PROFILING OF CARBOHYDRATES IN HONEY BY HILIC-MS

Significance of the Topic

Carbohydrate profiling of honey is critical for verifying authenticity, detecting adulteration and assessing quality. The detailed mono-, di- and tri-saccharide composition serves as a fingerprint for botanical and geographical origin of honey. Advanced analytical techniques provide reliable detection of minor sugars and support regulatory compliance.

Study Objectives and Overview

The study aimed to develop a robust HILIC-MS method using a XBridge BEH Amide XP column and QDa mass detector for comprehensive profiling and quantification of mono-, di- and tri-saccharides in honey and syrup samples. Method modifications targeted faster elution, improved separation efficiency and sensitive detection of oligosaccharides.

Methodology and Instrumentation

• Sample preparation: Stock solutions of 25 sugar standards at 10 mg/mL in acetonitrile-water (1/1 v/v) and stable isotope labeled fructose-13C6, glucose-13C6 and sucrose-13C6 as internal standards at 25 µg/mL. Honey and syrup samples were diluted in acetonitrile-water, filtered through 0.2 µm PVDF and spiked with internal standards.
• LC conditions: ACQUITY Arc System with column heater; XBridge BEH Amide XP column (3.0 × 10 mm, 2.5 µm) at 90 °C; 1 µL injection; flow rate 0.8 mL/min; gradient elution from 90:6:4 to 78:20:2 acetonitrile-water-methanol mixtures with 0.05% diethylamine and 5 mg/L guanidine hydrochloride over 25 min.
• MS parameters: ACQUITY QDa detector in ESI negative mode; cone voltage 5 V; detection of chloride adducts [M+Cl]− by single ion recording at m/z 215 for monosaccharides, 377 for disaccharides and 539 for trisaccharides.

Key Results and Discussion

Optimization of cone voltage revealed enhanced formation of chloride adduct ions at 5 V. Chromatograms showed baseline resolution of 14 disaccharides and 6 trisaccharides with distinct capacity factors. Comparative profiling of five honey and three syrup samples highlighted clear differences between groups. Clover honey exhibited a unique disaccharide pattern compared to other North American honeys. Quantitative analysis achieved limits of quantitation of 0.02% w/w for fructose, glucose and sucrose, linear calibration from 2 to 200 ppm, spike recoveries of 91-94% and relative standard deviations below 5%. Unknown saccharide peaks were detected, indicating potential novel carbohydrates requiring further study.

Benefits and Practical Applications of the Method

The proposed HILIC-MS method delivers high separation efficiency and sensitivity for comprehensive sugar profiling in honey. It enables botanical origin differentiation, adulteration detection with syrups and robust quality control in food analysis and regulatory environments. Minimal sample preparation and a 25-minute run time enhance laboratory throughput and reproducibility.

Future Trends and Potential Applications

Future developments may include extension to higher-order oligosaccharides, integration with machine learning for pattern recognition, coupling with isotopic ratio analysis for provenance verification, and automation for high-throughput screening. Such advances could broaden applications to other complex food matrices and metabolomics studies.

Conclusion

The modified HILIC-MS method demonstrates excellent resolution, sensitivity and quantification capabilities for mono-, di- and tri-saccharides in honey. Its ability to resolve minor carbohydrates and generate reproducible profiles makes it a valuable tool for authenticity testing, quality assessment and regulatory compliance in the honey industry.

Reference

• Doner LW 1977 The sugars of honey a review Journal of the Science of Food and Agriculture 28 443-456
• Codex Alimentarius Commission 1981 Reinforced Codex Standard for Honey Codex STAN 12-1981 Rev 1 (1987) Rev 2 (2001)
• Da Costa Leite JM et al 2000 Determination of oligosaccharides in Brazilian honeys of different botanical origin Food Chemistry 70 93-98
• Yang J Rainville P Liu K Pointer B 2019 Quantification of Mono and Disaccharides in Foods Waters application note 720006575en