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The review discussed the significant roles of nucleotides and nucleotide sugars and the problems in their quantification.The strategies for the quantification of nucleotides and nucleotide sugars included IP, IE and HILIC chromatography.The separation mechanisms, column categories and column choices of HILIC were specially evaluated.Nucleotides and their sugar conjugates are fundamental molecules in life, participating in processes of DNA/RNA composition, cell wall build-up, glycosylation reactions, and signal conduction. Therefore, the quantification of these compounds in biological samples significantly benefits the understanding of their functions. However, nucleotides and nucleotide sugars are extremely hydrophilic, causing bad retention and peak symmetry on regular C18 chromatographic columns. To solve this problem, ion-pair (IP) chromatography, ion-exchange (IE) chromatography and hydrophilic interaction chromatography (HILIC) were applied, of which differentiated mechanisms were utilized to increase the retention of the analytes on the stationary phases. IP-HPLC and HILIC were convenient for coupling with many kinds of detectors (ultraviolet, UV or mass spectrometry, MS). Combining these two kinds of techniques, the advantages of better separation and retention were increased, while disadvantages like irreversible adsorption by stationary phases were greatly decreased. Due to the high concentrations of nonvolatile buffer salts used, IE-HPLC was not suitable for MS detectors. Protein precipitation and solid phase extraction were the common methods for sample treatment in the analysis of nucleotides and nucleotide sugars. By carefully optimizing the LC-UV or LC-MS conditions, high sensitivities could be achieved, and the methods could be applied to the analysis of many kinds of biological samples (cells, tissues, plants, bacteria, etc.). Developing new analyzing techniques may help the utilization of nucleotides and nucleotide sugars in the diagnosis and therapy of diseases.