Underivatized amino acid analysis in wine by HILIC separation and mass detection

Importance of the topic

Amino acids are key components in grapes and wine, serving as nitrogen sources for yeast during fermentation and as precursors of aroma compounds that define the sensory profile of wine. Accurate monitoring of amino acid levels is essential for process control, quality assurance, and authentication of wine products.

Objectives and study overview

The study aimed to develop and validate a sensitive and reproducible method for analyzing 22 underivatized amino acids by hydrophilic interaction liquid chromatography with mass detection and to apply the method to quantify 17 amino acids in wine samples, with a focus on accurate proline measurement using an isotopically labeled internal standard.

Methodology and instrumentation used

The method features a simple sample preparation involving filtration and dilution without derivatization, reducing handling errors and analysis time. Separation was achieved on an amide HILIC column using a gradient of acetonitrile and aqueous ammonium formate buffer. Detection was performed on a single quadrupole MS operating in simultaneous positive and negative ion modes with selected ion monitoring. Key parameters such as buffer salt concentration, flow rate, and ion source settings were optimized to enhance peak shape, sensitivity, and selectivity.

Instrumentation used

• Vanquish Flex UHPLC system with quaternary pump, autosampler, and column compartment
• Accucore-150-Amide-HILIC column (2.1 × 150 mm, 2.6 µm)
• ISQ EM single quadrupole mass spectrometer with ESI source

Main results and discussion

Optimization identified 20 mM ammonium formate at pH 2.8 as the optimal buffer concentration, yielding sharp, symmetric peaks and robust retention. Calibration curves for all amino acids showed excellent linearity (R2 > 0.991) with limits of quantification in the low micromolar range. Repeatability tests demonstrated retention time RSD below 0.12% for most analytes and area RSD under 10%. Application to white wine samples enabled quantification of 17 amino acids, with proline and arginine present at the highest levels. Comparison of external versus internal calibration for proline revealed that the use of a deuterated internal standard reduced matrix effects and improved precision.

Benefits and practical applications of the method

• Avoids laborious and error prone derivatization steps
• Enables analysis of highly polar amino acids with minimal sample handling
• Offers high sensitivity and selectivity through SIM mass detection
• Delivers high reproducibility and robust quantification in complex matrices such as wine

Future trends and potential applications

• Extension to other complex food and fermentation matrices
• Use in wine authentication and geographic or varietal fingerprinting
• Integration with high throughput platforms for quality control
• Advancements in mass spectrometry allowing broader metabolite profiling

Conclusion

The developed HILIC-MS method provides a rapid, derivatization-free approach for the reliable quantification of underivatized amino acids in wine. Its high sensitivity, reproducibility, and compatibility with internal standards make it suitable for routine quality control, research, and authentication tasks in enological and food analysis laboratories.

References

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