Zborník príspevkov z vedeckej konferencie - Department of ...

Chemicals
All chemicals were of analytical reagent grade. Carbohydrate standards (D-glucose, D-fructose, D-mannitol) were
purchased from Merck (Bratislava, Slovakia) and carbohydrate standards (D-galactose, D-galactitol) were purchased from
CMS Chemicals (Bratislava, Slovakia). Derivatizations of carbohydrates were performed using hexamethyldisilane (HMDS)
with trimethylchlorsilane (TMCS) and pyridine as catalyst, all supplied from Sigma Aldrich (St. Louis, MO, USA).
Urine samples were obtained from Department of Laboratory Medicine of Comenius University Children´s Hospital,
Bratislava, Slovakia.
Sample preparation
Urine samples from patients were frozen immediately after collection and kept at –20 °C until analyzed. 100 l aliquots
of each urine sample were taken and were trimethylsilylated with 3 ml silylating reagent HMDS: TMCS: Pyridine in the
volume ratio of 1: 1: 1 at 70 °C for 70 min. 1 l portion of derivatized sample (model and sample solution) was injected to
the chromatograph.
GC – MS analysis
The analysis was performed on 6890 N/5973 N GC–MS Agilent Technologies (USA) by electron ionization at 70 eV
using an DB–Dioxin column 60 m x 250 m x 0.25 m (J&W Scientific, Folson, CA, USA). The injector was held at 300 °C
and was operated in splitless mode. The purge flow of 9.5 ml min -1 was started 2 min after the sample injection. The initial
column temperature was 90 °C, then the temperature was increased to 250 °C at rate of 10 °C.min -1 , and kept at the final
temperature of 250 °C for 5 min. High purity helium was used as carrier gas with inlet pressure of 200 kPa. MS data were
obtained in SIM-mode (m/z–73, 204, 205, 217, 319, 422, 435, 437). Transfer line temperature was 280 °C. Quadrupole
conditions were as follows: electron energy 70 eV and ion source temperature 230 °C.
Compound identification was performed by comparison with the chromatographic retention characteristics and mass spectra
of authentic standards, reported mass spectra and mass spectral library of GC-MS data system. Compounds were quantified
by SIM peak area, and converted to compound mass using calibration curves of galactitol and galactose.
Results and discussion
Figure 3 shows typical GC-MS chromatogram of fructose, glucose, galactose, mannitol and galactitol TMS derivates
obtained using silylation reagent (HMDS: TMCS: Pyridine: 1: 1: 1). Chromatographic analysis can be accomplished in less
than 16 min, without loss of resolution between critical pairs.
Abundance
25000000
20000000
15000000
10000000
5000000
Fructose
Mannitol
Galactitol
Glucose Galactose
Galactose
Glucose
0
12 13 14
Time[min]
15 16
Fig. 3. Total ion GC-MS chromatogram of TMS carbohydrate derivatives.
Due to the and configurations of the OH group on pyrano-ring galactose and glucose yields two major GC peaks,
whereas fructose produced three GC major peaks due to the and configurations of the OH group on pyrano- and furanorings.
These isomers are also present in urine samples and are commonly summed to report one value. The mass spectra of
saccharide with the pyrano-ring (5C) are characterized by the fragment ion of m/z 204 (galactopyranose as trimehtylsilyl, Fig.
4) and therewith the furano-rings (4C) are characterized by the m/z 437 fragment ion, whereas the fragmentation of alcohol
saccharides (galactitol and mannitol) are yield the m/z 319 (galactitol as TMS, Fig. 4).
These three fragments are used as key ions for identification of these two sugars as TMS derivatives.
Zborník príspevkov
z 18. medzinárodnej vedeckej konferencie
"Analytické metódy a zdravie loveka", ISBN 978-80-969435-7-9
- 118 -
hotel Falkensteiner, Bratislava
11. - 14. 10. 2010