Identification and Semiquantitation of Monoterpene Glycosides in Ripening Muscat of Alexandria Grapes

Significance of the Topic

Grape monoterpene glycosides are nonvolatile precursors of key aroma compounds that influence wine flavor and quality. During grape ripening and wine fermentation, these glycosides slowly hydrolyze to release floral and fruity monoterpenols, which are critical for varietal character, especially in Muscat, Riesling, and Gewürztraminer cultivars. Developing analytical methods to directly identify and semiquantitate these bound aroma compounds without chemical standards or derivatization addresses a significant challenge in wine chemistry and quality control.

Objectives and Study Overview

This study aimed to establish a workflow using ultrahigh-performance liquid chromatography coupled with accurate-mass quadrupole time-of-flight mass spectrometry (UHPLC-ESI-Q-TOF MS) and tandem MS for the untargeted identification and semiquantitation of monoterpene glycosides in Muscat of Alexandria grapes at different ripening stages. Key steps included building a custom compound database (PCDL), performing automated MS/MS for structural elucidation, and implementing a quantitative analysis method for relative profiling across fruit development.

Methodology and Instrumentation

Sample Preparation and Extraction

• Solid-phase extraction of grape skin and mesocarp to enrich monoterpene glycosides.
• Internal standard decyl-β-D-glucopyranoside spiked prior to extraction to normalize recovery and instrument variability.

Chromatography and Mass Spectrometry

• UHPLC system: Agilent 1290 Infinity binary pump, isocratic pump, autosampler, and column compartment; column: Poroshell 120 Phenyl-Hexyl (2.1×150 mm, 2.7 µm).
• Mobile phase: 0.1% acetic acid in water (A) and in acetonitrile (B) with a 0–25 min gradient from 5% to 90% B.
• Flow rate: 0.42 mL/min, column temperature: 40 °C, injection volume: 10 µL.
• Mass spectrometer: Agilent 6530 Accurate-Mass Q-TOF with dual Jet Stream ESI in negative mode; MS1 (m/z 100–1,700) and data-dependent MS/MS (m/z 50–1,000) at 3–4 spectra/s.
• Continuous internal calibration using purine (m/z 119.0362) and HP-0921 (m/z 980.0164).

Data Analysis Workflow

• Creation of a Personal Compound Database and Library (PCDL) containing predicted exact masses of monoterpenol glycosides (mono-, di-, trisaccharides, and malonylated derivatives).
• Untargeted find-by-formula search in MassHunter Qualitative Analysis to detect potential compounds within ±10 ppm mass error.
• Automated MS/MS acquisition and structural correlation using Agilent Molecular Structure Correlator (MSC) to confirm fragment patterns and assign putative structures.
• Updating PCDL entries with observed retention times and MS/MS spectra.
• Semiquantitation with MassHunter Quantitative Analysis: auto-integration of extracted ion chromatograms, retention time and S/N monitoring, and relative abundance calculation against the internal standard.
• Statistical evaluation using one-way ANOVA and pairwise t-tests (α=0.05) in R to assess changes across ripening stages.

Main Results and Discussion

The workflow identified and semiquantitated 18 monoterpene glycosides, including monoterpenol glucosides, hexose-pentose conjugates, dihexose-pentose compounds, hexose-deoxyhexose derivatives, and malonylated glucosides. Representative extracted ion chromatograms revealed distinct peaks corresponding to each glycoside. Mass errors for all identifications remained within ±1 ppm.

Semiquantitative profiling showed a pronounced increase in 15 of the 18 glycosides after veraison, indicating a surge in glycosylation and storage of aroma precursors during onset of ripening. Mature berries at commercial harvest displayed the highest diversity and abundance of these compounds, underscoring their accumulation in late development.

Practical Benefits and Applications

• Direct measurement of nonvolatile glycosides without hydrolysis or derivatization streamlines workflow and preserves native structures.
• Accurate-mass MS and automated MS/MS enable identification in the absence of commercial standards.
• Semiquantitative data guide viticultural and enological decisions by monitoring aroma precursor dynamics.
• Method adaptable for quality control in grape breeding, harvest timing, and wine authenticity assessments.

Future Trends and Opportunities

Advances in high-resolution MS and chromatography will expand coverage of grape secondary metabolites, enabling deeper metabolomic profiling. Integration of ion mobility, enhanced data-independent acquisition, and machine learning-driven spectral libraries will improve structural annotation and reduce false positives. Combining glycoside profiling with sensory studies will clarify precursor-aroma relationships and support targeted viticultural interventions.

Conclusion

An UHPLC-ESI-Q-TOF workflow with custom spectral libraries and automated MS/MS provides an efficient and robust approach for the identification and semiquantitation of monoterpene glycosides in grapes. The method delivers sensitive, reproducible data on aroma precursor dynamics without requiring reference standards or hydrolytic steps, offering valuable insights for grape and wine science.

References

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