Ion Pairing Free Mixed-Mode Chromatography for the Separation and Measurement of TCA Cycle Metabolites in Urine Samples of Breast Cancer Patients

Importance of the Topic

The tricarboxylic acid (TCA) cycle lies at the heart of cellular energy production and biosynthesis, converting carbohydrates, proteins and fats into ATP and key metabolic precursors. Monitoring TCA cycle intermediates in biofluids such as urine provides insight into metabolic alterations associated with disease states, including cancer. However, the small, polar organic acids involved are challenging to retain and separate by conventional reversed-phase liquid chromatography, and they often chelate metal surfaces, compromising sensitivity and reproducibility.

Objectives and Study Overview

This study presents an ion-pairing-free, mixed-mode anion exchange chromatography method for profiling TCA cycle metabolites in urine from healthy controls and breast cancer patients. The method employs a PREMIER CSH Phenyl-Hexyl column with MaxPeak High Performance Surface (HPS) technology to mitigate metal-analyte interactions, aiming to achieve robust chromatographic resolution of isobaric compounds without derivatization or ion-pairing reagents. Unsupervised principal component analysis (PCA) evaluates the method’s ability to discriminate sample groups.

Methodology and Instrumentation

Sample Preparation:

• Thaw urine samples on ice
• Add 100 µL urine to 300 µL water
• Vortex mix 30 s and centrifuge at 21 130 rcf, 10 min, 4 °C
• Transfer supernatant to silanized vials

Liquid Chromatography:

• System: ACQUITY I-Class PLUS
• Column: PREMIER CSH Phenyl-Hexyl, 1.7 µm, 2.1 × 100 mm
• Mobile phase A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile
• Flow rate: 0.4 mL/min; gradient 0–25% B over 4 min, 25–95% B over 3 min, wash and re-equilibration

Mass Spectrometry:

• System: Xevo G2-XS QTOF, negative ion mode
• Data processing: MassLynx and Progenesis QI

Key Results and Discussion

Chromatographic resolution effectively separated critical isobaric and isomeric pairs, including citric/isocitric acids and malic/fumaric acids, as well as overlapping fragments from itaconic and cis-aconitic acids. The MaxPeak HPS surface reduced metal-induced analyte losses, improving peak area recovery by over 100% for sensitive compounds. Analytical reproducibility over 462 injections showed tight retention time and peak area consistency (<1% RSD). PCA of replicate injections and individual samples demonstrated clear clustering of healthy control, breast cancer positive and QC pools, indicating method robustness and sample discrimination power.

Benefits and Practical Applications

This ion-pair-free approach streamlines sample preparation and eliminates buffer salts that can suppress MS sensitivity. The enhanced recoveries and reproducibility make the method well suited for high-throughput metabolomics, clinical biomarker studies, QA/QC workflows and longitudinal monitoring of metabolic health.

Future Trends and Opportunities

Ongoing developments may include integration with automated sample handling, expansion to other biofluids such as plasma and cerebrospinal fluid, coupling with ion mobility separation for further isomer resolution, and application of machine learning for data interpretation. Advances in column surface chemistries will continue to improve analyte recovery and extend method longevity.

Conclusion

A mixed-mode, ion-pairing-free LC-MS method using PREMIER CSH Phenyl-Hexyl with MaxPeak HPS reliably separates and quantifies TCA cycle metabolites in urine. The approach delivers high sensitivity, reproducibility and sample discrimination without complex derivatization or ion-pairing reagents, making it a powerful tool for clinical and research metabolomics.

Reference

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