 2050, 200 nm detection; fused silica capillary, 55/62
cm; voltage, 18.6 kV; separation carrier, 100 mmol/L boric
acid (pH 10.24); pressure injection, 3.44 kPa, 3 s.

X. Wang, Y. Chen / Carbohydrate Research 332 (2001) 191–196
195
Table 1
Regression equation and correlation coefficients for monosaccharides
Monosaccharide
Regression equation
Correlation coefficient (R)
RSD (n=5)
Arabinose
Fucose
Glucose
Galactose
Y=−0.0607+216.3037X
Y=−0.0062+82.8772X
Y=−0.0295+286.5016X
Y=0.0065+17.31372X
Y=0.0172+92.9369X
Y=0.0289+48.05302X
Y=0.0064+17.99916X
Y=0.0084+31.95348X
0.996
0.995
0.997
0.994
0.999
0.999
0.999
0.995
0.042
0.016
0.023
0.004
0.002
0.016
0.035
0.005
Mannose
N-acetylglucosamine (b)
Maltose (a)
Maltose (b)
method was applicable, considering that the
results were obtained by directly derivatizing
the sugar solution of different concentrations.
It is noticeable that the sugars, e.g., maltose,
which produces two peaks, can also be quanti-
tatively determined because either one or both
of their peaks could be used for calibration
purposes. The two-peak phenomenon did not
diminish the accuracy of the quantitative
analysis.
Application to component analysis.—The
applicability of this method was demonstrated
by analyzing the monosaccharide components
of laminaran extracted from kelp. The result-
ing CZE peaks were identified by spiking stan-
dard sugar-BHZ derivatives. Galactose,
mannose, fucose and glucose were determined
in laminaran hydrolysate with a molar ratio of
detection wavelengths were set at 200 nm for
P/ACE or 206 nm for the homemade system.
Separations were carried out in fused-silica
capillaries (Yongnian Optical Fiber Factory,
Hebei, China) of 50 mm i.d. and 375 mm o.d.
The effective length was 55 cm, and the total
length was 62 cm for P/ACE or 75 cm for the
homemade system. The applied electrical field
strength was 300 V/cm. Prior to each injec-
tion, the capillaries were sequentially flushed
with 0.1 M NaOH, 0.1 M HNO , water and
3
separation buffer for 2 min for reproduciblil-
ity. The capillary tubing was filled with 0.1 M
NaOH for overnight storage. The separation
buffer was boric acid adjusted to the desired
pH by adding KOH pellets. All the water used
was double-distilled water. The sample was
introduced to the anode end of the tubing by
gravity injection for 20 s at a 10 cm height on
the homemade CZE system or by pressure
0
.67:0.092:1:0.124, which were comparable
20
with those in the literature.
1
Precolumn derivatization of carbohydrates
with p-hydrazinobenzenesulfonic acid is a
rapid, simple and reproducible method that is
suitable for quantitative analysis. Although a
few sugars formed two hydrazone products
after derivatization, the method serves well for
quantitative analysis, as the derivatization is
quite reproducible. The two-peak phe-
nomenon might also be useful in component
identification.
injection for 2 s at 3.44 kPa on P/ACE. H
NMR experiments were conducted on a
Bruker ARX 400 MHz instrument. Mass
spectrometry was performed in a Kyky-ZHP-5
instrument (The Center of Science Instrument,
CAS; Beijing, China) using FAB as the ion
source and glycerol as the matrix.
Chemicals.—All sugars were purchased
from Sigma Chemical Co. (St. Louis, USA).
p-Aminobenzenesulfonic acid was a product
of Beijing Chemical Factory (Beijing, China).
Preparation of p-hydrazinobenzensulfonic
acid (BHZ).—BHZ was synthesized according
3
. Experimental
2
1
to the literature procedure, with modifica-
tions described hereafter. p-Aminobezenesul-
fonic acid (19 g) was dissolved in 25 mL of
20% NaOH, then 100 mL of 3.5% (w/v)
Apparatus.—Quantitative analysis was per-
formed on a Beckman CZE instrument model
P/ACE 2050 (Fullerton, USA) using the SYS-
TEM GOLD software. Other experiments were
performed on a homemade CZE system. The
NaNO was added. After cooling, the solution
2
was treated with 50 mL of 1.5 M H SO by
2
4

196
X. Wang, Y. Chen / Carbohydrate Research 332 (2001) 191–196
stirring at −5 °C for 20 min. The mixture was
filtered, the precipitate was suspended in 20
Acknowledgements
mL of concd HCl, and 32% NaHSO was
This work was financially supported by Na-
tional Science Foundation of China (No.
29825112) and The Chinese Academy of Sci-
ences (No. KJ951-A1-507). The Beckman
CZE instrument was kindly donated by the
Humboldt Foundation. We also appreciate Dr
Guiyun Xu (Center for Molecular Science,
Institute of Chemistry, Chinese Academy of
Sciences) for her kind donation of a sample of
laminaran, Professor Lipu Li for her assis-
3
added dropwise until the reaction solution
became clear. The solution was then acidified
with 20 mL of concd HCl, boiled and filtered
to yield approximately 9 g of p-hydrazi-
nobezenesulfonic acid. The product was iso-
lated as crystalline needles. The crude product
was purified by recrystallization until the elec-
trophorogram showed a single peak. Fresh
BHZ solution for derivatization was prepared
daily by dissolving in an equivalent amount of
Na CO solution.
1
tance with H NMR, and Professor An Zhu
for his helpful advice concerning the
manuscript.
2
3
Deri6atization of sugars with BHZ.—An
aliquot of 10 mL of 0.3–10 mM sugar solution
in a 2 M ammonium acetate solution of pHꢀ
3
.0–5.0 (adjusted by concentrated HCl) was
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2
3
2
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1
.