A rapid and sustainable method for the detection of extra-virgin olive oil adulteration using surfactant-free microemulsions and 3D spectrofluorimetry

Abstract

Due to the high standards in preparation, purity, and low production yields, extra-virgin olive oils (EVOO) is a high value product that makes adulteration with lower-quality oils a frequent practice. Since conventional quality control techniques rely on costly analytical instrumentation and complex methods and data treatment, there is a need for simpler, faster, and more sustainable analytical alternatives. The present work proposes the use of 3D spectrofluorimetry combined with surfactant-free microemulsions (SFMEs) as a sample preparation strategy for the detection of EVOO adulteration with soybean oil. These systems, composed of an oily phase, an aqueous phase, and a short-chain alcohol, generate microenvironments capable of enhancing the fluorescence of EVOO endogenous fluorophores with minimal sample consumption while reducing the risk of cross-contamination and inner filter effect. In this work, ternary phase diagrams were constructed with three different alcohols, with propan-1-ol being selected due to its ability to form SFMEs with higher water proportions, making it more suitable for EVOO analysis. Tests were then performed to determine proper excitation/emission wavelengths (λ_ex/λ_em) for SFMEs containing EVOO (290/320 nm) and soybean oil (300/335 nm). A compromise condition for SFME formation consisted of 25 μL of oily phase (sample:octan-1-ol, 1:3, v/v), 750 μL of water, and propan-1-ol to a final volume of 5.00 mL. Additionally, the influence of oil sampling was evaluated allowing decreasing down to 60% in relative standard deviation when reverse sample pipetting was used instead of direct sample pipetting, demonstrating its effectiveness in handling high-viscosity samples such as EVOO, regardless of the analyst's level of experience. For the qualitative evaluation, 24 samples comprising olive oils, soybean oils, and blended oils (soybean/EVOO) were analyzed by 3D spectrofluorimetry. Excitation-emission matrices (λ_ex: 200-350 nm and λ_em: 250-400 nm) were obtained for all samples prepared in SFMEs. Significant differences in fluorescence intensity and fluorescence fingerprint were observed when comparing EVOO and soybean oil samples. Among the olive oils, pronounced differences were observed in samples from other categories or without specification, showing the method's potential not only for distinguishing between soybean oil and EVOO, but also for differentiating distinct olive oil categories. A quantitative study with different levels of soybean oil adulteration (0, 25%, 50%, 75%, and 100%, v/v) in EVOO samples achieved a high coefficient of determination (R² = 0.9979) enabling identification down to 1% of adulteration (limit of detection of 0.97%) and precise and accurate quantification of levels of adulteration down to 3% (limit of quantification of 3.23%). Linear regression ANOVA showed statistical significance and response linearity (p < 0.05). Coefficients of variation (CV) obtained from the replicates of the curve were below 5%, demonstrating the potential applicability of the proposed method for quantitative analysis. Sustainability assessment using AGREE and Analytical Eco-Scale metrics resulted in satisfactory scores of 0.71/1.00 and 84/100, respectively, supporting that this method is not only simple and comprehensible but also an eco-friendly approach for the detection of EVOO adulteration.