**1. Introduction**

Virgin olive oil is obtained exclusively from the fruits of the olive tree, *Olea Europaea* L., by mechanical extraction processes at controlled thermal conditions which do not lead to chemical and physical deterioration of the oil, thus preserving its characteristic and distinctive properties [1]. Virgin olive oil is an edible oil greatly appreciated, which is an essential component of the Mediterranean diet. Due to the high content of mono-unsaturated fatty acids, such as the oleic acid, and bioactive minor compounds, extra-virgin olive oil is considered beneficial for human health [2–4]. International recognized institutions and approved regulations, such as the International Olive Council (IOC), *Codex Alimentarius*, and the European Commission, have established a commercial classification based on both chemical-physical and organoleptic properties of the final product. Virgin olive oils (VOOs) and extra-virgin olive oils (EVOOs) are those ones having the highest content of minor compounds with bioactive and nutritional properties (about 1%–2% of the total weight of olive oil) [3,4]. This class of chemical compounds can be divided into polar phenols and their derivatives, and non-polar compounds, such as squalene and other triterpenes, sterols, tocopherols, and pigments [1,2,4–8].

Pigments determine olive oil's distinctive colour. Their relative chemical composition varies during olive oil's life and it depends on many factors, such as the type of cultivar (genetic factor), the climatic and environmental conditions, the state of ripeness of the fruit at harvest, the storage and sampling of olives, the oil production process, and the storage conditions of the

final product [9]. Pigments, which are exclusively synthesized from plants and assimilated by humans only through the diet [10], can be divided in two main classes: Carotenoids and chlorophyll derivatives [9,11–13]. Olive oils contain a relatively rich variety of carotenoids (i.e., β-carotene, lutein, violaxanthin, neoxanthin, and other xanthophylls in minor percentages) and chlorophyll derivatives (i.e., chlorophylls A and B, pheophytins A and B, and other minor derivatives) [9,11–13]. Several works have demonstrated the potential health benefits of both carotenoids and chlorophylls' derivatives [1,2,10].

In the literature, there are many papers and reviews relating the concentration of main pigments and other derived quantities (i.e., the ratio between lutein and β-carotene, or the relative ratio between lutein and minor carotenoids), with olive oil authenticity and quality [9,11–28]. Moreover, several works demonstrated the usefulness of pigments' determination to reveal olive oil adulterations [29–31]. The identification and quantification of single pigments is usually performed by means of chromatographic methods, such as high performance liquid chromatographic with ultraviolet-visible detection (HPLC-DAD) [12,14,15,22,23,25–27]. On the other hand, the near UV-vis spectroscopic absorption technique has been used mainly to evaluate the total amount of carotenoids and the total amount of chlorophylls' derivatives from absorbance values obtained on olive oil samples diluted in cyclohexane, as first reported by Mínguez-Mosquera et al. [11,13]. Despite an initial treatment of the oil sample, implying a dilution in cyclohexane, this method is relatively simple, fast, and cheap. For these reasons, this simple spectroscopic method has been used in several works [11,13,32,33] to determine the total concentrations of carotenoids and chlorophylls in view of a chemical-physic characterization of olive oils and their quality.

A recent new spectroscopic method, based on the quantitative analysis of the whole absorption spectrum of olive oil samples in the near UV-vis range from 390 nm to 720 nm, has been developed to determine the concentration of four main pigments: β-carotene and lutein among the carotenoids, and pheophytin A and pheophytin B among chlorophylls' derivatives [17,19]. The advantage of this spectroscopic method is the very fast analysis and the absence of any sample treatment: Spectra are indeed acquired in bulk and they can be analysed by using a simple deconvolution procedure [17,18]. This spectroscopic approach has been recently tested on extra-virgin olive oil samples produced in several Mediterranean countries, from different cultivars, and it was validated by comparing it with the standard HPLC-DAD method [18], confirming its validity, goodness, and high reproducibility. Moreover, it was revealed to be useful in studying the effect of the different harvest years on the main pigments' content of several extra-virgin olive oils produced from a blend of three cultivars (*Moraiolo*, *Frantoio*, and *Leccino*) typical of Tuscany (Italy) [24].

Other methods based on the analysis of near UV-vis spectra of virgin and extra-virgin olive oils have been developed in the recent years, either in combination with multivariate chemometric approaches [16,28,34–37] or by using neural network to the spectral analysis [29,30,36]. These methods provided are very useful for olive oil quality and authentication purposes, but their application requires a relatively complex data treatment and/or specific software. On the other hand, the need for fast, non-destructive, and validated analytical methods is justified by the raising competition in the field of olive oil and the consumer demand of high standards and high quality products.

The present work focuses on the determination of the total amount of chlorophylls' derivatives, the total amount of carotenoids, and the total amount of pigments in several virgin and extra-virgin olive oil samples, either mono-cultivar or a blend of different cultivars, produced in Tuscany (Italy), in different harvest years. Two relatively simple near UV-vis spectroscopic approaches have been used: The first method, proposed by Mínguez-Mosquera et al. [11], applied to olive oil samples diluted in cyclohexane, and the second method, proposed by Domenici et al. [17], for the quantification of the four main pigments, applied to not fresh olive oils analysed in bulk. Results obtained by the two spectroscopic methods are then compared and discussed in view of their practical usefulness for a fast determination of pigments in virgin and extra-virgin olive oils.

#### **2. Materials and Methods**

In the following subsection, the olive oil samples investigated in this work, the experimental procedures, the analytical methods, and the mathematical tools employed are described.
