Enumeration of hydroxyl groups of sugars and sugar alcohols by aqueous-based acetylation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Article abstract of DOI:10.1002/rcm.6234

RATIONALE The properties of carbohydrates are determined in part by the number and stereochemical arrangement of their hydroxyl groups. To facilitate their analysis, rapid methods are needed to identify and enumerate hydroxyl groups in sugars and polyalcohols, especially methods that are water-based. METHODS The present report details the results of an alternative method for identification and enumeration of hydroxyl groups in aqueous media. It employs vinyl acetate to selectively derivatize hydroxyl groups in analytes, followed by analysis of the reaction mixtures by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS The method has been applied to several single and multi-component mixtures of monosaccharides and polyalcohols. The O-acetylated products were analyzed without chromatographic separation or purification by MALDI-TOF-MS. The mass spectra revealed consecutive ion peaks that are separated by 42 mass units as a consequence of displacement of one hydroxyl hydrogen by one acetyl group. CONCLUSIONS A rapid and aqueous-based method is described to enumerate the hydroxyl groups in carbohydrates. The number of ion peaks due to derivatized products is determined by MALDI-TOF-MS, and corresponds to the number of free hydroxyl groups in the analyte. The method is applicable to both single and multi-component mixtures. Published 2011. This article is a US Government work and is in the public domain in the USA. Published 2012. This article is a US Government work and is in the public domain in the USA.

Full text of DOI:10.1002/rcm.6234

Research Article
Received: 21 November 2011
Revised: 16 March 2012
Accepted: 18 March 2012
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2012, 26, 1372–1376
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6234
Enumeration of hydroxyl groups of sugars and sugar alcohols by
aqueous-based acetylation and matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry
Anthony Adeuya ,† and Neil P. J. Price
*
USDA-ARS-NCAUR, Renewable Product Technology Research Unit, 1815 North University Street, Peoria, IL 61604, USA
RATIONALE: The properties of carbohydrates are determined in part by the number and stereochemical arrangement of
their hydroxyl groups. To facilitate their analysis, rapid methods are needed to identify and enumerate hydroxyl groups
in sugars and polyalcohols, especially methods that are water-based.
METHODS: The present report details the results of an alternative method for identification and enumeration of
hydroxyl groups in aqueous media. It employs vinyl acetate to selectively derivatize hydroxyl groups in analytes,
followed by analysis of the reaction mixtures by matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF-MS).
RESULTS: The method has been applied to several single and multi-component mixtures of monosaccharides and
polyalcohols. The O-acetylated products were analyzed without chromatographic separation or purification by
MALDI-TOF-MS. The mass spectra revealed consecutive ion peaks that are separated by 42 mass units as a consequence
of displacement of one hydroxyl hydrogen by one acetyl group.
CONCLUSIONS: A rapid and aqueous-based method is described to enumerate the hydroxyl groups in carbohydrates.
The number of ion peaks due to derivatized products is determined by MALDI-TOF-MS, and corresponds to the number
of free hydroxyl groups in the analyte. The method is applicable to both single and multi-component mixtures.
Published 2011. This article is a US Government work and is in the public domain in the USA.
Carbohydrates and polyalcohols are of great importance in
both the pharmaceutical and food industries. For example,
polyalcohols are used as fillers in drugs and therefore are one
of the by-products from drug metabolism. Carbohydrates and
polyalcohols, like other hydroxyl group containing analytes,
are typically characterized by gas chromatography (GC)
and liquid chromatography (LC) based methods.[1–6] Analy-
sis by GC usually requires tedious and time-consuming con-
version of the hydrophilic polyalcohols into their lipophilic
derivatives that are suitable for in-line pre-separation by
GC.[3] Permethylation, peracetylation and trimethylsilylation
are commonly used for this purpose.[7–11] Matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF-MS) has extended the usefulness of mass spec-
trometry in recent years. This technique, unlike electron
impact (EI) ionization, generally gives molecular weight
information as sample fragmentation is minimized with the
matrix used for MALDI analysis.[12]
Studies have proposed hydroxyl group identification and
enumerating methods that include ion-molecule derivatiza-
tion reactions in a Fourier-transform ion cyclotron resonance
(FT-ICR) mass spectrometer and hydrogen/deuterium (H/D)
exchange by MALDI-TOF-MS.[13,14] These methods require
specialized instrumentation and a high degree of expertise.
We recently reported a rapid and facile method of identifying
and enumerating hydroxyl groups in sugars and polyalcohols
by trimethylsilyl derivation reaction coupled with MALDI-
TOF-MS.[15,16] The method can only be used in characterizing
dry samples because of the inability to prepare trimethylsilyl
derivatives in the presence of moisture.
The present report details the results of an alternative
method for identification and enumeration of hydroxyl groups
in aqueous media. This method employs a modified GC/MS
per-O-acetylation method procedure where vinyl acetate
selectively derivatized hydroxyl groups in analytes, followed
by analysis of the reaction mixture by MALDI-TOF-MS.
MALDI-TOF mass spectra of acetyl-derivatized analytes
revealed consecutive ion peaks that are separated by 42 Da.
This 42 mass unit is due to a net substitution of one hydroxyl
hydrogen atom by one acetyl group. The number of peaks
due to derivatized products corresponds to the number of free
hydroxyl groups in the analyte. This method was applied to
several single and multi-component mixtures of monosaccha-
rides and polyalcohols. In addition, like the results of previous
studies,[15,16] more information is obtained for each sugar or
polyalcohol due to the presence of the reactant (starting sugar
or polyalcohol), partial and fully derivatized product ions in
* Correspondence to: A. Adeuya, USDA-ARS-NCAUR,
Renewable Product Technology Research Unit, 1815 North
University Street, Peoria, IL 61604, USA.
E-mail: anthony.adeuya@fda.hhs.gov
†
Current address: 3900 NCTR Road, Jefferson, AR 72079,
USA.
Rapid Commun. Mass Spectrom. 2012, 26, 1372–1376
Published 2012. This article is a US Government work and is in the public domain in the USA.

Enumeration of hydroxyl groups by aqueous-based acetylation
the mass spectrum. This can be very valuable when multi-
component mixtures are being analyzed. This method can be
used in analysis of hydroxyl groups containing compounds
in food, drugs and environmental matrixes.
output. Measured mass spectra were an average of 200 laser
shots. The instrument was calibrated externally by a series
of malto-oligosaccharides.
RESULTS AND DISCUSSION
EXPERIMENTAL
MALDI-TOF-MS coupled with aqueous-based acetylation
has been used successfully in determining the number of free
hydroxyl groups in monosaccharides (glucose, ribose, galac-
tose) and polyalcohols (erythritol, mannitol, and xylitol).
The product mixture of each analyte derivative was detected
by using MALDI-TOF-MS in the positive ion mode as sodium
adducts represented as [M + Na]+ and [M–xH + xAc + Na]+
(where x is the number of hydroxyl or Acetyl (Ac) groups)
for the starting material and the acetyl derivatized product,
respectively. The presence of multiple hydroxyl groups in an
analyte results in consecutive derivatized species that are
separated by 42 Da in molecular mass, corresponding to the
mass increase as the result of displacement of one hydrogen
atom by one acetyl group.
Erythritol was derivatized with vinyl acetate as described
in the Experimental section and analyzed by MALDI-TOF-MS.
A total of five peaks, separated by 42 mass units, were observed
in the mass spectrum of the analyzed reaction mixture (Fig. 1(a)).
These peaks are due to the starting material (erythritol), three
partially derivatized erythritols and the fully acetyl deriva-
tized erythritol at m/z 145, 187, 229, 271, and 313, respectively.
Similarly, with xylitol the number of observed peaks due to
differently derivatized products corresponds to the number
of free hydroxyl groups in xylitol. There are five free hydroxyl
groups on xylitol and a total of six mass spectra peaks
were observed at m/z 175, 217, 259, 301, 343, and 385 due to
the starting material, xylitol, and the first through fifth
derivatized products, respectively. The peak at m/z 535
corresponds to the sodium adduct of the fully acetylated
species [xylitol–5 H + 5Ac + Na]+ (Fig. 1(b)).
Chemicals
All monosaccharides used were from the collection at
NCAUR. Erythritol, xylitol, vinyl acetate and the matrix
reagent (2,5-dihydroxybenzoic acid and acetonitrile) were
obtained from Sigma Aldrich (St. Louis, MO USA). Mannitol
was obtained from Fisher Scientific (Fair Lawn, NJ, USA). All
samples were used as received.
Derivatization reactions
Recently, Shi and coworkers reported the results of a study
that showed excellent total derivatization of hydroxyl
groups.[7] In this study, the reported method has been
modified to obtain all the partial and fully derivatizated
products of hydroxyl group containing analytes. In brief,
about 2 mg of sample were dissolved in a vial containing
20 mL of vinyl acetate and 100 mL of 3% Na3PO4 buffer
solution at room temperature (pH of approximately 12).
(For the mechanism of the reaction, see Scheme 1.) The
mixture was vortexed, allowed to stand for about 3 to
5 min and analyzed by MALDI-TOF-MS without purification
or extraction. For the multi-component study, about 1 mg
of each component was dissolved in 30 mL of vinyl acetate
with 150 mL of the buffer solution. Special care must be taken
when working with vinyl acetate. Its vapor causes eye
and respiratory irritations, and it is a fire hazard according
to the United States Occupational Safety and Health Admin-
istration (OSHA).[17]
The reactivity of mannitol towards vinyl acetates is similar
to that of erythritol and xylitol. As observed previously, the
number of observed peaks due to differently derivatized
products correlates with the number of free hydroxyl groups
in mannitol. Hence, mannitol has six free hydroxyl groups
and a total of seven mass spectra peaks were observed at
m/z 205, 247, 289, 331, 373, 415, and 457 due to the starting
material, mannitol, and the first through sixth derivatized
products, respectively. It is important to note that the
MALDI-TOF mass spectra revealed the fully derivatized
product peaks for both xylitol and mannitol (Fig. 1(c)) unlike
those obtained in a previous FT-ICR-MS study.[13] In addition,
there are no fragment ions due to ethane losses and the results
are therefore easier to interpret.
MALDI-TOF-MS
Acetylation reaction mixtures (about 0.5 mL) were spotted on
the MALDI-TOF MS stainless steel target followed by 2 mL of
a saturated matrix solution (2,5-dihydroxybenzoic acid, DHB,
in acetonitrile). The resulting cocrystal was allowed to dry at
room temperature and the target was introduced into the
mass spectrometer via the probe carrier. Measurements were
acquired in the positive ion reflectron mode on an Omniflex
time-of-flight mass spectrometer (Bruker Daltonics Inc., Billerica,
MA, USA). A 200 ns pulsed ion extraction was used with ion
sources 1 and 2 set to 19.00 kV and 13.6 kV, respectively. The
reflectron voltage was set to 20 kV. Ions were excited at
337 nm, typically at 65% of 150 mJ maximum laser power
Scheme 1. Proposed mechanism for the reaction of erythritol and vinyl acetate.
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A. Adeuya and N. P. J. Price
a
b
1500
1250
1000
750
500
250
0
+
[M+Na]
800
600
400
200
0
meso-Erythritol
Xylitol
+
[M+Na]
+
[M-5H+5Ac+Na]
+
[M-4H+4Ac+Na]
175
200
225
250
275
300
325
350
375
400
140
160
180
200
220
240
260
280
300
320
m/z
m/z
c
d
+
[Xylitol+Na]
1500
1250
1000
750
500
250
0
2500
2000
1500
1000
500
+
[M+Na]
Mannitol
+
[M-6H+6Ac+Na]
0
200 225 250 275 300 325 350 375 400 425 450
150
200
250
300
350
400
450
500
m/z
m/z
Figure 1. MALDI-TOF mass spectra of the reaction products of erythritol (a), xylitol (b), mannitol (c), and erythritol/
mannitol/xylitol mixture (d) with vinyl acetate. The peak denoted as [M + Na]+ represents the ion for the starting
polyalcohol (m/z 145, 175, 205 for erythritol, xylitol and mannitol, respectively). The other peaks are due to sodium
adducts of the acetyl derivatives. Unlabeled peaks are due to matrix and impurity ions.
The hydroxyl group enumeration method described above
can be applied to multi-component mixtures. This was shown
by treating a mixture containing erythritol, xylitol and manni-
tol with vinyl acetate and analyzing the resulting mixture by
MALDI-TOF-MS. The measured mass spectrum (Fig. 1(d))
reveals the same information as the measured mass spectra
for single components (Figs. 1(a), 1(b) and 1(c)). Hence, a total
of 18 peaks are evident for the mixture, 5, 6 and 7 for erythri-
tol, xylitol and mannitol, respectively.
intensities. Qualitatively, the same numbers of peaks were
obtained for corresponding trials. This observed variation
may be due to the varying ionization efficiency and a
well-known phenomenon of non-uniform co-crystalization
of analyte/matrix mixture on the MALDI target plate.[18]
CONCLUSIONS
MALDI-TOF-MS coupled with acetyl derivatization described
in this work may be used to identify and enumerate
the hydroxyl groups in monosaccharides and polyalcohols
in aqueous media. This approach is rapid, requires little
or no expertise in organic synthesis, and there is no need
for sample purification prior to mass spectrometry
measurements.
The results obtained for the three sugars examined (ribose,
fucose and glucose and a mixture containing all three) are
shown in Fig. 2. Like the results from the polyalcohols study,
it revealed peaks that are due to starting material, the
partially derivatized and fully derivatized products for each
sugar. Hence, five characteristic ions are observed for the
6-deoxysugar, fucose (m/z 187, 229, 271, 313 and 355); five
for ribofuranose (m/z 173, 215, 257, 299 and 341); and six
for glucopyranose (m/z 203, 245, 287, 329, 371 and 413).
Noticeably, the cyclic hemiacetal forms of these sugars are
retained during the reaction procedure, resulting in the
corresponding five- or six-membered cyclic O-acetylated
derivatives.
Acknowledgements
The authors would like to thank Karen Hughes for help
in preparing this manuscript. Trina Hartman is thanked for
technical assistance. Mention of trade names or commercial
products in this article is solely for the purpose of providing
specific information and does not imply recommendation or
endorsement by the U.S. Department of Agriculture.
Like the result of a previous study,[15] repeated acetylation
and MALDI-TOF-MS measurements for the single and
multi-component analyte mixtures showed varied peak
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Enumeration of hydroxyl groups by aqueous-based acetylation
a
b
400
300
200
100
0
+
[M+Na]
800
Fucose
Ribose
600
+
[ M-4H+4Ac+Na ]
400
+
[M+Na]
+
[ M-4H+4Ac+Na ]
200
0
180
200
220
240
260
280
300
320
340
180 200 220 240 260 280 300 320 340 360
m/z
m/z
c
d
+
[ M-5H+5Ac+Na ]
1000
800
600
400
200
0
800
600
400
200
0
Glucose
+
[M+Na]
200
225
250
275
300
325
350
375
400
425
175
200
225
250
275
300
325
350
375
400
m/z
m/z
Figure 2. MALDI-TOF mass spectra of the reaction products of fucose (a), ribose (b), glucose (c), and glucose/
fucose/ribose mixture (d) with vinyl acetate. The peak denoted as [M + Na]+ represents the ion due to a non-
derivatized or the starting monosaccharide (m/z 187, 173, 203 for fucose, ribose and glucose, respectively). The
other labeled peaks are due to sodium adducts of the acetyl derivatized products. Unlabeled peaks are due to
matrix and/or impurity ions.
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