Improving sensitivity for the quantification of TCA cycle analytes in human plasma by the ACQUITY™ Premier system solution

Importance of Topic

This work addresses challenges in quantifying tricarboxylic acid (TCA) cycle metabolites in human plasma. Due to their polar nature, low molecular weight and interactions with metal surfaces, key TCA analytes often suffer from poor chromatographic performance and low sensitivity. Reliable measurement of these metabolites is critical for studies in cell metabolism, disease biomarker discovery, and clinical diagnostics.

Study Objectives and Overview

The study evaluated the performance of the ACQUITY™ Premier System Solution, incorporating MaxPeak High Performance Surface technology, for improved sensitivity and peak shape in the analysis of TCA cycle intermediates. Using human plasma samples, the authors compared conventional UPLC columns against the new Premier LC and Premier CSH™ Phenyl-Hexyl column in a mixed-mode anion exchange method.

Methodology and Instrumentation

A targeted LC-MS/MS assay was developed on an ACQUITY™ Premier LC coupled to a Xevo TQ-S micro in negative ESI mode. Separation was performed on a 2.1×100 mm, 1.7 µm ACQUITY Premier CSH Phenyl-Hexyl column using 0.1% formic acid in water and acetonitrile. Sample preparation involved protein precipitation of plasma (25 µL plasma + 75 µL cold methanol), centrifugation, evaporation, and reconstitution. Stable isotope labeled internal standards were spiked into each sample. Calibration curves (0.1–500 µM) employed 1/x weighting and achieved linearity (r^2 > 0.99) for all analytes.

Results and Discussion

Compared to a conventional UPLC column, the Premier system delivered:

• Over 100-fold increase in signal for citric and isocitric acids.
• Reduced peak tailing for malic acid by 58%.
• Consistent performance across eight injections without prior column passivation.
• High linearity (r^2 ≥ 0.994) across low nanomolar to micromolar ranges.
• Quantitation of TCA cycle metabolites in healthy and breast cancer plasma samples, revealing elevated citric and succinic acid levels in cancer patients.

The MaxPeak HPS coatings minimized metal-analyte interactions, eliminating the need for mobile phase chelators and avoiding ion suppression or chromatographic alterations.

Benefits and Practical Applications

• Enhanced analytical sensitivity and robustness for polar organic acids.
• Simplified workflow by avoiding extensive system passivation or addition of chelating agents.
• Applicability to high-throughput clinical and research laboratories for reliable metabolite profiling.

Future Trends and Opportunities

Further applications of high-performance surface technologies may extend to other metal-sensitive analytes, including phosphorylated and sulfated metabolites. Integration into automated sample preparation and ultra-high-throughput platforms could accelerate large-scale clinical studies and real-time metabolic monitoring. Combining this approach with emerging ion mobility or high-resolution MS may deepen insights into metabolic flux and biomarker discovery.

Conclusion

The ACQUITY™ Premier System Solution, featuring MaxPeak HPS components and a mixed-mode CSH Phenyl-Hexyl column, significantly improves sensitivity, peak shape, and reproducibility for TCA cycle metabolite quantification in human plasma. This approach streamlines analysis by removing chelator additives and system passivation, offering a robust tool for metabolic research and clinical applications.

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