Analysis of the monosaccharide composition of water-soluble polysaccharides from Sargassum fusiforme by high performance liquid chromatography/electrospray ionisation mass spectrometry

Article abstract of DOI:10.1016/j.foodchem.2013.09.019

Sargassum fusiforme (hijiki) is the well-known edible algae, whose polysaccharides have been proved to possess interesting bioactivities like antitumor, antioxidant, antimicrobial and immunomodulatory activities. A facile and sensitive method based on hig

Full text of DOI:10.1016/j.foodchem.2013.09.019

Food Chemistry 145 (2014) 976–983
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Analysis of the monosaccharide composition of water-soluble
polysaccharides from Sargassum fusiforme by high performance
liquid chromatography/electrospray ionisation mass spectrometry
Xiaodan Wu a,b, Wei Jiang b, Jiajia Lu a, Ying Yu a, Bin Wu b,
⇑
a Center of Analysis and Measurement, Zhejiang University, Hangzhou 310058, People’s Republic of China
b Ocean College, Zhejiang University, Hangzhou 310058, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 June 2012
Received in revised form 28 August 2013
Accepted 4 September 2013
Available online 12 September 2013
Sargassum fusiforme (hijiki) is the well-known edible algae, whose polysaccharides have been proved to
possess interesting bioactivities like antitumor, antioxidant, antimicrobial and immunomodulatory activ-
ities. A facile and sensitive method based on high-performance liquid chromatography method of
pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) coupled with electrospray ioni-
sation mass spectrometry (HPLC/ESI–MS) has been established for the analysis of the monosaccharide
composition of polysaccharides in S. fusiforme. Monosaccharides have been converted into PMP-labelled
derivatives with aqueous ammonia as a catalyst at 70 °C for 30 min. The optimisation of the pre-column
derivatization process was studied. The LODs of the monosaccharides were in the range from 0.01 to
0.02 nmol. PMP-labelled mixture of monosaccharides has been well separated by a reverse-phase HPLC
and detected by on-line ESI–MS method under optimised conditions. The mobile phase of elution system
was chosen as acetonitrile (solvent A) and 20 mM aqueous ammonium acetate (solvent B) (pH 3.0) with
Zorbax XDB-C18 column at 30 °C for the separation of the monosaccharide derivatives. Identification of
the monosaccharides composition was carried out by analysis with mass spectral behaviour and chroma-
tography characteristics of 1-phenyl-3-methyl-5-pyrazolone (PMP) labelled monosaccharides. All PMP-
labelled derivatives display high chemical stabilities, whose regular MS fragmentation is specific for
reducing labelled sugars. The result showed that the S. fusiforme polysaccharide consisted of mannose,
glucose, galactose, xylose, fucose and glucuronic acid or galacturonic acid, or both uronic acids.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
HPLC–ESI–MS
Precolumn PMP derivatization
Monosaccharide composition
Sargassum fusiforme
1. Introduction
been used to treat human disease (Zhang & Wang, 1990), so the
water-soluble polysaccharide was focused on in the present study.
Sargassum fusiforme (hijiki), one of the well-known brown
seaweed and edible algae, has been applied as an important food
and therapeutic for thousands of years and widely cultivated in
China and Japan (Ji, Wang, Wu, & Ji, 2007; Mao, Li, Gu, Fang, & Xing,
2004; Yan, Chuda, Suzuki, & Nagata, 1999; Zhu, Ooi, Chan, & Ang,
2003). The antioxidant and antimicrobial properties of the sea-
weeds have been intensively studied (Lu, Yan, Xu, & Nan, 2010;
Yan et al., 1999). Recently, studies of polysaccharides from S. fusi-
forme have attracted more attention. Bioactivities including vibrio-
sis resistance, immunity enhancing effect (Huang, Zhou, & Zhang,
2006), antitumor activity against S180 in mice (Ji et al., 2007),
inhibitory activity to calcium oxalate (CaOxa) urinary stones
(Wu, Ouyang, Deng, & Chen, 2006) and antihyperlipidemia activity
(Mao et al., 2004) have been reported. As an important Traditional
Chinese Medicine, the water-soluble extract of S. fusiforme has
Monosaccharide composition analysis of polysaccharides is of
fundamental importance for the research on polysaccharide struc-
ture and its characteristics. A variety of chromatographic systems
such as gas chromatography (GC) (Akiyama, Yamazaki, & Tanamot-
o, 2011; Chen, Xie, Wang, Nie, & Li, 2009), high-performance liquid
chromatography (HPLC) (Gomis, Tamayo, & Alonso, 2001; Han,
Chen, Sun, Zhan, & Bi, 2009; Ikegami et al., 2008) and high perfor-
mance liquid chromatography-mass spectrometry (LC–MS) (Dye &
Yttri, 2005; Hammad, Derryberry, Jmeian, & Mechref, 2010) have
be used to separate and analyse monosaccharides.
Over the past decade, the development of LC/MS methods
dedicated to the analysis of sugars and monosaccharides in partic-
ular, has led to significant advances in terms of sensitivity and
specificity while maintaining speed and simplicity of implementa-
tion. However, LC–MS method is limited by loss of sensitivity
owing to the low ionisation efficiency of monosaccharides.
Therefore, the derivatization of monosaccharides is indispensable
to obtain highly sensitive detection. The reagent 1-phenyl-3-
⇑
Corresponding author. Tel./fax: +86 571 882 08540.
E-mail address: wubin@zju.edu.cn (B. Wu).
0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.09.019

X. Wu et al. / Food Chemistry 145 (2014) 976–983
977
methyl-5-pyrazolone (PMP), which was first developed in 1989 by
2.2. Extraction of the polysaccharide from the Sargassum fusiforme
the Honda’s group (Honda et al., 1989), is one of the popular labels
that react with reducing carbohydrate under mild condition,
requiring no acid catalyst and causing no desialylation and isomer-
ization (Dai et al., 2010; Zhang, Wang, Xie, Nie, & Huang, 2010).
Pre-column derivatization with PMP method was first developed
for the analysis of carbohydrates by high-performance liquid chro-
matography (HPLC) (Shen & Perreault, 1998), and later successfully
applied to capillary electrophoresis (CE) (Suzuki et al., 2001),
including capillary zone electrophoresis (CZE) (Honda, Suzuki,
Nose, Yamamoto, & Kakehi, 1991), micellar electrokinetic chroma-
tography (MEKC) (Janini & Issaq, 1992), and ion-exchange electro-
kinetic chromatography (IXEKC) (Honda, Toghi, Uegaki, & Honda,
1998).
Analyses of polysaccharide from S. fusiforme are extensive (Ji
et al., 2007; Mao et al., 2004; Zhu et al., 2003). However, the appli-
cation of analytical methods of PMP precolumn derivatization
high-performance liquid chromatography coupled with electro-
spray ionisation mass spectrometry on S. fusiforme have not been
reported. In the present study, a precolumn PMP derivatization
LC–ESI–MS method for simultaneous determination of six sugars
was established. The data on characteristic fragment ions of the
six PMP-labelled monosaccharides have been collected. All PMP-
labelled derivatives display high chemical stabilities, whose regu-
lar MS fragmentation is specific for reducing labelled sugars. The
procedure of the pre-column derivatization reaction was optimised
by examination of reaction time and neutralization condition of
the reaction system for six sugars. At the same time, the optimised
method and procedure were successfully applied in sugar compo-
sitional analysis of polysaccharide fractions isolated from S.
fusiforme.
The polysaccharide was isolated from Sargassum fusiforme by
hot-water extraction and ethanol precipitation (Wang & Luo,
2007; Yoshizawa, Enomoto, Todoh, Ametani,
& Kaminogawa,
1993). The dried S. fusiforme (50 g) were extracted with 95% alco-
hol and dried, then extracted with 100 ml distilled water at 95 °C
for 3 h. The water extracts were concentrated to 20 ml under a re-
duced pressure. Then 60 ml anhydrous alcohol was added slowly
by stirring to precipitate the polysaccharide which kept at 4 °C
overnight. Then polysaccharide precipitation were obtained by
centrifugation at 3000 rpm for 30 min and repeatedly washed
sequentially with possibly less amounts of ethanol, acetone and
ether, respectively. Residue was dried and the S. fusiforme polysac-
charide was obtained.
2.3. Hydrolysis of the polysaccharide
The polysaccharide sample (50 mg) was infiltrated with 4 M
TFA (4 ml) (Åman, McNeil, Franzén, & Darvill, 1981) in a sealed
flask (10 ml) and kept at ambient temperature for 15 min. Then
1 ml distilled water was added and kept in boiling water bath for
2 h. After that, 2 ml of distilled water was added and kept in boiling
water bath for 1 h. After being cooled to room temperature, the
reaction mixture was then centrifugated at 3000 rpm for 10 min.
The supernatant was collected and dried under a reduced pressure.
2.4. Derivatization procedure
The procedure employed for the derivatization of monosaccha-
rides was carried out according to the method of Daotian, F et al.
(Fu & Oneill, 1995) and modified by us. The hydrolysed polysaccha-
ride or monosaccharides were dissolved in 5 ml ammonia. Then,
2. Materials and methods
the 100
methanol solution (100
ture was allowed to react for 30 min at 70 °C, then cooled to ambi-
ent temperature and was neutralized with 20 l 1% glacial acetic
l
l solution was transferred into a clean tube, and 0.5 M
ll) of PMP was added and mixed. The mix-
2.1. Materials and reagents
l
D-Mannose,
L-rhamnose, D-glucose, D-xylose, D-galactose, and
acid. Water and chloroform (1.0 ml each) were added, and the mix-
ture was shaken vigorously. The chloroform layer was discarded,
and the extraction process was repeated three times. Then, the
supernatant was centrifugated at 10,000 rpm for 10 min, which
was collected for analysis directly or stored at À20 °C for later
LC/MS analysis. Scheme 1 shows the Glc derivatization reaction.
L
-fucose were purchased from the Shanghai Pharmaceutical Hold-
ing Co., Ltd. (Shanghai, China). Trifluoroacetic acid (TFA) was
obtained from Merck (Darmstadt, Germany). 1-Phenyl-3-methyl-
5-pyrazolone (PMP) was purchased from Major Chemicals Co.,
Ltd. (Hangzhou, China). Acetonitrile (HPLC-grade) was obtained
from Merck (E. Merck, Darmstadt, Germany). Water was purified
with a Mili-Q academic water purification system (Millipore, Bed-
ford, MA, USA). Other chemicals were of analytical reagent grade. S.
fusiforme was collected in Dongtou County, Zhejiang Province,
People’s Republic of China, in September 2009 and identified by
Prof. Changxi Zhang (Jinhua Medical College, Jinhua, People’s
Republic of China). A voucher specimen (Vs71) is maintained at
the Jinhua Medical College, Jinhua, People’s Republic of China.
2.5. Chromatography and mass spectrometry conditions
LC–MS analysis was conducted using an Agilent (Agilent Tech-
nologies, Palo Alto, CA, USA) 1100 series HPLC system consisting
of a G1312A binary pump, a G1322A degasser and an ALS
G1329A auto-sampler interfaced to an Agilent 1100 series
N
N
OH
OH
N
N
H
O
O
H
+
N
O
O
HO
HO
OH
HO
OH
OH
H
N
OH
H
OH
glucose
double PMP-labeled glucose
PMP
Scheme 1. Derivatization reaction of PMP and glucose.

978
X. Wu et al. / Food Chemistry 145 (2014) 976–983
G1946D mass spectrometer equipped with an electrospray ionisa-
tion (ESI) interface. The data acquisition software employed was
Agilent Chemstation version 10.02. An Agilent Zorbax XDB-C18
six repeated determination of the hydrolysed monosaccharides
from Sargassum fusiforme polysaccharide.
column (250 Â 4.6 mm, 5
lm) was used and kept at 30 °C. The mo-
bile phase consisted of 20 mM ammonium acetate (adjusted to pH
3. Results and discussion
3.0 with glacial acetic acid)–acetonitrile (85:15, v/v). The flow rate
was 1.0 ml/min. The injection volume was 20 lL. The settings of
3.1. Hydrolysis of polysaccharide
the mass spectrometer were as follows: the drying gas flow rate
was 12 L/min and a nebulizer pressure was 50 psi. The drying gas
temperature was set at 350 °C. The value of the fragmentor was
100 V with capillary voltage at 4000 V in positive ion scan mode
and 3500 V in negative ion scan mode. The scan range was set from
m/z 100 to 1000 amu.
H2SO4 is the mostly used hydrolysis agent in the studies of poly-
saccharide, which is difficult to remove and poses negative effect
on MS-based measurement. Since polysaccharides in Sargassum
fusiforme linked mainly by simple carbohydrate chains (Zhou, Hu,
Wu, Pan, & Sun, 2008), they could be essentially hydrolysed com-
pletely using trifluoroacetic acid (TFA) in mild condition. In the
present study, the results showed that TFA hydrolysis was suitable
to polysaccharide from S. fusiforme, and at least six monosaccha-
rides were obtained. TFA was removed in vacuo before samples
running in MS, so the contamination caused by TFA was avoided.
2.6. Method validation
The analytical method was validated according to the following
criteria: linearity, sensitivity, recovery, precision and reproducibil-
ity. The linearity of each analyte was evaluated by using a series of
standard solutions. The calibration curves were constructed based
on peak area versus concentrations of analyte standards. The
detection limits (LOQ) of each analyte was obtained by injecting
a standard mixture derivatized as mentioned above in the deriva-
tization procedure, followed by the comparison of peak height
with baseline noise level and a signal-to-noise ratio (S/N) of three
assigned the detection limit. The precisions detection was
examined by five repeated injections of a mixed standard solution.
In order to examine the recovery of the developed method, the
standards of each analyte at three different concentration levels
were spiked to samples and then derivatived by the optimised
derivatization method. The reproducibilities were examined by
3.2. Optimisation of the derivatization procedure
3.2.1. Effect of a catalyst on derivatization reaction
Although sodium hydroxide is typical catalysts for the derivati-
zation of carbohydrates in alkaline medium, the reaction produces
considerable amounts of inorganic salts in the resultant solution. In
the present study, ammonia was applied as the catalysts for the
derivatization, which can be removed by evaporating the solution
to dryness in vacuo, avoiding the production of inorganic salts.
3.2.2. Optimisation of derivatization conditions
The result of the analysis of reaction time effect on derivatiza-
tion was summarised in Fig. 1(a), which indicated that the labelling
Fig. 1. (a) Effect of reaction time on derivatization of glucose. (b) Effect of reaction temperature on derivatization of glucose. (c) Effect of PMP concentration on derivatization
of glucose.

X. Wu et al. / Food Chemistry 145 (2014) 976–983
979
Table 1
The calibration ranges, LODs and precision for five monosaccharides.
Analyte
Calibration curve
Range (nmol)
Correlation coefficient
LODs (nmol)
Precision (RSD,%)
Mannose
Glucose
Galactose
Xylose
Y = 383X-5.86
Y = 188X-4.6
Y = 256X-1.14
Y = 250X-15
0.1–10
0.1–10
0.1–10
0.1–10
0.1–10
0.9999
0.9999
0.9999
0.9999
0.9999
0.01
0.01
0.02
0.02
0.02
0.21
1.76
1.40
0.56
1.34
Fucose
Y = 218.7X-5.79
Table 2
Table 3
Recovery analysis of five monosaccharides in the sample of Sargassum fusiforme
polysaccharide (n = 5).
Reproducibility of five monosaccharides in the sample of Sargassum fusiforme
polysaccharide (n = 6).
Analyte
Mannose
Glucose
Galactose
Xylose
Content in
sample
(nmol)
Spiked
amount
(nmol)
Found
amount
(nmol)
Recovery
(%)
RSD
(%)
Content in sample
(nmol)
Mean value
(nmol)
Reproducibility RSD
(%)
Mannose
Glucose
Galactose
Xylose
0.88
0.87
0.85
0.87
0.87
0.87
0.87
0.94
2.46
0.29
2.45
1.27
1.39
0.75
1.99
1.61
0.44
0.47
1.20
0.14
1.19
0.25
2.5
10
0.65
2.62
9.33
85.1
87.3
88.9
2.29
0.99
0.91
0.25
2.5
10
0.69
3.02
11.59
86.1
101.9
111.2
3.64
2.83
2.56
0.95
0.95
0.95
0.93
0.94
0.92
0.25
2.5
10
1.42
3.60
11.77
87.4
96.0
105.7
2.91
1.90
1.10
0.25
2.5
10
0.36
2.48
9.58
88.6
93.6
94.40
1.98
1.07
0.78
2.46
2.47
2.43
2.44
2.49
2.46
Fucose
0.25
2.5
10
1.42
3.88
12.48
89.2
107.1
112.8
2.56
2.15
0.63
0.28
0.28
0.30
0.29
0.29
0.28
reaction completes in 30 min. To investigate the temperature effect
on derivatization, a series of test reactions with glucose as a model
saccharide were performed. The result of the effect of reaction
temperature of 50, 70, 90 and 110 °C on the peak area (propor-
tional to the yield of the derivative) is presented in Fig. 1(b). The
yield of labelled glucose increases with the increase of the temper-
ature within a range from 50 to 90 °C. Performing the reaction at
higher temperatures from 70 to 110 °C gives only little effect.
Therefore, performing of the labelling routine at 70 °C should be
recommended. To define the optimal amount of PMP on derivatiza-
tion reaction, a series of derivatization experiments with different
molar ratios of PMP to Glc were carried out. The results were
presented in Fig. 1(c). The yield of the reaction is very low at
PMP concentration of 0.1 mol/L and dramatically increases with
the PMP concentration of 0.3 mol/L. Then, the growth of the peak
area slows down, and the graph reaches the saturation state at
0.5 mol/L PMP. In the present work, 0.5 mol/L PMP was chosen in
the derivatization reaction.
Fucose
2.39
2.41
2.47
2.49
2.46
2.47
factory. The recoveries are summarised in Table 2. Good recoveries
from 85% to 113% were obtained. The result of reproducibility anal-
ysis was summarised in Table 3. The relative standard deviations
(RSDs) values fell within 0.75–1.99%.
3.4. HPLC separation of PMP-monosaccharides derivatives
The HPLC separation conditions were adjusted and tested for a
mixture of 6 PMP-labelled monosaccharides (mannose, rhamnose,
glucose, galactose, xylose and fucose). Since no nonvolatile inor-
ganic salts are allowed in the MS-based measurement methods,
the mobilephase of elution system was chosen as acetonitrile
(solvent A) and 20 mM aqueous ammonium acetate (solvent B)
(pH 3.0). Six PMP-labelled monosaccharides were separated
successfully in the order of peak 1 for mannose, peak 2 for rham-
nose, peak 3 for glucose, peak 4 for galactose, peak 5 for xylose
and peak 6 for fucose. In this study, the good separation of the
six monosaccharide derivatives was achieved within 45 min (see
Fig. 2). In the process of mobilephase evaluation, we found that
the concentration of ammonium acetate affect the separation of
PMP-labelled monosaccharides. The mobilephase of elution
3.3. Results of method validation
According to Section 2.6, the calibration curves were
constructed based on peak area versus concentrations of analyte
standards. Table 1 is the summary of calibration curves, linear
ranges and limit of detection. Good linearities were found in the
ranges of 0.1–10 nmol for each analyte. The results of detection
limits analysis showed that the LODs of the monosaccharides were
in the range from 0.01 to 0.02 nmol (Table 1), indicating that the
sensitivity of the method was satisfactory. The results of precisions
detection were summarised in Table 1, showing that the relative
standard deviations (RSDs) of peak areas were all under 2.0% for
each analyte, which indicated that the method precision was satis-

980
X. Wu et al. / Food Chemistry 145 (2014) 976–983
MSD1 TIC, MS File (SUGAR\SUGAR020.D+BSB) API-ES, Neg, Scan, Frag: 100
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
1
2
4
5
6
ESI(-)
3
0
10
20
30
40
50
60
min
VWD1 A, Wavelength=245 nm (SUGAR\SUGAR020.D)
mAU
1600
1400
1200
1000
800
600
400
200
0
1
2
5
4
3
6
0
10
20
30
40
50
60
min
MSD1 TIC, MS File (SUGAR\SUGAR035.D+BSB) API-ES, Pos, Scan, Frag: 100
500000
400000
300000
200000
100000
0
5
4
ESI(+)
1
3
2
0
10
20
30
40
50
min
VWD1 A, Wavelength=245 nm (SUGAR\SUGAR035.D)
mAU
1750
1500
1250
1000
750
1
2
4
5
500
3
6
250
0
0
10
20
30
40
50
min
Fig. 2. Positive and negative total ion current chromatogram (TIC) and HPLC chromatogram of six PMP-labelled monosaccharides.

X. Wu et al. / Food Chemistry 145 (2014) 976–983
981
Table 4
3.6. LC–ESI–MS analysis of monosaccharide composition of
polysaccharides in Sargassum fusiforme
MS data of double PMP-labelled saccharide.
Saccharides
tR(min)
[M–H]À
[M+Cl]À
Fragment ions (m/z)
When HPLC and MS are combined on-line, the total ion current
chromatogram (TIC) of the molecular ion peaks and HPLC spectrum
(see Fig. 3) of the separated PMP-labelled monosaccharides are ob-
tained simultaneously. In the present study, seven PMP-labelled
compounds were separated, among which six PMP-labelled mono-
saccharides were identified under conditions as described in Sec-
tion 2.5 by means of HPLC–ESI–MS/MS combination technique.
Peaks 1, 4, 5, 6 and 7 could be unambiguously identified as
PMP-labelled mannose, glucose, galactose, xylose and fucose based
on the comparison of retention times and MS data with those of
authentic PMP-labelled monosaccharides. Peak 2 gave negative
ions at m/z 523.1 [M–H]À and 349.2 [M–PMP–H]À. The mass differ-
ence between the fragments of the compound and those of authen-
tic PMP-labelled glucose was 14 Da, and the molecular weight was
194 Da, which indicated that peak 2 might be a PMP-labelled
GlcUA or GalUA. Similar ions at m/z 509.2, 505.2, 335.1, 331.1 as
those of PMP-labelled authentic glucose were observed at peak 4
based on the above analyses for the reference standards in the
HPLC–ESI–MS experiments. Two negative ions at m/z 509.2 and
335.1 were consistent with fragments of [M–H]À and [M–PMP–
H]À of PMP-labelled glucose. However, peak 4 also gave negative
ions at m/z 505 [M–H]À and 331 [M–PMP–H]À. Peak 4 was obvi-
ously the mixture of a PMP-labelled glucose and another unknown
compound. The mass difference between the unknown compound
and the authentic PMP-labelled glucose was 4 Da, which indicated
that peak 4 might exist another byproduct derivative from glucose
lactone. From analysis of the fragmentation pattern and the
molecular weight of 176 Da, this compound could be tentatively
identified as ascorbic acid. Although, ascorbic acid widely
Mannose
Rhamnose
Glucose
Galactose
Xylose
11.6
16.7
28.5
30.6
35.9
43.1
509.2
493.2
509.2
509.2
479.2
493.2
545.2
529.2
545.2
545.5
515.2
529.2
180
194
208
180, 176
180
Fucose
164
system, acetonitrile and 20 mM aqueous ammonium acetate, is the
best condition for separating samples, especially for separating Glu
and Gal. According to the results of our experiments and other re-
ports (Abdel-Hamid, Novotny, & Hamza, 2001; Kim, Yoo, Han, Lee,
& Lee, 2003), MS can tolerate MS-friendly mobile phases consisting
of ammonium acetate of 20 mM concentration.
3.5. LC–ESI–MS characterisation
All PMP-labelled monosaccharides were characterised by LC–
ESI–MS method (positive-ion and negative-ion mode, see Fig. 2).
Although both ion modes has high response, negative-ion mode
was chosen, since more fragments were obtained by negative-ion
mode. For example, PMP-labelled glucose comprises peaks of m/z
514, 509, 371, 335, 299 and 263 correspondent to [M+Cl]À, [M–
H]À, [M–PMP+Cl]À, [M–PMP–H]À, [M–2PMP+Cl]À and [M–2PMP–
H]À by negative-ion mode, whereas, only m/z 511 [M+H]+ was
detected by positive-ion mode. The numerical data are collected
in Table 4, which allow suggesting the fragmentation mode for
PMP-labelled monosaccharides.
MSD1 TIC, MS File (SUGAR\SUGAR031.D+BSB) API-ES, Neg, Scan, Frag: 100
1000000
800000
600000
400000
200000
0
0
10
20
30
40
50
min
VWD1 A, Wavelength=245 nm (SUGAR\SUGAR031.D)
mAU
140
120
100
80
60
40
20
0
4
7
5
3
1
2
6
0
10
20
30
40
50
min
Fig. 3. The total ion current chromatogram and HPLC spectrum of PMP-labelled monosaccharide composition of polysaccharides.

982
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Table 5
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Peak tR(min) [M–H]À
No.
Fragment Mw
ions (m/z)
Saccharides
1
2
11.6
12.5
509.2
523.2
335.1
349.1
180
194
Mannose
Glucuronic acid or
Galacturonic acid
Unknown
3
4
18.2
28.3
537.2
509.2,
505.2
509.2
479.2
493.2
363.2
335.1,
331.1
335.1
341.1
319.1
208
180,
176
180
150
164
Glucose, L-ascorbic acid
Dye, C., & Yttri, K. E. (2005). Determination of monosaccharide anhydrides in
atmospheric aerosols by use of high-performance liquid chromatography
combined with high-resolution mass spectrometry. Analytical Chemistry, 77,
853–1858.
5
6
7
30.8
36.2
43.4
Galactose
Xylose
Fucose
Fu, D. T.,
oligosaccharides
chromatography. Analytical Biochemistry, 227, 377–384.
Gomis, D. B., Tamayo, D. M., Alonso, J. M. (2001). Determination of
monosaccharides in cider by reversed-phase liquid chromatography. Analytica
Chimica Acta, 436, 173–180.
Hammad, L. A., Derryberry, D. Z., Jmeian, Y. R., & Mechref, Y. (2010). Quantification
&
Oneill, R. A. (1995). Monosaccharide composition analysis of
and glycoproteins by high-performance liquid
&
distribute in nature, its existence in this case might be a byproduct
from PMP label reaction. Peak 3 afforded negative ions at m/z 537.2
[M–H]À, 363.2 [M–PMP–H]À revealing that the molecular weight
could be 208 Da. However, its exact structure needs to be con-
firmed in future work. A total of six peaks were identified or tenta-
tively identified. From above evidence, polysaccharides from
Sargassum fusiforme mainly compose of mannose, glucose, galact-
ose, xylose, fucose, and glucuronic acid or galacturonic acid, or
both uronic acids (Table 5).
of
monosaccharides
through
multiple-reaction
monitoring
liquid
chromatography/mass spectrometry using an aminopropyl column. Rapid
Communications in Mass Spectrometry, 24, 1565–1574.
Han, J., Chen, X. H., Sun, L. X., Zhan, Z. H.,
& Bi, K. S. (2009). Analysis of
monosaccharide compositions in Heterosmilax japonica polysaccharide by
precolumn derivation HPLC. Journal of Chinese medicinal materials, 32, 893–895.
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ultraviolet-absorbing and electrochemically sensitive 1-phenyl-3methyl-5-
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4. Conclusion
Studies on polysaccharide from S. fusiforme are extensive (Ji
et al., 2007; Mao et al., 2004; Zhu et al., 2003). However, analytical
methods of PMP precolumn derivatization high-performance li-
quid chromatography coupled with electrospray ionisation mass
spectrometry have not been reported. In the present study, a facile
and sensitive method based on precolumn derivatization HPLC/
ESI–MS has been established for the analysis of the monosaccha-
ride composition of water-soluble polysaccharides in S. fusiforme.
All PMP-labelled derivatives display high chemical stability, their
regular MS fragmentation is specific for reducing labelled sugars.
The suggested method for monosaccharide composition analysis
with PMP pre-column derivatization by on-line HPLC–ESI–MS
was tested for a monosaccharide composition prepared from natu-
ral S. fusiforme water-soluble polysaccharide, which provides a
rapid, reproducible, accurate, and economic alternative for the
identification of monosaccharides from S. fusiforme. Capillary elec-
trophoresis (CE) is the well studied method for PMP-labelled
monosaccharides, which has an advantage over HPLC in the
separation of PMP-labelled monosaccharides since there is no
packing material nor metal plumbing involved which cause metal
oxidation of PMP-labelled monosaccharides and sample adsorption
or degradation. Relatively low sensitivity and reproducibility are
two major factors limiting the application of CE in the analysis of
monosaccharides, which could be overcome by on-line HPLC–
ESI–MS method.
Huang, X., Zhou, H.,
& Zhang, H. (2006). The effect of Sargassum fusiforme
polysaccharide extracts on vibriosis resistance and immune activity of the
shrimp, Fenneropenaeus chinensis. Fish and Shellfish Immunology, 20, 750–757.
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efficient analysis of underivatized carbohydrates using monolithic-silica-based
capillary hydrophilic interaction (HILIC) HPLC. Analytical and Bioanalytical
Chemistry, 391, 2533–2542.
Janini, G. M., & Issaq, H. J. (1992). Micellar electrokinetic capillary chromatography:
basic considerations and current trends. Journal of Liquid Chromatography, 15,
6–7.
Ji, Y., Wang, C., Wu, T., & Ji, C. (2007). Effect of Sargassum fusiforme polysaccharides
on the complex mobility of erythrocytes in tumor-bearing organisms using high
performance capillary electrophoresis. Chinese Journal of Chromatography, 25(3),
322–325.
Kim, H., Yoo, J.-Y., Han, S. B., Lee, H. J., & Lee, K. R. (2003). Determination of ambroxol
in human plasma using LC–MS/MS. Journal of Pharmaceutical and Biomedical
Analysis, 32, 209–216.
Lu, Y., Yan, M.-C., Xu, H.-X., & Nan, C.-R. (2010). Antimicrobial activities of extracts
from Sargassum fusiforme and Enteromorpha clathrata against aquatic animal
pathogen. Marine Sciences, 10, 6.
Mao, W. J., Li, B. F., Gu, Q. Q., Fang, Y. C., & Xing, H. T. (2004). Preliminary studies on
the chemical characterization and antihyperlipidemic activity of polysaccharide
from the brown alga Sargassum fusiforme. Hydrobiologia, 512, 263–266.
Shen, X. D., & Perreault, H. (1998). Characterization of carbohydrates using a
combination of derivatization, high-performance liquid chromatography and
mass spectrometry. Journal of Chromatography A, 811, 47–59.
Suzuki, S., Tanaka, R., Takada, K., Inoue, N., Yashima, Y., Honda, A., et al. (2001).
Analysis of sialo-N-glycans in glycoproteins as 1-phenyl-3-methyl-5-
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Wang, Z.,
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Acknowledgement
polysaccharide purified from Gynostemma pentaphyllum Makino.
Carbohydrate Polymers, 68, 54–58.
Wu, X. M., Ouyang, J. M., Deng, S., & Chen, Y. (2006). Inhibition of the crystal growth
and aggregation of calcium oxalate by algae sulfated polysaccharide in-vitro.
Chinese Chemical Letters, 17, 97–100.
This work was supported by NSFC (Nos. 81273386 and
30801430), the Ocean Pilot Foundation (No. 2012HY016B) and SFZJ
(No. J20110305).
Yan, X., Chuda, Y., Suzuki, M., & Nagata, T. (1999). Fucoxanthin as the Major
Antioxidant in Hijikia fusiformis,
a Common Edible Seaweed. Bioscience,
Biotechnology, and Biochemistry, 63, 605–607.
Yoshizawa, Y., Enomoto, A., Todoh, H., Ametani, A., & Kaminogawa, S. (1993).
Activation of murine macrophages by polysaccharide fractions from marine
algae (Porphyra yezoensis). Bioscience, Biotechnology, and Biochemistry, 157,
1862–1866.
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