Abstract

Abstract: Sugars and sugar alcohols are known as highly polar compounds. Four analytes were taken into consideration for comparison of classical and modern approach of separation of highly polar substances. Mannitol was chosen as an example of simple sugar alcohol, sucrose and lactulose as examples of disaccharides and structural isomers, raffinose as an example of complex trisaccharide. Raffinose was also used as internal standard for quantitaion. HILIC Hydrophilic Interactions Liquid Chromatography) was a technique of choice due to its advantages of separation of highly polar compounds. Mobile phase used is mainly organic component (>80%) with amount of polar solvent. Stationary phase in chromatographic column consist silica gel modified with functional groups.Classical approach was undertaken on Supelco LC-NH2 column and separation phenomena occurs when water and analytes compete to form hydrogen bonds with the amino phase of analytical column. Modern approach was undertaken on ZIC-HILIC column and separation is achieved by hydrophilic partitioning mechanism and by weak electrostatic interactions. Tandem mass spectrometer (MS/MS) was chosen as a detector. MS/MS is characterized by unique ability to monitor the transition of pseudomolecular ion and fragment ion of analyte in Multiple Reaction Monitoring (MRM) mode. In case of saccharides, there are two main types of fragmentation: break of glycosidic bond between two rings of sugar and/or break of ring through the one of the bonds of oxygen atom.Optimization of chromatographic parameters is crucial to obtain satisfactory separation to differentiate between structural isomers. Optimization of parameters of monitored ion transition and operation parameters of MS/MS is required to obtain high intensity of peaks and repeatability.

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