Mono and disaccharides, by GC and GC-MS

 
 

   Determination of sugars in plants or other bio-samples is essential as these compounds are involved in many important biological processes. In general, analyses of sugars are carried out using chromatographic techniques such as HPLC, ion chromatography and gas chromatography.

 

   Only the GC-MS analyses of sugars are offered in ZABAM at present

 

   Gas chromatographic analysis of mono and disaccharides requires conversion of sugars into their volatile derivatives. Conversion of sugars into alditol acetates and trimethylsilyl (TMS) ethers have been the most widely used methods for GC analysis of sugars.

   "Alditol acetate" method involves reduction of sugars with sodium borohydride following conversion of polyols to polyacetate esters.

  

 

 

    TMS ethers of sugars can be prepared from a number of commercially available silylation reagents.

 

 

    

    Both derivatization methods have disadvantages. Preparation of TMS ethers may result in as many as four derivatives for each sugar that is caused by anomer formation and ring isomerisation. On the other side, glucose, for instance, can not be unequivocally determined in the presence of fructose using "alditol acetate" derivatization method because on reduction glucose yields glucitol, whereas fructose yields a mixture of mannitol and glucitol.  

 

 

   GC-MS analysis of TMS derivatives

 

   Mixtures of TMS ethers of mono and disaccharides can be successfully analyzed in many cases. There are many sugars commercially available as D-anomers. However, when added to water a D-sugar is converted to other stereoisomers as a result of anomerization at anomeric carbon. TMS ethers are prepared then from the dry D-sugar and a mixture of all isomers after the evaporation of water.

A number and ratio of possible isomers for the particular sugar will be observed in the GC-MS chromatogram. Many carbohydrates can not be identified relying only on the MS analysis because of the great similarity of their mass spectra; in those cases use of standards is necessary for the correct identification of a carbohydrate in a sample.

However, EI mass spectra of aldoses and ketoses, for example glucose and fructose, contain different fragment ions and may be identified sucessfully.



Examples:

GC-MS chromatograms of D-glucose TMS ether and sample of glucose prepared after evaporation of water.

 


GC-MS chromatograms of D-fucose TMS ether and sample of fucose prepared after evaporation of water.

 


GC-MS chromatograms of D-arabinose TMS ether and sample of arabinose prepared after evaporation of water.

 


   GC-MS chromatogram of TMS ethers of some carbohydrates

 

Sample

TMS ethers of carbohydrates

Chromatography

Gas chromatography

carrier gas – helium

GC column: Thermo TR 5MS, 30m.

Ionization

EI

Detection

Polaris Q ion trap (Thermo Scientific)

 
   

   Although retention time of some sugars, like sucrose and lactose is essentially the same, these two carbohydrates still can be determined using mass spectrometric detection. The EI mass spectra of sucrose and lactose are qualitatively different. The different fragment ions can be used for identification and quantitation of compounds.

 

 

EI mass spectra of TMS ethers of sucrose and lactose 

 


 

Recommended Literature

 

V.G. Zaikin, J.M. Halket, "Derivatization in mass spectrometry -1. Silylation", Eur. J. Mass Spectrom, 2003, 9, 1.

V.G. Zaikin, J.M. Halket, "Derivatization in mass spectrometry -2. Acylation", Eur. J. Mass Spectrom, 2003, 9, 421.

 
 

Laboratory for Mass Spectrometry, ZABAM,

Faculty of Agriculture Food and Environment, Rehovot, Israel