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
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Metabolite Standards
2.3. U(H)PLC-MRM-MS
2.4. Method Validation
2.5. Manual Extraction from Serum
2.6. Semi-Automated Extraction from Serum and Plasma
3. Results
3.1. U(H)PLC-MRM-MS Method Development
3.2. Linearity, Lower Limit of Detection (Llod), and Lower Limit of Quantification (Lloq)
3.3. Within-Run and between-Run Accuracy and Precision
3.4. Semi-Automated Extraction and Sample Preparation of Human Serum and Plasma
3.5. Manual Extraction and Sample Preparation Versus Semi-Automated Workflow
3.6. Optimization of the Semi-Automated Extraction for Serum Samples
3.7. Optimization of the Semi-Automated Extraction for Plasma Samples
3.8. Recovery
3.9. Intra-Assay Variability and Inter-Assay Precision
3.10. Autosampler Stability
3.11. Analysis of NIST SRM 1950 Reference Plasma Sample
3.12. Automated Extraction and Analysis of Serum Samples from a Prostate Cancer Study
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Metabolite | LLOD [µM] | LLOQ [µM] |
---|---|---|
3-hydroxykynurenine | 0.097 | 0.292 |
Alanine | 0.140 | 0.420 |
Arginine | 0.147 | 0.441 |
Asparagine | 0.179 | 0.536 |
Aspartic acid | 0.141 | 0.424 |
Glutamine | 0.111 | 0.334 |
Glutamic acid | 0.280 | 0.841 |
Glycine | 0.822 | 2.467 |
Histidine | 0.184 | 0.552 |
Indole-3-acetic acid | 0.185 | 0.555 |
Isoleucine | 0.124 | 0.372 |
Kynurenine | 0.128 | 0.385 |
Kynurenic acid | 0.053 | 0.158 |
Leucine | 0.142 | 0.425 |
Lysine | 0.155 | 0.465 |
Methionine | 0.114 | 0.342 |
Nicotinic acid | 0.082 | 0.245 |
Nicotinamide | 0.075 | 0.226 |
Ornithine | 0.121 | 0.363 |
Phenylalanine | 0.178 | 0.534 |
Proline | 0.086 | 0.257 |
Serine | 0.198 | 0.594 |
Threonine | 0.127 | 0.381 |
Tryptophan | 0.097 | 0.292 |
Tyrosine | 0.167 | 0.501 |
Valine | 0.123 | 0.368 |
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Kipura, T.; Hotze, M.; Hofer, A.; Egger, A.-S.; Timpen, L.E.; Opitz, C.A.; Townsend, P.A.; Gethings, L.A.; Thedieck, K.; Kwiatkowski, M. 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. Metabolites 2024, 14, 370. https://doi.org/10.3390/metabo14070370
Kipura T, Hotze M, Hofer A, Egger A-S, Timpen LE, Opitz CA, Townsend PA, Gethings LA, Thedieck K, Kwiatkowski M. 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. Metabolites. 2024; 14(7):370. https://doi.org/10.3390/metabo14070370
Chicago/Turabian StyleKipura, Tobias, Madlen Hotze, Alexa Hofer, Anna-Sophia Egger, Lea E. Timpen, Christiane A. Opitz, Paul A. Townsend, Lee A. Gethings, Kathrin Thedieck, and Marcel Kwiatkowski. 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" Metabolites 14, no. 7: 370. https://doi.org/10.3390/metabo14070370