Improved Metabolomic Analysis Using an Iron-Free Flow Path

Importance of the Topic

Metabolomics provides a comprehensive view of cellular physiology by quantifying small molecules in biological systems. High-performance liquid chromatography coupled with mass spectrometry is the leading technology for metabolomics due to its sensitivity and broad coverage. However, conventional stainless steel flow paths can cause adsorption and poor peak shapes for phosphorylated metabolites, which limits data quality and robustness.

Objectives and Study Overview

This application note compares the Agilent 1290 Infinity II Bio LC system, featuring an iron-free biocompatible flow path, against the standard Agilent 1290 Infinity II LC with stainless steel components. The main goals were to evaluate adsorption effects, chromatographic peak shape, resolution of isobaric compounds, retention time reproducibility, and robustness in complex yeast metabolite extracts.

Methodology and Instrumentation

Saccharomyces cerevisiae cultures were grown under glucose limitation and subjected to a transient starvation stimulus. Samples were rapidly quenched in cold methanol, extracted using a methanol-water-chloroform protocol, and analyzed by hydrophilic interaction chromatography on a PEEK-lined Poroshell HILIC-Z column with an ammonium acetate buffer at pH 9. Detection was performed on a high-resolution Q-TOF mass spectrometer in negative mode. Ten replicate injections of standards and complex extracts were evaluated for adsorption, tailing factors, resolution, and retention time stability.

Instrumentation Used

• Agilent 1290 Infinity II Bio LC System: Bio Flexible Pump, Bio Multisampler with Sample Thermostat, Multicolumn Thermostat with Bio Heat Exchanger and Thermal Equilibration Devices
• Agilent 1290 Infinity II LC System: Flexible Pump, Multisampler with Sample Thermostat, Multicolumn Thermostat with Quick Connect Heat Exchanger and Thermal Equilibration Devices
• Agilent 6546 LC/Q-TOF mass spectrometer operated in negative low mass range

Main Results and Discussion

The iron-free Bio LC delivered dramatically lower adsorption for ATP and GTP (<3% area loss between injections) compared to >30% loss on the stainless steel system. Tailing factors for phosphorylated metabolites improved by 0.1–2.7 units on the Bio LC. Critical isobaric pairs such as G6P/F6P were baseline separated only with the Bio LC. Complex yeast extracts showed outstanding retention time precision (RSD ~0.1%) across ten injections, and dynamic profiling of adenylate energy charge during starvation illustrated consistent quantification of AMP, ADP, and ATP.

Benefits and Practical Applications

• Minimized adsorption and superior peak shapes for labile phosphorylated compounds
• Enhanced resolution of isobaric metabolites critical to pathway analysis
• Exceptional retention time reproducibility for high-throughput studies
• Reduced need for column passivation or mobile phase additives
• Improved robustness and ease of use in routine metabolomics workflows

Future Trends and Potential Applications

Advances in iron-free flow path technology are expected to extend to a wider range of labile analytes and complex matrices. Integration with automated sample preparation and ultra-high-throughput workflows will further increase throughput in large-scale studies. Coupling with emerging ion mobility and advanced data analytics promises deeper metabolic coverage and real-time monitoring of cellular processes.

Conclusion

The Agilent 1290 Infinity II Bio LC system outperforms its stainless steel counterpart in metabolomic analysis of phosphorylated metabolites, delivering reduced adsorption, improved peak quality, and excellent reproducibility. Its biocompatible flow path offers a robust solution for routine and high-demand metabolomics applications without the need for special treatments or additives.

References

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