Please use this identifier to cite or link to this item: http://hdl.handle.net/1893/36611
Appears in Collections:Senior Management Team Journal Articles
Peer Review Status: Refereed
Title: Automated Liquid Handling Extraction and Rapid Quantification of Underivatized Amino Acids and Tryptophan Metabolites from Human Serum and Plasma Using Dual-Column U(H)PLC-MRM-MS and Its Application to Prostate Cancer Study
Author(s): Kipura, Tobias
Hotze, Madlen
Hofer, Alexa
Egger, Anna-Sophia
Timpen, Lea E
Opitz, Christiane A
Townsend, Paul A
Gethings, Lee A
Thedieck, Kathrin
Kwiatkowski, Marcel
Contact Email: catherine.stewart@stir.ac.uk
Keywords: amino acids
tryptophan metabolites analysis
automation
LC-MS
mixed-mode chromatography
prostate cancer
Issue Date: 30-Jun-2024
Date Deposited: 13-Jan-2025
Citation: Kipura T, Hotze M, Hofer A, Egger A, Timpen LE, Opitz CA, Townsend PA, Gethings LA, Thedieck K & Kwiatkowski M (2024) Automated Liquid Handling Extraction and Rapid Quantification of Underivatized Amino Acids and Tryptophan Metabolites from Human Serum and Plasma Using Dual-Column U(H)PLC-MRM-MS and Its Application to Prostate Cancer Study. <i>Metabolites</i>, 14 (7), Art. No.: 370. https://doi.org/10.3390/metabo14070370
Abstract: Amino acids (AAs) and their metabolites are important building blocks, energy sources, and signaling molecules associated with various pathological phenotypes. The quantification of AA and tryptophan (TRP) metabolites in human serum and plasma is therefore of great diagnostic interest. Therefore, robust, reproducible sample extraction and processing workflows as well as rapid, sensitive absolute quantification are required to identify candidate biomarkers and to improve screening methods. We developed a validated semi-automated robotic liquid extraction and processing workflow and a rapid method for absolute quantification of 20 free, underivatized AAs and six TRP metabolites using dual-column U(H)PLC-MRM-MS. The extraction and sample preparation workflow in a 96-well plate was optimized for robust, reproducible high sample throughput allowing for transfer of samples to the U(H)PLC autosampler directly without additional cleanup steps. The U(H)PLC-MRM-MS method, using a mixed-mode reversed-phase anion exchange column with formic acid and a high-strength silica reversed-phase column with difluoro-acetic acid as mobile phase additive, provided absolute quantification with nanomolar lower limits of quantification within 7.9 min. The semi-automated extraction workflow and dual-column U(H)PLC-MRM-MS method was applied to a human prostate cancer study and was shown to discriminate between treatment regimens and to identify metabolites responsible for discriminating between healthy controls and patients on active surveillance.
DOI Link: 10.3390/metabo14070370
Rights: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Licence URL(s): http://creativecommons.org/licenses/by/4.0/

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