LC/MS Analysis of Free Amino Acids on Agilent InfinityLab Poroshell 120 HILIC 1.9 μm Columns

Importance of the Topic

Free amino acids serve as key biomarkers in fields ranging from metabolic research to food quality control. Rapid and reliable quantitation of these small molecules is essential for diagnostics, nutritional analysis, and pharmaceutical development.

Objectives and Study Overview

This application examines the performance of an Agilent InfinityLab Poroshell 120 HILIC 1.9 µm column combined with LC/MS to separate and quantify twelve free amino acids. Special emphasis is placed on achieving baseline chromatographic resolution between isobaric leucine and isoleucine within an 11-minute run time.

Methodology and Instrumentation

The study employed the following setup:

• Liquid chromatography: Agilent 1290 Infinity binary pump, high-performance autosampler, thermostatted column compartment; Poroshell 120 HILIC (2.1×150 mm, 1.9 µm).
• Mobile phases: A = 10 mM ammonium formate (pH 3) in water; B = 10 mM ammonium formate (pH 3) in acetonitrile:water (9:1).
• Gradient: 100–95 % B over 5 min, then 95–60 % B over 6 min; flow rate 0.4 mL/min; column at 15 °C; autosampler at 4 °C.
• Mass spectrometry: Agilent 6460 triple quadrupole with Jet Stream; positive ion mode; MS2 SIM acquisition with compound-specific fragmentor voltages and dwell times.
• Sample: Mixed amino acid standard (12 components) diluted 1:10 in acetonitrile; injection volume 5 µL.

Main Results and Discussion

The HILIC column delivered high efficiency under 550 bar, separating all twelve amino acids in 11 minutes. LC/MS detection differentiated analytes by mass fragments, with chromatographic resolution of leucine and isoleucine measured at Rs = 1.9. Key findings include:

• Clear separation of isobaric analytes, enabling accurate quantitation.
• Fast analysis compatible with high-throughput workflows.
• Stable retention and reproducible peak shapes across runs.

Benefits and Practical Applications

The demonstrated method offers:

• Speed: Sub-15-minute cycle times enhance laboratory throughput.
• Resolution: Baseline separation of critical isobaric pairs ensures data reliability.
• Sensitivity: MS detection permits low-level quantitation suitable for trace analysis.
• Versatility: Applicable to food testing, pharmaceutical QA/QC, metabolomics, and environmental studies.

Future Trends and Potential Applications

Advancements may include:

• Even smaller particle HILIC phases for sub-minute separations.
• Integration with high-resolution mass spectrometry for untargeted profiling.
• Automated sample preparation and data processing in regulated environments.
• Expansion to other polar metabolite classes and large-scale screening.

Conclusion

The Agilent InfinityLab Poroshell 120 HILIC 1.9 µm column, paired with a streamlined LC/MS setup, provides a robust, high-efficiency platform for rapid free amino acid analysis. Its ability to resolve challenging isobaric pairs in under 11 minutes highlights its value in diverse analytical contexts.

References

• A. Gratzfeld-Hugsen, E. Naegele. Maximizing efficiency using Agilent Poroshell 120 Columns. Agilent Technologies Application Note, 5990-5602EN, 2016.
• V. R. Meyer. Practical High Performance Liquid Chromatography, 4th Edition, p. 34. Wiley, 2004.