C H A P T E R 3 5

SUGARS (MONOSACCHARIDES)

35.1. GC SEPARATION OF DERIVATIZED SUGARS

A. Monosaccharides
1. Preparation of the TMS derivative: Add 0.5 mL of TRI-Sil Z reagent
(trimethylsilylimidazole in pyridine) to 1–5 mg of the sample. (This
derivatizing preparation does not react with amino groups and
tolerates the presence of water.) Heat in a sealed vial at 60 °C until
the sample is dissolved. An alternate method is to let the reaction
mixture stand at room temperature for at least 30 minutes (or
overnight). This procedure is not appropriate for amino sugars.
2. GC separation of TMS derivatives: arabinose, fucose, xylose,
mannose, galactose, α-glucose, and β-glucose: 30-m DB-1 column,
60–250 °C at 8 °C min�1.
B. Amino sugars (glucosamine and galactosamine as TMS derivatives)
1. Preparation of TMS derivative: To derivatize the amino sugars as well
as the non-amino sugars, substitute TRI-Sil TBT or TRI-Sil/BSA
(Formula P) reagent for TRI-Sil Z and follow the procedure given in
Section 35.1.A.1.
2. GC separations of amino sugars as TMS derivatives: 30-m DB-1701
column, 70–250 °C at 4 °C min�1. (Depending on the amounts
present, complete GC separation may not be achieved.)
C. Sugar alcohols (as acetates)
1. Preparation of the acetate derivative: Evaporate the extract to
dryness. Add 50 µL of three parts acetic anhydride and two parts
pyridine. Heat at 60 °C for 1 hour. Evaporate to dryness with clean
dry nitrogen and dissolve the residue in 25 µL of ethyl acetate.
2. GC separation of the acetate derivatives: Rhamnitol, fucitol, ribitol,
arabinitol, mannitol, galacitol, glucitol, and inositol: 30-m CP-Sil 88
column at 225 °C for 60 minutes.
D. Reduced sugars (as acetates)
1. Preparation of the acetate derivative: Concentrate the aqueous
mixture of saccharides to approximately 0.5 mL in a 20–50 mL
container. Reduce the saccharides by adding 20 mg of sodium
borohydride that has been dissolved carefully into 0.5 mL of water
and let the reducing mixture stand at room temperature for at least
Gas Chromatography and Mass Spectrometry � 2010 by Academic Press. Inc.
DOI: 10.1016/B978-0-12-373628-4.00035-6 All rights reserved.

407
408 Chapter 35

1 hour. Destroy the excess sodium borohydride by adding acetic

acid until the gas evolution stops. Evaporate the solution to dryness
with clean nitrogen. Add 10 mL of methanol and evaporate the
solution to dryness. Acetylate with 0.5 mL (three parts acetic
anhydride and two parts pyridine) overnight. Evaporate to a
syrupy residue and add 1 mL of water. Evaporate again to dryness
to remove the excess acetic anhydride. Dissolve the residue in 250 µL
methylene chloride.
2. GC separation of reduced and acetylated sugars: 30-m CP-Sil 88
column at 225 °C.

35.2. MASS SPECTRAL INTERPRETATION

A. Mass spectra of TMS derivatives of sugars
1. Molecular weight
In general, to deduce the molecular weight of the TMS derivative
of sugars, add to 105 the highest mass observed (loss of
(CH3)SiOH + •CH3).
• 333.1374 is the highest mass observed for arabinose, ribose,
ribulose, xylose, lyxose, and xylulose.
• 347.1530 is the highest mass observed for fucose and rhamnose.
• 435.1875 is the highest mass observed for sorbose, allose, altrose,
galactose, gulose, idose, and mannose.
2. Fragmentation
DeJongh et al. [1] have described the fragmentation of the TMS
derivatives of sugars. Comparison of GC retention times together
with the mass spectra is sufficient to identify the sugars. The mass
spectra suggest certain structural features. For instance, peaks at m/z
191, 204, and 217 suggest a TMS hexose. If the peak at m/z 204 is
more intense than the peak at m/z 217, the hexose is the pyranose
form; but if the m/z 217 peak is most intense, then it is a furanose
(Figure 35.1). Aldohexoses can be differentiated from ketohexoses
by the peaks at m/z 435 for aldohexoses versus m/z 437 for
ketohexoses.

m/z Ion structure

191 (CH3)3SiOCH=OSi(CH3)3 +
204 (CH3)3SiOCH=CHOSi(CH3)3 +
217 (CH3)3SiOCH=CH−CH=OSi(CH3)3 +
Sugars (Monosaccharides) 409

204
100

O Si

73
O

50 O Si
191 Si
O

147 O
Si
217
O
Si
103 129

59 157 231 257 305 345 361 379 435

0
60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540

Figure 35.1 EI mass spectrum of TMS–α-glucose.

B. Sample mass spectrum

C. Mass spectra of amino sugars as TMS derivatives
1. Molecular ion: By plotting mass chromatograms for the m/z value of
the M+• peak and the accurate m/z value 362.1639, the presence of
these amino sugars can be determined.

Highest m/z value peak observed

Glucosamine 393.1643
Galactosamine 467.2375

2. Fragmentation: The TMS derivatives of amino sugars have their base

peak at m/z 131.0765.
D. Mass spectra of sugar alcohol as acetates
1. Molecular ion: Chemical ionization using ammonia as reagent gas
establishes the molecular weights of sugar acetates.
2. Fragmentation: Biemann et al. [2] have shown the fragmentation of
alditol acetates. The acetates have intense ions at m/z 43, 103, and
145. They also appear to lose 42 Da, 59 Da, 60 Da, and 102 Da from
their molecular ions
CH2OC(O)CH3

CHOC(O)CH3 145 103

CHOC(O)CH3 217 157 115

CHOC(O)CH3 289 127 187

etc.
410 Chapter 35

The mass spectrum of alditol acetates are easy to interpret as they fragment
at each C–C bond as shown.

REFERENCES
1. DeJongh, D. C., Radfon, T., Hribar, J. D., et al., (1969). Analysis of derivatives of
carbohydrates by GC/MS. J. Am. Chem. Soc., 91, 1728.
2. Biemann, K., Schnoes, H. K., McCloskey, J. A. (1963). Applications of mass spectro­
metry to structure problems. Carbohydrates and their derivatives. Chem. Ind. (London),
448, 449.