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Liquid chromatographic analysis of nucleosides and their mono‐, di‐ and triphosphates using porous graphitic carbon stationary phase coupled with electrospray mass spectrometry
Author(s) -
Xing Jinsong,
Apedo Atsu,
Tymiak Adrienne,
Zhao Ning
Publication year - 2004
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.1524
Subject(s) - chemistry , diethylamine , ammonium acetate , chromatography , mass spectrometry , electrospray , acetonitrile , resolution (logic) , electrospray ionization , nucleotide , high performance liquid chromatography , organic chemistry , artificial intelligence , computer science , biochemistry , gene
Analysis of nucleosides and nucleotides is desirable in many biological studies, but the task is analytically challenging due to the high polarity of the analytes. In this study, resolution of mixtures containing nucleosides and their mono‐, di‐ and triphosphates was achieved using a porous graphitic carbon (PGC) stationary phase, Hypercarb, under conditions suitable for liquid chromatography/mass spectrometry (LC/MS). Different organic mobile phases and modifiers were evaluated and the separation of 16 nucleosides and nucleotides was optimized using gradient elution with a water/acetonitrile mobile phase containing ammonium acetate and diethylamine as modifiers. The ammonium acetate concentration proved to be critical for retention and diethylamine was found to improve the peak shapes of di‐ and triphosphates for mass spectrometric detection. A variety of silica‐based columns designed for polar compound separation were also tested using optimized LC conditions and compared with results obtained with the Hypercarb column. Only the Hypercarb column provided separations suitable for accurate quantitation of mixed nucleosides and their phosphates. Copyright © 2004 John Wiley & Sons, Ltd.