Routine Quantitation of 17 Underivatized Amino Acids by LC/MS

Significance of the Topic

The nutraceutical market is rapidly expanding, driven by growing consumer interest in dietary supplements and health‐conscious lifestyles. Amino acids, as fundamental building blocks of proteins and key components in muscle synthesis, glycemic regulation, and cognitive function, require accurate quantitation to ensure label integrity and regulatory compliance. The development of a reliable, high‐throughput method for underivatized amino acids addresses industry needs for speed, cost‐effectiveness, and robustness in quality control.

Study Objectives and Overview

This application note describes a method for simultaneous quantitation of 17 underivatized amino acids using liquid chromatography coupled to single‐quadrupole mass spectrometry (LC/MS). The aims were to:

• Establish a direct analysis workflow without chemical derivatization.
• Achieve baseline separation of isobaric amino acids (L‐leucine and L‐isoleucine).
• Demonstrate linearity, precision, and accuracy over a range relevant to nutraceutical formulations.
• Validate performance in both aqueous standards and real supplement matrices.

Methodology and Instrumentation

An Agilent 1290 Infinity II LC system equipped with a HILIC-Z column (3.0 × 150 mm, 2.7 µm) was coupled to an Agilent InfinityLab LC/MSD iQ single-quadrupole mass spectrometer. Key parameters included:

• Mobile phase A: 0.1% formic acid in water; B: acetonitrile; D: 10 mM ammonium formate in water.
• Gradient elution over 14 minutes at 0.6 mL/min, with column temperature maintained at 25 °C.
• Single‐ion monitoring (SIM) for each amino acid using optimized fragmentor voltages (65–120 V) and unit resolution.
• Calibration range: 10 to 1,000 pmol/µL using a load‐on‐column approach to reduce volumetric errors.

Key Results and Discussion

In solvent, all 17 amino acids exhibited excellent linearity (r² > 0.99) over the targeted range. Retention times were reproducible (RSD < 0.4%) and concentration precision was better than 2% RSD for most analytes. Accuracy across the calibration range averaged between 100% and 102%. The method successfully resolved L‐leucine and L‐isoleucine peaks. Application to two supplement formulations (one containing branched‐chain amino acids only, and another with a mixed amino acid blend) confirmed negligible matrix effects, recoveries of 100–107%, and precision under 3% RSD.

Benefits and Practical Applications

This workflow offers several advantages for routine quality control:

• Rapid analysis: single injection for 17 analytes without derivatization.
• Cost savings: elimination of reagent-intensive derivatization steps.
• Versatility: applicable to aqueous standards and complex supplement matrices.
• Regulatory compliance: high accuracy and reproducibility suitable for label verification.

Future Trends and Opportunities

The increasing demand for personalized nutrition and complex supplement formulations will drive further enhancements in amino acid analysis. Potential developments include high‐resolution mass spectrometry for broader analyte coverage, automated sample preparation for ultra-high throughput, and integration with data analytics platforms to support trend monitoring in product quality and consumer safety.

Conclusion

The described Agilent LC/MS method provides a sensitive, accurate, and robust solution for the routine quantitation of 17 underivatized amino acids in nutraceutical products. Its high throughput and minimal sample preparation make it a valuable tool for industrial and regulatory laboratories seeking efficient label verification and quality assurance.

References

1. Future Market Insights Inc. Food Amino Acids Market to be Worth US$ 19.6 Billion by 2033. GlobeNewswire; March 21, 2023.
2. AminoAcidsguide.com. Overview of Amino Acid Types and Functions; January 24, 2023.
3. Cleveland Clinic. Amino Acids: Functions and Health Benefits; December 22, 2021.
4. Kapalka GM. Nutritional and Herbal Therapies for Children and Adolescents. Academic Press; 2010. ISBN 9780080958019.
5. Shimadzu Corporation. Analytical Methods for Amino Acids. Shimadzu Technical Report.