Analysis of underivatized amino acids in wines by solid phase extraction and HILIC-MS

Significance of the topic

Analyzing underivatized amino acids in wine provides critical insights into nitrogen nutrition, fermentation progress and overall wine quality. Amino acid profiles reflect grape variety, geographic origin and vintage, and can aid winemakers and quality control laboratories in characterizing and differentiating wine samples.

Objectives and study overview

This application demonstrates a straightforward solid phase extraction (SPE) protocol coupled with hydrophilic interaction liquid chromatography–mass spectrometry (HILIC-MS) for reliable cleanup and quantification of 17 underivatized amino acids in white and red wines. The aim was to achieve high sensitivity, reproducibility and minimal matrix interference without derivatization.

Methodology and instrumentation

The workflow comprises:

• Sample preparation by SPE on HyperSep C18 cartridges to remove pigments, lipids and proteins while eluting polar amino acids under acidic conditions.
• Chromatographic separation on a Thermo Scientific Accucore Amide HILIC column (2.1×150 mm, 2.6 µm) using a gradient of acetonitrile/ammonium formate (pH 2.8) and water/ammonium formate.
• Mass detection on an ISQ EM single quadrupole spectrometer in electrospray ionization mode, operating in SIM (single ion monitoring) for enhanced sensitivity and selectivity.
• Calibration over 1–500 µM for 17 amino acids with R2 > 0.997 and repeatability assessed by retention time RSD <0.14 % and peak area RSD <9.3 %.

Instrumentation used

• Thermo Scientific Vanquish Flex Quaternary UHPLC system with split sampler and column compartment
• Thermo Scientific ISQ EM Single Quadrupole Mass Spectrometer
• HyperSep C18 SPE cartridges (1000 mg/6 mL) and 24-port SPE vacuum manifold
• Supporting labware: centrifuge, vortex mixer, pH meter, autosampler vials and pipettes

Main results and discussion

Method performance:

• Linearity: R2 > 0.997 for all amino acids, with twelve compounds ≥0.999.
• Repeatability: retention time RSD < 0.14 % and area RSD < 3 % for most analytes; higher RSD for Asp (9.2 %) and Glu (7.9 %) due to lower signal-to-noise.
• SPE precision: peak area RSD <6 % for 17 amino acids in test wines.
• Recovery in wine matrices: spiked-sample recoveries between 71 % and 113 % for 13 amino acids; Arg, His and Lys exhibited matrix effects and may require isotopic internal standards.

Application to six Italian wines:

• Quantified 16 amino acids (excluding proline due to concentration >500 µM calibration limit). Proline was the most abundant across all samples.
• White wines exhibited higher levels of leucine and tyrosine compared to red wines.
• Certain amino acids (e.g., His in one red wine; Ser and Val in another) fell below detection limits.

Benefits and practical applications of the method

• Avoids derivatization, reducing sample handling and potential artifacts.
• SPE cleanup enhances robustness by minimizing ion suppression from complex wine matrices.
• SIM mode on a single quadrupole delivers sensitivity comparable to more complex instruments.
• Fast analysis (<22 min) with reliable separation of isomeric pairs (Leu/Ile).
• Suitable for routine QC, varietal authentication and fermentation monitoring in enological laboratories.

Future trends and possibilities

• Integration of isotopically labeled internal standards to correct for matrix effects and improve accuracy for challenging analytes.
• Expansion to broader polar metabolite profiling by switching to tandem MS or high-resolution instruments.
• Automation of SPE and HILIC-MS workflows for high-throughput screening in industrial settings.
• Data-driven approaches using amino acid fingerprints for classification of origin, vintage and varietal blends.

Conclusion

The presented SPE-HILIC-MS protocol offers a rapid, reproducible and sensitive strategy for quantifying underivatized amino acids in wines without extensive sample derivatization. Its robust cleanup, excellent linearity and precision make it well suited for routine enological analysis and research applications.

References

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5. Thermo Scientific Application Note 73151. Underivatized amino acid analysis in wine by HILIC‐MS.
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