Nucleotide and Nucleotide Sugar Analysis by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry on Surface-Conditioned Porous Graphitic Carbon

We examined the analysis of nucleotides and nucleotide sugars by chromatography on porous graphitic carbon with mass spectrometric detection, a method that evades contamination of the MS instrument with ion pairing reagent At first, adenosine triphosphate (ATP) and other triphosphate nucleotides exhibited very poor chromato graphic behavior on new columns and could hardly be eluted from columns previously cleaned with trifluoro acetic acid Satisfactory performance of both new and older columns could, however, be achieved by treatment with reducing agent and, unexpectedly, hydrochloric acid Over 40 nucleotides could be detected in cell extracts including many isobaric compounds such is ATP, deoxy guanosine diphosphate (dGTP), and phospho-adenosine-5'-phosphosulfate or 3',5'-cyclic adenosine 5'-monophos-phate (AMP) and its much more abundant isomer 2',3'-cylic AMP A fast sample preparation procedure based on solid-phase extraction on carbon allowed detection of very short lived analytes such as cyndine 5 -monophos-phate (CMP) 2-keto deoxy-octulosonic a id In animal cells and plant tissues, about 35 nucleotide sugars were detected, among them rarely considered metabolites such as undine 5' diphosphate (UDP) L arabinopyranose, UDP-L-arabinofuranose, guanosine 5'-diphosphate (GDP) L-galactofuranose, UDP-L-rhamnose, and adenosine diphos phate (ADP)-sugars Surprisingly, UDP-arabinopyranose was also found m Chinese hamster ovary (CHO) cells Due to the unique structural selectivity of graphitic carbon, the method described herein distinguishes more nucleotides and nucleotide sugars than previously reported approaches