An optimized LC-ESI-MS/MS assay for erythrocyte 6-TG and 6-MMPD: Addressing critical methodological considerations for thiopurine metabolite monitoring

IF 2.5 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B Pub Date : 2026-01-15 Epub Date: 2025-11-26 DOI:10.1016/j.jchromb.2025.124872

Ya-Min Chu , Yuan-Yuan Zhang , Ya-Hui Hu , Jie Wang , Hong-Li Guo , Jin Xu , Yong-Jun Fang , Jing Xu , Xuan-Sheng Ding , Feng Chen

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引用次数: 0

Abstract

Thiopurines, a class of antimetabolites widely used in patients with inflammatory bowel disease (IBD) and acute lymphoblastic leukemia (ALL), are associated with two predominant adverse effects: delayed myelotoxicity and hepatotoxicity. The erythrocyte-based active metabolites 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine nucleotides (6-MMPN) serve as biomarkers reflecting systemic drug exposure and predicting both therapeutic efficacy and adverse reactions. In this study, we successfully developed and validated a rapid, sensitive, and accurate liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analytical method for simultaneous quantification of erythrocyte 6-TG and 6-MMPD concentrations. During method development and validation, we focused on addressing the following five key challenges currently of significant interest in the field: 1) Erythrocytes proved superior to whole blood as biological matrix; 2) Perchloric acid was essential in sample pretreatment; 3) Dual mobile phase additives (HCOOH+NH₄Ac) enhanced chromatographic peak shape and detection sensitivity; 4) Incomplete hydrolysis of 6-TGN and 6-MMPN occurred with heating durations < 60 min; 5) The linear correlation coefficient of the 6-MMPD (6-MMP derivative, 4-amino-5-(methylthio)carbonyl imidazole) standard curve reflected the stable conversion efficiency of 6-MMP. The method exhibited excellent linearity over the concentration ranges of 0.06–50 μmol/L for 6-TG (r = 0.9906) and 0.18–150 μmol/L for 6-MMPD (r = 0.9914). Selectivity, carry-over, intra- and inter-batch accuracy and precision, reproducibility, recovery, matrix effect, and stability all complied with the acceptance criteria outlined in the US Food and Drug Administration (FDA) Bioanalytical Method Validation Guidance (2018). The validated method was successfully employed to quantify 6-TG and 6-MMPD in samples obtained from 20 patients with IBD or ALL.

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红细胞6-TG和6-MMPD的优化LC-ESI-MS/MS分析：解决硫嘌呤代谢物监测的关键方法学问题。

硫嘌呤是一类广泛用于炎症性肠病（IBD）和急性淋巴细胞白血病（ALL）患者的抗代谢物，它与两种主要的不良反应相关：延迟性骨髓毒性和肝毒性。红细胞活性代谢物6-硫鸟嘌呤核苷酸（6-TGN）和6-甲基巯基嘌呤核苷酸（6-MMPN）可作为反映全身药物暴露的生物标志物，预测治疗疗效和不良反应。在本研究中，我们成功建立并验证了一种快速、灵敏、准确的液相色谱-电喷雾电离-串联质谱（LC-ESI-MS/MS）同时定量红细胞6-TG和6-MMPD浓度的分析方法。在方法开发和验证期间，我们专注于解决以下五个关键挑战，目前该领域的重大兴趣：1)红细胞被证明优于全血作为生物基质；2)样品预处理必须使用高氯酸；3)双流动相添加剂（HCOOH+NH4Ac）增强了色谱峰形和检测灵敏度；4) 6-TGN和6-MMPN在加热时间< 60 min时发生不完全水解；5) 6-MMPD （6-MMP衍生物，4-氨基-5-（甲基硫）羰基咪唑）标准曲线的线性相关系数反映了6-MMP稳定的转化效率。6-TG在0.06 ~ 50 μmol/L （r = 0.9906）和6-MMPD在0.18 ~ 150 μmol/L （r = 0.9914）的浓度范围内具有良好的线性关系。选择性、结转、批内和批间准确度和精密度、重现性、回收率、基质效应和稳定性均符合美国食品和药物管理局（FDA）生物分析方法验证指南（2018）中概述的验收标准。该方法被成功用于定量20例IBD或ALL患者样品中的6-TG和6-MMPD。

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来源期刊

Journal of Chromatography B 医学-分析化学

CiteScore

5.60

自引率

3.30%

发文量

306

审稿时长

44 days

期刊介绍： The Journal of Chromatography B publishes papers on developments in separation science relevant to biology and biomedical research including both fundamental advances and applications. Analytical techniques which may be considered include the various facets of chromatography, electrophoresis and related methods, affinity and immunoaffinity-based methodologies, hyphenated and other multi-dimensional techniques, and microanalytical approaches. The journal also considers articles reporting developments in sample preparation, detection techniques including mass spectrometry, and data handling and analysis. Developments related to preparative separations for the isolation and purification of components of biological systems may be published, including chromatographic and electrophoretic methods, affinity separations, field flow fractionation and other preparative approaches. Applications to the analysis of biological systems and samples will be considered when the analytical science contains a significant element of novelty, e.g. a new approach to the separation of a compound, novel combination of analytical techniques, or significantly improved analytical performance.