Separation and Detection of TCA Cycle Metabolites and Related Compounds in Human Urine by UPLC-MS/MS for Clinical Research

Importance of the topic

The citric acid cycle is a central metabolic pathway that converts carbohydrates, fats, and proteins into energy and biosynthetic precursors. Monitoring TCA cycle metabolites in urine provides insights into cellular metabolism, disease states, and therapeutic interventions. A rapid, reliable analytical method enhances clinical research and diagnostic workflows.

Objectives and study overview

The study aimed to develop a mixed-mode reversed-phase UPLC-MS/MS method for quantifying ten TCA cycle and related polar metabolites in human urine without derivatization or ion-pairing reagents. The goal was to achieve rapid separation, high reproducibility, and compatibility with clinical research requirements.

Methodology and instrumentation

• Column: ACQUITY UPLC CSH Phenyl-Hexyl, 2.1 x 100 mm, 1.7 μm, at 80 °C
• Mobile phase: water and acetonitrile each containing 0.1% formic acid; gradient from 2% to 15% organic over 3 minutes
• Flow rate: 0.4 mL/min; injection volume: 3 μL
• Detection: Xevo TQ-S micro tandem mass spectrometer in negative electrospray mode with MRM transitions for each analyte

Main results and discussion

The method achieved baseline resolution of ten analytes in under three minutes, including critical isobaric pairs such as citric/isocitric and malic/fumaric acids. Retention time reproducibility across column lots showed RSDs below 1.3%. Analysis of tenfold diluted human urine demonstrated clear detection of target metabolites with acceptable peak shapes and quantitative performance.

Practical benefits and applications

This approach eliminates the need for derivatization and ion-pairing reagents, reducing sample preparation time and potential contamination. Its short runtime and robust performance make it well suited for high-throughput metabolomics, clinical research, and quality control in pharmaceutical and biomedical laboratories.

Future trends and potential applications

Future developments may include integration with high-resolution mass spectrometry for untargeted profiling, expansion to other polar metabolite classes, and adaptation to additional biofluids. Automation and methodological refinements could further increase throughput and data quality.

Conclusion

A rapid, reproducible, and reagent-minimal UPLC-MS/MS method for TCA cycle metabolite analysis in urine has been developed and validated, offering significant improvements in speed and simplicity for clinical metabolic profiling.

Reference

• 1. Murray RK, Bender DA et al. Harper’s Illustrated Biochemistry, 28th ed. McGraw-Hill, New York, 2009, Chapters 16–18.
• 2. Yuan M et al. Nature Protocols, 2012;7:872–881.
• 3. Luo B et al. Journal of Chromatography A, 2006;1147:153–164.
• 4. Van Dam JC et al. Analytica Chimica Acta, 2002;460:209–218.
• 5. Patel DP et al. PLoS One, 2017;12:1–16.
• 6. Tan B et al. Analytical Biochemistry, 2014;465:134–147.