Untargeted fluorescence spectroscopy for the quantification of added caramel (E-150d) in presence of natural caramel in Aceto Balsamico di Modena PGI

Synthetic caramel (E-150d) is a permitted colorant in Aceto Balsamico di Modena with Protected Geographical Indication (ABM PGI), used for colour stabilization and requiring label declaration. However, caramel-like compounds also occur naturally due to the cooked must use in balsamic vinegar production, when cooked grape must is used as one of the raw materials. Currently, no official method distinguishes between added and natural caramel in ABM PGI. To address this, we investigated excitation-emission fluorescence spectroscopy as a rapid analytical tool. Three-way Excitation-Emission Matrices (EEMs) were acquired for various ABM samples and their raw materials, including those spiked with synthetic caramel and commercial products. Parallel Factor Analysis (PARAFAC) was applied to the EEM data to identify and resolve fluorescent components associated with both natural and synthetic caramel. The scores from the PARAFAC model were then used to build a Partial Least Squares Regression (PLS) model for quantifying the added caramel concentration. The developed method showed good accuracy in quantifying the synthetic caramel content in spiked samples and a blind test of commercial ABM, with satisfactory reproducibility over time. This study provides a promising untargeted fluorescence-based approach for the detection and quantification of synthetic caramel in ABM PGI, offering a valuable tool for quality control and authenticity verification.