Liquid chromatographic analysis of nucleosides and their mono-, di- and triphosphates using porous graphitic carbon stationary phase coupled with electrospray mass spectrometry
Liquid chromatographic analysis of nucleosides and their mono-, di- and triphosphates using porous graphitic carbon stationary phase coupled with electrospray mass spectrometry
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DOI：
10.1002/rcm.1524
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发表时间：
2004-01-01
影响因子：
2
通讯作者：
Zhao, N
中科院分区：
文献类型：
--
作者：
Xing, JS;Apedo, A;Zhao, N
核苷和核苷酸的分析在许多生物学研究中是期望的，但是由于分析物的高极性，该任务在分析上具有挑战性。在这项研究中，含有核苷及其单，二和三磷酸盐的混合物的分辨率实现了使用多孔石墨碳（PGC）固定相，Hypercarb，在适用于液相色谱/质谱（LC/MS）的条件下。考察了不同有机移动的相和改性剂，优化了16种核苷和核苷酸的梯度洗脱分离条件，以乙酸铵和二乙胺为改性剂的水/乙腈移动的相为流动相。乙酸铵浓度被证明是关键的保留和二乙胺被发现，以改善质谱检测的二磷酸和三磷酸的峰形。各种硅基柱设计的极性化合物分离也进行了测试，使用优化的LC条件，并与Hypercarb柱获得的结果进行了比较。只有Hypercarb色谱柱提供了适用于准确定量混合核苷及其磷酸盐的分离。版权所有（C）2004约翰威利父子有限公司。
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 (C) 2004 John Wiley Sons, Ltd.