A non-targeted metabolomics approach for the investigation of honey adulteration by HRMS

Importance of the Topic

Authentic honey commands a premium price and offers health benefits linked to its antioxidant, antimicrobial, and anti-inflammatory properties. However, fraudulent addition of sugar syrups undermines quality, misleads consumers, and poses health and economic risks. Rapid, robust screening methods capable of detecting a broad range of adulterants are essential for regulatory control, food safety, and quality assurance in the honey supply chain.

Objectives and Study Overview

This study aimed to develop a non-targeted metabolomics workflow to detect common sugar syrups in honey using high-resolution accurate-mass (HRAM) mass spectrometry. By combining two chromatographic strategies and a simple “dilute-and-shoot” sample preparation, the method sought to simultaneously screen for oligosaccharides, polysaccharides, and characteristic phytochemicals without complex extraction steps.

Methodology and Instrumentation

The workflow comprised:

• Sample preparation: 1 g honey diluted with water and methanol, vortexed, centrifuged, then directly injected.
• Chromatography: Method 1 employed a reversed-phase Accucore aQ column; Method 2 used an Accucore amide HILIC column for polar analytes. Gradient elution separated di- and oligosaccharides as well as phenolics.
• Mass spectrometry: Thermo Scientific Vanquish Flex UHPLC coupled to an Orbitrap Exploris 240 MS with heated electrospray ionization. Full-scan with data-dependent MS² (ddMS²) acquisition at resolutions up to 240 000 enabled accurate mass measurements within 5 ppm and fast polarity switching.
• Data processing: Thermo Xcalibur and Compound Discoverer software performed peak picking, formula assignment, isotopic evaluation, retention time alignment, and mzCloud/ChemSpider spectral matching (score >65%).

Main Results and Discussion

The combined methods detected over 200 compounds, including sugar oligomers (DP 3–18), monosaccharides, amino acids, and flavonoids. High resolution (240 000) maintained >15 data points per peak, ensuring precise quantitation even during rapid polarity switches. MS² spectra differentiated positional isomers such as naringenin and pinobanksin. Principal component analysis of six market samples clustered adulterated honey separately from genuine products. Marker oligomers DP 6 (maltohexaose) and DP 16 (maltohexadecaose) were elevated in adulterated samples, consistent with isotope-ratio mass spectrometry findings.

Benefits and Practical Applications

The protocol’s simplicity and speed (20–22 min runs) suit high-throughput quality control. Non-targeted detection captures unexpected adulterants and authentic components in a single acquisition. Accurate mass and MS² confirmation strengthen confidence in results, supporting food safety laboratories, industrial QA/QC, and regulatory agencies.

Future Trends and Potential Applications

Advances may include expanded spectral libraries, machine-learning for marker discovery, integration with isotope methods, and full-automation for real-time monitoring. Broader application across diverse food matrices can enhance fraud detection and traceability in the agri-food sector.

Conclusion

A dilute-and-shoot UHPLC-HRMS approach with complementary reversed-phase and HILIC separations, combined with non-targeted data processing, allows comprehensive screening of honey adulteration. High resolution, fast polarity switching, and MS² confirmation deliver reliable identification of sugar syrups and native compounds, providing an efficient tool for honey authentication.

References

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