**1. Introduction**

The health properties of phenolic compounds contained in extra virgin olive oil (EVOO) have been clearly established by many scientific papers [1–3]. On this basis, according to the European Food Safety Authority (EFSA), since 2012 European regulation has allowed the use of a health claim in olive oil labeling [4]. The health claim states as follows: "Olive oil polyphenols contribute to the protection of blood lipids from oxidative stress" and can be applied to olive oils containing at least 5 mg of hydroxytyrosol (OHTyr) and its derivatives (e.g., oleuropein complex and tyrosol) per 20 g of product.

F.; Fortunato, S.; Rotondi, P.; Bellumori, M.; Cecchi, L.; Cosma, P.; Mulinacci, N.; Caponio, F. Paving the Way to Food Grade Analytical Chemistry: Use of a Natural Deep Eutectic Solvent to Determine Total Hydroxytyrosol and Tyrosol in Extra Virgin Olive Oils. *Foods* **2021**, *10*, 677. https://doi.org/10.3390/ foods10030677

**Citation:** Paradiso, V.M.; Longobardi,

Academic Editor: Francesca Venturi

Received: 4 March 2021 Accepted: 16 March 2021 Published: 22 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Based on this regulation, scholars focused their attention on analytical issues related to the assessment of the suitability of oils to be labelled with the health claim. Several analytical methods have been proposed, usually comprising an extraction step followed by separation by means of liquid chromatography, gas chromatography, or capillary electrophoresis, and a suitable detection method.

Irrespective of the separation and detection methods, the quantification step of the target compounds remains a critical issue. In fact, OHTyr and tyrosol (Tyr) are present in EVOO in their free form, as well as esterified in several derivatives [5]. Therefore, the quantitation of each derivative is difficult to obtain. A widely adopted approach to overcome this difficulty is to carry out a hydrolysis step prior to separation and analysis, in order to quantify, as free forms, the total of free and linked OHTyr and Tyr. This approach has been adopted either directly on the oil or on the phenolic extract, with quite satisfactory performances [6–9].

Nevertheless, the adoption of separation methods for the analytical determination, though being affordable and sensitive, requires expensive equipment, toxic/pollutant reagents, and trained operators. The availability of easy, less expensive, and operator- and environment-friendly methods, rather than being an alternative, could be a valid analytical complement, useful for screening purposes, even in oil mills or bottling plants as a quality monitoring or a decision supporting tool [10]. Some efforts in this direction have been made. The Folin–Ciocalteu assay, a widely used spectrophotometric method, has provided good preliminary results [11].

In addition, natural deep eutectic solvents (NADES) have been proposed as solvents for easy screening methods. NADES are green solvents consisting of mixtures of one or more hydrogen bond acceptor–donor pair that, in appropriate molar ratios, generate strong intermolecular interactions [12]. Compared to deep eutectic solvents (DES), NADES are obtained from molecules naturally present in living organisms as metabolites [13]. They are being increasingly used for analytical purposes [12,14]. NADES have been successfully applied in the analysis of EVOO phenolic compounds as extraction media prior to liquid chromatography [15], electrochemical analysis [16], and spectrophotometric analysis [10,17]. In one case, NADES extraction and direct spectrophotometric analysis allowed assessing the amount of OHTyr and Tyr derivatives determined as the sum of the free and linked forms determined by high-performance liquid chromatography (HPLC), thus providing a useful tool to label EVOO according to the EU health claim [10].

In view of a better harmonization of the green screening methods with the candidate official methods related to the health claim, there is still the need for easy and green methods allowing assessing total OHTyr and Tyr as free forms, as determined by emerging hydrolysis methods. The present research was therefore aimed at setting up the spectrophotometric determination of total OHTyr and Tyr, free and linked, after extraction with a NADES composed of lactic acid, glucose, and water.
