Routine Quantitation of 17 Underivatized Amino Acids by LCMS

Importance of the Topic

Underivatized amino acid analysis by liquid chromatography–mass spectrometry (LC–MS) offers a rapid and cost-effective alternative to traditional derivatization workflows. In nutritional science, quality control, and industrial label verification, accurate quantitation of free amino acids without chemical modification simplifies sample preparation, reduces reagent consumption, and minimizes matrix-related variability.

Objectives and Study Overview

This work presents a validated method for routine quantitation of 17 underivatized amino acids in aqueous solutions and dietary supplement matrices. Key goals included achieving robust sensitivity, high throughput (single injection), and minimal matrix effects, using commercially available standards and supplements.

Methodology and Instrumentation

All analyses were performed on an Agilent 1260 Prime Infinity II LC coupled to an Agilent InfinityLab LC/MSD iQ mass spectrometer. Chromatographic separation employed a HILIC-Z column (3.0 × 150 mm, 2.7 µm) at 25 °C, with a quaternary gradient of 0.1% formic acid in water (A), acetonitrile (B), and 10 mM ammonium formate (C). The flow rate was 0.6 mL/min over a 14 min run time plus 8 min re-equilibration. Electrospray ionization in positive mode was used, with a capillary voltage of 3500 V, gas temperature of 325 °C, nebulizer at 50 psi, and nitrogen sheath gas at 11 L/min. Sample injection volume was 1 µL.

Main Results and Discussion

Linearity and Range

• Seventeen amino acids showed linear responses from 10 to 1000 pmol/µL (equivalent to 0.75–174 ppm) with correlation coefficients (R²) above 0.99.
• Retention times ranged from 4.37 min (histidine) to 8.18 min (cysteine), with retention time RSD below 0.4% for all analytes.

Precision and Accuracy

• Instrumental precision (concentration RSD) was below 2% for most analytes, with accuracy within 98–103% of nominal values.
• Baseline resolution of isomeric leucine and isoleucine was achieved without derivatization.

Matrix Analysis

• Supplement #1 (leucine, isoleucine, valine mix) and Supplement #2 (nine-amino-acid blend) exhibited recoveries of 100–112%, indicating negligible matrix effects.
• Spike-recovery experiments confirmed quantitative performance for complex supplement formulations.

Benefits and Practical Applications

This method streamlines quantitative analysis in food, feed, and supplement quality control by eliminating derivatization steps. Single-injection quantitation of multiple underivatized amino acids reduces analysis time, reagent costs, and potential sample losses, supporting high-throughput industrial and research laboratories.

Future Trends and Opportunities

Advances in high-resolution mass spectrometry and novel stationary phases may further enhance sensitivity and selectivity for challenging amino acids and complex matrices. Integration with automated sample handling and data processing pipelines will expand the applicability of underivatized LC–MS workflows in metabolomics, clinical diagnostics, and real-time process monitoring.

Conclusion

The presented LC–MS method using an Agilent 1260 Prime Infinity II LC and iQ mass spectrometer delivers reliable, sensitive, and accurate quantitation of 17 underivatized amino acids in aqueous and supplement matrices. Its robustness, linearity, and minimal matrix interference make it a valuable tool for routine analytical chemistry applications.

References

1. Hui Zhao, Doug Postl, Mike Van Amburgh, et al. Quantitation of Amino Acids in Soy Flour, Dried Cow’s Milk Powder, and Corn Silage by Triple Quadrupole LC/MS/MS. Agilent Technologies, Inc.; Cornell University, Dept. of Animal Science. 5994-3044EN.
2. Shimadzu Corporation. Analytical Methods for Amino Acids. Shimadzu Application Notes.