by GC/MS. These derivatives have been compared for the
quantitative evaluation of 13C distribution into isotopomers of 13C-
labeled aldoses and ketoses. In addition, the fragmentation
pathways for 15 hexoses, pentoses, and amino sugars of biological
origin have been assessed. Finally, a new type of carbohydrate
derivative (dialkyldithioacetal acetate) has been developed for GC/
MS and optimized for use with several thiolating agents (1,2-
dithioethane, 1,2-dithiopropane, 1,3-dithiopropane, and British anti-
Lewisite). Electron impact ionization of these acyclic derivatives
generates a dominant, well-resolved base peak arising from C1-
C2 bond cleavage and series of fragment ions with charge
retention at the C1 thiol groups. These derivatives are well suited
for measurement of isotopic enrichment into the anomeric carbon
of a large number of different aldose sugars and will facilitate the
global analysis of metabolic flux in carbohydrate pathways.
with acetic anhydride: pyridine-dichloromethane (1:1:1 by vol-
ume, 60 °C, 1 h).20 Extractions and injections were as for the alditol
acetates.
Dialkyldithioacetal acetates. Optimized conditions for the
preparation of diethyldithioacetal acetates were as follows. Monosac-
charide stocks (10 µL ) 1 mg) were evaporated to dryness and
treated with ethanethiol (100 µL) plus trifluoroacetic acid (50 µL)
at room temperature for 15 min. Peracetylation was achieved by
adding acetic anhydride (300 µL) and continuing the reaction at
room temperature for a further 30 min. The excess reagents were
removed by evaporation on an air manifold (in a fume hood!),
and the residues were partitioned between ethyl acetate-water
(1:2 v/v). Other dialkyldithioacetals were prepared by replacing
ethanethiol with by the appropriate thioalkane and, after evapora-
tion, were peracetylated with acetic anhydride-pyridine-dichlo-
romethane (1:1:1 by volume, room temperature, 3 h), re-
evaporated, and partitioned with ethyl acetate-water (1:2 v/v).
The ethyl acetate layers was analyzed by GC/MS (1-µL injection
) 1 µg of underivatized monosaccharide).
Gas Chromatography/Mass Spectrometry. GC/MS analy-
ses were performed on a Hewlett-Packard 5890 Series II gas
chromatograph equipped with a HP 7673A autoinjector. The GC
was interfaced with a HP 5972 Series mass-selective detector
configured in electron impact (EI) mode. Chromatography was
accomplished with a fused-silica capillary HP-1 column (25 m ×
0.2 mm). Helium was used as the carrier. The oven temperature
was ramped over a linear gradient from 150 to 250 °C at 4 °C/
min. Chromatography of the higher cyclic thioacetal acetates
required higher oven temperatures, typically 200-300 °C at 4 °C/
min. Injector and detector/interface temperatures were 275 and
300 °C, respectively. Mass spectra were recorded over the range
m/z 60-550. Mass spectra were recorded in positive ion mode.
Integrals of extracted ion data were used to calculate isotopic
enrichments (13C/12C ratios). Data processing was done off-line
using a Hewlett-Packard Chemstation.
METHODS AND MATERIALS
Materials. D D D
-[1-13C]Glucose, -[U-13C]glucose, and -[U-13C]-
fructose were from Aldrich (Milwaukee, WI) and other reagents
from Sigma-Aldrich. 1,2-Dithioethane and 1,3-dithiopropane were
from Lancaster Synthesis Inc. (Pelham, NH). Monosaccharide
stock solutions were prepared at 100 mg‚mL-1 in water and stored
at -20 °C. Hydroxylamine-4-(dimethylamino)pyridine (DMAP)
reagent was prepared by dissolved hydroxylamine hydrochloride
(100 mg) and DMAP (100 mg) in dry pyridine (3 mL). The reagent
was stable for one week when stored in the dark at 4 °C.
Aldononitrile Acetates. Aldonitrile acetate derivatives were
prepared according to the procedure of Guerrant and Moss,17 with
reference to the GC/MS studies of Seymour et al,18 Neiderer et
al,21 and Szafranek et al.22 Aqueous stock solutions (10 µL, 100
mg‚mL-1) of the standard sugars were evaporated to dryness in
screw-capped reaction tubes. Hydroxylamine-DMAP reagent (200
µL) was added and reacted on a hot block (60 °C) for 30 min.
The reactions were cooled and without evaporating were treated
with acetic anhydride (100 µL, 60 °C) to complete the peracety-
lation. The reactions were quenched after 30 min by the addition
of water (2 mL) and extracted by partitioning with ethyl acetate
(1 mL). The upper, organic layer was used directly for GC/MS
analysis (1-µL injection, equivalent to 1 µg of the underivatized
sugar).
Deuterioalditol Acetates. Alditol acetates were prepared
according to Merkle and Poppe,19 but substituting sodium boro-
deuteride in the reduction step to affect incorporation of deuterium
at the anomeric carbons. Peracetylation was achieved with acetic
anhydride-pyridine-dichloromethane (1:1:1 by volume, 60 °C,
1 h). Reactions were evaporated under air and partitioned with
dichloromethane-water (3 mL, 1:2 v/v). The lower, organic layer
was analyzed by GC/MS (typically 1 µL ) 1 µg of the underiva-
tized sugar).
RESULTS AND DISCUSSION
Peracetates. D-Glucose, D D
-[1-13C]glucose, and -[U-13C]glucose
were peracetylated and subjected to GC/MS analysis in EI mode.
Molecular ions (i.e., m/z 390, 391, 396) were not observed, but
three prominent fragmentation pathways were evident (Figure 1).
For unlabeled
generated a 2,3,4,6-tetraacetylglucose oxonium ion, [M - 59]+,
at m/z 331. For
-[1-13C]glucose this ion was evident at m/z 332,
i.e., 1 amu greater than the unlabeled ion, and predominantly at
m/z 336 for
-[U-13C]glucose. Fragmentation of unlabeled
D-glucose, loss of acetate from the anomeric carbon
D
D
D-
glucose peracetate also gave rise to abundant ions at m/z 242
and 157, respectively (Figure 1). M/z 242 arises from [C5H5O3-
(acetyl)3]+ and contains carbons 2-6 of the original glucose
molecule (Figure 1). The corresponding ion from
D
-[1-13C]glucose
Peracetates. Monosaccharide stocks (10 µL ) 1 mg) were
evaporated to dryness on an air manifold (40 °C) and peracetylated
has the same mass (i.e., m/z 242), but for
D
-[U-13C]glucose is 5
mass units higher at m/z 247, evidence for loss of carbon 1 and
retention of carbons 2-6. The m/z 242 ion fragments further by
consecutive losses of ketene (42 amu) and acetic anhydride (102
amu) radicals, to generate m/z 200 and 98. Corresponding ions
(17) Guerrant, G. O.; Moss, C. W. Anal. Chem. 1984, 56, 633-638.
(18) Seymour, F. R.; Chen, E. C. M.; Bishop, S. H. Carbohydr. Res. 1979, 73,
19-45.
(19) Merkle, R. K.; Poppe, I. Methods Enzymol. 1994, 230, 1-15.
(20) Biemann, K.; DeJongh, D. C.; Schnoes, H. K. J. Am. Chem. Soc. 1963, 85,
1763-1770.
are observed at m/z 200 and 98 for
for the loss of carbon 1) and at m/z 204 (+4) and 103 (+5) for
-[U-13C]glucose, further supporting the retention of carbons 2-6
D
-[1-13C]glucose (evidence
(21) Niederer, I. B.; Manzardo, G. G. G.; Amado, R. Carbohydr. Res. 1995, 278,
181-194.
D
(22) Szafranek, J.; Pfaffenberger, C. D.; Horning, E. C. Carbohydr Res. 1974,
38, 97-105.
on this fragment.
Analytical Chemistry, Vol. 76, No. 22, November 15, 2004 6567