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Letters to the Editor |
1 Departamento de Química, Universidade Federal, de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte-MG, 31270-901, Brazil, Fax 55-31-3499-5700 E-mail drhtcl{at}yahoo.com
To the Editor:
Monosaccharides are usually analyzed by use of automatic monitors or enzymatic immunoassays. However, gas chromatography (GC) is an accurate and precise technique for galactose quantification (1), and it is regarded as a reference method for glucose (2). GC procedures require a long derivatization time in 2 consecutive reactions of 60–90 min to generate the aldonitrile pentaacetate derivative. Fast derivatization techniques are often requested today because the bottleneck for sample throughput has moved from analysis to sample preparation. Recently, we dramatically decreased the derivatization time for sugars (mono- and disaccharides) in GC analysis (3) by using trimethylsilyl-oxime derivatives. This derivative, however, gives 2 peaks in the chromatogram, which is acceptable for the glucose–fructose pair but not for the glucose–galactose pair.
In this study, we optimized the aldonitrile pentaacetate derivativization step, using microwave-assisted conditions to obtain single-peak derivatives for each sugar. Plasma from healthy adults was used as a model matrix for the recovery experiments.
Glucose and galactose were from Sigma. Plasma samples were prepared as follows: we deproteinized 200 µL of plasma with 500 µL of methanol. After centrifugation at 5000g, the supernatant was withdrawn and evaporated to dryness under a stream of nitrogen. We added 100 µL of hydroxylamine hydrochloride (20 g/L in pyridine) to the vial, vortex-mixed it for 30 s, and then reacted the mixture for 2 min in a microwave oven (200 W, 25% of total exit power). The final step was to add 100 µL of acetic anhydride and allow the reaction to proceed for 6 min. We directly injected 1 µL (split ratio 1:25) into a chromatograph (Varian 3380) equipped with a BP-10 Column (SGE). The column temperature started at 120 °C for 1 min, then was increased to 280 at 10 °C/min rate. The injector and detector (flame ionization detection) were at 280 °C.
Optimal reaction times were 2 min for the oxime reaction and 6 min for the acetylation and aldonitrile formation.
Although galactose and glucose are enantiomers differing in only one optical center, the BP-10 column easily achieved the desired baseline separation (Fig. 1
). Calibration curves constructed either with aqueous solutions or by standard additions to plasma showed no differences in slope.
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Recovery, calculated with the aqueous calibrators, was 97%–101% for glucose and 92%–104% for galactose. The CV of 5% (n = 3) for both sugars is attributable mainly to the irradiation step because of a lack of homogeneity in domestic ovens. The detection limit was 5 µmol/L (signal-to-noise ratio = 3) for both sugars without modification of the split ratio.
This study shows that a simple microwave oven can be used to accelerate sample preparation in GC analysis of monosaccharides in plasma. Flame ionization detection enables easy measurement of glucose and can achieve the detection limits necessary for galactose. The present procedure is an alternative for laboratories that lack automated glucose analyzers and for others that do galactose screening with methods such as thin-layer chromatography (4)(5).
References
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