Analysis of Amino Acid Content in Commercially Available Supplements

Importance of the Topic

Energy supplements often claim precise amino acid contents, making analytical verification essential for product safety and regulatory compliance. Reliable quantitation methods help confirm label claims and detect potential interferences from complex supplement matrices.

Objectives and Study Overview

The study aimed to measure amino acid levels in commercial energy supplements—including powdered drink mixes and tablets—and compare results to labeled values. Pre-column derivatization with HPLC/UHPLC separation and UV/MS detection was employed to achieve accurate quantification.

Methodology and Instrumentation

• Sample Preparation: Tablets were ground and extracted in 0.1 N HCl, shaken, centrifuged, and supernatant collected. Drink mixes were dissolved in 0.1 N HCl, stirred, centrifuged, and diluted (1:10 to 1:100) based on concentration.
• Derivatization: Amino acids were tagged with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) following a standardized protocol.
• Used Instrumentation:
  • LC Systems: Arc HPLC and ACQUITY Arc UHPLC with column heating.
  • Detectors: 2489 UV detector (260 nm) and QDa mass detector for peak confirmation.
  • Column: AccQ•Tag Ultra C18 (2.5 µm, 4.6 × 150 mm) at 43 °C (hydrolysate) or 50 °C (food/feed).
  • Mobile Phases: AccQ•Tag Ultra eluents A/B and water, with a multi-step gradient program for optimal separation.

Results and Discussion

• Label Compliance: Total amino acid content for all samples matched the claimed 5000 mg per serving (or per five tablets) within acceptable tolerances.
• Separation Challenges: Initial co-elution of citrulline and glutamic acid in some drink mixes was resolved by adjusting column temperature and using QDa for selective detection.
• Quantitative Findings: Detailed individual amino acid profiles for one sample correlated closely with label values, confirming method reliability.

Benefits and Practical Applications

• Pre-column derivatization minimizes matrix effects, improving quantitation accuracy.
• Temperature-controlled separations combined with MS confirmation ensure clear resolution of critical analytes.
• The workflow is adaptable for routine quality control of dietary supplements and food products.

Future Trends and Potential Applications

• Method expansion to a broader range of amino acids and complex formulations.
• Incorporation of high-resolution MS could enhance sensitivity for trace analysis.
• Automation of sample prep may increase throughput in industrial QA/QC settings.

Conclusion

The AQC derivatization HPLC/UHPLC method effectively quantifies amino acid content in diverse energy supplements, ensuring label accuracy and regulatory compliance. Chromatographic adjustments and MS-based confirmation establish a robust protocol for quality assurance.

References

1. Smriga M. International Regulations on Amino Acid Use in Foods and Supplements and Recommendations to Control Their Safety Based on Purity and Quality. J Nutr. 2020;150:2602S–2605S.
2. Roberts A. The Safety and Regulatory Process for Amino Acids in Europe and the United States. J Nutr. 2016;146(Suppl):2635S–2642S.
3. Waters. Amino Acid Standard Kits Care and Use Manual. 72000663EN.
4. Martin K, Berthelette KD, Hong P. Instrument Considerations for Reliable Amino Acid Analysis Using AccQ•Tag Ultra C18 2.5 µm Column. Application Note 720007678EN; 2022.