Authentication of the geographical origin of Italian multifloral honey: multi-analytical characterization of the composition and pesticide residues to distinguish the production area

Verifying the geographical origin of honey has become a key issue for food quality assurance, consumer safeguards and regulatory enforcement, particularly following recent European regulation mandating verifiable origin labeling. This task is particularly challenging for multifloral honey, as its chemical composition is shaped by local biodiversity, environmental factors, and agricultural activities. In this work, an integrated multi-analytical strategy was employed to characterize Italian multifloral honeys and identify indicators linked to their origin. Seventy-five multifloral honey samples collected from Northern, Central, and Southern Italy were examined using a combination of analytical techniques, i.e., the untargeted profiling of volatile organic compounds (VOCs) via dielectric barrier discharge ionization high-resolution mass spectrometry (DBDI-HRMS), quantification of sugars and amino acids by quantitative nuclear magnetic resonance (q-NMR), elemental analysis by total reflection X-ray fluorescence (TXRF), and and targeted assessment of pesticide residues. In parallel, several thousand melissopalynological analyses performed on multifloral Italian honey collected during 2015–2024 were statistically analyzed to retrieve the pollen species that characterize the Northern, Central and Southern Italian macro-areas. The statistical evaluation revealed clear regional patterns in pollen counts, VOCs, mineral composition, and specific sugars and amino acids, highlighting the influence of local flora, geological background, and environmental conditions. DBDI-HRMS exhibited a high ability to differentiate between Italian macro-regions, with cresol, santene, and acetoaminophenone having a pivotal function in demonstrating the bees' collection of chestnut, rhododendron, and linden nectars in the honey produced in Northern Italy, which is in alignment with the results of our pollen counts. Moreover, our DBDI-HRMS findings indicate that methyl-syringate is a typical marker of Central and Southern honeys which had the highest counts of citrus pollen. Similarly, our melissopalynological assessment revealed that Italian Southern honey is distinguished by its high prevalence of citrus pollen. Q-NMR analysis identified distinctive trends in minor carbohydrates and amino acids. Honey from Southern Italy exhibited high proline, leucine, and isoleucine concentrations. These are quality indicators related to specific botanical origins, such as citrus and eucalyptus, which are prevalent in this region as demonstrated by our melissopalynological data. Honey samples originated from central and southern Italy presented significantly elevated bromine levels, probably due to the contribution of marine aerosol, and strontium concentrations revealed moderate increase along the north–south gradient. Within this integrated framework, pesticide residues were interpreted as indicators of environmental exposure and agricultural context rather than as direct geographical markers. Levels of pesticide residues, were generally minimal and largely compliant with regulatory thresholds, confirming the overall high quality of Italian honey. Overall, 62 out of 75 honeys were either completely residue-free or contained only trace amounts of pesticides. The low concentrations of acaricides, such as amitraz, fluvalinate, and coumaphos, reflects controlled use in hive treatments. The occasional detection of agricultural pesticides, such as propamocarb and chlorphenvinphos, suggests localized crop-related inputs and environmental drift consistent with regional land use. . Collectively, the combination of diverse analytical datasets provides a solid approach for the geographical authentication of multifloral honey, supporting origin verification and the development of future classification models.