Comparative evaluation of phenyl isothiocyanate derivatization and "dilute-and-shoot" methods for HPLC-MS/MS-based targeted metabolomics analysis of amine-containing metabolites in plasma samples.

IF 3.8 2区 化学 Q1 BIOCHEMICAL RESEARCH METHODS
Kangkang Xu, Markus Aigensberger, Franz Berthiller, Heidi E Schwartz-Zimmermann
{"title":"Comparative evaluation of phenyl isothiocyanate derivatization and \"dilute-and-shoot\" methods for HPLC-MS/MS-based targeted metabolomics analysis of amine-containing metabolites in plasma samples.","authors":"Kangkang Xu, Markus Aigensberger, Franz Berthiller, Heidi E Schwartz-Zimmermann","doi":"10.1007/s00216-025-06079-5","DOIUrl":null,"url":null,"abstract":"<p><p>Metabolomics, the study of small molecule metabolites in biological systems, is essential for disease diagnosis and biomarker discovery. A key consideration in developing targeted metabolomics methods using HPLC-MS/MS for human or animal plasma is whether to employ derivatization of amino acids, amino acid-related compounds, and biogenic amines. Derivatization with phenyl isothiocyanate (PITC) enhances ionization and LC-separation, but complicates sample preparation and introduces potential errors. This study focuses on (1) the validation of a PITC derivatization method employing reversed-phase (RP) HPLC-MS/MS analysis and (2) a comparison of two analytical approaches for targeted metabolomics analysis of animal and human plasma: the PITC derivatization-based RP-LC-MS/MS method and a \"dilute-and-shoot\" approach using hydrophilic interaction chromatography (HILIC)-MS/MS and RP-LC-MS/MS analysis. The derivatization method was validated for porcine plasma, assessing limits of detection, lower limits of quantification (LLOQs), linearity, repeatability, recovery, and trueness. Derivatization reduced LLOQs for derivatized compounds in pure solvent solutions but, due to higher dilution factors, resulted in similar LLOQs for derivatized compounds and higher LLOQs for non-derivatized compounds in plasma compared to the \"dilute-and-shoot\" method. Derivatization improved chromatographic separation of isomers and reduced carryover but introduced challenges such as matrix effects, coelution with impurities, and calibration issues. The \"dilute-and-shoot\" method performed better for non-derivatized compounds and was less error-prone. Both methods were applied to plasma from various species, demonstrating comparable concentrations for most metabolites. The results also emphasize the importance of using different approaches for cross-validation. Above all, this study highlights the strengths and limitations of both the derivatization method and the \"dilute-and-shoot\" approach, providing guidance for their application in targeted metabolomics.</p>","PeriodicalId":462,"journal":{"name":"Analytical and Bioanalytical Chemistry","volume":" ","pages":""},"PeriodicalIF":3.8000,"publicationDate":"2025-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Analytical and Bioanalytical Chemistry","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1007/s00216-025-06079-5","RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0

Abstract

Metabolomics, the study of small molecule metabolites in biological systems, is essential for disease diagnosis and biomarker discovery. A key consideration in developing targeted metabolomics methods using HPLC-MS/MS for human or animal plasma is whether to employ derivatization of amino acids, amino acid-related compounds, and biogenic amines. Derivatization with phenyl isothiocyanate (PITC) enhances ionization and LC-separation, but complicates sample preparation and introduces potential errors. This study focuses on (1) the validation of a PITC derivatization method employing reversed-phase (RP) HPLC-MS/MS analysis and (2) a comparison of two analytical approaches for targeted metabolomics analysis of animal and human plasma: the PITC derivatization-based RP-LC-MS/MS method and a "dilute-and-shoot" approach using hydrophilic interaction chromatography (HILIC)-MS/MS and RP-LC-MS/MS analysis. The derivatization method was validated for porcine plasma, assessing limits of detection, lower limits of quantification (LLOQs), linearity, repeatability, recovery, and trueness. Derivatization reduced LLOQs for derivatized compounds in pure solvent solutions but, due to higher dilution factors, resulted in similar LLOQs for derivatized compounds and higher LLOQs for non-derivatized compounds in plasma compared to the "dilute-and-shoot" method. Derivatization improved chromatographic separation of isomers and reduced carryover but introduced challenges such as matrix effects, coelution with impurities, and calibration issues. The "dilute-and-shoot" method performed better for non-derivatized compounds and was less error-prone. Both methods were applied to plasma from various species, demonstrating comparable concentrations for most metabolites. The results also emphasize the importance of using different approaches for cross-validation. Above all, this study highlights the strengths and limitations of both the derivatization method and the "dilute-and-shoot" approach, providing guidance for their application in targeted metabolomics.

基于HPLC-MS/ ms的血浆样品含胺代谢物靶向代谢组学分析中异硫氰酸苯衍生化和“稀释-射击”方法的比较评价
代谢组学研究生物系统中的小分子代谢物,对疾病诊断和生物标志物发现至关重要。在使用HPLC-MS/MS开发针对人或动物血浆的靶向代谢组学方法时,一个关键的考虑因素是是否使用氨基酸、氨基酸相关化合物和生物胺的衍生化。与异硫氰酸苯酯(PITC)衍生化增强了电离和lc分离,但使样品制备复杂化并引入潜在的误差。本研究的重点是:(1)采用反相(RP) HPLC-MS/MS分析的PITC衍生化方法的验证;(2)比较两种用于动物和人血浆靶向代谢组学分析的分析方法:基于PITC衍生化的RP- lc -MS/MS方法和使用亲水相互作用色谱(HILIC)-MS/MS和RP- lc -MS/MS分析的“稀释-射击”方法。对猪血浆衍生化方法进行了验证,评估了检测限、定量下限(lloq)、线性、重复性、回收率和准确性。衍生化降低了衍生化化合物在纯溶剂溶液中的lloq,但由于更高的稀释系数,与“稀释-射击”方法相比,衍生化化合物在血浆中的lloq相似,非衍生化化合物的lloq更高。衍生化改善了同分异构体的色谱分离,减少了携带,但也带来了一些挑战,如基质效应、杂质共洗脱和校准问题。“稀释-射击”方法对非衍生化化合物表现更好,并且不易出错。这两种方法都应用于不同物种的血浆,显示出大多数代谢物的浓度相当。结果还强调了使用不同方法进行交叉验证的重要性。总之,本研究突出了衍生化方法和“稀释-射击”方法的优势和局限性,为它们在靶向代谢组学中的应用提供了指导。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
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.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:604180095
Book学术官方微信