Isoprenoids composition and colour to differentiate virgin olive oils from a specific mill

1 ISOPRENOIDS COMPOSITION AND COLOUR TO DIFFERENTIATE 1 VIRGIN OLIVE OILS FROM A SPECIFIC MILL 2 Running title: Colour and isoprenoids in olive oils 3 4 Paula Mapelli-Brahma, Dolores Hernanz-Vilab, Carla M. Stincoa, Francisco J. 5 Herediaa, Antonio J. Meléndez-Martíneza* 6 7 a Food Colour & Quality Lab. Department of Nutrition & Food Science. Universidad de Sevilla. 8 Facultad de Farmacia, 41012. Sevilla, Spain. 9 b Department of Analytical Chemistry. Universidad de Sevilla. Facultad de Farmacia, 41012. Sevilla, 10 Spain. 11 12 *Author to whom correspondence should be addressed. 13 Antonio J. Meléndez-Martínez 14 Área de Nutrición y Bromatología. Universidad de Sevilla. 15 Facultad de Farmacia. 41012. Sevilla, Spain. 16 TELEPHONE: ++34 95455 7017 17 FAX: ++ 34 95455 7017 18 e-mail: [email protected] 19 2 ABSTRACT 20 The objective of this work was verify if, based on the concentration of some key 21 isoprenoids and the colour, olive oils from a specific mill could be differentiated from those 22 from other mills by linear discriminant analysis. The isoprenoids studied were β-carotene, 23 lutein, α-tocopherol, β-sitosterol, pheophytin a and squalene, which are isoprenoids of interest 24 in food science and nutrition. 88 % of correct classification was obtained by linear 25 discriminant analysis and, therefore, a good differentiation was achieved. This fact can reveal 26 the impact of the conditions of elaboration of each particular mill on the isoprenoid content of 27 the olive oil and therefore on its colour, which is a characteristic of foods with a high effect 28 on consumers. 29 KEYWORDS 30 Carotenoids; β-sitosterol; squalene; tocopherol. 31 32 CHEMICAL COMPOUNDS STUDIED IN THIS ARTICLE 33 β-carotene (PubChem CID: 5280489); lutein (PubChem CID: 5281243); pheophytin a 34 (PubChem CID: 459387); β-sitosterol (PubChem CID: 222284); squalene (PubChem CID: 35 638072); α-tocopherol (PubChem CID: 14985). 36 3 1. Introduction 37 The nutritional quality of olive oil concerns increasingly to both consumers and health 38 systems. Olive oil consumption has been associated with a lower risk of developing diseases 39 like myocardial infarction, atherosclerosis and certain cancers, among others (Fitó, De la 40 Torre, & Covas, 2007; Pérez-Jiménez, Ruano, Pérez-Martínez, López-Segura, & López-41 Miranda, 2007). Until recently, most of the protective effects were associated almost 42 exclusively to the high proportion of unsaturated fatty acids (Pérez-Jiménez et al., 2007). 43 However, in recent years it is becoming increasingly evident the importance that minor 44 constituents of the unsaponifiable fraction, such as isoprenoids and phenolic compounds, have 45 on the benefits associated with olive oil consumption (Covas et al., 2006). Within the 46 isoprenoid fraction, there are a series of compounds like α-tocopherol, β-sitosterol, squalene, 47 carotenoids and chlorophylls, which are of great importance. Indeed, altogether, they are 48 related to the nutritional and sensory quality of the product. 49 Tocopherols exhibit vitamin E activity. They are considered potent lipophilic 50 antioxidants that can protect membrane lipids from oxidation, although non-antioxidant 51 biological actions of these compounds have also been postulated (Azzi, 2007). They are 52 commonly found in vegetable oils, fats and some vegetables like broccoli, celery and 53 tomatoes (Monge-Rojas & Campos, 2011). The main tocopherol in virgin olive oil (VOO) is 54 α-tocopherol (Haddam et al., 2014). Among phytosterols, the most abundant in olive oil is β-55 Sitosterol (Haddam et al., 2014). It has a recognized effect on lowering cholesterol 56 concentrations by interfering in the absorption of cholesterol in the intestinal tract (Trautwein 57 et al., 2003). Some studies suggest that the high concentration of squalene in olive oil could 58 be one of the factors contributing to the anti-carcinogenic effect associated to the intake of it 59 (Reddy & Couvreur, 2009). The major olive oil carotenoids are β-carotene and lutein 60 (Giuffrida, Salvo, Salvo, Cossignani, & Dugo, 2011). β-carotene is a provitamin A carotenoid 61 and continues eliciting interest as a health-promoting carotenoid. The study of the health 62 4 benefits of lutein is particularly important for its roles in the macula lutea and the brain 63 (Johnson, 2004; Krinsky & Johnson, 2005). During the extraction of olive oil, chlorophylls 64 lose the coordinated magnesium and are transformed into the respective pheophytin 65 derivatives, which are consequently the major chlorophyll pigments in them (Giuffrida et al., 66 2011). Unlike carotenoids, there are not many studies on the possible health benefits of 67 chlorophylls (Moyano, Heredia, & Meléndez-Martínez, 2010). 68 The colour of foods is an attribute closely related to their acceptability or rejection by 69 consumers, and therefore is very important in economic terms. Generally, the consumers 70 associate the observed colours with possible aromas and tastes of the product (Moyano et al., 71 2010). The characteristic colour of an olive oil depends on the concentration and the ratio of 72 the different pigments. These are classified in chlorophyll pigments, responsible for greenish 73 hues, and carotenoids, responsible for yellow hues (Giuffrida et al., 2011). Autooxidation of 74 olive oil results in losses of pigments with the concomitant decolouration (Aparicio-Ruiz & 75 Gandul-Rojas, 2014). 76 In this study, both some key isoprenoids and the colour of different VOO marketed in 77 Andalusia were determined. The main objective was to evaluate if the concentration of these 78 isoprenoids and the colour parameters can be useful to differentiate the samples from a 79 specific mill (SM) from those from other mills (OM). 80 81 2. Materials and methods 82 2.1. Reagents and standards 83 HPLC-grade reagents, i.e. methanol (MeOH) and methyl tert-butyl ether (MTBE), and 84 α-tocopherol, β-sitosterol, β-carotene, squalene, chlorophyll a and b standards were supplied 85 by Sigma-Aldrich (Steinheim, Germany). Pheophytin a and b were obtained from 86 chlorophylls a and b respectively by acidification with 0.1 mol/L methanolic hydrochloric 87 acid (Sievers & Hynninen, 1977). Lutein was isolated from a saponified extract of green 88 5 leaves by open column chromatography according to recommended procedures (Rodríguez-89 Amaya, 2001). 90 2.2. Samples 91 Thirty samples of extra virgin olive oil (EVOO) produced in different parts of 92 Andalusia were analysed. Fifteen of them (OM) were EVOO corresponding to common 93 brands retailed in Spain and were purchased at a local supermarket. The rest (SM) were 94 EVOO obtained with different olive varieties in an oil mill located in Palma del Rio 95 (Oleopalma S.A.T., Palma del Río, Córdoba, Spain) from 2015 harvest. SM samples were 96 obtained at an industrial scale. They were taken from the deposits from where the samples 97 that are eventually bottled and marketed are kept. Both, OM and SM sets includes samples 98 from different olive cultivars and geographical origins, within Andalucía. However, while 99 OM were processed in different mills, the SM set includes oils obtained according to the same 100 technological procedures. Information on the olive variety, geographic origin and type of 101 packing as well as the companies of both groups of samples is summarised in Supplementary 102 Table 1. Although in most cases the conditions of production of olive oil, i.e. temperature, 103 extraction time, etc., were not indicated, it seems sensible to assume that these conditions vary 104 between the different mills. 105 2.3. Isoprenoid analysis 106 2.3.1. Extraction and saponification 107 The samples were extracted in triplicate by using the protocol described by Mínguez-108 Mosquera, Gandul-Rojas, & Gallardo-Guerrero (1992) with some modifications. The method 109 is based on a liquid-liquid distribution so that there is a partition of the compounds between 110 N,N-dimethylformamide (DMF) and hexane. In the hexane phase, which is subsequently 111 saponified, triglycerides, carotenes (mainly β-carotene), squalene and β-sitosterol are retained 112 and in the DMF phase, xanthophylls, tocopherols and chlorophyll pigments are extracted. 113 6 Three-gram aliquots of the EVOO were dissolved directly in 30 mL of DMF saturated 114 with MgCO3 and treated with five successive 10 mL portions of hexane. The mixtures were 115 centrifuged at 3000 rpm for 5 min. at 4 °C. The two phases were transferred to different 116 separatory funnels. 117 Eighty mL of Na2SO4 solution (2 g/100 mL in water at approximately 0 °C), 15 mL of 118 hexane and 15 mL of diethyl ether were added to the separatory funnel containing the DMF 119 phase. This solution was shaken and kept approximately 20 min. Then, the lower aqueous 120 phase was discarded. Afterward, the ether phase was washed three times with the Na2SO4 121 solution and was concentrated to dryness in a rotary evaporator at 30 ºC. 122 The hexane phase was saponified by adding 20 mL of methanolic KOH (35 g/100 mL) 123 and 30 mL diethyl ether. The reaction mixture was maintained with mechanical shake 124 overnight, in darkness and at room temperature. Then, the organic phase was washed with 125 water and a solution of 10 g/100 mL NaCl until neutrality of the waste water. Finally, the 126 remaining water was removed with the Na2SO4 solution. The organic phase was concentrated 127 to dryness by rotary evaporation at 30 ºC. 128 The two extracts obtained from each sample (DMF and hexane extracts) were kept in 129 the freezer under a nitrogen atmosphere. Prior to injection in the HPLC system, the two 130 extracts were re-dissolved in 300 µL of ethyl acetate and filtered through a nylon membrane 131 (13 mm ´ 0.45 µm) (Billerica, MA, USA). The injection volume was 20-μL. In samples 132 where the concentrations of squalene and β-sitosterol were out of the linearity range of the 133 detector, the hexane phase was further diluted with approximately 1.5 mL of ethyl acetate and 134 20 µL were injected. 135 2.3.2. Qualitative and quantitative analysis 136 The analysis of isoprenoids was carried out by High Performance Liquid 137 Chromatography (HPLC) with external calibration. The concentrations of the standards were 138 determined spectrophotometrically using the specific extinction coefficients in ethanol 139 7 (Britton, 1995). To prepare the stock solutions the standards were dissolved in ethyl acetate. 140 HPLC analyses were carried out on an Agilent Technologies 1100 system (Agilent 141 Technologies, Palo Alto, CA, USA). A C30 YMC column (3 µm, 150 cm ´ 4.6 mm) 142 (Wilmington, NC, USA) kept at 20 ºC and a flow rate of 1 mL/min were used. The diode 143 array detector was set at 450, 410, 290, 215 and 210 nm for the detection of carotenoids, 144 pheophytins, tocopherols, squalene and β-sitosterol, respectively. The gradient was: 0 min: 85 145 % MeOH + 15 % MTBE; 20 min: 68 % MeOH + 32 % MTBE; 25 min: 85 % MeOH + 15 % 146 MTBE. MeOH contained a small proportion of ammonium acetate (0.1 g/100 mL) in order to 147 protect the carotenoids during the chromatographic analysis and to improve the recovery of 148 them from the column. 149 The identification of the isoprenoids was made by comparison of their spectroscopic 150 and chromatographic features with those of standards. For chlorophyll pigments and 151 derivatives, the characteristic peak ratio was that between the absorbance of the Soret band (I) 152 and the absorption maximum in the red region (II). For carotenoids, the height of the largest 153 wavelength absorption band (III) was expressed as a percentage of that of the middle 154 absorption band (II) (Rodríguez-Amaya, 2001). To identify the cis isomers of carotenoids the 155 hypsochromic shifts of the absorption maxima relative to those of the corresponding all-trans 156 isomers and the intensity of the cis peak (measured as DB/DII) have been taken into account 157 (Rodríguez-Amaya, 2001). 158 2.4. Colour measurement 159 Colour measurements were made on a Hewlett Packard 8453 UV–Vis diode array 160 spectrophotometer (Palo Alto, CA). Samples, without dilution, were filtered through paper 161 filter. The entire visible spectra of the filtered samples in 5-mm pathlength quartz cuvettes 162 were recorded. n-Hexane was used as blank reference due to its transparency and lipophilicity 163 (Moyano, Meléndez-Martínez, Alba, & Heredia, 2008a). The colour parameters under CIE 164 Illuminant D65 and 1964 Standard Colourimetric Observer were obtained by means of the 165 8 software CromaLab© (Heredia, Álvarez, González-Miret, & Ramírez, 2004). The colour 166 parameters of the CIELAB space (CIE, 1978), i.e. L* (lightness), a* (ranging from green to 167 red), and b* (ranging from blue to yellow), and the angular coordinates, i.e. C*ab (chroma, the 168 quantitative expression of colourfulness) and hab (hue angle, the qualitative expression of 169 colour), which are more related to the psychophysical characteristics of colour, were 170 calculated. 171 2.5. Statistical analysis 172 Data processing was performed using the IBM SPSS Statistics 20® software (SPSS 173 Inc., 2012). Analysis of variance (ANOVA) was performed in order to determine significant 174 differences (P < 0.05) in the concentrations of compounds between OM and SM. Linear 175 discriminant analysis (LDA) was performed to explore the possibility of classification of the 176 two groups of samples according to their isoprenoid composition and colour. The forward 177 stepwise and the “leave one out” methods were employed. 178 179 3. Results and discussion 180 3.1. Isoprenoid analysis 181 The main isoprenoids of interest in food science and nutrition present in olive oils, i.e. 182 β-carotene, lutein, α-tocopherol, β-sitosterol, pheophytin a and squalene were analysed. 183 Considering the retention time of each compound and the phase in which it was extracted 184 (hexane or DMF), it can be readily inferred that there were no co-elution problems (Table 1 185 and Supplementary Fig. 1). The HPLC method used is also appropriate for the analysis of 186 some carotenoid isomers, such as two cis isomers of β-carotene that were detected in almost 187 all the samples. These isomers have been identified as the 9-cis isomer and the 13-cis or 15-188 cis isomer. 9-cis-Lutein has also been detected in some samples. Although some cis isomers 189 of carotenoids have also been detected by other authors, they are not reported in the majority 190 of studies. The study of the presence of cis isomers of carotenoids in olive oil should be 191 9 encouraged as they could provide interesting information for the comparison of different 192 processing conditions or to compare the age of different oils. Zeaxanthin and antheraxanthin, 193 which are common olive oil carotenoids (Aparicio-Ruiz, Mínguez-Mosquera, & Gandul-194 Rojas, 2011; Gandul-Rojas, Cepero, & Mínguez-Mosquera, 2000), were also detected. 195 However, they were not analysed as their concentrations were very low. 196 There are many studies (Criado, Motilva, Goñi, & Romero, 2007; Giuffrida et al., 197 2011) aiming specifically at analysing the pigments in VOO by using the extraction method 198 developed by Mínguez-Mosquera et al. (1992). On the other hand, there are also studies that 199 focus on some of the isoprenoids analysed in this study by using other techniques and 200 methodologies such as GC, capillary electrophoresis, spectrophotometry, and direct injection 201 in HPLC, among others (Cañabate Díaz et al., 2007; Galeano-Díaz, Acedo-Valenzuela, & 202 Silva-Rodríguez, 2012; Karabagias et al., 2013; Manzi, Panfili, Esti, & Pizzoferrato, 1998; 203 Puspitasari-Nienaber, Ferruzzi, & Schwartz, 2002; Sivakumar, Bati, Perri, & Uccella, 2006). 204 However, extraction and HPLC methods appropriate for the determination of all the 205 isoprenoids determined in the present study are not well-described in the literature. Some of 206 the methods mentioned above could be useful for the simultaneous analysis of all these 207 isoprenoids but it would require studies to confirm it. According to a study conducted by 208 Lietz and Henry (1997), an alternative to the basic hydrolysis could be an enzymatic 209 hydrolysis using the non-specific Candida cylindracea lipase such that losses of palm oil 210 carotenoids and tocopherols due to alkaline hydrolyses are reduced. In this work the 211 enzymatic hydrolysis is performed directly without prior separation of chlorophylls. In this 212 sense, it is not clear if this approach is also appropriate for the analysis of chlorophylls and its 213 derivatives, as the phytol group in them may also be hydrolysed by the enzyme. 214 3.2. Isoprenoids content 215 The mean concentrations and the calibration curves are presented in Table 2 and 216 Supplementary Table 2, respectively. The results of the ANOVA indicated that OM had 217 16 Fitó, M, De la Torre, R., & Covas, M.I. (2007). Olive oil and oxidative stress. 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Department of Nutrition & Food Science. Universidad de Sevilla. 8 Facultad de Farmacia, 41012. Sevilla, Spain. 9 b Department of Analytical Chemistry. Universidad de Sevilla. Facultad de Farmacia, 41012. Sevilla, 10 Spain. 11 12 *Author to whom correspondence should be addressed. 13 Antonio J. Meléndez-Martínez 14 Área de Nutrición y Bromatología. Universidad de Sevilla. 15 Facultad de Farmacia. 41012. Sevilla, Spain. 16 TELEPHONE: ++34 95455 7017 17 FAX: ++ 34 95455 7017 18 e-mail: [email protected] 19 2 ABSTRACT 20 The objective of this work was verify if, based on the concentration of some key 21 isoprenoids and the colour, olive oils from a specific mill could be differentiated from those 22 from other mills by linear discriminant analysis. The isoprenoids studied were β-carotene, 23 lutein, α-tocopherol, β-sitosterol, pheophytin a and squalene, which are isoprenoids of interest 24 in food science and nutrition. 88 % of correct classification was obtained by linear 25 discriminant analysis and, therefore, a good differentiation was achieved. This fact can reveal 26 the impact of the conditions of elaboration of each particular mill on the isoprenoid content of 27 the olive oil and therefore on its colour, which is a characteristic of foods with a high effect 28 on consumers. 29 KEYWORDS 30 Carotenoids; β-sitosterol; squalene; tocopherol. 31 32 CHEMICAL COMPOUNDS STUDIED IN THIS ARTICLE 33 β-carotene (PubChem CID: 5280489); lutein (PubChem CID: 5281243); pheophytin a 34 (PubChem CID: 459387); β-sitosterol (PubChem CID: 222284); squalene (PubChem CID: 35 638072); α-tocopherol (PubChem CID: 14985). 36 3 1. Introduction 37 The nutritional quality of olive oil concerns increasingly to both consumers and health 38 systems. Olive oil consumption has been associated with a lower risk of developing diseases 39 like myocardial infarction, atherosclerosis and certain cancers, among others (Fitó, De la 40 Torre, & Covas, 2007; Pérez-Jiménez, Ruano, Pérez-Martínez, López-Segura, & López-41 Miranda, 2007). Until recently, most of the protective effects were associated almost 42 exclusively to the high proportion of unsaturated fatty acids (Pérez-Jiménez et al., 2007). 43 However, in recent years it is becoming increasingly evident the importance that minor 44 constituents of the unsaponifiable fraction, such as isoprenoids and phenolic compounds, have 45 on the benefits associated with olive oil consumption (Covas et al., 2006). Within the 46 isoprenoid fraction, there are a series of compounds like α-tocopherol, β-sitosterol, squalene, 47 carotenoids and chlorophylls, which are of great importance. Indeed, altogether, they are 48 related to the nutritional and sensory quality of the product. 49 Tocopherols exhibit vitamin E activity. They are considered potent lipophilic 50 antioxidants that can protect membrane lipids from oxidation, although non-antioxidant 51 biological actions of these compounds have also been postulated (Azzi, 2007). They are 52 commonly found in vegetable oils, fats and some vegetables like broccoli, celery and 53 tomatoes (Monge-Rojas & Campos, 2011). The main tocopherol in virgin olive oil (VOO) is 54 α-tocopherol (Haddam et al., 2014). Among phytosterols, the most abundant in olive oil is β-55 Sitosterol (Haddam et al., 2014). It has a recognized effect on lowering cholesterol 56 concentrations by interfering in the absorption of cholesterol in the intestinal tract (Trautwein 57 et al., 2003). Some studies suggest that the high concentration of squalene in olive oil could 58 be one of the factors contributing to the anti-carcinogenic effect associated to the intake of it 59 (Reddy & Couvreur, 2009). The major olive oil carotenoids are β-carotene and lutein 60 (Giuffrida, Salvo, Salvo, Cossignani, & Dugo, 2011). β-carotene is a provitamin A carotenoid 61 and continues eliciting interest as a health-promoting carotenoid. The study of the health 62 4 benefits of lutein is particularly important for its roles in the macula lutea and the brain 63 (Johnson, 2004; Krinsky & Johnson, 2005). During the extraction of olive oil, chlorophylls 64 lose the coordinated magnesium and are transformed into the respective pheophytin 65 derivatives, which are consequently the major chlorophyll pigments in them (Giuffrida et al., 66 2011). Unlike carotenoids, there are not many studies on the possible health benefits of 67 chlorophylls (Moyano, Heredia, & Meléndez-Martínez, 2010). 68 The colour of foods is an attribute closely related to their acceptability or rejection by 69 consumers, and therefore is very important in economic terms. Generally, the consumers 70 associate the observed colours with possible aromas and tastes of the product (Moyano et al., 71 2010). The characteristic colour of an olive oil depends on the concentration and the ratio of 72 the different pigments. These are classified in chlorophyll pigments, responsible for greenish 73 hues, and carotenoids, responsible for yellow hues (Giuffrida et al., 2011). Autooxidation of 74 olive oil results in losses of pigments with the concomitant decolouration (Aparicio-Ruiz & 75 Gandul-Rojas, 2014). 76 In this study, both some key isoprenoids and the colour of different VOO marketed in 77 Andalusia were determined. The main objective was to evaluate if the concentration of these 78 isoprenoids and the colour parameters can be useful to differentiate the samples from a 79 specific mill (SM) from those from other mills (OM). 80 81 2. Materials and methods 82 2.1. Reagents and standards 83 HPLC-grade reagents, i.e. methanol (MeOH) and methyl tert-butyl ether (MTBE), and 84 α-tocopherol, β-sitosterol, β-carotene, squalene, chlorophyll a and b standards were supplied 85 by Sigma-Aldrich (Steinheim, Germany). Pheophytin a and b were obtained from 86 chlorophylls a and b respectively by acidification with 0.1 mol/L methanolic hydrochloric 87 acid (Sievers & Hynninen, 1977). Lutein was isolated from a saponified extract of green 88 5 leaves by open column chromatography according to recommended procedures (Rodríguez-89 Amaya, 2001). 90 2.2. Samples 91 Thirty samples of extra virgin olive oil (EVOO) produced in different parts of 92 Andalusia were analysed. Fifteen of them (OM) were EVOO corresponding to common 93 brands retailed in Spain and were purchased at a local supermarket. The rest (SM) were 94 EVOO obtained with different olive varieties in an oil mill located in Palma del Rio 95 (Oleopalma S.A.T., Palma del Río, Córdoba, Spain) from 2015 harvest. SM samples were 96 obtained at an industrial scale. They were taken from the deposits from where the samples 97 that are eventually bottled and marketed are kept. Both, OM and SM sets includes samples 98 from different olive cultivars and geographical origins, within Andalucía. However, while 99 OM were processed in different mills, the SM set includes oils obtained according to the same 100 technological procedures. Information on the olive variety, geographic origin and type of 101 packing as well as the companies of both groups of samples is summarised in Supplementary 102 Table 1. Although in most cases the conditions of production of olive oil, i.e. temperature, 103 extraction time, etc., were not indicated, it seems sensible to assume that these conditions vary 104 between the different mills. 105 2.3. Isoprenoid analysis 106 2.3.1. Extraction and saponification 107 The samples were extracted in triplicate by using the protocol described by Mínguez-108 Mosquera, Gandul-Rojas, & Gallardo-Guerrero (1992) with some modifications. The method 109 is based on a liquid-liquid distribution so that there is a partition of the compounds between 110 N,N-dimethylformamide (DMF) and hexane. In the hexane phase, which is subsequently 111 saponified, triglycerides, carotenes (mainly β-carotene), squalene and β-sitosterol are retained 112 and in the DMF phase, xanthophylls, tocopherols and chlorophyll pigments are extracted. 113 6 Three-gram aliquots of the EVOO were dissolved directly in 30 mL of DMF saturated 114 with MgCO3 and treated with five successive 10 mL portions of hexane. The mixtures were 115 centrifuged at 3000 rpm for 5 min. at 4 °C. The two phases were transferred to different 116 separatory funnels. 117 Eighty mL of Na2SO4 solution (2 g/100 mL in water at approximately 0 °C), 15 mL of 118 hexane and 15 mL of diethyl ether were added to the separatory funnel containing the DMF 119 phase. This solution was shaken and kept approximately 20 min. Then, the lower aqueous 120 phase was discarded. Afterward, the ether phase was washed three times with the Na2SO4 121 solution and was concentrated to dryness in a rotary evaporator at 30 ºC. 122 The hexane phase was saponified by adding 20 mL of methanolic KOH (35 g/100 mL) 123 and 30 mL diethyl ether. The reaction mixture was maintained with mechanical shake 124 overnight, in darkness and at room temperature. Then, the organic phase was washed with 125 water and a solution of 10 g/100 mL NaCl until neutrality of the waste water. Finally, the 126 remaining water was removed with the Na2SO4 solution. The organic phase was concentrated 127 to dryness by rotary evaporation at 30 ºC. 128 The two extracts obtained from each sample (DMF and hexane extracts) were kept in 129 the freezer under a nitrogen atmosphere. Prior to injection in the HPLC system, the two 130 extracts were re-dissolved in 300 µL of ethyl acetate and filtered through a nylon membrane 131 (13 mm ´ 0.45 µm) (Billerica, MA, USA). The injection volume was 20-μL. In samples 132 where the concentrations of squalene and β-sitosterol were out of the linearity range of the 133 detector, the hexane phase was further diluted with approximately 1.5 mL of ethyl acetate and 134 20 µL were injected. 135 2.3.2. Qualitative and quantitative analysis 136 The analysis of isoprenoids was carried out by High Performance Liquid 137 Chromatography (HPLC) with external calibration. The concentrations of the standards were 138 determined spectrophotometrically using the specific extinction coefficients in ethanol 139 13 controversy in this respect (Di Giovacchino, Sestili, & Di Vincenzo, 2002). In this regard, it is 295 important to consider that the extraction of lutein and other xanthophylls, which are more 296 polar than carotenes, may be influenced by the addition of water in the three-phase system. 297 Besides, the use of sodium chloride as adjuvant seems to affect the pigment content and thus 298 the colour of the resulting oil (Pérez, Romero, Yousfi, & García, 2008). It has also been 299 reported that the filtered oils have a lower amount of chlorophylls than the unfiltered ones 300 (Bottino, Capannelli, Mattei, Rovellini, & Zunin, 2008). Research is needed in order to 301 evaluate if the amount of carotenoids also decreases. 302 303 4. Conclusions 304 Although the development of the chromatographic method was not the main objective 305 of this work, it is worth noting that with this HPLC method it have been able to analyse lutein, 306 β-carotene, pheophytin a, β-sitosterol, squalene and α-tocopherol, which are isoprenoids of 307 interest due to their nutritional and / or sensory importance. Moreover, the developed HPLC 308 method allows detection of some cis isomers of carotenoids. Overall, the concentrations of 309 lutein and squalene were significantly higher in the OM than in the SM (P < 0.05). The 310 comparison of pigments concentration data between the two groups of samples is consistent 311 with the results obtained by comparing colour parameters. Despite the chemical composition 312 of olive oils is dependent of numerous factors it has been found that some key isoprenoids and 313 colour parameters can be useful to differentiate the samples obtained in a particular mill from 314 those obtained in others. More specifically, 89 % of correct classifications were obtained by 315 LDA. These results highlight the importance that the processing conditions have in the 316 extraction of olive constituents and therefore in the composition of the virgin olive oils. 317 318 ACKNOWLEDGEMENTS 319 14 AJMM acknowledges funding from the Spanish State Secretariat of Research, Development 320 and Innovation (Ministry of Economy and Competitiveness, project ref. AGL2012-37610, co-321 funded by FEDER). We gratefully thank Oleopalma S.A.T. for providing samples, the 322 technical staff of Biology Service (SGI, Universidad de Sevilla) for the services offered and 323 J.M. Hernández Hierro for his help in the statistical analysis. The authors are members of the 324 IBERCAROT network, funded by CYTED (ref. 112RT0445). 325 CONFLICT OF INTEREST 326 All authors declare that there are no conflicts of interest. 327 328 REFERENCES 329 Abdalle, A.E. M., El-Difrawy, E.A., & Abdelneem, Y.F. (2008). 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