Capillary electrophoresis-electrospray-mass spectrometry of nucleosides and nucleotides: Application to phosphorylation studies of anti-human immunodeficiency virus nucleosides in a human hepatoma cell line
Capillary electrophoresis-electrospray-mass spectrometry of nucleosides and nucleotides: Application to phosphorylation studies of anti-human immunodeficiency virus nucleosides in a human hepatoma cell line
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DOI：
10.1002/elps.200410423
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发表时间：
2005-04-01
期刊：
影响因子：
2.9
通讯作者：
Dovichi, NJ
中科院分区：
文献类型：
--
作者：
Liu, CC;Huang, JS;Dovichi, NJ
我们报告了使用毛细管电泳-电喷雾电离-质谱（CE-ESI-MS）来测定抗逆转录病毒双脱氧核苷（ddN）、其核苷酸以及一组核糖核苷和核糖核苷酸。一种用于分离最常用的抗逆转录病毒ddN的CE系统已经基于具有挥发性电解质的碱性缓冲液开发，该电解质适合于在未经处理的毛细管柱中进行ESI-MS检测。研究并比较了正离子化模式和负离子化模式的灵敏度和稳定的电喷雾性能。一个14-化合物的核苷和核苷酸的混合物在一个单一的毛细管区带电泳分离与不同的洗脱顺序：电渗流，ddNs，单核苷酸，二核苷酸，和三核苷酸在不到18分钟的剖析。在ESI-MS中的核苷和核苷酸的裂解途径已被解释。检测浓度限为100至200 nm，进样体积约为10 nL。该技术已被用于检测天然存在的核苷酸和研究拉米夫定（3 TC）在人肝癌细胞系Hep G2中的代谢。3 TC与细胞孵育10 h后，检测到3 TC及其代谢产物3 TC-单磷酸、3 TC-二磷酸和3 TC-三磷酸。
We report the use of capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) for the determination of antiretroviral dideoxynucleosides (ddNs), their nucleotides, and a set of ribonucleosides and ribonucleotides. A CE system for separation of most commonly used antiretroviral ddNs has been developed based on a basic buffer with a volatile electrolyte suitable for ESI-MS detection in an untreated capillary column. Positive and negative ionization modes are investigated and compared for sensitive and stable electrospray performance. A 14-compound mixture of nucleosides and nucleotides is profiled in a single capillary zone electrophoresis separation with a distinct elution order: electroosmotic flow, ddNs, mono-nucleotides, dinucleotides, and trinucleotides in less than 18 min. The fragmentation pathways of the nucleosides and nucleotides in ESI-MS have been interpreted. Concentration limits of detection are 100 to 200 nm with an injection volume of similar to 10 nL. This technique has been used to detect naturally occurring nucleotides and to study the metabolism of lamivudine (3TC) in the human hepatoma cell line Hep G2. 3TC and its metabolites 3TC-monophosphate, 3TC-diphosphate, and 3TC-triphosphate were detected after 10 h of incubation of 3TC with the cells.