Native amino acid analysis in wine by HILIC separation and detection with single quadrupole mass spectrometry

Significance of the Topic

Amino acid profiling in wine is critical for understanding yeast nutrition during fermentation, controlling flavor development and ensuring quality assurance. Quantifying native, underivatized amino acids simplifies sample handling and reduces analytical errors compared to derivatization approaches.

Objectives and Study Overview

This study aimed to develop a rapid, reproducible HILIC-based LC method coupled with single quadrupole mass detection for 22 underivatized amino acids. The method was then applied to quantify 17 amino acids in white wine and evaluate proline as a diagnostic marker using internal standard calibration.

Methodology

A hydrophilic interaction liquid chromatography (HILIC) separation was performed on an Accucore™ 150-Amide-HILIC column (2.1×150 mm, 2.6 µm) with a gradient of acetonitrile/ammonium formate (200 mM, pH 2.8) and water/ammonium formate at 0.4 mL/min. Sample preparation involved simple dilution of standards and wine in 0.1 N HCl. Detection used selected ion monitoring (SIM) in both positive and negative ESI modes. Linearity and repeatability were evaluated over mM to sub-mM ranges.

Used Instrumentation

• Thermo Scientific™ Vanquish™ Flex UHPLC system
• Thermo Scientific™ Vanquish™ ISQ Single Quadrupole Mass Detector
• Accucore™ 150-Amide-HILIC column
• Thermo Scientific™ Chromeleon™ software v7.2.8 for data processing

Main Results and Discussion

All amino acids except five co-eluting pairs were baseline-separated within 22 minutes, including Leu/Ile isomers (Rs > 5). Calibration curves exhibited excellent linearity (R2 > 0.991) across concentration ranges from 0.1 to 500 mM. Application to white wine enabled detection of 17 amino acids; proline quantification via internal standard (Pro-2,2,5-d3) improved precision compared to external calibration and reduced matrix effects.

Benefits and Practical Applications

• Avoids time-consuming and error-prone derivatization steps
• Delivers robust, reproducible quantification of native amino acids
• Applicable to wine QA/QC, fermentation monitoring and food analysis

Future Trends and Potential Applications

Expansion of this approach to other fermented beverages and food matrices could streamline high-throughput amino acid profiling. Integration with high-resolution or tandem MS may further enhance specificity. Automation of sample handling and data processing will support large-scale quality control in industry.

Conclusion

A straightforward HILIC-SQ mass spectrometry method was established for sensitive analysis of 22 native amino acids. The technique offers high throughput, excellent reproducibility and minimal sample preparation, making it suitable for routine wine analysis and broader applications in food and beverage testing.

References

1. P. Lehtonen, Determination of amines and amino acids in wine – A review, American Journal of Enology and Viticulture, 47 (1996) 127–133.
2. E. M. Sanders and C. S. Ough, Determination of free amino acids in wine by HPLC, American Journal of Enology and Viticulture, 36 (1985) 43–46.
3. V. Gokmen, A. Serpen, B. A. Mogol, Rapid determination of amino acids in foods by hydrophilic interaction liquid chromatography coupled to high-resolution mass spectrometry, Analytical and Bioanalytical Chemistry, 403 (2012) 2915–2922.