Olive oil, a globally significant product, exhibits a complex aroma profile influenced by various factors including cultivar, ripening stage, environment, and processing methods. Understanding the native volatile compound profile is crucial for quality control and sustainable supply. Existing methods, such as SAFE and HS-SPME, face limitations due to the strong matrix effect of the oil. OA-LLE, previously developed by the authors, offers an efficient method for isolating volatile compounds from various food matrices, but when applied to EVOO it results in extracts contaminated with non-volatiles. This paper proposes a combined approach using OA-LLE followed by SAFE (OA-LLE + SAFE) to enhance the extraction efficiency and purity of volatile compounds in EVOO, ultimately providing a more accurate representation of its aroma profile. The hypothesis is that this two-step process will effectively isolate a wider range of aroma compounds from EVOO than using either method alone.

Numerous studies have investigated the aroma profile of olive oil, highlighting the influence of various factors such as cultivar, ripening stage, environment, and processing methods. The importance of C5 and C6 aliphatic compounds as key aroma contributors has been established. Existing extraction methods, including SAFE and HS-SPME, have been employed, but limitations related to matrix effects and incomplete extraction of volatile compounds have been recognized. The authors' previous work demonstrated the effectiveness of OA-LLE for isolating volatile compounds from other complex matrices, forming the basis for the proposed combined approach.

The study employed four extraction methods: OA-LLE, SAFE, HS-SPME, and the novel OA-LLE + SAFE. A model study was conducted to optimize the OA-LLE process, specifically investigating the effect of water addition to the methanol solution on the efficiency of liquid-liquid extraction with dichloromethane. Extra virgin olive oil (EVOO) samples from different cultivars and origins were analyzed. The OA-LLE + SAFE method involved performing OA-LLE to obtain a dichloromethane layer containing most volatile compounds, then subjecting this layer to SAFE to remove non-volatiles. A modified OA-LLE x 3 + SAFE procedure was also tested, where OA-LLE was performed three times on a larger sample (15g), combining the dichloromethane layers before SAFE to potentially enhance concentration. GC-MS analysis was used to identify and quantify the volatile compounds in the extracts. Statistical analyses, including principal component analysis (PCA), were used to compare the performance of the extraction methods. Authentic standards were used for identification and quantification, using retention indices and mass spectral data. Boiling points were obtained from PubChem and ChemSpider databases.

OA-LLE alone extracted 63 aroma compounds from 5g of EVOO, but the extracts were dark green due to non-volatile contaminants. SAFE and HS-SPME extracted significantly fewer compounds (20 and 23 respectively). The OA-LLE + SAFE method yielded 41 aroma compounds from 5g of EVOO, producing colorless and transparent extracts. The total amount of aroma compounds in OA-LLE + SAFE extracts was significantly higher than in SAFE extracts (43.3 ± 1.7 µg/200 mL vs 30.4 ± 6.6 µg/200 mL). PCA analysis showed that OA-LLE + SAFE extracted a broader range of compounds, including some with higher boiling points, compared to SAFE and HS-SPME which primarily isolated low boiling point compounds. The OA-LLE x 3 + SAFE method further enhanced the extraction, yielding 59 aroma compounds from 15g of Hojiblanca EVOO, along with 45 and 39 compounds from Mission and Lucca EVOOs respectively, demonstrating significant improvement in the extraction of both volatile and semi-volatile compounds. The number of aroma compounds with retention indices exceeding 2000 was substantially increased by the combined method.

The results demonstrate the superior performance of the OA-LLE + SAFE method for extracting aroma compounds from EVOO. The combination effectively overcomes the limitations of individual methods, improving extraction efficiency and eliminating the contamination of non-volatiles. The ability to isolate a wider range of volatile and semi-volatile compounds provides a more comprehensive picture of the EVOO aroma profile. The increased yield of compounds with higher boiling points, which are typically less readily extracted, highlights the effectiveness of the combined approach. This approach could be applied to other oil-based food products to enhance their aroma analysis.

This study successfully developed a novel and efficient extraction method (OA-LLE + SAFE) for analyzing the aroma profile of EVOO, significantly improving the extraction efficiency and purity of volatile compounds. The OA-LLE x 3 + SAFE approach further enhances the yield and enables the isolation of trace compounds and semi-volatiles with higher boiling points. Future research could explore the application of this method to other edible oils and the use of advanced analytical techniques such as GCxGC-TOF-MS to further characterize the aroma compounds and link them to sensory attributes.

The study primarily focused on three EVOO cultivars. Further research is needed to validate the method's generalizability across a wider range of EVOO types and other edible oils. While the combined method successfully removed many non-volatiles, trace amounts might still remain. The sensory evaluation of extracted aromas was not performed in this study.