Quantification of deoxythioguanosine in human DNA with LC-MS/MS, a marker for thiopurine therapy optimisation.

IF 3.8 2区 化学 Q1 BIOCHEMICAL RESEARCH METHODS
Analytical and Bioanalytical Chemistry Pub Date : 2024-12-01 Epub Date: 2024-10-13 DOI:10.1007/s00216-024-05581-6
Björn Carlsson, Louise Karlsson, Andreas Ärlemalm, Sophie Sund, Malin Lindqvist Appell
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引用次数: 0

Abstract

In the treatment of diseases such as acute childhood leukaemia (ALL) and inflammatory bowel disease (IBD), the thiopurines azathioprine, 6-mercaptopurine, and 6-thioguanine are used. Thiopurines are antimetabolites and immunomodulators used to maintain remission in patients. They are all prodrugs and must be converted into the competing antimetabolites thioguanosine triphosphate and deoxythioguanosine triphosphate for final incorporation into RNA or DNA. The current therapeutic drug monitoring (TDM) method measures the sum of the formed metabolites in the sample, after acidic hydrolysis at high temperature. In this work, the goal is to measure these drugs closer to their pharmacological endpoints, once incorporated into DNA. After extracting DNA from whole blood, followed by DNA hydrolysis, 2'-deoxythioguanosine (dTG) and the complementary natural nucleobase 2'-deoxycytidine (dC) were measured. Chromatographic separation on a HSS T3 column followed by mass spectrometric detection was performed in multi-reaction monitoring (MRM) mode on a Xevo TQ-XS with ESI in positive mode, within 5 min. The concentration range for dTG was 0.04-5 nmol/L, and for dC, 0.1-12.5 µmol/L. The lower limit of detection was determined to a concentration of 0.003 nmol/L for dTG and 0.019 µmol/L for dC. The intra- and inter-assay imprecision for the quality controls ranged between 3.0 and 5.1% and between 8.4 and 10.9%, respectively. Sample stability for up to 4 years is shown. In summary, a sensitive method to quantify the thiopurines incorporated into DNA as dTG has been developed and will be used in further clinical studies for a better understanding of the mode of action of the thiopurines and the use of this method in TDM.

利用 LC-MS/MS 对人类 DNA 中的脱氧硫代鸟苷进行定量,作为优化硫嘌呤疗法的标记。
在治疗急性儿童白血病(ALL)和炎症性肠病(IBD)等疾病时,会用到硫嘌呤类药物硫唑嘌呤、6-巯基嘌呤和 6-硫鸟嘌呤。硫嘌呤类药物是抗代谢药和免疫调节剂,用于维持患者病情的缓解。它们都是原药,必须转化为竞争性抗代谢物三磷酸硫鸟苷和三磷酸脱氧硫鸟苷,才能最终掺入 RNA 或 DNA 中。目前的治疗药物监测(TDM)方法测量的是样品在高温酸性水解后形成的代谢物的总和。在这项工作中,我们的目标是在这些药物与 DNA 结合后,更接近其药理终点进行测量。从全血中提取 DNA 后进行 DNA 水解,然后测量 2'-deoxythioguanosine (dTG) 和互补天然核苷酸 2'-deoxycytidine (dC)。在 HSS T3 色谱柱上进行色谱分离,然后在 Xevo TQ-XS 上以多反应监测(MRM)模式进行质谱检测,ESI 为正离子模式,检测时间为 5 分钟。dTG 的浓度范围为 0.04-5 nmol/L,dC 的浓度范围为 0.1-12.5 µmol/L。dTG 的检测下限为 0.003 nmol/L,dC 的检测下限为 0.019 µmol/L。质量控制的测定内和测定间不精确度分别为 3.0%至 5.1%和 8.4%至 10.9%。样本稳定性长达 4 年。总之,我们已开发出一种灵敏的方法来定量检测以 dTG 形式结合到 DNA 中的硫嘌呤,并将用于进一步的临床研究,以更好地了解硫嘌呤的作用模式并将此方法用于 TDM。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
8.00
自引率
4.70%
发文量
638
审稿时长
2.1 months
期刊介绍: Analytical and Bioanalytical Chemistry’s mission is the rapid publication of excellent and high-impact research articles on fundamental and applied topics of analytical and bioanalytical measurement science. Its scope is broad, and ranges from novel measurement platforms and their characterization to multidisciplinary approaches that effectively address important scientific problems. The Editors encourage submissions presenting innovative analytical research in concept, instrumentation, methods, and/or applications, including: mass spectrometry, spectroscopy, and electroanalysis; advanced separations; analytical strategies in “-omics” and imaging, bioanalysis, and sampling; miniaturized devices, medical diagnostics, sensors; analytical characterization of nano- and biomaterials; chemometrics and advanced data analysis.
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