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.

Analytical Chemistry Laboratory Chemistry Department National and Kapodistrian University of Athens Panepistimiopolis Zographou 15771 Athens Greece

Attiki Honey SA Kryoneri Attikis 14568 Athens Greece

Bruker Daltonik GmbH Fahrenheitstraße 4 28359 Bremen Germany

Department of Food Analysis and Nutrition Faculty of Food and Biochemical Technology University of Chemistry and Technology Prague Technická 5 16628 Prague Czech Republic

Division of Engineering and Food Science School of Applied Science Abertay University Bell Street Dundee DD1 1HG UK

Food Chemistry Laboratory Department of Chemistry National and Kapodistrian University of Athens Panepistimiopolis Zographou 15771 Athens Greece

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