Quantitation of TCA Cycle with Automated Sample Prep, Reproducible HILIC Chromatography and Ion Funnel Triple Quadrupole

Importance of the Topic

The tricarboxylic acid (TCA) cycle is a fundamental metabolic pathway that drives cellular energy production and biosynthesis. Its dynamic regulation is implicated in various physiological and pathological states, including cancer metabolism and immune response.

Aims and Overview of the Study

This study aimed to establish a robust, high-throughput workflow for absolute quantitation of TCA cycle metabolites in complex biological samples. The workflow integrates automated sample preparation, metal-free hydrophilic interaction chromatography (HILIC), and a sensitive triple quadrupole mass spectrometer with an ion funnel interface.

Methodology and Instrumentation

• Sample Preparation: Automated extraction using the Bravo Metabolomics Sample Prep Platform with Captiva EMR-Lipid SPE plates for plasma and cell samples.
• Chromatography: Agilent Poroshell 120 HILIC-Z column (2.1x150 mm, 2.7 micron) on the 1290 Infinity II Bio LC system, employing a high-pH ammonium acetate buffer and acetonitrile gradient.
• Mass Spectrometry: Agilent 6495D LC/TQ with 4th generation iFunnel technology. MRM transitions were optimized via MassHunter Acquisition 12.1 and database-driven source parameter tuning.

Main Results and Discussion

• Expanded MRM Database: Addition of 13C-labeled TCA metabolite transitions to a database covering over 500 polar compounds.
• Enhanced Sensitivity: Fragile iFunnel mode improved signal response by up to 124 percent compared to standard settings.
• Quantitative Performance: Calibration curves demonstrated linear dynamic ranges spanning six orders of magnitude (1-100000 nM) with R2 values >=0.995 and RSDs below 10 percent for labeled internal standards.
• Matrix Application: All twelve targeted TCA metabolites were detected and quantified reliably in bovine plasma and K562 cell extracts within calibration ranges.

Benefits and Practical Applications

The presented workflow offers high sensitivity, reproducibility, and dynamic range for targeted metabolomics. It supports absolute quantitation of key metabolic markers in biomedical research, clinical studies, and quality control laboratories.

Future Trends and Potential Applications

• Integration with isotope tracing and flux analysis for deeper metabolic pathway insights.
• Coupling with high-resolution MS and multi-omics platforms for comprehensive systems biology studies.
• Automation and data analytics pipelines powered by machine learning for accelerated method development and interpretation.
• Expansion to clinical diagnostics and personalized medicine through robust biomarker quantitation.

Conclusion

The combination of automated sample preparation, reproducible HILIC chromatography, and advanced ion funnel triple quadrupole detection establishes a reliable end-to-end workflow for absolute quantitation of TCA cycle metabolites in diverse biological matrices.

References

1. Yamamoto T et al. Biochem Biophys Res Commun. 2021;584:53-59.
2. Yannell K et al. An End-to-End Targeted Metabolomics Workflow. Agilent Application Note 5994-5628EN. 2023.
3. Van de Bittner GC et al. Automated Dual Metabolite + Lipid Sample Preparation for Mammalian Cell Samples. Agilent Application Note 5994-5065EN. 2022.
4. Sartain M et al. Automated Low-Volume Plasma Metabolite Extraction with the Agilent Bravo Platform. Agilent Application Note 5994-2156EN. 2020.
5. Yannell K et al. Mastering HILIC-Z Separation for Polar Analytes. Agilent Application Note 5994-5949EN. 2023.