Optimization and comparison of amino group derivatization reagents for sensitive and isomer-selective LC-MS/MS analysis of tyrosine-derived biomarkers of oxidative and nitrosative stress

IF 5.6 1区化学 Q1 CHEMISTRY, ANALYTICAL

Talanta Pub Date : 2025-07-17 DOI:10.1016/j.talanta.2025.128596

Yusuke Iwasaki , Hironori Saito , Kangrong Chen , Hu Leqi , Natsuki Koshiishi , Keisuke Mochizuki , Rie Ito , Hiroshi Akiyama

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Abstract

Reactive oxygen species and reactive nitrogen species play roles in the pathogenesis of numerous diseases, but their presence is often assessed indirectly via analysis of specific biomarkers. Tyrosine-derived compounds such as ortho-tyrosine, meta-tyrosine, 3-chlorotyrosine, and 3-nitrotyrosine are considered markers of oxidative and nitrosative stress. However, these biomarkers are typically present in extremely low concentrations (in the low nanomolar range) and have structural isomers, making accurate quantification challenging. The aim of this study was to improve the sensitivity and chromatographic separation of these analytes using various commercially available derivatization reagents targeting amino groups. Selected reaction monitoring transitions were optimized by flow injection analysis of derivatized standards, and structural isomers were evaluated using LC-MS/MS. A Box-Behnken design and response surface methodology were employed to determine the optimal derivatization conditions for each reagent. Among the reagents tested, diethyl ethoxymethylenemalonate was most effective, enabling complete isomer separation and approximately 1000-fold signal enhancement compared with non-derivatized analytes, with a limit of quantification reaching 5 nM. The results of this study highlight the importance of selecting an appropriate derivatization strategy for sensitive and selective quantification of tyrosine-based biomarkers and offer a promising approach for the accurate assessment of oxidative and nitrosative stress in biological samples.

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用于酪氨酸衍生氧化和亚硝化应激生物标志物敏感和异构体选择性LC-MS/MS分析的氨基衍生试剂的优化和比较

活性氧和活性氮在许多疾病的发病机制中发挥作用，但它们的存在往往是通过分析特定的生物标志物间接评估的。酪氨酸衍生的化合物，如正酪氨酸、间酪氨酸、3-氯酪氨酸和3-硝基酪氨酸被认为是氧化和亚硝化应激的标志。然而，这些生物标志物通常以极低的浓度（在低纳摩尔范围内）存在，并且具有结构异构体，这使得准确的定量具有挑战性。本研究的目的是提高这些分析物的灵敏度和色谱分离使用各种市售衍生试剂针对氨基。衍生化标准品流动注射分析优化反应监测转变，LC-MS/MS评价结构异构体。采用Box-Behnken设计和响应面法确定每种试剂的最佳衍生化条件。在所测试的试剂中，乙氧基甲基灌肠酸二乙酯最有效，可以完全分离异构体，与未衍生化的分析物相比，信号增强约1000倍，定量限达到5 nM。本研究的结果强调了选择合适的衍生化策略对酪氨酸生物标志物的敏感性和选择性定量的重要性，并为准确评估生物样品中的氧化和亚硝化应激提供了一种有前途的方法。

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来源期刊

Talanta 化学-分析化学

CiteScore

12.30

自引率

4.90%

发文量

861

审稿时长

29 days

期刊介绍： Talanta provides a forum for the publication of original research papers, short communications, and critical reviews in all branches of pure and applied analytical chemistry. Papers are evaluated based on established guidelines, including the fundamental nature of the study, scientific novelty, substantial improvement or advantage over existing technology or methods, and demonstrated analytical applicability. Original research papers on fundamental studies, and on novel sensor and instrumentation developments, are encouraged. Novel or improved applications in areas such as clinical and biological chemistry, environmental analysis, geochemistry, materials science and engineering, and analytical platforms for omics development are welcome. Analytical performance of methods should be determined, including interference and matrix effects, and methods should be validated by comparison with a standard method, or analysis of a certified reference material. Simple spiking recoveries may not be sufficient. The developed method should especially comprise information on selectivity, sensitivity, detection limits, accuracy, and reliability. However, applying official validation or robustness studies to a routine method or technique does not necessarily constitute novelty. Proper statistical treatment of the data should be provided. Relevant literature should be cited, including related publications by the authors, and authors should discuss how their proposed methodology compares with previously reported methods.