GC-MS analysis of mono and disaccharides
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Mono- and disaccharides are traditionally analyzed either using the ion or HILIC liquid chromatography coupled with pulsed amperometric or refractive index detectors, or GC-MS analysis after chemical derivatization (trimethylsilylation, per-acetylation, or other). Separation of complex mixtures of saccharides using liquid chromatographic methods may involve long analysis times and require a high degree of expertise. Gradient methods however are unfavorable for refractive index detectors. Many saccharides converted into semi-volatile compounds are readily separated on capillary gas chromatographic columns and can be correctly identified in biological samples if corresponding standards are available. However quantitative analysis is not so straightforward using the gas-chromatographic methods. The aldose and ketose monosaccharides exist in solutions as equilibrium mixtures of tautomeric forms, - hemiacetals or hemiketals. The equilibrium depends on many factors, - temperature, pH, etc. In the analysis of real biological samples the composition of the matrix is unknown, and the ratio between tautomers obtained in the calibration samples may not be the same as in unknown samples. A variety of internal standards used in the course of sample preparation usually cannot solve the problem of inaccuracy.
The following instruments were used through the years for the analysis of mono and disaccharides:
GC-MS ion trap Polaris-Q (Thermo Scientific). Discontinued.
GC-MS single quadrupole (7860 coupled with 5973C, Agilent). Sugars can still be analyzed in scan or SIM modes.
From now and further, - the GC-MS/MS consists of the gas chromatograph Trace 1300 coupled with the triple-quadrupole mass spectrometer TSQ 9000 (Thermo Scientific). Nitrogen (>99.999) was used as a collision gas. For quantitative analysis, data were acquired in positive EI MRM mode. The acquisition was controlled, and data was analyzed using Xcalibur and TraceFinder software respectively.
Carbohydrates cannot be analyzed "as is" by the gas chromatography. These compounds are essentially non-volatile due to strong hydrogen bonding and will decompose (caramelize) in the GC inlet at high temperatures. To analyze sugars by GC, the labile hydrogens of hydroxyl groups must be replaced by functional groups that will improve volatility. The most popular derivatization of carbohydrates for GC analysis is trimethylsilylation, peracetylation, and pertriflouracetylation.
Trimethylsilylation of glucose.
However, in real life, derivatized aldoses or ketoses will not be detected as a single chromatographic peak but rather as a mixture of two or more isomeric tautomers.
Tautomers of glucose.
The GC-MS chromatogram of commercially available crystallized D-glucose TMS ether (the crystal of D-glucose was derivatized without prior dissolution in water). Separated on Rxi-5MS capillary column (30 m, 0.25 mm, 0.25 µm, Restek); carrier gas, - helium.
The GC-MS chromatogram of isomeric glucose TMS derivatives (after a crystal of D-glucose was dissolved in water following evaporation and derivatization). Separated on Rxi-5MS capillary column (30 m, 0.25 mm, 0.25 µm, Restek); carrier gas, - helium.
Fructose exists in solution in the forms of five and six-membered rings and all these isomers are separated and detected in GC analysis i.e., there will be detected already several chromatographic peaks.
Other sugars in the aquatic environment.
Structures of xylose in water with relative amounts at 27°C.
From: "The effect of sodium chloride concentration on the mutarotation and structure of D-xylose in water: Experimental and theoretical investigation", Carbohydr. Res. 2020, 489, 107941.
Structures of arabinose in water with relative amounts at 31°C.
From: "The composition of reducing sugars in dimethyl sulfoxide solution". Carbohydr. Res. 1994, 263, 1−11.
The GC-MS chromatogram of crystallized D-arabinose TMS ether (a crystal of D-arabinose was derivatized without prior dissolution in water). Separated on Rxi-5MS capillary column (30 m, 0.25 mm, 0.25 µm, Restek); carrier gas, - helium.
The GC-MS chromatogram of isomeric arabinose TMS ethers (after a crystal of D-arabinose was dissolved in water following evaporation and derivatization). Separated on Rxi-5MS capillary column (30 m, 0.25 mm, 0.25 µm, Restek); carrier gas, - helium.
Four isomers of arabinose were detected though the ratio is quite different from that determined in water as shown in the above publication.
Tautomeric isomerization may seriously complicate the analysis of complex mixtures.
The number of tautomers can be reduced through the oximation reaction of aldoses and ketoses. For example, although only about 0.25% of glucose exists in the open form in water solution (the rest is closed in cyclic forms), the aldehyde (open form) may be converted into oxime, and the reaction is over in half an hour (two isomeric oximes namely, syn and anti, are obtained).
Gas chromatographic separation of syn and anti isomers of glucose and fructose oximes after trimethylsilylation (DB-35MS capillary column (25 m, 0.2 mm, 0.33 µm, Agilent), carrier gas, - hydrogen).
The EI mass spectra of TMS ethers of oximes have characteristic higher m/z ions which allow using isotopically labeled internal standards for quantitative analysis.
Positive EI mass spectra of fructose oxime TMS (above) and fructose-13C6 oxime TMS (below).
The MS/MS product ion spectra of m/z 307.2 of fructose oxime TMS (above) and m/z 310.2 of fructose-13C6 oxime TMS (below). Although ion m/z 310.2 presents in small amounts also in the mass spectrum of unlabeled glucose, it has a different isotopic composition (due to 29Si and 30Si isotopes) than the m/z 310.2 detected in labeled fructose (all 13C6). Thus, the MS/MS spectra are distinctive for labeled and unlabeled sugars and can be used for quantitative MRM analysis.
The EI MRM chromatograms of fructose oxime TMS and fructose-13C6 oxime TMS which was used as an internal standard for the quantitative analysis of fructose.
Calibration curves obtained using isotopically labeled compounds as internal standards for GC-MS analysis of carbohydrates (fructose-13C6, glucose-13C6, sorbitol-13C6 )
Gas chromatographic separation of trimethylsilylated oximes of mono- and disaccharides on VF5-MS (25 m, 0.2 mm, 0.33 µm, Agilent) capillary column using hydrogen as a carrier gas (GC-MS/MS TSQ 9000 (Thermo Scientific)). Note: better separation of TMS derivatives of sugars is achieved using GC columns with a lesser percent of phenyl groups.
Gas flow, - 0.8 ml/min (constant); temp. program: 130 °C (0.5min) then 8°/min to 195°C, then 3°/min to 204°C, then 12°/min to 290°C (3 min).
Conclusions
Accurate and reliable quantification of sugars by the GC-MS is challenging due to the unpredictable tautomerism of aldose and ketose carbohydrates in aqua media and unknown matrix effects that may play a role in the course of sample preparation and analysis. The addition of isotopically labeled analogues of carbohydrates, which undergo identical chemical transformations and reactions during sample preparation and analysis was the only reliable method (i.e. the isotope dilution method) for accurate quantification of carbohydrates.
Structures of mono- and disaccharides commonly analyzed in TSABAM
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