**2. Extraction of carotenoids and sample preparation for analysis**

It is challenging to define a general and common method for carotenoid extraction. The optimal extraction depends not only on the polarity of the carotenoids, but also on the characteristics of the matrix (moisture content, presence of cell wall, etc.). Additional parameters to take into consideration are the environmental and health hazards issues (with some solvents), time consuming techniques, costs, or even feasibility. In any case, the analytical parameters that should be determined for the development of an analytical method are precision, accuracy, specificity, linearity, and limits of detection and quantification. The present section discusses in detail the advantages or disadvantages of each method and its applicability.

Liquid-liquid extraction or solvent extraction, and partitioning method is the more widespread technique applied for carotenoid extraction. The selection of the organic solvent nature is not a simple task. The polarity of the solvent and the characteristics of the matrix are determining parameters for the efficiency of the extraction. Typically, nonpolar solvents are used for nonpolar carotenoids (hexane for β-carotene extraction), while polar solvents are suitable for polar carotenoids (acetone for lutein extraction). However, the selection of the proper solvent or mixture of solvent will subsequently require the optimization for each specific matrix. Thus, the hexane and the ethanol/hexane (4:3) are the solvents most frequently applied, although numerous alternatives have been assayed: acetone, ethanol, petroleum ether, tetrahydrofuran, diethyl ether, methanol, etc. The solvent or solvents mixture could be supplemented with antioxidants to avoid degradation and transformation of carotenoids during extraction. Butylated hydroxytoluene is the most well-known, although other antioxidants, such as pyrogallol and ascorbic acid, are also utilized.

MAE and UAE, or to join UAE with solvent extraction methods to enhance the efficiency of the conventional system [28]. UAE utilizes wave sounds of 20 kHz–100 MHz, generating cavitation in the medium through compression and expansion cycles, and consequently, the production of bubbles. The collapse of the bubbles induces disruption of the cell walls and the release of the cellular content. The main benefit of this technique is the extraction at low temperatures.

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PLE was developed first time in 1996 [29] and since then it has been successfully applied to different matrices. This methodology utilizes solvents at high temperature and pressure in the ranges of 50–200°C and 35–200 bar, respectively [23]. The high temperatures help in the cell wall disruption process, while the high pressure forces the contact between the solvent and the disrupted matrix [30]. A recent modification introduced in PLE technique is the pressurized hot water extraction (PHWE) allowing the claim of *green* recovery of bioactive compounds from leaves of *Stevia rebaudiana* (Bertoni), with positive results for carotenoids. Nevertheless, the application of different processing conditions including different temperatures (100, 130, and 160°C), static extraction times (5 and 10 min), and cycle numbers (1, 2, and 3)

The strategy followed by the PEF is to locate the sample matrix between two electrodes and apply intermittent high electric frequencies with intense electric fields during nanoseconds or milliseconds periods of time. When the transmembrane potential exceeds 1 V, the charged molecules initiate the repulsion, generating pores in the membranes [28], process that could be reversible or permanent. Recently, PEF has been validated as an enhancer of the carotenoid bioaccessibility [32], setting the processing parameters to increase the carotenoid amount

Since its discovery in 1879 [33], SFE has been applied with a great success mostly at industrial scale. The basis of this technique consists on to leave the substance at temperature and pressure conditions beyond its critical point. At this stage, the supercritical fluid exhibits high diffusivity and low viscosity, features of the gas phase, but develops a high solvation power, a feature characteristic of the liquid phase. Usually, the solvent is carbon dioxide as the critical temperature and pressure are relatively low. SFE can be applied as the traditional *off-line* mode, which means that the extracted analyte is collected in an external collector or in the more recent *on-line* mode, when the extracted analyte is directly transferred to the chromatographic column [34]. Following this approach, it is possible to efficiently extract the targeted carotenoids from red Habanero peppers (*Capsicum chinense* Jacq.) [35]. In comparison with the *off-line* systems, the *on-line* approach seems to improve the run-to-run precision, enables the

setting of batch-type applications, and reduces the risk of sample contamination.

The EAE performs the disruption of the cell walls by the activity of different enzymes as pectinases, cellulose, or hemicellulose. Two different strategies are applied: the cell walls and the polysaccharide-protein colloids are the targets of the enzymatic activity of the aqueous-EAE. The alternative method is the cold-EAE, where the enzymes only hydrolyze the cell walls [30].

Independently of the extraction method, sometimes the matrix required a saponification step prior the analysis. Such reaction should be considered as a clean-up process, which eliminates lipids and chlorophylls, facilitating the separation in the HPLC column. On the contrary, if saponification is necessary, it should proceed with caution as several carotenoids are prone to

did not improve the carotenoid recuperation [31].

incorporated into the micellar phase from tomato fruits.

Recent reviews have recapitulated the different extraction methods postulated for carotenoids [14–16]. One specific case is the esterified carotenoids [15]. Chemical hydrolysis cannot be applied if the aim is to determine the nature of fatty acids involved in the esterification, while the presence of triacylglycerides in the sample produces interferences in the subsequent chromatographic analysis. Therefore, the extraction methods mean the application of similar protocols applied for the extraction of free carotenoids, but the oily-dense nature of some matrices containing carotenoid esters requires clean-up steps. Different approaches have been set up, including the utilization of open column chromatography on silica gel [17] or magnesium oxide [18], enzymatic hydrolysis [19], solid-phase extraction [20], or even the use of semi-preparative isolation on C18 column and subsequent identification [21].

Although the most common method for the isolation of the carotenoid profile is solvent extraction, during the last decade, new techniques have raised becoming popular because of their *green* features and environmentally friendly practices. They include the microwaved-assisted extraction (MAE), ultrasound-assisted extraction (UAE), pressurized liquid extraction (PLE), pulse electric field-assisted extraction (PEF), supercritical fluid extraction (SFE), and enzymeassisted extraction (EAE). The development of these techniques is mainly motivated by the specific difficulties for the extraction of lipophilic content from microalgae and seaweeds species. The cell walls and the extracellular material with different polysaccharide distributions, such as agars, carrageenans, fucoidans, and alginates, limit the mass transfer of carotenoids during conventional extraction processes. The biological characteristics of those species have aimed the development of alternative methodologies to optimize the efficiency of the extraction process.

The development of MAE was first reported in 1986 [22]. During the MAE, the nonionizing electromagnetic radiations heat the extraction medium, transferring the analytes from their cellular environment. In addition, the heat evaporates the water inside the cells, increasing the pressure that increases the cell porosity and breaks cell membranes, to finally improve the solvent penetration [23]. MAE can be developed in open vessels at low temperatures, or in closed systems at high-temperature extractions. The advantage of the microwave is that heating is simultaneously produced in the whole sample, unlike classical conductive heating methods [24]. To achieve an efficient extraction, MAE requires an optimization process in function of the matrix properties, being significant the determination of the solvent-sample ratio, solid-liquid ratios, microwave power, and delivered energy equivalents [25]. This optimization process is performed by the application of experimental design with response surface methodology. MAE has been satisfactorily applied in the extraction of carotenoids from fruits [26], wastes, and by-products [27] or seaweeds [23].

UAE is recognized as the lowest effective method among the nonconventional methods for carotenoid extraction. Consequently, the trend is to combine several *green* methodologies as MAE and UAE, or to join UAE with solvent extraction methods to enhance the efficiency of the conventional system [28]. UAE utilizes wave sounds of 20 kHz–100 MHz, generating cavitation in the medium through compression and expansion cycles, and consequently, the production of bubbles. The collapse of the bubbles induces disruption of the cell walls and the release of the cellular content. The main benefit of this technique is the extraction at low temperatures.

proper solvent or mixture of solvent will subsequently require the optimization for each specific matrix. Thus, the hexane and the ethanol/hexane (4:3) are the solvents most frequently applied, although numerous alternatives have been assayed: acetone, ethanol, petroleum ether, tetrahydrofuran, diethyl ether, methanol, etc. The solvent or solvents mixture could be supplemented with antioxidants to avoid degradation and transformation of carotenoids during extraction. Butylated hydroxytoluene is the most well-known, although other antioxi-

Recent reviews have recapitulated the different extraction methods postulated for carotenoids [14–16]. One specific case is the esterified carotenoids [15]. Chemical hydrolysis cannot be applied if the aim is to determine the nature of fatty acids involved in the esterification, while the presence of triacylglycerides in the sample produces interferences in the subsequent chromatographic analysis. Therefore, the extraction methods mean the application of similar protocols applied for the extraction of free carotenoids, but the oily-dense nature of some matrices containing carotenoid esters requires clean-up steps. Different approaches have been set up, including the utilization of open column chromatography on silica gel [17] or magnesium oxide [18], enzymatic hydrolysis [19], solid-phase extraction [20], or even the use of

Although the most common method for the isolation of the carotenoid profile is solvent extraction, during the last decade, new techniques have raised becoming popular because of their *green* features and environmentally friendly practices. They include the microwaved-assisted extraction (MAE), ultrasound-assisted extraction (UAE), pressurized liquid extraction (PLE), pulse electric field-assisted extraction (PEF), supercritical fluid extraction (SFE), and enzymeassisted extraction (EAE). The development of these techniques is mainly motivated by the specific difficulties for the extraction of lipophilic content from microalgae and seaweeds species. The cell walls and the extracellular material with different polysaccharide distributions, such as agars, carrageenans, fucoidans, and alginates, limit the mass transfer of carotenoids during conventional extraction processes. The biological characteristics of those species have aimed the development of alternative methodologies to optimize the efficiency of the extraction process. The development of MAE was first reported in 1986 [22]. During the MAE, the nonionizing electromagnetic radiations heat the extraction medium, transferring the analytes from their cellular environment. In addition, the heat evaporates the water inside the cells, increasing the pressure that increases the cell porosity and breaks cell membranes, to finally improve the solvent penetration [23]. MAE can be developed in open vessels at low temperatures, or in closed systems at high-temperature extractions. The advantage of the microwave is that heating is simultaneously produced in the whole sample, unlike classical conductive heating methods [24]. To achieve an efficient extraction, MAE requires an optimization process in function of the matrix properties, being significant the determination of the solvent-sample ratio, solid-liquid ratios, microwave power, and delivered energy equivalents [25]. This optimization process is performed by the application of experimental design with response surface methodology. MAE has been satisfactorily applied in the extraction of carotenoids from

UAE is recognized as the lowest effective method among the nonconventional methods for carotenoid extraction. Consequently, the trend is to combine several *green* methodologies as

semi-preparative isolation on C18 column and subsequent identification [21].

dants, such as pyrogallol and ascorbic acid, are also utilized.

20 Progress in Carotenoid Research

fruits [26], wastes, and by-products [27] or seaweeds [23].

PLE was developed first time in 1996 [29] and since then it has been successfully applied to different matrices. This methodology utilizes solvents at high temperature and pressure in the ranges of 50–200°C and 35–200 bar, respectively [23]. The high temperatures help in the cell wall disruption process, while the high pressure forces the contact between the solvent and the disrupted matrix [30]. A recent modification introduced in PLE technique is the pressurized hot water extraction (PHWE) allowing the claim of *green* recovery of bioactive compounds from leaves of *Stevia rebaudiana* (Bertoni), with positive results for carotenoids. Nevertheless, the application of different processing conditions including different temperatures (100, 130, and 160°C), static extraction times (5 and 10 min), and cycle numbers (1, 2, and 3) did not improve the carotenoid recuperation [31].

The strategy followed by the PEF is to locate the sample matrix between two electrodes and apply intermittent high electric frequencies with intense electric fields during nanoseconds or milliseconds periods of time. When the transmembrane potential exceeds 1 V, the charged molecules initiate the repulsion, generating pores in the membranes [28], process that could be reversible or permanent. Recently, PEF has been validated as an enhancer of the carotenoid bioaccessibility [32], setting the processing parameters to increase the carotenoid amount incorporated into the micellar phase from tomato fruits.

Since its discovery in 1879 [33], SFE has been applied with a great success mostly at industrial scale. The basis of this technique consists on to leave the substance at temperature and pressure conditions beyond its critical point. At this stage, the supercritical fluid exhibits high diffusivity and low viscosity, features of the gas phase, but develops a high solvation power, a feature characteristic of the liquid phase. Usually, the solvent is carbon dioxide as the critical temperature and pressure are relatively low. SFE can be applied as the traditional *off-line* mode, which means that the extracted analyte is collected in an external collector or in the more recent *on-line* mode, when the extracted analyte is directly transferred to the chromatographic column [34]. Following this approach, it is possible to efficiently extract the targeted carotenoids from red Habanero peppers (*Capsicum chinense* Jacq.) [35]. In comparison with the *off-line* systems, the *on-line* approach seems to improve the run-to-run precision, enables the setting of batch-type applications, and reduces the risk of sample contamination.

The EAE performs the disruption of the cell walls by the activity of different enzymes as pectinases, cellulose, or hemicellulose. Two different strategies are applied: the cell walls and the polysaccharide-protein colloids are the targets of the enzymatic activity of the aqueous-EAE. The alternative method is the cold-EAE, where the enzymes only hydrolyze the cell walls [30].

Independently of the extraction method, sometimes the matrix required a saponification step prior the analysis. Such reaction should be considered as a clean-up process, which eliminates lipids and chlorophylls, facilitating the separation in the HPLC column. On the contrary, if saponification is necessary, it should proceed with caution as several carotenoids are prone to modification, transformation, or degradation in the presence of KOH. In any case, recently, it has been highlighted the importance of the gap between comprehensive extraction protocols in plant metabolomics studies and method validation [36]. Classically, the determination of specific secondary metabolites has used targeted methods for very specific purposes. But modern metabolomics consist on a data-driven approach with predictive power to determine all measurable metabolites without any preselection. Consequently, classical extraction methods cannot be strictly applied in metabolomics studies and requires validation to new purposes.

at high back pressure (as an example, HPLC works at 35–40 MPa, while UPLC can reach more than 100 mPa). The advantages of the new UPLC systems are the higher sensitivity, lower run times, and lower consumption of the mobile phases. Years ago, the commercially available columns for UPLC were packed with C18, but in the last 5 years also C30 columns with less than 2 μm of particle size are accessible. In Ref. [47], the performance of a conventional C<sup>30</sup> (3 μm particle size) column with the novel sub-2-micron C30 was compared in the analysis of 31 different carotenoids. The results have showed a better compound resolution and shorter

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Mono-dimensional (single-column or one-dimensional) chromatography can be considered the most common methodology used for carotenoid analysis. However, a recent development [48] was the application of two-dimensional chromatography to elucidate the carotenoid pattern in citrus. In this technique, the carotenoids are analyzed by two *on-line* chromatographic systems, increasing the separation power and peak capacity [49]. This modality of chromatography should be applied in the analysis of complex matrices or when the carotenoid profile is intricate. The best performance is achieved when both systems operate with complementary selectivity, that is, normal-phase × reversed-phase. A recent optimization implements UPLC in the second-dimensional chromatography increasing the resolution [50]. Even so, the practical performance of this methodology must resolve different challenges including solvent immiscibility or the peak focusing at the head of the second column [49]. To overcome these drawbacks, the normal phase applied in the first-dimensional chromatography could be replaced by supercritical fluid chromatography [35] increasing the identification power.

In this sense, a rapid ultra-performance convergence chromatography (UPC<sup>2</sup>

the determination of seven fat-soluble vitamins (vitamin A: retinol, retinyl acetate; vitamin D: ergocalciferol, cholecalciferol; vitamin E: α-tocopherol; and vitamin K: phylloquinone, menaquinone) and three carotenoids (lutein, lycopene, and β-carotene) was developed [51]. Carotenoids were separated within 3 min with an HSS C-18 SB column (3.0 × 100 mm, 1.8 μm) under isocratic conditions with a mobile phase of carbon dioxide and ethanol (75:25, v/v).

Another parameter to take note is the influence of the injection conditions in the resolution of the chromatography [52]. The authors proposed that the nature of the injection solvent could be responsible of distorted or false peaks. Parameters as the polarity index, solvent strength, injection volume, solute concentration, or solvent selectivity should be optimized before a

The main solvents utilized as mobile phases in the carotenoid separation are acetonitrile and methanol. The first solvent presents low absorbance in the range of UV light and low back pressure in the column. On the contrary, methanol is less expensive and less toxic [14]. In addition, those solvents are commonly mixed with less polar solvents as dichloromethane, ethyl acetate, tetrahydrofuran, etc., to improve the resolution power. Some methods also include ammonium acetate or trimethylamine to enhance the recovery. Mixtures of those solvents can be applied in isocratic mode, allowing reproducible retention times and relatively stable baselines [39]. As an example, the isocratic HPLC method with a resolution time of 12 min has been developed for the simultaneous detection of eight xanthophylls: 13-*cis*-lutein, 13′-*cis*lutein, 13-*cis*-zeaxanthin, all-*trans*-lutein, all-*trans*-zeaxanthin, all-*trans*-canthaxanthin, all*trans*-β-apo-8′-carotenoic acid ethyl ester, and all-*trans*-β-apo-8′-carotenal [53]. The separation

) method for

analysis time with the novel particle size.

chromatographic method is developed.
