Improved Analytical Sensitivity and Chromatographic Peak Shape for the Quantification of TCA Cycle Analytes in Human Plasma using the ACQUITY PREMIER System Solution

Importance of the Topic

The tricarboxylic acid (TCA) cycle is central to cellular energy metabolism and signaling. Quantifying its intermediates in plasma provides critical insights into physiological and pathological states. However, the small, polar nature of these metabolites, their metal sensitivity, and isobaric interferences pose analytical challenges. Addressing these issues enhances our ability to monitor disease biomarkers and support metabolic research.

Objectives and Overview of the Study

This study aimed to develop a fast, simple, and sensitive LC-MS/MS method for the quantification of TCA cycle and related organic acids in human plasma. Key goals included improving chromatographic peak shape and analytical sensitivity by mitigating metal–analyte interactions, while avoiding complex mobile phase additives.

Methodology and Instrumentation

Sample Preparation:

• Individual analyte stocks prepared at 50 mM and combined to a 2.5 mM working solution.
• Stable isotope-labeled internal standards mixed in aqueous solution.
• Plasma (25 µL) spiked with internal standards, precipitated with cold methanol, centrifuged, dried, reconstituted, and filtered into silanized vials.

Calibration Curve:

• Serial dilutions from 2.5 mM to 0.5 µM.
• Standards and internal standards mixed in silanized vials to match plasma matrix dilution.
• Final measured calibration range: 0.033–167 µM.

Chromatography and Mass Spectrometry:

• Mixed-mode anion exchange separation using a CSH Phenyl-Hexyl column (2.1 × 100 mm, 1.7 µm) at 50 °C.
• Mobile phases: 0.1% formic acid in water (A) and in acetonitrile (B), simple reversed-phase gradient.
• ESI negative mode tandem quadrupole detection with MRM transitions for each compound.

Instrumentation Used

• Waters ACQUITY PREMIER UPLC System with MaxPeak High Performance Surface Technology
• ACQUITY PREMIER CSH Phenyl-Hexyl Column, 2.1 × 100 mm, 1.7 µm
• Xevo TQ-S micro Triple Quadrupole Mass Spectrometer
• MassLynx v4.2 and TargetLynx software for data acquisition and quantification

Main Results and Discussion

The use of MaxPeak HPS technology significantly increased peak areas and improved peak shapes for metal-sensitive analytes. Compared with a standard system, isocitric and citric acids showed 41× and 5× higher peak areas, respectively, while 3-phosphoglyceric acid increased over 100×. Malic acid peak tailing decreased by 58% at 10% height. The PREMIER setup delivered >100-fold higher sensitivity for citric acid on the first injection, reducing equilibration time. Calibration curves were linear across the entire concentration range with 1/x weighting, and back-calculated concentrations in control and breast cancer plasma samples demonstrated high retention time stability and reproducibility.

Benefits and Practical Applications

• Rapid and straightforward method without specialized mobile phase modifiers.
• Enhanced analytical sensitivity and peak shape through metal interaction mitigation.
• Reduced system equilibration time improves sample throughput.
• Applicable to biomarker studies, clinical research, and quality control in metabolomics.

Future Trends and Opportunities

Advances may include integration with high-resolution MS for untargeted profiling, automation of sample preparation, and expansion to additional polar metabolites. Further refinement of surface technologies could allow broader application in challenging matrices and low-abundance analyte detection.

Conclusion

The ACQUITY PREMIER System Solution combined mixed-mode anion exchange chromatography and MaxPeak HPS surfaces to overcome metal-analyte interactions, delivering a sensitive, robust assay for TCA cycle metabolites in plasma. This approach simplifies method development and enhances analytical performance without complex additives.

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