DETERMINATION OF [13C]GALACTOSE ENRICHMENT IN PLASMA
219
analysis require an initial derivatization step.6,7,11–23 We
utilized the pentaacetylaldononitrile derivative of galac-
tose, which has been used previously for the determination
of glucose by GC/MS.11,13–18 Acetylation of the hydroxyl
groups improves the volatility of the analyte, and con-
version of the aldehyde functional group eliminates the
center of asymmetry at C-1. Previous reports indicated
that the peracetylaldonitrile derivatives have better chro-
matographic behavior than peracylated alditols, another
class of derivative commonly employed for the analysis
of aldoses.15
injection liner was replaced with a Restek Open-Top Uni-
liner packed with deactivated glass-wool packing (Restek,
Bellefonte, PA, USA). Separation of the galactose and
glucose derivatives was achieved on a 30 m ð 0.25 mm
i.d. HP-5MS column with a film thickness of 0.25 µm
(Hewlett-Packard). The gas chromatograph was operated
in the constant flow mode with the flow-rate of the helium
carrier gas set to 25 cm sꢀ1. Helium was used as the
carrier gas after passing through a moisture trap (R &
D Separations) and an oxygen filter (Hewlett-Packard)
before entering the gas chromatograph. The temperature
program started at 80 °C, which was held for 2 min, and
then ramped at 30 °C minꢀ1, to 180 °C, which was then
held for 19 min.ꢀF1inally, the column was cleaned by ramp-
ing at 30 °C min to 300 °C and holding that temperature
for 1 min.
EXPERIMENTAL
Materials
Mass spectrometry
Galactose, glucose, hydroxylamine hydrochloride, pyri-
dine, high-performance liquid chromatographic (HPLC)-
grade water, HPLC-grade methylene chloride and HPLC-
grade ethyl acetate were obtained from Fisher Scientific
(Fairlawn, NJ, USA). Acetic anhydride was obtained from
Supelco (Bellefonte, PA, USA). [1-13C]galactose was pur-
chased from Omicron Biochemicals (South Bend, IN,
USA). Grade 4 methane (<5 ppm O2), argon (99.999%)
and grade 6 helium were supplied by BOC Gases (River-
ton, NJ, USA).
The gas chromatograph was connected by a transfer line
kept at 250 °C to a TSQ 7000 triple-quadrupole mass spec-
trometer (Finnigan, San Jose, CA, USA) equipped with a
Finnigan EI/CI 7000 ion source. The mass spectrometer
was operated in the positive ion mode with the ion source
configured for CI (PCI). The PCI conditions were 5.8 Torr
of methane (1 Torr D 133.3 Pa), a 220 eV electron energy
and a 400 µA filament current. Methane was passed
through a moisture filter (R & D Separations) and an oxy-
gen filter (Lab Clear, Oakland, CA, USA) before entering
the mass spectrometer. The collision cell contained argon
at 2.0 mTorr and was set to a collision energy of 15 eV.
The mass spectrometer, configured in the selected reac-
tion monitoring (SRM) scan mode, monitored the tran-
sitions m/z 328 (MHC ꢀ CH3COOH/ ! 106 (C6H4NO)
for galactose and m/z 329 (MHC ꢀ CH3COOH/ ! 107
(12C513C1NO) for [1-13C]galactose. In contrast to typical
SRM experimental conditions, the mass resolution of the
first quadrupole was kept at unit resolution to minimize
the possibility of cross-talk between the two monitored
transitions. The data system was controlled using Finni-
gan ICIS 8.3.0 software and Finnigan LCQuan V1.2 was
used to calculate peak areas.
Extraction and derivatization
Plasma samples were extracted and derivatized by a mod-
ification of the methods of Pfaffenberger et al.17 and
Tserng and Kalhan.11 Methanol (1 ml) was added to a
plasma sample (100 µl) contained in a 2 ml polypropy-
lene Eppendorf tube (VWR, Bridgeport, NJ, USA) in
order to precipitate the plasma proteins. Each sample was
then centrifuged at 8000 rpm for 10 min in an Eppen-
dorf Model 5415 C microcentrifuge. The supernatant from
each sample was transferred to a 10 ml screw-capped
conical-bottomed glass centrifuge tube (VWR) and dried
under nitrogen. After drying, hydroxylamine hydrochlo-
ride (2 mg, 0.029 mmol) in pyridine (100 µl) was added
to each sample, and the samples were kept at 90 °C for
30 min on a heating block (VWR). Acetic anhydride
(75 µl) was then added to each sample and heating con-
tinued for another hour. Each sample was purified by
adding water (1.5 ml) and methylene chloride (0.3 ml),
vortex mixed and then centrifuged at 5000 rpm for 10 min
at 15 °C in a Beckman Model GS-6KR centrifuge. The
methylene chloride layer was removed and dried under
nitrogen in 1.5 ml Eppendorf tubes. Finally, the samples
were reconstituted in ethyl acetate (40 µl) and transferred
to 0.7 ml conical-bottomed sample tubes (Chromatogra-
phy Research Supplies, Addison, IL, USA) ready for
injection into the GC/MS system.
Calibration curves
Standard solutions were prepared in dialyzed plasma con-
taining 4 mM glucose and 50 µM galactose. [1-13C]Galac-
tose was added to the plasma to give APEs of 4.76, 9.09,
16.67, 20.00, 33.33, 50.00, 60.00, 66.67, 75.00, 88.89 and
92.31%. The samples were processed as described above,
and the APEs were calculated from the peak areas of the
SRM profiles m/z 329 ! 107 for [13C]galactose (PA-13C)
and m/z 328 ! 106 for galactose (PA-12C) using the fol-
lowing equation:
.PA-13C//.PA-12C/ ꢀ 0.07139
APE D
ð 100
1 C [.PA-13C//.PA-12C/ ꢀ 0.07139]
Gas chromatography
Splitless injections of 2 µl were made on an HP 5890 gas
chromatograph (Hewlett-Packard, Palo Alto, CA, USA)
with the injector held at 250 °C. The standard HP splitless
The determined APE was then plotted against the calcu-
lated APE and the linear regression equation calculated
within the range 3.5–92%.
Copyright 2000 John Wiley & Sons, Ltd.
J. Mass Spectrom. 35, 218–223 (2000)