Development of a nucleotide sugar purification method using a mixed mode column & mass spectrometry detection

•A chromatographic separation method was developed for the analysis of nucleotide sugars, nucleoside di- and triphosphates and sugar-phosphates.•A mixed mode column was utilized for the separation while various mobile phase conditions such as pH, buffer concentration and organic modifier were explored.•Negative ion mode mass spectrometry was adopted for detection of the sugar phosphate.•The semi-preparative separation method was developed to prepare 30mg of the nucleotide sugar.•19F NMR was utilized to determine purity of the purified fluorinated nucleotide sugar and found to be 99% pure.