离子对液相色谱-质谱应用于核苷和核苷酸的同时测定

Ion-Pairing Liquid Chromatography Coupled with Mass Spectrometry for the Simultaneous Determination of Nucleosides and Nucleotides

Adenosine and its corresponding nucleotides adenosine 5′-monophosphate (AMP), adenosine 5′-diphosphate (ADP) and adenosine 5′-triphosphate (ATP) are important biomolecules that provide energy and substrates for various cellular biochemical processes[1]. There have been strong demands for sensitive and reliable analysis of these nucleotides because the determination of their levels in cells may provide valuable information for understanding cellular energy metabolism. Analysis of adenosine nucleotides is a big challenge because these chemicals exist together with high cellular background in samples. Moreover, nucleotides are extremely polar due to the presence of multiple phosphate groups that may interfere with chemical determination. In particular, the nucleotide phosphates are not retained under conventional reversed-phase chromatographic conditions because of their extremely high polarity[2]. The most common approach for quantitative analysis of nucleotide phosphates in cellular extracts is to determine indirectly the corresponding parent nucleosides resulted from enzymatic dephosphorylation after the nucleotides were separated by using an anion-exchange solid phase extraction. The generated nucleosides were analyzed by using liquid chromatography with ultra violet absorption detection (LC/UV), radio-immunoassay and liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS)[3-6]. Because nucleotides are not retained under conventional reversed-phase conditions, ion-suppression high performance liquid chromatography (HPLC)[7], capillary electrophoresis[8] and ion-pairing HPLC[9-11] have been employed to circumvent the poor retention problem. The high selectivity of MS and MS/MS techniques provides great separation and detection power, making them attractive alternatives for the trace analysis of nucleotides. The application of negative ion electrospray ionization (ESI)-MS to nucleotide analysis has been reviewed[12,13]. The use of negative ion ESI-MS seemed to be a logical starting point for nucleotide phosphate analysis due to the existing phosphate groups. However, the application of positive ion ESI-MS coupled with ion-pairing HPLC for the quantitative analysis of intracellular nucleotides has recently been explored. Cai and co-workers[2]reported the selection of most abundant MS-MS quantitative ions of 6 ribo- and ribodeoxy-nucleotides by using ion-pairing HPLC coupled with positive ESI-MS. Fung et al.[11] discussed the advantage of positive ion mode in comparing with negative ion mode for the simultaneous determination of nucleotides and nucleosides when dimethylhexylamine (DMHA) was used as the ion-pairing agent. Recently, Cai and co-workers[14] also reported an assay of ion-pairing HPLC coupled with positive ion electrospray ionization-time of flight mass spectrometry (ESI-TOFMS) for determining cellular levels of ATP. This presentation describes the method development and applications of the ion-pairing HPLC and positive ion ESI-MS for the simultaneous determination of nucleosides and nucleotides.