Honey is a highly consumed commodity due to its potential health benefits upon certain consumption, resulting in a high market price. This fact indicates the need to protect honey from fraudulent acts by delivering comprehensive analytical methodologies. In this study, targeted, suspect and non-targeted metabolomic workflows were applied to identify botanical origin markers of Greek honey. Blossom honey samples (n = 62) and the unifloral fir (n = 10), oak (n = 24), pine (n = 39) and thyme (n = 34) honeys were analyzed using an ultra-high-performance liquid chromatography hybrid quadrupole time-of-flight mass spectrometry (UHPLC-q-TOF-MS) system. Several potential authenticity markers were revealed from the application of different metabolomic workflows. In detail, based on quantitative targeted analysis, three blossom honey markers were found, namely, galangin, pinocembrin and chrysin, while gallic acid concentration was found to be significantly higher in oak honey. Using suspect screening workflow, 12 additional bioactive compounds were identified and semi-quantified, achieving comprehensive metabolomic honey characterization. Lastly, by combining non-targeted screening with advanced chemometrics, it was possible to discriminate thyme from blossom honey and develop binary discriminatory models with high predictive power. In conclusion, a holistic approach to assessing the botanical origin of Greek honey is presented, highlighting the complementarity of the three applied metabolomic approaches.

The determination of volatile compounds is essential for the chemical characterisation of honey's aroma and its correlation to its sensory profile and botanical origin. The present study describes the development, optimization and validation of a new, simple and reliable method for the determination of volatile compounds in honey using headspace solid-phase microextraction combined with gas chromatography/mass spectrometry (HS-SPME-GC-MS). The optimization of the SPME conditions showed that the ratio of honey: water (2:1) and the incubation temperature (60 °C) are the most critical parameters. Gas chromatography was performed with medium polar Varian CP-Select 624 column and the experimental Retention Index for a number of compounds was determined as an additional identification feature for suspect analysis. The simultaneous use of four internal standards chlorobenzene, benzophenone, 2-pentanol and 4-methyl-2-pentanone and matrix matched calibration enhanced method accuracy achieving recoveries 73–114 % and repeatability ranging between 3.9 and 19 % relative standard deviations. Furthermore, the superiority of the HS-SPME to static head space technique was verified exhibiting four-to nine-fold higher sensitivity. Target and suspect screening were applied to 30 Greek honey samples and 53 volatile compounds belonging to different chemical classes, such as alkanes, aldehydes, ketones, alcohols, and esters were identified with quantified concentrations ranging between 3.1 μg kg⁻¹ (Limonene) up to 20 mg kg⁻¹ (Benzeneacetaldehyde). Among the new findings is the detection of Myrtenol in Greek pine honey and 2,3-butanediol in Greek oak honey. The developed analytical protocol can be a valuable tool in order to chemically characterize honey based on the volatile content.