Use of electrospray ionization ion-trap tandem mass spectrometry and principal component analysis to directly distinguish monosaccharides

Article abstract of DOI:10.1002/rcm.6219

RATIONALE Carbohydrates are good source of drugs and play important roles in metabolism processes and cellular interactions in organisms. Distinguishing monosaccharide isomers in saccharide derivates is an important and elementary work in investigating saccharides. It is important to develop a fast, simple and direct method for this purpose, which is described in this study. METHODS Stock solutions of monosaccharide with a concentration of 400 μM and sodium chloride at a concentration of 10 μM were made in water/methanol (50:50, v/v). The samples were subjected to electrospray ionization ion-trap tandem mass spectrometry (ESI-MS) and the detected [2M + Na - H₂O]⁺ ions were further investigated by tandem mass spectrometry (MS/MS), followed by applying principal component analysis (PCA) on the obtained MS/MS data sets. RESULTS The MS/MS spectra of the [2M + Na - H₂O]⁺ ions at m/z 365 for hexoses and m/z 305 for pentoses yielded unambiguous fragment patterns, while rhamnose can be directly identified by its ESI-MS [M + Na] ⁺ ion at m/z 187. PCA showed clustering of MS/MS data of identical monosaccharide samples obtained from different experiments. By using this method, the monosaccharide in daucosterol hydrolysate was successfully identified. CONCLUSIONS A new strategy was developed for differentiation of the monosaccharides using ESI-MS/MS and PCA. In MS/MS spectra, the [2M + Na - H ₂O]⁺ ions yielded unambiguous distinction. PCA of the archived MS/MS data sets was applied to demonstrate the spatial resolution of the studied samples. This method presented a simple and reliable way for distinguishing monosaccharides by ESI-MS/MS. Copyright

Full text of DOI:10.1002/rcm.6219

Research Article
Received: 12 October 2011
Revised: 20 February 2012
Accepted: 6 March 2012
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2012, 26, 1259–1264
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6219
Use of electrospray ionization ion-trap tandem mass spectrometry
and principal component analysis to directly distinguish
monosaccharides
1
1
2
1
1
3
1
*
*
Bing Xia , Yan Zhou , Xin Liu , Juan Xiao , Qing Liu , Yucheng Gu and Lisheng Ding
1Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization, Chengdu Institute of Biology, Chinese
Academy of Sciences, Chengdu 610041, P.R. China
2Beijing Entry-Exit Inspection and Quarantine Bureau, Beijing 100026, P.R. China
3Syngenta Jealott’s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, UK
RATIONALE: Carbohydrates are good source of drugs and play important roles in metabolism processes and cellular
interactions in organisms. Distinguishing monosaccharide isomers in saccharide derivates is an important and elemen-
tary work in investigating saccharides. It is important to develop a fast, simple and direct method for this purpose, which
is described in this study.
METHODS: Stock solutions of monosaccharide with a concentration of 400 mM and sodium chloride at a concentration of
10 mM were made in water/methanol (50:50, v/v). The samples were subjected to electrospray ionization ion-trap tan-
dem mass spectrometry (ESI-MS) and the detected [2M + Na – H2O]+ ions were further investigated by tandem mass
spectrometry (MS/MS), followed by applying principal component analysis (PCA) on the obtained MS/MS data sets.
RESULTS: The MS/MS spectra of the [2M + Na – H2O]+ ions at m/z 365 for hexoses and m/z 305 for pentoses yielded
unambiguous fragment patterns, while rhamnose can be directly identified by its ESI-MS [M + Na]+ ion at m/z 187.
PCA showed clustering of MS/MS data of identical monosaccharide samples obtained from different experiments. By
using this method, the monosaccharide in daucosterol hydrolysate was successfully identified.
CONCLUSIONS: A new strategy was developed for differentiation of the monosaccharides using ESI-MS/MS and PCA.
In MS/MS spectra, the [2M + Na – H2O]+ ions yielded unambiguous distinction. PCA of the archived MS/MS data sets
was applied to demonstrate the spatial resolution of the studied samples. This method presented a simple and reliable
way for distinguishing monosaccharides by ESI-MS/MS. Copyright © 2012 John Wiley & Sons, Ltd.
Carbohydrates are important compounds and play important
roles in a wide variety of metabolism progresses and cellular
interactions in organisms.[1,2] They are also an important
source of drugs.[3–5] In natural products, saccharides and their
derivatives including glycosides and glycolipids account for a
large percentage of identified second metabolites. In recent
years, research has been ongoing to determine the molecular
structures of carbohydrates and saccharide moieties in
natural products such as monosaccharide composition,
anomeric configuration, their sequence, and linkages.[6]
Among them, distinguishing the monosaccharide isomers in
carbohydrates and natural products is the most elementary
and important task.
and absence of measurable coupling constants, as well as the
substantial quantity of sample required. Chromatographic
methods are also widely used to separate and identify
monosaccharides[7–9] but they are time-consuming and need
pre- or post-column derivatization due to the low sensitivity
of detecting underivatized saccharide samples. On the other
hand, electrospray ionization mass spectrometry (ESI-MS)
and tandem mass spectrometry (MS/MS) offer precise mass
spectra for chemicals and non-covalent complexes with high
sensitivity. They have been adopted in qualitative distinction
and quantitation (to some degree) of monosaccharide stereo-
isomers.[10–13] Although these methods showed good results
in the characteristic differentiation of diastereomeric mono-
saccharides, they usually associated with specific reagents.
In this study, we report a simple, direct and additional
assistant reagent-free ESI-MS/MS method for the differentia-
tion of monosaccharides for the first time. Eight very common
D-sugar isomers commonly present in natural products
including four hexose isomers [glucose (Glc), galactose
(Gal), mannose (Man), and fructose (Fru)], three pentose
[ribose (Rib), arabinose (Ara) and xylose (Xyl)] and one
desose [rhamnose (Rha)] (see Supplementary Scheme 1,
Supporting Information) have been subjected to ESI-MS. An
interesting product ion of [2M + Na – H2O]+ was detected
and further investigated by MS/MS. Principal component
Many analytical methods have been introduced for the
determination of monosaccharides. As the key tool in
molecular structure identification, the nuclear magnetic
resonance (NMR) method encountered certain limitations in
identifying monosaccharides due to overlap of proton signals
* Correspondence to: Y. Zhou or L. S. Ding, Key Laboratory of
Mountain Ecological Restoration and Bioresource Utiliza-
tion, Chengdu Institute of Biology, Chinese Academy of
Sciences, Chengdu 610041, P.R. China.
E-mail: zhouyan@cib.ac.cn; lsding@cib.ac.cn
Rapid Commun. Mass Spectrom. 2012, 26, 1259–1264
Copyright © 2012 John Wiley & Sons, Ltd.

B. Xia et al.
analysis was applied and clearly demonstrated the spatial
resolution of these studied monosaccharides (hexoses and
pentoses). The application of this method in distinguishing
monosaccharide in a natural glycoside is also described.
range was set to m/z 100–500 with an isolation width of 2.0
m/z units. A stable sample flow of 5 mL/min was introduced
into the mass spectrometer by syringe pump.
RESULTS AND DISCUSSION
EXPERIMENTAL
ESI-MS and ESI-MS/MS of monosaccharides
Chemicals
In our work, ESI-MS spectra of monosaccharides in water/
methanol (50:50, v/v) were obtained. Typical full ESI-MS
spectra of hexose and pentose can be seen in Fig. 1. A remark-
able phenomenon was observed in our study that sodiated
monosaccharide dimer ([2M + Na]+, m/z 383) and dimer
dehydrate ([2M + Na – H2O]+ , m/z 365) ions were formed in
the ESI process; this phenomenon was also repeatable on
the LTQ Orbitrap LX instrument and these ions were
confirmed to be sodiated monosaccharide dimers and dimer
dehydrates according to the HR-MS data.
All monosaccharide standards (Glc, Gal, Man, Fru, Rib, Ara,
Xyl and Rha) used in this study were purchased from Kelong
Chemical Reagent (Chengdu, China) with purities over 98%.
Daucosterol was separated and identified by our group. Stock
solutions of monosaccharide with a concentration of 400 mM
and sodium chloride at a concentration of 10 mM were made
in water/methanol (50:50, v/v).
Glycoside hydrolysis
A source CID test was performed to inspect the origin of
the [2M + Na – H2O]+ ions by raising the cone voltage from
20 to 60 V in step sizes of 10 V. The signal intensity of
[2M + Na – H2O]+ ions increased and that of [2M + Na]+ ions
decreased with increasing the cone voltage. CID MS/MS
experiments were also employed for the [2M + Na]+ ions
and peaks with a mass loss of 18 Da were observed. The
results confirm that [2M + Na – H2O]+ was formed from the
dehydration process of [2M + Na]+, and might be formed via
Daucosterol (0.5 mg) dispersed in MeOH (5 mL) was heated
with 2 mol/L HCl (6 mL) under reflux for 15 h. The reaction
mixture was diluted with water (15 mL) and extracted with
CHCl3 (3 ꢀ 15 mL) to remove the aglycon. The aqueous layer
was directly subjected to ESI-MS analysis.
Mass spectrometry
ESI-MS and ESI-MS/MS analyses were performed on a
Finnigan LCQDECA (San Jose, CA, USA) ion-trap mass
spectrometer equipped with an ESI source. The ESI spray
voltage was held at 4.5 kV, and the spray was stabilized with
nitrogen sheath gas. The heated capillary temperature was set
to 200 ꢁC. Helium was introduced into the ion trap as the
collision gas for collision-induced dissociation (CID). The
maximum ion injection time of 50 ms and three ’microscans’
per analytical scan was set. The instrument was operated in
the positive ion mode with the experimental parameters as
follows: sheath gas flow rate, 35 arbitrary units (arb);
capillary voltage, 30 V.
The CID MS/MS experiments were carried out by
increasing the normalized collision energy from 10% to 40%
with a step size of 5%, and the mass range was set to m/z
100–500. Samples were continuously introduced into the ESI
source chamber for analysis with a syringe pump at a flow
rate of 5 mL/min. To deposit insoluble substances, all samples
were centrifuged before analysis at a rotate rate of 5000 rpm
for 10 min. All experiments were repeated at least three times
on different days. Each spectrum used in analysis was the
result of at least 30 scan accumulations.
(a)
High-resolution ESI-MS/MS (HR-ESI-MS/MS) was
performed on a LTQ Orbitrap XL mass spectrometer (Thermo
Fisher Scientific, USA) to verify and help interpret the results
obtained from the low-resolution ESI ion-trap mass
spectrometer. The mass analyzer was calibrated before
measurements according to the manufacturer’s instructions
to ensure operation within the <3 ppm instrument
specifications. The instrument was operated in the positive
ion mode with the experimental parameters as follows:
sheath gas flow rate, 30 arb; auxiliary gas flow rate, 8 arb;
(b)
ꢁ
capillary voltage, 30 V; capillary temperature, 200 C; spray
voltage, 4.5 kV. A normalized collision energy of 30% was
adopted to perform CID MS/MS experiments and the mass
Figure 1. ESI-MS spectra of (a) glucose and (b) ribose in a
methanol/water solution of NaCl.
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Rapid Commun. Mass Spectrom. 2012, 26, 1259–1264

Use of ESI-MS and PCA to distinguish monosaccharides
a similar way to the formation of protonated disaccharides
from monosaccharide proton-bound dimer dehydration
mentioned by Zapfe and Mu.[14] Although [2M+ Na – H2O]+
ions can be easily observed during MS/MS of [2M + Na]+, the
signal intensity was low and the CID procedure mainly
produces sodiated molecules, as was reported by March and
Stadey.[15] In addition, MS/MS spectra of [M + Na]+ ions
showed a slight difference between Gal, Glc and Man as was
reported by Zhu and Sato.[12] Thus [2M + Na]+ and [M + Na]+
ions were not chosen to distinguish monosaccharide isomers.
Formation of non-covalent dimers during ESI of saccharides
has already been observed and was found to be very often a
concentration-related phenomenon.[16,17] To find the proper
concentration of monosaccharide samples, LOD (limit of
detection) tests for the [2M + Na – H2O]+ ions were performed
for all monosaccharide samples by observing the absence of
[2M + Na – H2O]+ ions with a sample concentration increase
in steps of 50 mM starting from 50 mM; the LOD tests showed
that samples with concentration of 350 mM were capable of
forming [2M + Na – H2O]+ ions for all samples on the Finnigan
LCQDECA instrument. To obtain better [2M+ Na – H2O]+ ion
signal intensity, a sample concentration of 400 mM was finally
set in our experiments.
easily recognized from hexoses (Glc, Man and Gal) according to
its relative abundance ratio of ions at m/z 347 to 275
(1.41 ꢂ 0.042 for Glc, 0.68 ꢂ 0.023 for Man and 0.59 ꢂ 0.012 for
Gal). Although the MS/MS spectra for Man and Gal seemed
quite similar to each other at first glance, they maintained
different relative abundance ratios of ions at m/z 275 to 245
(2.47 ꢂ 0.024 for Man and 3.77 ꢂ 0.016 for Gal) and thus could
also be distinguished. As for the three pentoses (Rib, Ara and
Xyl), they could be distinguished from each other by
comparing the relative abundance ratios of ions at m/z 245 to
215 (0.40 ꢂ 1.23 for Ara, 1.09 ꢂ 0.12 for Rib and 6.93 ꢂ 0.034
for Xyl).
Fragmentation mechanism
The [2M + Na – H2O]+ ions are in essence sodium-ionized
disaccharides, so the fragmentation pattern of the MS/MS
spectrum of [2M + Na – H2O]+ ions can be explained as that
of the disaccharides. It is clear that the ions at m/z 347 and
287 were formed due to loss of water of hexoses and pentoses,
respectively, and the other fragment ions may arise from the
precursor ion as shown in Supplementary Fig. 2 (see
Supporting Information).
The mass spectra of hexoses and pentoses are shown in Fig. 1;
in the case of hexoses, the ions [M+ Na]+ (m/z 203), [2M + Na]+
(m/z 383) and [2M + Na – H2O]+ (m/z 365) were observed, while
pentoses showed ions at m/z 173 ([M+ Na]+), m/z 323
([2M + Na]+) and m/z 305 ([2M+ Na – H2O]+), and for desose
they were m/z 187 ([M + Na]+), m/z 351 ([2M + Na]+) and
m/z 333 ([2M + Na – H2O]+). As the only desose, Rha can be
identified directly by its quasi-molecular ion from the MS
mode. To distinguish different hexoses and pentoses, MS/MS
was adopted to investigate the dimer dehydrate ions.
Breakdown curves of the MS/MS ions were generated for the
dimer dehydrate ions of all the monosaccharides (see
Mechanisms of the generation of MS/MS fragment ions
from the precursor [2M + Na – H2O]+ ions of hexoses (except
for Fru) and pentoses have been proposed. Glucose and
arabinose were chosen as examples to demonstrate the
fragmentation pathways for hexoses and pentoses,
respectively. Supplementary Scheme
2 (see Supporting
Information) shows the fragmentation mechanism of glucose. In
the MS/MS spectra, a loss of 60 Da (C2O2H4, HOCH = CHOH)
was observed from [2Glc + Na – H2O] at m/z 365, which
was formed due to the fragmentation from m/z 365 ! 305.
A further fragmentation from m/z 305 ! 275 and m/z
275 ! 245 showed the successively losses of 30 Da (HCHO).
In addition, the fragmentation mechanism of arabinose
can be found in Supplementary Scheme 3 (see Supporting
Information). Similar to glucose, a loss of 60 Da (C2O2H4,
HOCH = CHOH) from the precursor ion [2Ara + Na – H2O]+
at m/z 305 gave the fragment ion at m/z 245, which was further
fragmented and produced the ion at m/z 215 corresponding to a
loss of 30 Da (HCHO). All these results have been confirmed by
HR-ESI-MS/MS obtained from a LTQ Orbitrap LX mass
Supplementary Fig. 1, Supporting Information). With
a
collision energy lower than 25%, the relative abundances of
the product ions of these dimer dehydrate ions were low
compare to that of their precursor ions (base peaks), while the
breakdown curves for all but the precursor ions became flat
when the precursor ions were fragmented with a collision
energy higher than 30%. This means that a better MS/MS
spectra reproducibility could be obtained when the
experiments were carried out with a collision energy higher than
30%, and this is why the product ions of [2M + Na – H2O]+ and
30% normalized collision energy were chosen for differentiation
of monosaccharides.
spectrometer (Supplementary Table
1
(see Supporting
Information) and Table 2), and conform to the results reported
by Hofmeister et al.[18] and Salpin and Tortajada.[17]
The MS/MS spectra generated from [2M + Na – H2O]+ ions
at m/z 365 (hexoses) and 305 (pentoses) presented a series of pro-
duct ions as shown in Fig. 2(a) (for four hexoses) and Fig. 2(b)
(for three pentoses). The hexose isomers and pentose isomers
gave similar product ions with different relative abundant
ratios, respectively. The product ions were detected at m/z
185, 203, 245, 275, 305 and 347 for hexose isomers (except for
Fru), and m/z 173, 215, 245 and 287 for pentose isomers. It
should be noticed that, as a ketose, Fru presented a special
fragmentation pattern; the lack of peaks at m/z 245 and 305
made it different from the other three hexoses. Although
[2M + Na – H2O]+ ions of hexoses (Glc, Man and Gal) and
pentoses (Rib, Ara and Xyl) showed similar fragmentation
patterns, the relative abundances of their product ions showed
the potential to distinguish the saccharide isomers. Glc could be
Principal component analysis
PCA was employed to analyze the MS/MS data obtained
from the [2M + Na – H2O]+ ions of different monosaccharide
isomers (for hexoses they were Glc, Man and Gal, while, for
pentose, they were Rib, Ara and Xyl) with the software
SIMICA-P + (version 11.0, Umetric, Umea, Sweden). Intensi-
ties of the MS/MS ions of [2M + Na – H2O]+ (m/z 185, 245,
275, 305 and 345 for hexose isomers and m/z 173, 215, 245
and 287 for pentose isomers) were chosen as components
for PCA. Good spatial resolution was obtained in the
PCA plot (Fig. 3), which showed clustering of MS/MS data
of identical monosaccharide samples obtained from
different experiments.
Rapid Commun. Mass Spectrom. 2012, 26, 1259–1264
Copyright © 2012 John Wiley & Sons, Ltd.
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B. Xia et al.
Glc
Gal
Man
Fru
(a)
Rib
Ara
Xyl
(b)
Figure 2. Positive ESI-MS/MS spectra of (a) four hexose isomers (Glc, Gal, Man and Fru) and (b) three
pentose isomers (Rib, Ara and Xyl) generated from the precursor ion [2M + Na – H2O]+ (m/z 365).
Fragmentation energy was 30%, label threshold was set to 3%.
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Rapid Commun. Mass Spectrom. 2012, 26, 1259–1264

Use of ESI-MS and PCA to distinguish monosaccharides
of these studied monosaccharides and the clustering with that
of the glycoside sample. This method presented a simple and
reliable way for distinguishing monosaccharides without
derivatization by direct injection to ESI-MS/MS analysis.
Glc
Man
Gal
SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article.
Acknowledgements
(a)
This work was supported by grants from the National Natural
Sciences Foundation of China (No. 30973634; 21072185), Innova-
tion Key Program (KSCX2-EW-J-22) and 2010 Technological
Talents Program of the Chinese Academy of Sciences granted to
Dr. Y. Zhou, and the Syngenta PhD fellowship awarded to B. Xia.
Ara
Xyl
Rib
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