Evaluation of a fully automated method for the measurement of glycerol in wine

Significance of the topic

Glycerol is a major non-volatile fermentation by-product in wine, influencing mouthfeel, viscosity and perceived sweetness. Typical concentrations are around 5 g/L but can reach 15–20 g/L depending on fermentation conditions and sulfite management. Reliable, high-throughput and automatable glycerol determination is useful for quality control, process monitoring during winemaking and proficiency testing in regulatory laboratories.

Objectives and study overview

This application note evaluated a fully automated enzymatic photometric assay kit for glycerol implemented on the Thermo Scientific Arena 20XT discrete analyzer. The primary aims were to assess analytical performance (accuracy, precision, linear range) and to compare results with two established methods: the FOSS WineScan FT120 (infrared-based) and an accredited enzymatic reference method used by ALKO, Inc. The study included 53 commercial wine samples (white, rosé, red) and five proficiency test samples analyzed by the accredited reference laboratory.

Methodology

Principle of the assay:

• Enzymatic conversion sequence using glycerokinase (GK), ADP-dependent hexokinase (ADP-HK) and glucose-6-phosphate dehydrogenase (G6P-DH).
• Formation of NADH (measured at 340 nm) provides the photometric signal proportional to glycerol concentration.

Assay workflow on the Arena 20XT:

• Sample volume: 5 µL (no pretreatment required).
• Initial incubation: sample mixed with 100 µL buffer containing D-glucose and the enzyme mix (GK, ADP-HK, G6P-DH) at 37 °C for 120 s, followed by a blank reading.
• Reaction initiation: addition of 25 µL reagent containing NAD+ and ATP, incubation 420 s, absorbance read at 340 nm.
• Calibration: two-point linear calibration with two replicates; stated measuring range 0.07–30.00 g/L.

Samples and controls:

• 53 wine samples: 17 white, 3 rosé, 33 red.
• Two-level water-based glycerol controls prepared from glycerol standard.

Used instrumentation

• Thermo Scientific Arena 20XT discrete analyzer (primary platform evaluated).
• Thermo Scientific Gallery and Gallery Plus discrete analyzers (method adapted for these platforms as noted).
• FOSS WineScan FT120 (comparative analyzer).

Main results and discussion

Accuracy and comparability:

• Correlation between Arena 20XT and WineScan FT120: Pearson r = 0.964, indicating strong agreement across the sample set.
• Comparison with the accredited enzymatic method (ALKO, Inc.) on five proficiency test samples showed an exact correlation coefficient of 1.000 for those samples, and Arena results fell within the stated proficiency tolerances. Example proficiency results (Arena): white wine 5.22 g/L (tolerance 5.4 ± 0.8), red wine 6.82 g/L (7.1 ± 1.1), sparkling wine 5.61 g/L (6.0 ± 0.9), aromatized wine 8.33 g/L (8.7 ± 1.3).

Precision and repeatability:

• Within-run standard deviations measured on a water-based standard and wine samples (range 4.25–10.39 g/L) were between 0.036 and 0.099 g/L, demonstrating good repeatability appropriate for routine QC.

Calibration and dynamic range:

• Two-point linear calibration produced reliable quantitation over the declared range (0.07–30.00 g/L), covering both typical and elevated glycerol concentrations encountered in winemaking.

Practical considerations:

• The assay requires minimal sample volume and no sample pretreatment, simplifying integration into routine workflows.
• Endpoint photometry at 340 nm is a robust, well-established detection mode for dehydrogenase-coupled enzymatic assays.

Benefits and practical applications

The automated discrete Arena workflow provides several advantages for wine laboratories:

• High throughput and walk-away automation enable multiple assays on the same sample batch, increasing laboratory productivity.
• Low sample volume and minimal preparation reduce consumables and handling errors.
• Analytical performance (accuracy and repeatability) is comparable to established infrared and enzymatic reference methods, making it suitable for QC, production monitoring and participation in inter-laboratory comparisons.

Future trends and potential applications

• Integration of glycerol assays into multi-analyte panels on discrete analyzers can further streamline routine wine analysis (e.g., combined monitoring of sugars, organic acids and fermentation by-products).
• Miniaturized and multiplexed enzymatic assays with improved reagent stability could reduce costs and expand onsite testing in wineries.
• Coupling discrete enzymatic assays with digital laboratory information systems will facilitate trend analysis, process control and regulatory reporting.

Conclusion

The Thermo Scientific glycerol enzymatic test run on the Arena 20XT discrete analyzer demonstrated accurate, precise and linear performance across a broad concentration range relevant to enology. Results correlated strongly with the FOSS WineScan FT120 and matched accredited enzymatic reference determinations for proficiency samples. The method's minimal sample handling, low volume requirement and automation capability make it well suited for routine wine quality control and laboratory proficiency testing.

References

1. Ribéreau-Gayon P, Glories Y, Maujean A, Dubourdieu D. Handbook of Enology, Volume 2: The Chemistry of Wine Stabilization and Treatments. John Wiley & Sons; 2000.
2. O’Kennedy K. Glycerol: The Myth. newworldwinemaker.com; 2010.
3. Patterson T. Many Roads to Mouthfeel. Wines & Vines; [date not specified].
4. Organisation Internationale de la Vigne et du Vin (OIV). Compendium of International Methods of Wine and Must Analysis. 2009.