Authentication of honey origin by stable isotope method (δ13C) based on combination of elemental analysis and liquid chromatography hyphenated with isotope ratio mass spectrometry (EA-LC/IRMS)

Abstract

This study introduces the first δ¹³C analysis for individual sugars, such as glucose and fructose, and proposes a method combining elemental analysis and isotope ratio liquid chromatography-mass spectrometry (EA/LC-IRMS) to enhance isotopic techniques for verifying honey authenticity. A total of 612 honey samples were analyzed, including 465 from Vietnam, 60 from Cambodia, 42 from India, 39 from Thailand, and 5 Manuka samples from Australia. Honey δ¹³C values ranged from −29.77 to −23.84 ‰, while protein δ¹³C values ranged from −28.74 to −23.18 ‰. The study observed a strong correlation between individual sugar δ¹³C ratios, which changed significantly with sugar adulteration, even at low levels. Using δ¹³C values for glucose and fructose, along with δ¹³C (‰) max and % C₄ differences, enables reliable detection of added C₃ beet sugar and C₄-based products such as cane sugar, cane syrup, iso-glucose syrup, and high-fructose corn syrup (HFCS) (DL = 1–10 %). Statistical methods, including analysis of variance (ANOVA), Tukey's honest significance test (Tukey HSD), and principal component analysis (PCA), grouped honey samples based on their origins. This novel isotopic method surpasses existing techniques in sensitivity and analysis speed. Importantly, it is the first approach developed to detect beet sugar adulteration in Vietnamese honey, ensuring product authenticity and quality control.