机构：[1]Chinese Acad Med Sci, Inst Mat Med, Dept Pharmacol, Beijing 100050, Peoples R China;[2]Peking Union Med Coll, Beijing 100050, Peoples R China;[3]Beijing Neurosurg Inst, Beijing 100050, Peoples R China;研究所北京市神经外科研究所首都医科大学附属天坛医院[4]Chinese Acad Med Sci, Inst Mat Med, Dept Pharmacol, 1 Xian Nong Tan St, Beijing 100050, Peoples R China
A reversed-phase ion-pair high-performance liquid chromatographic method for the direct and simultaneous determination of ribonucleoside triphosphates and their corresponding deoxyribonucleoside triphosphates in trichloroacetic acid cell extracts is presented. Using this system, high resolution of nine acid-soluble compounds, including ADP, CTP, dCTP, GTP, UTP, dGTP, dTTP, ATP and dATP in 16 normal or tumor cell lines, is obtained. The method is based on an extraction of nucleotides from cells with a solution of 6% trichloroacetic acid followed by neutralization with the addition of 5 M K2CO3 just prior to HPLC analysis. Chromatographic separations were performed using a Symmetry C-18 3.5 mum (150 x 4.6 mm) column (Waters) equipped with a NovaPak(18) Sentry guard column with UV detection at 254 nm. The HPLC columns were kept at 27 C. The mobile phase was delivered at a flow-rate of 1.0 ml/min, with the following stepwise gradient elution program: A-B (60:40) at 0 min-->4(40:60) at 30 min-->4(40:60) at 60 min. Solvent A contained 10 mM tetrabutylammonium hydroxide, 10 mM KH2PO4 and 0.25% MeOH, and was adjusted to pH 6.9 with 1 M HCl. Solvent B consisted of 5.6 mM tetrabutylammonium hydroxide, 50 mM KH2PO4 and 30% MeOH, and was neutralized to pH 7.0 with 1 M NaOH. The calibration curves (r > 0.99) of the components in cell extracts were established with their aqueous standards. The average within-day precision for the nine compounds was 0.9%, and the average day-to-day precision was 5.0%. The detection limits (pmol) of the nine reagents were 1.39 (ADP), 4.32 (CTP), 15.5 (dCTP), 2.38 (GTP), 4.42 (UTP), 9.45 (dGTP), 14.6 (dTTP), 2.44 (ATP) and 11.8 (dATP). The recovery of this method for the standards ranged from 82.4 to 120.5%. The results for the detection of nucleotide pools in 16 normal and tumor cell lines were presented. In conclusion, this simplified analytical method enables the simultaneous quantitation of NTP and dNTP in cell or tissue extracts and may represent a valuable tool for the detection of minute alterations of intracellular NTP/dNTP pools induced by anticancer/antiviral drugs and diseases. (C) 2002 Elsevier Science B.V. All rights reserved.
语种：
外文
被引次数：
WOS：
中科院(CAS)分区：
出版当年[2002]版：
无
最新[2023]版：
大类|3 区医学
小类|3 区生化研究方法3 区分析化学
JCR分区：
出版当年[2001]版：
Q2CHEMISTRY, ANALYTICALQ2BIOCHEMICAL RESEARCH METHODS
最新[2023]版：
Q2BIOCHEMICAL RESEARCH METHODSQ2CHEMISTRY, ANALYTICAL
第一作者机构：[1]Chinese Acad Med Sci, Inst Mat Med, Dept Pharmacol, Beijing 100050, Peoples R China;[2]Peking Union Med Coll, Beijing 100050, Peoples R China;[3]Beijing Neurosurg Inst, Beijing 100050, Peoples R China;[4]Chinese Acad Med Sci, Inst Mat Med, Dept Pharmacol, 1 Xian Nong Tan St, Beijing 100050, Peoples R China
通讯作者：
通讯机构：[1]Chinese Acad Med Sci, Inst Mat Med, Dept Pharmacol, Beijing 100050, Peoples R China;[2]Peking Union Med Coll, Beijing 100050, Peoples R China;[3]Beijing Neurosurg Inst, Beijing 100050, Peoples R China;[4]Chinese Acad Med Sci, Inst Mat Med, Dept Pharmacol, 1 Xian Nong Tan St, Beijing 100050, Peoples R China
推荐引用方式(GB/T 7714)：
Huang D,Zhang YH,Chen XG.Analysis of intracellular nucleoside triphosphate levels in normal and tumor cell lines by high-performance liquid chromatography[J].JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES.2003,784(1):101-109.doi:10.1016/S1570-0232(02)00780-8.
APA：
Huang, D,Zhang, YH&Chen, XG.(2003).Analysis of intracellular nucleoside triphosphate levels in normal and tumor cell lines by high-performance liquid chromatography.JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES,784,(1)
MLA：
Huang, D,et al."Analysis of intracellular nucleoside triphosphate levels in normal and tumor cell lines by high-performance liquid chromatography".JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES 784..1(2003):101-109
