Advanced characterization of raw honey authenticity using high-resolution sugar profiling and stable carbon isotope ratio analysis

Raw honey is a complex biological matrix whose quality attributes are dictated by minimal processing and preserved enzymatic activity. This study presents a comprehensive biochemical and isotopic characterization of 150 authentic samples collected from two distinct ecological regions of Türkiye (Tokat and Hakkari/Yüksekova). A multi-layered analytical approach was employed, encompassing physicochemical parameters, enzymatic profiles (invertase and diastase), high-resolution sugar composition via HPLC-RID, and molecular authentication using Stable Carbon Isotope Ratio Analysis (SCIRA). The findings revealed exceptional enzymatic potency, with invertase activity reaching 194.7 U/kg and proline content exceeding 1000 mg/kg, serving as definitive markers of biological ripeness and the absence of thermal stress. High-resolution sugar profiling identified unique “nectar fingerprints”—particularly erlose levels reaching 3.3% in high-altitude samples—which effectively distinguish genuine matrices from industrial design syrups. SCIRA molecularly confirmed sample purity, with Δδ¹³C values remaining within legal thresholds, verifying 0% C4-sugar adulteration across the tested population. Furthermore, multivariate statistical tools (PCA and HCA) successfully discriminated samples based on their geographical terroir, separating mineral-rich Tokat honeys from high-fructose Hakkari honeys. A strong positive correlation ( r = 0.82) between invertase and proline was also identified, suggesting a synergistic relationship in honey maturation. Conclusively, these findings establish a robust analytical framework for quality grading and demonstrate the utility of a “Biological Quality Index” (BQI) to safeguard premium honey products in the global market.