Widely targeted metabolomics of hydrophilic compounds in wine using two LC-MS/MS methods: Comparison of different types and producing regions

Significance of the Topic

Widely targeted metabolomics of hydrophilic compounds in wine offers deep insight into the chemical determinants of flavor, quality and fermentation performance. By profiling key metabolites including amino acids, organic acids and short chain fatty acids, producers and researchers can evaluate raw material characteristics, optimize fermentation processes and ensure consistent product quality.

Objectives and Overview

The study aimed to compare metabolite profiles of six red wines from different grape varieties and producing regions using two LC-MS/MS approaches. A newly developed ionization device was evaluated for its ability to enhance sensitivity and robustness. Key goals were to identify characteristic metabolites linked to grape variety and origin, and to assess the performance gains from the novel ionization unit.

Methodology and Instrumentation

Samples of six red wines were processed by centrifugation and filtration. For organic and short chain fatty acids, samples underwent derivatization with 3-nitrophenylhydrazine and carbodiimide reagents. Hydrophilic metabolites were analyzed after dilution. Two LC-MS/MS methods were used:

• Analysis of 23 organic and short chain fatty acids by UHPLC on a C18 column (Mastro C18) with MRM detection in positive and negative electrospray modes.
• Analysis of 97 hydrophilic compounds on an F5 column (Discovery HS F5) using MRM detection.

Both methods were run on Shimadzu systems. An IonFocus Unit was tested to improve ion transmission by focusing electrospray ions and excluding matrix contaminants.

Used Instrumentation

• UHPLC systems: Nexera X3 and Nexera X2
• Mass spectrometers: LCMS-8060 and LCMS-8060NX
• Ionization device: IonFocus Unit (ESI with adjustable focus voltage)

Main Results and Discussion

The IonFocus Unit increased signal intensity for most hydrophilic compounds, with optimal focus voltage at ±2 kV. In organic acid analysis, six short chain fatty acids and 14 organic acids were detected across all samples. Principal component analysis (PCA) of these acids grouped wines by producing region:

• French wines showed higher tartaric and succinic acids, indicating extended malolactic fermentation.
• US wines were richer in malic and pyruvic acids.
• Chile and Australia exhibited similar profiles.

For hydrophilic metabolites, 60 out of 97 compounds were quantified. Amino acids predominated, reflecting varietal and regional differences. PCA separated samples by grape variety:

• Cabernet Sauvignon and Merlot profiles were distinguished by elevated proline and hydroxyproline in Cabernet and higher phenylalanine, leucine and lysine in Merlot.
• Pinot Noir displayed higher alanine, contributing to sweetness and umami.

Benefits and Practical Applications

This approach enables differentiation of wines by variety and origin through metabolite markers. Enhanced sensitivity from the IonFocus Unit supports reliable detection of low-abundance compounds in complex wine matrices. Applications include raw material screening, fermentation monitoring, quality control and product authentication.

Future Trends and Potential Applications

• Integration with other omics platforms for comprehensive food quality assessment.
• Automation and high-throughput screening in winery laboratories.
• Real-time process monitoring using rapid LC-MS workflows.
• Machine learning for predictive modeling of flavor outcomes based on metabolite signatures.

Conclusion

The combination of widely targeted LC-MS/MS and the IonFocus ionization device yields highly sensitive, robust analysis of hydrophilic wine metabolites. The method successfully discriminates wines by grape variety and region, offering a powerful tool for wine evaluation, quality assurance and process control.