Methods for the Analysis of Underivatized Amino Acids by LC/MS

Importance of Topic
Underivatized amino acids analysis plays a key role in food quality control, metabolomics profiling, and life science research. Their polar characteristics challenge conventional reversed-phase separation, making HILIC-MS approaches essential for accurate quantitation and resolution of isobaric compounds such as leucine and isoleucine.

Objectives and Study Overview
This work aimed to develop and optimize a streamlined HILIC-MS method for direct analysis of underivatized amino acids in complex matrices. Two stationary phases, Poroshell 120 HILIC-Z and HILIC-OH5, were evaluated for chromatographic performance and sensitivity.

Methodology and Instrumentation
Sample Preparation
Standards of 16 amino acids were dissolved in water at 0.25 mM and injected without further pretreatment.

Chromatographic Conditions

• Columns: Agilent Poroshell 120 HILIC-Z and HILIC-OH5 (2.1 × 100 mm, 2.7 µm)
• Mobile Phases: 20 mM ammonium formate, pH 3 (10% stock) in water (A) or acetonitrile (B)
• Gradient: 100% B to 70% B at 10 min, return to 100% B at 11 min; total run time 16 min
• Flow Rate: 0.8 mL/min; Column Temp: 30 °C; Injection: 0.25 µL

Mass Spectrometry

• Instrument: Agilent 6470 triple quadrupole with Jet Stream ESI source
• Mode: Positive ionization; Sheath gas 11 L/min (400 °C); Dry gas 7 L/min (300 °C); Nebulizer 45 psi; Capillary voltage 3500 V
• dMRM Transitions: Optimized for each amino acid, fragmentor (25–75 V) and collision energy (5–28 V)

Main Results and Discussion
Both HILIC phases achieved baseline separation of all amino acids, including the challenging leucine/isoleucine pair (selectivity 1.08). HILIC-Z delivered superior peak shape for basic residues (e.g., aspartic acid, histidine), while HILIC-OH5 improved resolution of early eluting analytes (tyrosine, valine, proline). Retention times ranged from 2.1 to 6.5 min, demonstrating a total cycle of 16 min.

Benefits and Practical Applications

• No derivatization step simplifies sample workflow and reduces potential analyte losses.
• Fast analysis time and high chromatographic resolution enhance throughput in food testing and metabolomic studies.
• Wide applicability to life science, QA/QC, and clinical research due to compatible HILIC-MS workflow.

Future Trends and Potential Applications

• Integration with high-resolution MS for simultaneous untargeted metabolomics and targeted amino acid profiling.
• Miniaturization through microflow or nanoflow HILIC to reduce sample consumption and enhance sensitivity.
• Application in real-time process monitoring in bioprocessing, fermentation, and food quality control.

Conclusion
The optimized HILIC-MS method provides a rapid, robust, and sensitive solution for underivatized amino acid analysis, overcoming the limitations of reversed-phase techniques and enabling precise separation of isobaric compounds.

Reference
No external references were provided in the source document.