Profiling of Carbohydrates in Honey by HILIC-MS

Importance of the Topic

Honey is a high-value natural product whose quality and authenticity depend on its minor carbohydrate content. While fructose and glucose dominate, di- and trisaccharides offer fingerprints of botanical and geographical origin and reveal potential adulteration.

Objectives and Study Overview

This application note presents a modified HILIC-MS method for comprehensive profiling and quantification of mono-, di-, and trisaccharides in honey and syrup samples. The goal is to achieve superior separation efficiency, sensitive detection, and reliable quantitation within a rapid runtime.

Methodology

Honey and commercial syrup samples were diluted in a 1:1 acetonitrile–water mixture, filtered, and spiked with stable isotope–labeled internal standards. Standards spanning 2–200 µg/mL were prepared similarly. Chromatographic separation was performed on an amide-phase HILIC column under a gradient of acetonitrile, water, and methanol with diethylamine and guanidine additives. A 25-minute gradient program resolved mono-, di-, and trisaccharides into distinct retention windows. Detection employed negative-mode ESI single-ion recording of chloride adduct ions at specific m/z values.

Used Instrumentation

• UHPLC system with column heater
• Amide-phase HILIC column
• Single-quadrupole mass detector (negative ESI)
• Chromatography data software

Main Results and Discussion

Optimized cone voltage (5 V) maximized chloride adduct signal. The method delivered fourteen well-resolved peaks in honey with flat baselines, including several tentatively identified di- and trisaccharides and a few unknowns. Comparative profiling across five honeys and three syrups revealed distinct carbohydrate patterns linked to botanical and geographical origins. In quantitative analyses, fructose and glucose showed linear ranges with R²>0.99, LOQ ~0.02 g/100 g, and RSDs below 9%. Spike recoveries in maple syrup exceeded 90%.

Benefits and Practical Applications

This HILIC-MS approach offers high separation efficiency, stable column performance, and selective MS detection for rapid profiling and quantification in a single 25-minute run. It supports honey quality assessment, origin authentication, and adulteration detection in industrial and research laboratories.

Future Trends and Potential Applications

Expanding the library of carbohydrate standards and integrating high-resolution MS can improve structural elucidation of isomers. Automation and advanced data analytics, including machine learning, may enable large-scale origin classification and quality screening across diverse honey types.

Conclusion

The modified HILIC-MS method on an amide column with MS detection provides an efficient and reliable solution for profiling and quantifying honey carbohydrates. Its robustness, selectivity, and throughput make it an attractive tool for quality control and authenticity studies.

References

1. Doner LW. The Sugars of Honey: A Review. J Sci Food Agric. 1977;28:443–456.
2. Codex Alimentarius Commission. Revised Codex Standard for Honey. Codex STAN 12-1981 Rev. 2. 2001.
3. Da Costa Leite JM et al. Determination of Oligosaccharides in Brazilian Honeys of Different Botanical Origin. Food Chem. 2000;70:93–98.
4. Yang J, Rainville P, Liu K, Pointer B. Quantification of Mono and Disaccharides in Foods. Waters Application Note 720006575EN. 2019.