Researchers have used phenolic-focused, untargeted ion mobility liquid chromatography-mass spectrometry to identify season-associated chemical patterns in Scottish honey.
The analysis distinguished spring, summer, and heather honey profiles, including differences between spring and summer samples collected from the same apiaries. This natural variation may need to be accounted for when developing chemical reference profiles to assess honey authenticity.
Renata Garbellini Duft and Julian L. Griffin of the University of Aberdeen analyzed 39 honey samples. These included five Scottish heather honeys, 11 spring honeys, and 12 summer honeys, along with blended, infused, and non-Scottish commercial products.
A paired subset contained spring and summer honey collected from approximately nine of the same Aberdeenshire apiaries. This design reduced differences related to location and producer, allowing the researchers to examine seasonal variation more directly.
Sample preparation used acidified water followed by repeated acetonitrile extraction. The high sugar content of honey created phase separation, allowing phenolic and other semi-polar metabolites to move into the acetonitrile-rich phase while reducing the sugar content of the final extract. The protocol used less than 2 mL of solvent per sample.
Extracts were analyzed by ultra-high-performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry in negative electrospray ionization mode. Ion mobility added drift-time and collision cross-section information to accurate mass, retention time, and tandem mass spectra.
The researchers used a collision cross-section tolerance of ±1.5 percent to support compound annotations. However, they noted that this range may not distinguish closely related structural isomers, meaning collision cross-section should be considered supporting rather than definitive evidence.
Quality-control samples, blanks, calibration standards, and a dilution series were distributed throughout the analytical run. After blank filtering, drift correction, and quality-control assessment, 1,738 features met the final precision and linearity requirements. The mean mass error for reference standards was 1.82 parts per million, while the mean absolute collision cross-section deviation was 0.6 percent.
The researchers annotated 51 features: four were confirmed with authentic standards, 41 received Metabolomics Standards Initiative Level 2 annotations, and six received Level 3 assignments.
Compounds that helped distinguish spring and summer honeys included galangin, quercetin, sebacic acid, tricoumaroyl spermidine, suberic acid, and luteolin-7-glucuronide.
Heather honey markers included 3-phenyllactic acid, abscisic acid, 4-hydroxycinnamaldehyde, and kaempferol-related compounds.
Exploratory analyses also found differences among Scottish, non-Scottish, blended, and infused products. However, several comparator groups contained only one or a few samples. The models were therefore intended for marker discovery rather than as validated authentication classifiers.
The study did not include an independent validation cohort or evaluate complete extraction recovery using internal standards. Broader sampling, additional authentic standards, and external validation will be needed before the workflow can support routine traceability or regulatory testing.
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