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.
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| Název: | 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. |
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| Autoři: | Xu K; Institute of Bioanalytics and Agro-Metabolomics, Department of Agricultural Sciences, BOKU University, Konrad-Lorenz-Straße 20, 3430, Tulln, Austria.; Christian Doppler Laboratory for Innovative Gut Health Concepts of Livestock, 1210, Vienna, Austria., Aigensberger M; Institute of Bioanalytics and Agro-Metabolomics, Department of Agricultural Sciences, BOKU University, Konrad-Lorenz-Straße 20, 3430, Tulln, Austria.; Christian Doppler Laboratory for Innovative Gut Health Concepts of Livestock, 1210, Vienna, Austria., Berthiller F; Institute of Bioanalytics and Agro-Metabolomics, Department of Agricultural Sciences, BOKU University, Konrad-Lorenz-Straße 20, 3430, Tulln, Austria.; Christian Doppler Laboratory for Innovative Gut Health Concepts of Livestock, 1210, Vienna, Austria., Schwartz-Zimmermann HE; Institute of Bioanalytics and Agro-Metabolomics, Department of Agricultural Sciences, BOKU University, Konrad-Lorenz-Straße 20, 3430, Tulln, Austria. heidi.schwartz@boku.ac.at.; Christian Doppler Laboratory for Innovative Gut Health Concepts of Livestock, 1210, Vienna, Austria. heidi.schwartz@boku.ac.at. |
| Zdroj: | Analytical and bioanalytical chemistry [Anal Bioanal Chem] 2025 Nov; Vol. 417 (26), pp. 5859-5872. Date of Electronic Publication: 2025 Aug 30. |
| Způsob vydávání: | Journal Article; Comparative Study |
| Jazyk: | English |
| Informace o časopise: | Publisher: Springer-Verlag Country of Publication: Germany NLM ID: 101134327 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1618-2650 (Electronic) Linking ISSN: 16182642 NLM ISO Abbreviation: Anal Bioanal Chem Subsets: MEDLINE |
| Imprint Name(s): | Original Publication: Heidelberg : Springer-Verlag, 2002- |
| Výrazy ze slovníku MeSH: | Tandem Mass Spectrometry*/methods , Metabolomics*/methods , Isothiocyanates*/chemistry , Amines*/blood , Amines*/metabolism, Animals ; Chromatography, High Pressure Liquid/methods ; Humans ; Swine ; Limit of Detection ; Reproducibility of Results ; Liquid Chromatography-Mass Spectrometry |
| Abstrakt: | Competing Interests: Declarations. Ethics approval: The methods and protocols followed in this experiment were approved by the institutional ethics and animal welfare committee of the University of Veterinary Medicine Vienna and the Austrian national authority according to the law for animal experiments 2012—Tierversuchsgesetz (BMWFW 2022-0855.741). Conflict of interest: The authors declare no competing interests. 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. (© 2025. The Author(s).) |
| References: | Metabolites. 2021 Jun 25;11(7):. (PMID: 34201929) Anal Chem. 2010 Jul 1;82(13):5527-33. (PMID: 20524683) Nat Rev Mol Cell Biol. 2016 Jul;17(7):451-9. (PMID: 26979502) PLoS One. 2011 Feb 16;6(2):e16957. (PMID: 21359215) Nat Rev Mol Cell Biol. 2012 Mar 22;13(4):263-9. (PMID: 22436749) Metabolites. 2018 Aug 05;8(3):. (PMID: 30081599) Anal Chem. 2017 Jul 18;89(14):7667-7674. (PMID: 28581703) Anal Bioanal Chem. 2016 Sep;408(22):6079-91. (PMID: 27370688) Anal Chim Acta. 2023 Oct 23;1279:341791. (PMID: 37827685) Commun Biol. 2020 Jan 22;3(1):39. (PMID: 31969651) Metabolomics. 2017;13(10):122. (PMID: 28943831) Anal Chem. 2017 Jan 3;89(1):656-665. (PMID: 27959516) Metabolomics. 2019 Jul 9;15(7):103. (PMID: 31289941) Metabolites. 2023 Jan 31;13(2):. (PMID: 36837833) Anal Chem. 2020 Aug 4;92(15):10627-10634. (PMID: 32634308) Molecules. 2025 Feb 05;30(3):. (PMID: 39942809) Anal Chim Acta. 2020 Aug 15;1125:144-151. (PMID: 32674760) J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Sep 1;966:34-47. (PMID: 24815365) J Chromatogr A. 2023 May 24;1697:463985. (PMID: 37062154) |
| Grant Information: | CDL-LiveGut Christian Doppler Forschungsgesellschaft |
| Contributed Indexing: | Keywords: Amino acids; Biogenic amines; Derivatization; High-performance liquid chromatography; Mass spectrometry; Method comparison |
| Substance Nomenclature: | 0 (Isothiocyanates) 0 (Amines) |
| Entry Date(s): | Date Created: 20250829 Date Completed: 20251016 Latest Revision: 20251019 |
| Update Code: | 20251019 |
| PubMed Central ID: | PMC12532724 |
| DOI: | 10.1007/s00216-025-06079-5 |
| PMID: | 40883495 |
| Databáze: | MEDLINE |
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Nájsť tento článok vo Web of Science | Abstrakt: | Competing Interests: Declarations. Ethics approval: The methods and protocols followed in this experiment were approved by the institutional ethics and animal welfare committee of the University of Veterinary Medicine Vienna and the Austrian national authority according to the law for animal experiments 2012—Tierversuchsgesetz (BMWFW 2022-0855.741). Conflict of interest: The authors declare no competing interests.<br />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.<br /> (© 2025. The Author(s).) |
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| ISSN: | 1618-2650 |
| DOI: | 10.1007/s00216-025-06079-5 |