Determination of sugars in honey - comparison of refractive index and light scattering detection

Significance of the Topic

Honey is a widely consumed natural food product whose sugar composition reflects its botanical origin and quality parameters.
Ensuring accurate determination of key carbohydrates in honey supports authenticity assessment, labeling compliance and consumer safety.
Quality control of honey sugar profile using advanced chromatographic methods enhances industry standards and regulatory adherence.

Objectives and Study Overview

This study aimed to evaluate the performance of refractive index (RID) and evaporative light scattering detection (ELSD) for quantifying sugars in honey.
The research compared detection sensitivity, calibration range and quantitative agreement between AZURA RID 2.1L and SEDEX LT100 ELSD systems.
Three samples were analyzed: a proprietary KNAUER honey, a commercial blossom honey and agave nectar as a substitute matrix.

Methodology

The separation was performed using an isocratic HILIC protocol on a Eurokat Pb polymer column at 75°C and 0.8 mL/min with water as mobile phase.
Sample preparation involved dissolving honey in water, followed by dilution factors of 1:100 for ELSD and 1:10 for RID prior to injection.
Calibration curves covered 0.03–1.50 mg/mL for ELSD and 0.30–3.00 mg/mL for RID using mixed standards of fructose, glucose, sucrose and maltose.

Instrumentation Used

• AZURA P 6.1L isocratic pump
• AZURA AS 6.1L autosampler
• AZURA CT 2.1 column thermostat
• AZURA RID 2.1L refractive index detector (20 Hz, extended dynamic range)
• SEDEX LT100 ELSD (nitrogen 3.5 bar, nebulizer 65 °C, dynamic gain)
• Vertex Plus Eurokat Pb column (10 µm, 300 × 8 mm ID)
• ClarityChrom 7.4.2 chromatography workstation

Main Results and Discussion

Both RID and ELSD successfully resolved major honey carbohydrates: fructose, glucose, sucrose and maltose.
ELSD demonstrated superior sensitivity, detecting down to 0.03 mg/mL, while RID provided a broader linear range up to 3.00 mg/mL without signal saturation.
Quantitative results from both detectors were in close agreement, with total sugar contents of approximately 77% in KNAUER honey and 73% in blossom honey.
Agave nectar showed a distinct profile, with fructose levels about 1.3 times higher than honey samples.
Minor discrepancies in sucrose and maltose quantification were attributed to different detector response functions and calibration approaches.

Benefits and Practical Applications

• Ability to authenticate honey origin and detect adulteration by comparing sugar profiles
• Enhanced quality control combining complementary detectors for sensitivity and dynamic range
• Method adaptability to other natural products and sugar-containing matrices
• Straightforward sample preparation and robust HILIC separation using water-only eluent

Future Trends and Potential Applications

Advancements in detector technology may further improve sensitivity and selectivity for low-abundance oligosaccharides.
Integration of mass spectrometric detection could enable comprehensive profiling of sugar isomers and atypical carbohydrates.
Automated data processing using machine learning could streamline authentication workflows and adulteration screening.
Expansion of validated methods to a wider range of natural sweeteners and food matrices is anticipated.

Conclusion

The comparative analysis demonstrated that both ELSD and RID are suitable for reliable determination of honey sugars.
ELSD offers higher sensitivity at low concentrations, while RID delivers a large linear range facilitating direct sample injection.
Combining both detection strategies enhances method robustness for honey quality control and differentiation of substitutes like agave nectar.

References

1. AID Zucker, Sirupe, Honig, Zuckeraustauschstoffe und Süßstoffe, Nr. 1157
2. Honigverordnung vom 16. Januar 2004 (BGBI I S. 92)
3. Codex Alimentarius Commission, GB18796-2005, 2005