Quantitative Analysis of Underivatized Amino Acids in Plant Matrix by Hydrophilic Interaction Chromatography (HILIC) with LC/MS Detection

Significance of the topic

Amino acids serve as fundamental building blocks of proteins and key metabolites in plant physiology. Accurate quantitation of underivatized amino acids in complex matrices such as plant extracts enables insights into nutritional quality, stress responses, and metabolic pathways.

Objectives and overview

This study presents a rapid and sensitive LC/MS method for quantifying 23 underivatized amino acids in cucumber leaf tissue using hydrophilic interaction chromatography (HILIC) coupled with triple quadrupole detection. It evaluates method linearity, sensitivity, reproducibility, and matrix effects.

Methodology and instrumentation

The separation employs an Agilent InfinityLab Poroshell 120 HILIC-Z column with a pH 3 ammonium formate buffer and acetonitrile gradient, achieving complete separation in 15 min. Sample extracts are prepared by acid extraction of frozen cucumber powder followed by dilution with internal standards. Quantitation uses seven-point calibration (50–10000 ng/mL) with isotope-labeled standards.

• LC system: Agilent 1260 Infinity binary pump, autosampler, thermostatted column compartment, ultralow dispersion kit
• Column: Poroshell 120 HILIC-Z, 2.1×100 mm, 2.7 µm
• MS: Agilent 6470 triple quadrupole with Jet Stream ESI in positive mode

Main results and discussion

The method provides baseline resolution of all target analytes including isobaric pairs leucine/isoleucine and threonine/homoserine. Calibration curves display excellent linearity (R2 > 0.995), and limits of detection reach low ng/mL levels. Reproducibility tests (n=15) show relative standard deviations below 3% for response and retention time. Spike recovery in cucumber extract ranges from 77% to 127%, confirming robustness against matrix effects.

Benefits and practical applications

This workflow eliminates derivatization, streamlines analysis time, and ensures high throughput for plant metabolomics, food quality testing, and agricultural chemistry applications.

Future trends and potential applications

Advances may include coupling this HILIC method with high-resolution mass spectrometry for broader metabolite profiling, miniaturized sample preparation, and automation strategies to extend applications to diverse plant species and stress studies.

Conclusion

The described HILIC-LC/MS protocol delivers rapid, sensitive, and reproducible quantitation of underivatized amino acids in plant matrices, demonstrating its suitability for routine analysis in research and quality control.

Used instrumentation

Agilent 1260 Infinity binary pump, Agilent 1260 autosampler, thermostatted column compartment, ultralow dispersion kit, Agilent 6470 triple quadrupole LC/MS with Jet Stream ESI, Poroshell 120 HILIC-Z column.

References

1. A. Kennedy, A. Bivens. Methods for the Analysis of Underivatized Amino Acids by LC/MS. Agilent Technologies Application Note, 5991-8582EN, 2017.