Analysis of plasma nucleotides in rat by micellar electrokinetic capillary chromatography/electrospray ionization mass spectrometry
Analysis of plasma nucleotides in rat by micellar electrokinetic capillary chromatography/electrospray ionization mass spectrometry
复制标题
DOI：
10.1002/rcm.6245
复制
发表时间：
2012-06-30
影响因子：
2
通讯作者：
Jung, Byung Hwa
中科院分区：
文献类型：
--
作者：
Bhowmik, Salil Kumar;Jung, Byung Hwa
目的：建立胶束电动毛细管色谱/电喷雾电离质谱（MEKC/ESI-MS）同时定量分析大鼠血浆中9种内源性核苷酸的方法。方法选择最佳分离条件，考察不同pH、流动缓冲液和表面活性剂的浓度。选择含有表面活性剂十二烷基三甲基溴化铵（2mm, pH 3.5）的乙酸铵（20 mM）作为胶束运行缓冲液。血浆样品是用2mm EDTA在60%乙醇中沉淀蛋白质制备的。采用毛细管电泳(CE)/质谱（MS）和选择正离子离子监测（SIM）模式对样品进行分析。毛细管电泳采用反极性硅胶毛细管柱进行。结果核酸的检出限和定量限分别为0.055 μ M和2.020 μ M。所有分析物的校准曲线均为线性（R2 >0.99），准确度和精密度在+/- 15%以内。本方法应用于大鼠口服对乙酰氨基酚（1000mg /kg/天）后血浆核苷酸分析，评价肝毒性条件下血浆核苷酸水平的变化。结论GTP水平降低和胞嘧啶核苷酸水平升高与肝毒性有关，肝毒性与血浆中核苷酸水平变化密切相关。版权所有：John Wiley & Sons， Ltd
RATIONALE The aim of this study was to establish a simultaneous quantitative analysis method of nine endogenous nucleotides in rat plasma using micellar electrokinetic capillary chromatography/electrospray ionization mass spectrometry (MEKC/ESI-MS). METHODS To select the optimum conditions for separation of the nucleotides, various pH, concentrations of running buffers and surfactants were tested. Ammonium acetate (20 mM) containing the surfactant dodecyltrimethylammonium bromide (2 mM, pH 3.5) was selected as the micellar running buffer. The plasma samples were prepared by precipitating the proteins with 2 mM EDTA in 60% ethanol. The samples were analyzed using capillary electrophoresis (CE)/MS and selected ion monitoring (SIM) mode with positive ionization. CE was performed using a silica capillary column in reversed polarity mode. RESULTS The limits of detection (LODs) and limits of quantification (LOQs) of the nucleotides ranged from 0.055 and 2.020 mu M, respectively. The calibration curves were linear (R2 >0.99) for all analytes, and the accuracy and precision were within +/- 15%. The developed method was applied to the analysis of nucleotides in rat plasma that was collected after oral administration of acetaminophen (1000 mg/kg/day) to evaluate the changes in plasma nucleotide levels under hepatotoxic conditions. CONCLUSIONS Decreased level of GTP and increased level of cytosine nucleotides were found to be associated with liver toxicity, which led to the conclusion that liver toxicity is closely related to changes in nucleotide levels in plasma. Copyright (C) 2012 John Wiley & Sons, Ltd.