Separation and Analysis of TCA Cycle Analytes by Mixed-Mode Chromatography Coupled with Mass Spectrometry

Significance of the Topic

The tricarboxylic acid (TCA) cycle is central to cellular energy production and biosynthesis, generating ATP and precursors for amino acids, lipids, and nucleotides. Quantitative analysis of TCA intermediates provides insights into metabolic regulation under physiological and pathological states, including cancer. However, the small, polar nature of these organic acids poses challenges for conventional reversed-phase LC methods, necessitating alternative chromatographic strategies.

Objectives and Study Overview

This study aimed to develop a robust, mixed-mode liquid chromatography–mass spectrometry (LC–MS) method for simultaneous analysis of TCA cycle metabolites and related organic acids in urine without sample derivatization or ion-pairing reagents. The method was applied to urine samples from healthy individuals and breast cancer patients to assess metabolic differences and instrument performance.

Methodology and Instrumentation

• Sample preparation: Four control and four breast cancer urine samples were diluted, centrifuged, and analyzed in five replicates. System suitability was verified using neat and spiked standards at 100 μM.
• Chromatography: Separation was achieved on an ACQUITY UPLC CSH Phenyl-Hexyl column (2.1 × 100 mm, 1.7 μm) using a 0.1% formic acid water–acetonitrile gradient at 50 °C column temperature and 5 °C sample temperature. The gradient ranged from 100% aqueous to 95% organic over 7 minutes.
• Mass spectrometry: A Xevo G2-XS QTof, operated in negative electrospray ionization mode, acquired data in MS

Main Results and Discussion

• Chromatographic separation: Critical isobaric pairs—citric/isocitric acids and malic/fumaric acids—were baseline resolved with resolution values of 6.0 and 3.8 (FWHM), respectively.
• Reproducibility: QC samples clustered tightly in principal component analysis, demonstrating high analytical precision.
• Biological findings: PCA distinguished breast cancer from healthy samples, with significant changes in key metabolites (isocitric acid, citric acid, 2-hydroxyglutaric acid, lactic acid, succinic acid, cis-aconitic acid) across sample groups.

Benefits and Practical Applications

The mixed-mode LC–MS approach offers:

• Elimination of derivatization and ion-pairing reagents for streamlined workflows.
• Reliable quantitation of polar organic acids relevant to metabolic research and clinical diagnostics.
• Adaptability to urine metabolomics for disease biomarker discovery.

Future Trends and Potential Applications

Advancements in mixed-mode stationary phases and high-resolution mass spectrometry will further enhance detection sensitivity and selectivity of small, polar metabolites. Integration with multivariate statistical tools and AI-driven data analysis will refine biomarker identification for precision medicine. Potential applications include longitudinal metabolic profiling, therapeutic monitoring, and expanded coverage of central carbon and ancillary pathways.

Conclusion

A mixed-mode LC–MS method without derivatization or ion-pairing was established for robust analysis of TCA cycle analytes in urine. The approach achieved high chromatographic resolution, reproducibility, and discrimination between healthy and disease states, demonstrating its utility for metabolomics and clinical research.

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