| Autor: |
Kipura T; Institute of Biochemistry and Center for Molecular Biosciences Innsbruck, University of Innsbruck, A-6020 Innsbruck, Austria., Hotze M; Institute of Biochemistry and Center for Molecular Biosciences Innsbruck, University of Innsbruck, A-6020 Innsbruck, Austria., Hofer A; Institute of Biochemistry and Center for Molecular Biosciences Innsbruck, University of Innsbruck, A-6020 Innsbruck, Austria., Egger AS; Institute of Biochemistry and Center for Molecular Biosciences Innsbruck, University of Innsbruck, A-6020 Innsbruck, Austria., Timpen LE; Institute of Biochemistry and Center for Molecular Biosciences Innsbruck, University of Innsbruck, A-6020 Innsbruck, Austria., Opitz CA; German Cancer Research Center (DKFZ), Division of Metabolic Crosstalk in Cancer and the German Cancer Consortium (DKTK), DKFZ Core Center Heidelberg, 69120 Heidelberg, Germany., Townsend PA; Division of Cancer Sciences, Manchester Cancer Research Center, Manchester Academic Health Sciences Center, University of Manchester, Manchester M20 4GJ, UK.; School of Biosciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK., Gethings LA; School of Biosciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK.; Waters Corporation, Wilmslow SK9 4AX, UK., Thedieck K; Institute of Biochemistry and Center for Molecular Biosciences Innsbruck, University of Innsbruck, A-6020 Innsbruck, Austria.; Freiburg Materials Research Center (FMF), Albert-Ludwigs-University of Freiburg, 79104 Freiburg, Germany.; Department of Pediatrics, Section Systems Medicine of Metabolism and Signaling, University of Groningen, University Medical Center Groningen, 9700 RB Groningen, The Netherlands.; Department Metabolism, Senescence and Autophagy, Research Center One Health Ruhr, University Hospital Essen, University Duisburg-Essen, 45147 Essen, Germany., Kwiatkowski M; Institute of Biochemistry and Center for Molecular Biosciences Innsbruck, University of Innsbruck, A-6020 Innsbruck, Austria. |
| Abstrakt: |
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. |
