Metabolomics-based authentication of acacia honey against C3 and C4 sugar adulteration

Acacia (Robinia pseudoacacia) honey is frequently adulterated with low-cost alternatives via direct syrup addition or in-hive sugar feeding. We focused on sugar-feeding adulterants, which is difficult to distinguish from nectar-based honey. Stable carbon isotope ratio analysis (SCIRA) is an established method for assessing honey authentication; however, its applicability is limited to detecting C4 plant-derived sugar adulteration. Therefore, we developed a metabolomics-based multi-marker strategy to discriminate acacia honey from beet-sugar (C3)- and cane-sugar (C4)-fed honey. Thirteen metabolites were prioritized using combined multivariate and univariate criteria. Subsequently, 1716 panels of seven-marker (₁₃C₇) were evaluated for discrimination performance. 291 panels achieved 100% accuracy in an independent validation set. In blending scenarios, acacia honeys spiked with 20% beet-sugar-fed honey and 20% cane-sugar-fed honey were successfully identified, outperforming SCIRA (60%) and beet-sugar marker 3-methoxytyramine (3-MT) (30%). These findings establish metabolomic panels as a robust marker for acacia honey authentication, extending beyond current reference methods.