MetaboQuan-R for the Analysis of Tryptophan and its Metabolites

Importance of the Topic

The metabolism of tryptophan and its downstream products is central to neurological function, immune regulation, and cardiometabolic health. Rapid, high-throughput quantification of these analytes supports large-scale studies in biomedical and clinical research, enabling efficient biomarker discovery and monitoring of metabolic pathways.

Objectives and Study Overview

This application note presents a streamlined UPLC-MS/MS workflow for simultaneous quantification of tryptophan and seven key metabolites in human urine and plasma. The method aims to deliver sub-three-minute analysis per injection, facilitating high sample throughput and ease of integration into multi-omics pipelines.

Methodology and Instrumentation

Sample Preparation:

• Urine: 1:10 dilution in water, direct injection of 1 µL.
• Plasma: Protein removal and SPE on Oasis PRiME HLB µElution plate, elution in methanol, 1:1 dilution, 1 µL injection.

Chromatography:

• ACQUITY UPLC I-Class System with CORTECS T3 (2.1 × 30 mm, 2.7 µm).
• Gradient from 0 to 70% isopropanol/formic acid over 0.95 min, total cycle <3 min.
• Column at 60 °C, flow rate 0.45 mL/min.

Mass Spectrometry:

• Xevo TQ-S micro in positive ESI mode.
• MRM transitions optimized for specificity and sensitivity.
• Source at 150 °C, desolvation at 650 °C, capillary voltage 2.0 kV.

Informatics:

• Method import via Quanpedia in MassLynx.
• Quantification with TargetLynx processing scripts.

Results and Discussion

The method resolved eight analytes within three minutes with baseline separation. In urine, all targets except melatonin were detected; plasma analysis confirmed tryptophan, serotonin, and kynurenine at physiologically relevant levels. Single and multiple MRM transitions provided comparable sensitivity, while the rapid gradient ensured clear peak shapes and retention time reproducibility.

Benefits and Practical Applications

This approach offers:

• High throughput for large cohort studies.
• Flexibility to switch between metabolomics and other targeted assays on the same platform.
• Minimal method development effort due to generic LC-MS configuration.

Its applicability spans clinical research, pharmacokinetic profiling, nutritional studies, and quality control in biomanufacturing.

Future Trends and Applications

Integration into fully automated multi-omics workflows will accelerate biomarker validation. Advances in ionization techniques and high-resolution MS may further enhance sensitivity and selectivity, while expanding metabolite coverage. Cloud-based data analysis and AI-driven interpretation will support real-time decision-making in clinical settings.

Conclusion

The described rapid UPLC-MS/MS method enables robust quantification of tryptophan and key metabolites in biological fluids. Its speed, flexibility, and ease of use make it a valuable tool for targeted metabolomics and multi-omics research.