Profiling of Endogenous Metabolites Using Time-of-Flight LC/MS with Ion‑Pair Reverse Phase Chromatography

Significance of the Topic

This study presents an ion-pair reversed-phase LC coupled to time-of-flight MS approach that addresses critical challenges in metabolomics profiling of endogenous compounds, such as the retention of ionic species, reproducible separation of isomers, and comprehensive coverage in a single run.

Objectives and Study Overview

• Develop a robust IP-RP LC/TOF MS method using tributylamine as ion-pairing agent to analyze various metabolite classes.
• Optimize mobile phase composition, pH, and gradient to achieve baseline separation of isomeric metabolites.
• Evaluate method performance with 19 representative standards spanning amino acids, organic acids, sugars, phosphates, nucleotides, energy/redox compounds, and CoA derivatives.

Methodology and Instrumentation

• Chromatography: Agilent ZORBAX RRHD Extend 80Å C18 column (2.1 × 150 mm, 1.8 µm) with SB-C8 guard; mobile phases A (97% water/3% MeOH, 5 mM TBA, pH adjusted with acetic acid) and B (MeOH with 5 mM TBA); nonlinear gradient from 0 to 99% B over 22 min; flow rate 0.25 mL/min; column temperature 40 °C; injection volume 5 µL; autosampler at 4 °C.
• Ionization: negative electrospray using tributylamine-containing mobile phases for stable pH and retention.

Used Instrumentation

• Agilent 1290 Infinity LC system with binary pump, autosampler with thermostat, column compartment and seal wash.
• Agilent 6230 TOF MS with Dual ESI source, capillary voltage 3500 V, dry gas 13 L/min at 250 °C, nebulizer 35 psi, fragmentor 130 V, skimmer 60 V; mass range 60–1600 m/z, acquisition rate 1.5 spectra/s; continuous reference mass infusion for high mass accuracy.

Main Results and Discussion

• Detection and separation of 38 endogenous metabolites—including amino acids, organic acids, sugars, sugar phosphates, nucleosides, nucleotides, energy/redox metabolites, and CoA derivatives—in a 22 min run.
• Baseline resolution of biologically relevant isomer pairs: citrate/isocitrate, D-glucose-6-phosphate/α-D-glucose-1-phosphate, leucine/isoleucine, and fumaric/maleic acid.
• pH optimization showed that pH 5.8 provided the best overall chromatographic selectivity and signal response across metabolite classes; variations in pH affected retention times and intensities of phosphorylated, energy and redox metabolites.
• Retention time reproducibility for 19 representative compounds demonstrated CVs below 0.5%, confirming method robustness for quantitative applications.

Benefits and Practical Applications

• Comprehensive analysis of polar and anionic metabolites in a single method.
• Fast throughput with 22 min run time and excellent chromatographic resolution.
• Reliable separation of critical isomers to enhance identification accuracy in discovery and targeted studies.
• High reproducibility and robustness suitable for QA/QC workflows and large-scale metabolomics investigations.

Future Trends and Applications

• Integration with automated sample preparation and advanced data-processing pipelines to accelerate metabolite discovery.
• Exploration of alternative ion-pair reagents and column chemistries to extend metabolite coverage.
• Coupling with high-resolution MS platforms (e.g., Q-TOF, Orbitrap) for increased mass accuracy and MS/MS capabilities.
• Application in targeted biomarker quantitation for clinical diagnostics, environmental monitoring, and food safety.

Conclusion

This IP-RP LC/TOF MS method on Agilent instrumentation provides a fast, reproducible, and comprehensive solution for endogenous metabolite profiling, overcoming key chromatographic challenges and ensuring robust separation of critical isomers.

References

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3. Zhang T, et al. Resolving power in liquid chromatography–mass spectrometry analysis of metabolites. Analytical Chemistry. 2012;84:1994–2001.
4. Lu W, et al. Metabolite profiling using liquid chromatography–mass spectrometry. Analytical Chemistry. 2010;82:3212–3221.