Deep Eutectic Solvents Application in Food Analysis

Current trends in Analytical Chemistry are focused on the development of more sustainable and environmentally friendly procedures. However, and despite technological advances at the instrumental level having played a very important role in the greenness of the new methods, there is still work to be done regarding the sample preparation stage. In this sense, the implementation of new materials and solvents has been a great step towards the development of “greener” analytical methodologies. In particular, the application of deep eutectic solvents (DESs) has aroused great interest in recent years in this regard, as a consequence of their excellent physicochemical properties, general low toxicity, and high biodegradability if they are compared with classical organic solvents. Furthermore, the inclusion of DESs based on natural products (natural DESs, NADESs) has led to a notable increase in the popularity of this new generation of solvents in extraction techniques. This review article focuses on providing an overview of the applications and limitations of DESs in solvent-based extraction techniques for food analysis, paying especial attention to their hydrophobic or hydrophilic nature, which is one of the main factors affecting the extraction procedure, becoming even more important when such complex matrices are studied.


Introduction
Nowadays, humans are living in a globalized world, where it is necessary to guarantee the supply of food to a population of around 7900 million people, as well as to ensure its safe consumption, which becomes a difficult and essential task. The fact that each country has its own food regulations clearly complicates this scenario. In this sense, the development of new analytical methodologies that allow the effective and reliable analysis of foods, including the determination of pathogenic microorganisms and contaminants that can cause food poisoning or trigger food-related illnesses, are essential to guarantee the safety and quality of the food consumed around the world.
Current trends at both industrial and academia levels, are focused on the development and application of sustainable processes from an economical and environmental point of view. In this context, the development of new analytical processes is marked by the principles of the Green Analytical Chemistry (GAC), which emerged from the Green Chemistry principles in the 1990s, looking for a balance between an improvement in the quality of the results and the creation of more sustainable analytical procedures [1,2]. There is no doubt that the rapid development of analytical instrumentation, both in the miniaturization of the systems and in the improvement of sensitivity and selectivity, has contributed enormously in this regard. However, sample preparation still plays a very

Application of Hydrophilic Deep Eutectic Solvents
Since the first DES was synthesized in 2003 by Abbott et al. [7] and until 2015, most of the DESs reported in the literature were generally made up of hydrophilic compounds and, as a consequence, were soluble in water. Despite that fact clearly limits their application in certain sample preparation approaches, this type of DESs is still very useful for the extraction of different compounds of interest through simple, green and efficient procedures.
Among the main properties of hydrophilic DESs, their density values greater than that of water stand out [19]. Furthermore, and as mentioned before, these kinds of DESs are also characterized by their miscibility with polar solvents, such as water or methanol (MeOH), which is due to the hydrophilic nature of their components that contain highly electronegative groups and can form hydrogen bonds through special cases of dipoledipole interactions [20]. Table 1 shows works in which hydrophilic DESs have been used for the extraction and determination of a great variety of analytes in different food samples. Most of the DESs shown in the table were prepared by simple mixing of their components with constant stirring and heating at temperatures below 100 • C until a transparent and homogeneous mixture was obtained, and in neither case a purification process was needed. The reproducibility of the method was validated by analysing all samples in different laboratories by ICP-OES. For comparison, a CAD was used for the determination of analytes in all samples. [11] ChCl:oxalic acid (1:3) (700 µL)

As and Sb
Waste, mineral, well, tap, and river water, honey and rice (125 mL) Water samples were filtered. Rice samples were dried, ground and homogenized VA-DLLME HG-AAS 0.0075-0.0156 µg/L

MeHg and total Hg
Fish (tuna, salmon, trout, mackerel, whiting and anchovy), seafood (shrimp) and lake, dam, well and waste water (2.5 mL) Edible parts of fish were homogenised, oven-dried and frozen. Water samples were filtered and concentrated by evaporation UA-DLLME UV-Vis 0.25-0.92 µg/L 90-104% (2-5%) Different pretreatment for each Hg species. NADES phase contained 10% water (v/v). ACN was used as aprotic solvent. NADES acts as a reactive pH-controlled zwitterionic surfactant. [14] Molecules 2021, 26  The DES was mixed with 18% water (v/v). The extraction with NADES has less penalty points of AES than other techniques. [24] Molecules 2021, 26    GC-MS was used as a reference procedure. In situ DES formation between analytes (HBDs) and ChCl (HBA) supported in a hydrophilic porous membrane.

Cr(III) and
Cr(VI) Tap, river and mineral water, and rice and sausage (10 mL) Food samples were digested with HCl and all samples were filtered PAN was used as a complexing agent. [64] The extraction capacity of the DESs was compared with the one of the EtOH-water medium. ChCl:urea DES provided better results, but DES with ethylene glycol provided the best performance in terms of recovery.
However, and as it has been previously mentioned, the miscibility of hydrophilic DESs with water generally limits their direct application to aqueous samples. For this reason, in these cases, a dispersing or emulsifying agent (generally tetrahydrofuran, THF, or acetonitrile, ACN) is usually added to obtain a cloudy solution, achieving the separation of the two liquid phases after a shaking and centrifugation step in DES-based DLLME procedures [12,40,42,[44][45][46][47][48][49][50][51][52]54,56]. Nonetheless, the addition of this solvent has negative consequences since it may reduce the environmental friendliness and increases the laboratory hazards. On the other hand, its properties, such as viscosity or density, are those that will allow better or worse retention of the analytes [1,6]. In general, hydrophilic DESs tend to have a relatively high viscosity, which makes an effective mass transfer in the extraction processes difficult, so a common practice to reduce their viscosity to suitable values consists in the addition of a known amount of water to hydrophilic DESs using the heating method [15,21,24,25,29,31,33,35,37], in which known concentrations of the three components (HBD, HBA and water) are mixed under constant stirring in a water bath (generally at 50 • C) until a homogeneous and transparent liquid is obtained. In certain cases, water has even been replaced by an organic solvent, such as ethanol (EtOH) or MeOH [43].
Both viscosity and density values vary depending on the components of DES. For example, in the case of viscosity, DESs containing ChCl as HBA are more viscous when HBD is an acid (values up to 14,480 cP in ChCl:citric acid, 1:1 [74]) than when it is an alcohol (values lower than 400 cP [75]) due to the greater presence of hydrogen bonds between HBA and HBD. In addition, within the acids, citric acid contributes to a higher viscosity DESs, with values up to 437,768 cP (glucose:citric acid 1:1 [76]). Furthermore, the effect of the addition of water can be clearly observed in ChCl:citric acid (1:1) DES, whose viscosity decreases to 4080.8 cP [76] when amounts less than 50% of water are added, otherwise the eutectic properties would be lost, and dissolution of the individual DES components in water would occur. On the other hand, the change in the molar ratio also affects the viscosity of DES. In this way, in some cases if the proportion of ChCl is decreased, the viscosity is increased, such as ChCl:glycerol, which increases from 234 cP (1:1) to 301 cP (1:2), or ChCl:fructose, where it increases from 28.31 cP (1:1) to 72.42 cP (1:2) [75]. In the case of density, it can be ranged between 1.0 and 1.5 g/mL. The different values will depend on the molecular weight of the components, so for example, ChCl:maltose (1:2) will have a higher density (1.431 g/mL [77]) than ChCl:1,2-propanediol (1:2, 1.04 g/mL [78]), as if changing the HBA by one of higher molecular weight such as citric acid (citric acid:glucose, 1:1, 1.442 g/mL [76] compared to ChCl:glucose (1:1, 1.27 g/mL [79]). Furthermore, the addition of water decreases the density of DESs, for example, when adding 40% water to citric acid:glucose DES, the density decreases to a value of 1.246 g/mL [76]. From a procedural point of view, it is worth mentioning the work of Shishov and co-workers [60], in which the authors synthesized in situ different deep eutectic mixtures (DEMs) based on the combination of the analytes (phenols, HBDs) and ChCl (HBA) supported in a hydrophilic porous membrane. For this, square membranes (10 × 10 mm) were picked by syringe needle, as can be seen in Figure 1, and impregnated with a ChCl solution. After drying in an incubator, it was placed in a vial containing the sample mixed with hexane and shaken. After that, the syringe needle with the membrane was withdrawn, the hexane was evaporated and was introduced into a vial containing ultra-pure water. Then, it was shaken to promote analytes desorption, since this membrane type allows microextraction from organic sample phase and back-extraction of the analytes into aqueous phase. Finally, the aqueous phenol solution obtained was injected in the high-performance liquid chromatography (HPLC) system coupled to a fluorescence detector (FD). Several types of membrane were studied, being the poly(vinylidene fluoride-co-tetrafluoroethylene) the one that provided maximum extraction recovery. This membrane-based microextraction was used for the separation of six phenols from smoked sausages and smoked fish samples, showing good selectivity due to the formation of DEMs between ChCl and analytes. Good sensitivity with limits of detection (LODs) between 0.3 and 1.0 µg/kg and high extraction capacity with extraction recovery values ranging from 70 to 80% were obtained.
alcohol (values lower than 400 cP [75]) due to the greater presence of hydrogen bonds between HBA and HBD. In addition, within the acids, citric acid contributes to a higher viscosity DESs, with values up to 437,768 cP (glucose:citric acid 1:1 [76]). Furthermore, the effect of the addition of water can be clearly observed in ChCl:citric acid (1:1) DES, whose viscosity decreases to 4080.8 cP [76] when amounts less than 50% of water are added, otherwise the eutectic properties would be lost, and dissolution of the individual DES components in water would occur. On the other hand, the change in the molar ratio also affects the viscosity of DES. In this way, in some cases if the proportion of ChCl is decreased, the viscosity is increased, such as ChCl:glycerol, which increases from 234 cP (1:1) to 301 cP (1:2), or ChCl:fructose, where it increases from 28.31 cP (1:1) to 72.42 cP (1:2) [75]. In the case of density, it can be ranged between 1.0 and 1.5 g/mL. The different values will depend on the molecular weight of the components, so for example, ChCl:maltose (1:2) will have a higher density (1.431 g/mL [77]) than ChCl:1,2-propanediol (1:2, 1.04 g/mL [78]), as if changing the HBA by one of higher molecular weight such as citric acid (citric acid:glucose, 1:1, 1.442 g/mL [76] compared to ChCl:glucose (1:1, 1.27 g/mL [79]). Furthermore, the addition of water decreases the density of DESs, for example, when adding 40% water to citric acid:glucose DES, the density decreases to a value of 1.246 g/mL [76]. From a procedural point of view, it is worth mentioning the work of Shishov and co-workers [60], in which the authors synthesized in situ different deep eutectic mixtures (DEMs) based on the combination of the analytes (phenols, HBDs) and ChCl (HBA) supported in a hydrophilic porous membrane. For this, square membranes (10 × 10 mm) were picked by syringe needle, as can be seen in Figure 1, and impregnated with a ChCl solution. After drying in an incubator, it was placed in a vial containing the sample mixed with hexane and shaken. After that, the syringe needle with the membrane was withdrawn, the hexane was evaporated and was introduced into a vial containing ultra-pure water. Then, it was shaken to promote analytes desorption, since this membrane type allows microextraction from organic sample phase and back-extraction of the analytes into aqueous phase. Finally, the aqueous phenol solution obtained was injected in the high-performance liquid chromatography (HPLC) system coupled to a fluorescence detector (FD). Several types of membrane were studied, being the poly(vinylidene fluoride-co-tetrafluoroethylene) the one that provided maximum extraction recovery. This membrane-based microextraction was used for the separation of six phenols from smoked sausages and smoked fish samples, showing good selectivity due to the formation of DEMs between ChCl and analytes. Good sensitivity with limits of detection (LODs) between 0.3 and 1.0 μg/kg and high extraction capacity with extraction recovery values ranging from 70 to 80% were obtained.  Hydrophilic DESs have also been combined with other materials and used in the extraction of the compounds of interest. For example, a magnetic nanofluid (MNF) consisting of a DES (ChCl:thiacetamide, 1:2 molar ratio) based magnetic multi-walled carbon nanotubes (MWCNTs) was successfully applied by Shirani et al. [71], combining the excellent properties of DESs with the magnetic features of magnetic nanomaterials. In this work, the authors sonicated the mixture of magnetic MWCNTs and the previously synthesized DES to obtain a homogenous black gel called DES-MNF. Then, the sample and the DES-MNF were mixed by rapidly suctioning and dispensing repeatedly for six times with a syringe, after which a turbid solution was obtained. The upper phase was eliminated by retaining the DES-MNF with an external magnet and a 1 M nitric acid solution was added to desorb the analytes. Finally, 10 µL of the supernatant solution were injected in an electrothermal atomic absorption spectroscopy (ETAAS) system. The proposed method showed high extraction capacity and good sensitivity for the determination of Cd, Pb, Cu and As from walnut, rice, tomato paste, spinach, orange juice, black tea and water samples.
In addition to the previously mentioned combinations, similar to what is made with ILs, DESs-based polymeric sorbents can also be synthesized and have been applied in food analysis. These poly(DES)s have emerged as promising alternatives to conventional sorbents used in solid-phase extraction (SPE) techniques, as they combine the properties of DESs and those of porous materials. As an example, Abdolhosseini et al. [68] prepared a DES of tetrabutylammonium bromide (TBABr) and acrylic acid (1:2 molar ratio) and polymerized it under solventless condition, through a cost-efficient and energysaving photopolymerization process. DES polymerization consisted of mixing under a nitrogen atmosphere and at room temperature for 60 min the previously prepared DES, ethylene glycol dimethacrylate (used as crosslinker) and 2-hydroxy-4 -(2-hydroxyethoxy)-2-methylpropiophenone (used as photoinitiator) in a 100:10:1 weight ratio. The resulting homogeneous mixture was exposed to UV light and washed to remove any unreacted monomers. The synthesized polymeric DES was used for preconcentration of lead from vegetables such as onion, celery, carrot and tomato, as well as from mineral water samples through a dispersive SPE procedure, to later proceed to its quantification by flame atomic absorption spectroscopy. The results showed that the polymeric DES allowed obtaining acceptable selectively, low LOD (2 µg/L) and high stability since it can be reused 16 times without a significant reduction in the recovery.
As it can be seen, Table 1 also compiles several works in which hydrophilic NADESs have been used for the extraction of a wide variety of analytes. In order to demonstrate that their application constitutes a greener and more environmentally friendly alternative extraction procedure, in some of these works, its greenness has been evaluated according to the penalty points of an analytical eco-scale [80] calculated by considering hazards, amount of reagents, energy and waste, just like López et al. [24] did, who only obtained two penalty points in their developed method for the extraction of three free seleno-amino acids in lyophilized samples of seleno-biofortified sheep milk and cow milk powder and determination by liquid chromatography (LC)-inductively coupled plasma-mass spectrometry (ICP-MS). Another method used to evaluate the toxicity of hydrophilic NADESs has been bacterial growth inhibition. In this way, Huang et al. [30] performed a culture with two Gram-positive (S. aureus and L. monocytogenes) and two Gram-negative (E. coli and S. enteritidis) bacteria, which were incubated in a nutrient agar medium with a filter paper soaked with each of the thirteen NADESs they tested to extract rutin from tartary buckwheat hull. It was shown that none of the NADESs led to a decrease in the growth of bacteria with the exception of glycerol:L-arginine NADES, because, despite the fact that the individual components are nontoxic and were approved by the European Food Safety Authority [81,82], there occurs a charge delocalization as a result of a hydrogen bond, which makes the eutectic mixture toxic [83].
The great extraction capacity shown by the hydrophilic DESs together with the sophisticated detection techniques used, have allowed to obtain low LODs, in the order of µg/L or µg/kg in most cases as can be seen in Table 1, or even ng/L or ng/kg [12,13,39,42,46,65,71].

Applications of Hydrophobic Deep Eutectic Solvents
Despite the previously mentioned limitations of hydrophilic DESs, it was not until 2015 that van Osch et al. [84] presented some DESs with hydrophobic properties for the first time. These DESs were characterized by the immiscibility of their two components with water, resulting in a low water content after being mixed with this solvent (approx. 1.8 wt%) and a low leaching of the quaternary ammonium salts (approx. 1.9 wt%). These hydrophobic solvents consisted of a long chain alkyl quaternary ammonium salt (e.g., tetrabutylammonium chloride (N 4444 Cl), methyltrioctylammonium chloride (N 8881 Cl), tetraheptylammonium chloride (N 7777 Cl), tetraoctylammonium chloride (N 8888 Cl), methyltrioctylammonium bromide (N 8881 Br) and tetraoctylammonium bromide (N 8888 Br)) and poorly soluble carboxylic acids (e.g., decanoic acid), and their extraction capacity was evaluated by extracting volatile fatty acids from diluted aqueous solutions. Since then, multiple HDESs based on neutral compounds have also been proposed, including combinations of monoterpenes with fatty acids [85], tetraalkylammonium halides with fatty acids and alcohols [86,87], fatty acids with fatty acids [88], and monoterpenes with monoterpenes [17]. Many of the HDESs have also been designed and classified following the same classification that had been previously proposed and that was already used for hydrophilic DESs (type I, II, III and IV), but due to their need to be stable in the aquatic environment, they are mainly grouped into type III (a combination of a quaternary salt (HBA) with a HBD) and type IV (a combination of metal chloride with HBD) [89].
The main difference between hydrophilic and hydrophobic DESs lies in the presence of long alkyl chains or cycloalkyl groups, which reduces the effect of hydrophilic zones (e.g., charges of the salts) and hydrophilic groups (e.g., carboxylate and hydroxyl groups) [1,90]. These eutectic mixtures have unique properties of density, acidity, polarity, viscosity and volatility, which provide a good extraction capability through a careful selection of their components [89]. As a consequence, it has been found that the extraction efficiency of HDESs depends to a great extent on their immiscibility with water as a function of the difference in density. Thus, in contrast to hydrophilic DESs, HDESs generally have lower density values than water [19], since the increase in the length of the alkyl chain of the salt components results in a decrease in density (within 0.80-1.10 g/mL) [91], although, for example, DESs containing fluorinated alcohol (e.g., hexafluoroisopropanol, HFIP) are generally denser than water (around 1.5 g/mL) [59]. In addition, it is necessary to take into account that the greater the difference in density between DES and water, the more easily the separation between the two phases will occur [91].
On the other hand, most HDESs have a melting temperature below 25 • C, which allows them to be used as solvents or reaction media in different applications at room temperature [90,92]. However, it should be noted that increasing the alkyl chain of the component acting as HBD, like carboxylic acids, increases the melting point of HDESs, while increasing the alkyl chain of the ammonium salt results in a lower melting point [90]. It is also important to highlight that the hydrophobicity of DESs is also affected by the structure of their individual components in such a way that the longer the alkyl chain of the components (both in HBA and HBD), the lower the solubility in the aqueous phase of each of them as well as of the DES [89]. Regarding the viscosity of HDESs, it is usually high because the hydrogen bonds that are established between its components, decrease the movement of the HDES molecules. However, these eutectic mixtures, like the hydrophilic ones, show a wide range of viscosity (between 2.6 and 5985.0 cP), since it depends on the components that make up the HDES, especially the one that acts as HBA, which allows us to design solvents for specific tasks depending on their handling capacity [90]. Among them, it is possible to differentiate between neutral HDESs (such as menthol:decanoic acid 1:2, 27.7 cP) that are less viscous than ionic HDESs (such as N 4444 Cl:decanoic acid 1:2, 265.3 cP), and within the latter, the HDESs that contain the bromide anion (such as N 8881 Br:decanoic acid 1:2, 576.5 cP) are more viscous than those that contain the chlorine anion (such as N 8881 Cl:decanoic acid 1:2, 472.6 cP) [91]. Likewise, as in hydrophilic DESs, an increase in temperature leads to a decrease in viscosity [93,94].
As mentioned above, HDESs are mainly characterized by their immiscibility in the aqueous phase. For this reason, it is necessary to study the stability of HDESs in contact with water, so that there is no leaching or loss of its components towards the aqueous phase, as well as that their water content is practically zero [84]. Many of the HDESs formed through the combination of a hydrophobic and a hydrophilic component have been found to be unstable in water. This is because the hydrophilic component tends to leach into the aqueous phase. For example, Florindo et al. [95] showed that the DES formed by DLmenthol:dodecanoic acid (2:1, molar ratio) was stable in contact with water compared to DL-menthol:acetic acid (1:1, molar ratio) and N 4444 Cl:octanoic acid (1:2, molar ratio) when comparing the 1H NMR spectra of each one of them. As a consequence, Shishov et al. [6] proposed a new term for these unstable HDESs in aqueous phase: "quasi-hydrophobic DES", since it would not be appropriate to consider them as HDESs. In most of the works collected in this review, a study has not been carried out to verify if the extraction is due to a HDES or to one of its components as consequence of the leaching of the other one, as it was verified, for example, in the study carried out by Ortega-Zamora et al. [96]. That is why in this section both the HDESs and the quasi-hydrophobic DESs that have been used for the analysis of food samples, have been grouped and are shown in Table 2.         In general, all the DESs included in the table have been synthesized following the same guidelines as for hydrophilic DESs: mixing the components while heating with constant stirring until a homogeneous mixture is obtained. In addition, although most HDESs used in food analysis are made up of two components, some ternary DESs have been designed, which show numerous advantages over traditional DESs, such as a lower viscosity and melting point, and even a better extraction efficiency in some cases [17,114]. It is the case of the work of Shishov and co-workers [103], in which different quaternary ammonium salts, carboxylic acids and medium chain fatty acids were studied as components of a DES. The best results were obtained with the DES formed by TBABr:malonic acid:hexanoic acid (1:1:1 molar ratio) and it was used in the sequential extraction of sulfonamides from chicken samples through a DLLME followed by HPLC-UV. First, an attempt was made to carry out the extraction using a DES formed by TBABr and hexanoic acid but, due to its high viscosity and lack of acidic media, a high mass-transfer from the solid phase did not occur. However, with the introduction of a third component, in this case a carboxylic acid, an increment in the extraction efficiency was observed due to the formation of hydrogen bonds between hexanoic acid and the analytes. The introduction of hexanoic acid not only has benefits during the extraction process, but also produces a decrease of DES viscosity, enabling its direct injection in the chromatographic system. Some HDESs have even been mixed with other materials, such as Fe 3 O 4 magnetic nanoparticles (m-NPs) to form a nanoferrofluid, which speeds up the sample preparation procedure and makes it more sustainable to perform the preconcentration of various analytes in complex food samples before their injection in different chromatographic systems [17,120]. For its preparation, the Fe 3 O 4 m-NPs and the DES are separately synthesized and then mixed under constant stirring until a homogeneous fluid is obtained: the DES-based nanofluid [17,120]. As an example, Fan et al. [17] synthesized one of these nanofluids based on a ternary HDES composed of menthol, borneol and camphor in a 5:1:4 molar ratio. They used it for the extraction of 14 PAHs in 12 kinds of coffee samples after four different roasting conditions, which were separated and determined by HPLC-FD. LODs in the order of ng/L for all analytes and recovery values between 91.3 and 121%, showed the excellent performance of the methodology, which allowed verifying that the content of PAHs in the samples of coffee was modified depending on the temperature and time conditions of the roasting of its beans.
Currently, studies on the synthesis and application of HDESs for the extraction of a great variety of analytes from food matrices have expanded rapidly, which has led to an increase in the number of articles published in recent years. As can be seen in Table 2, several HDESs have been used for the extraction of both organic (phthalic acid esters [18,96,99], dyes [97,104,110,117], PAHs [17,85], sterols [98], pesticides [100,105,112,122,124,126,127], herbicides [102], insecticides [125], preservatives [101], pigments [86], antibiotics [87,103,108,114], fluorescent whitening agents [106], vitamins [107], mycotoxins [16], bisphenols [115], perfluoroalkyl substances [120] and terpenes [121]) and inorganic compounds (Co, Cd, Ni, As, V and Pb [109,111,113,116,118,119,123]) from aqueous phases (water [96,113,118,124], soft drinks [18,85,86,96,110], infusions [18,86,102,124], coffee [17], dairy products [86,99,108,111,114,126], fruit juices [16,86,87] and wine [116]). However, sauces [97,104], oils [97,100,120], egg yolk [97,125], jelly [110,117], honey [105] and solid food (meat [103], fish [106], flours [107], spices [121], dried fruits [112,119], vegetables [98,113,118,122,123] and fruits [101,115,118]) samples have also been analysed using HDESs. It is important to mention that, due to the complexity of some of the studied matrixes, different previous treatments have been needed in most cases. In this way, water [96,113,118,124], infusions [18,86,124], and soft drinks [18,85,86,96,110] were analysed without additional treatment or after degasification or filtration, while others such as honey [105] or fruit juices [16,86] were analysed after dilution with water and, in some cases, filtration. Likewise, procedures such as lyophilization or a previous extraction with an organic solvent (e.g., n-hexane, ACN, MeOH or acetone) were very useful in the treatment of egg yolk, olive oil, chili sauce, honey and some fruit juices, although it may be contradictory with the development of green sample preparation procedures using DESs. As a specific example, a hydrophilic DES has even been used in the treatment of oil samples to reduce the matrix effect [100]. In those cases, in which the matrix is more complex, more laborious procedures previous to the extraction of the compounds of interest are needed. For example, in the case of milk or yogurt, a deproteinization is usually carried out with ACN [114], although (NH 4 ) 2 SO 4 [108] has also been employed. When it comes to solid samples such as dried fruits, cereals, onion, parsley or even dairy products (milk, yogurt, cheese, etc.), they are usually digested with H 2 O 2 :HNO 3 (1:3, v/v) [109], although in some cases only HNO 3 is used [119,123], before the application of microextraction techniques. However, if a previous step such as QuEChERS (quick, easy, cheap, effective, rugged and safe) [112], is carried out before the extraction technique in dried fruits for example, it would only be necessary to grind and homogenize them.
Nowadays, the above-mentioned low solubility of HDESs in aqueous samples has allowed their large application as extraction solvents in microextraction methods, complying with the principles of GAC. Among the different variants of the LPME, DLLME constitutes once more one of the preferred options for the application of HDES for the analysis of samples of diverse nature, including water [96], soft drinks [85,110], infusions [124], honey [105], dried fruits [119], flour [107], fruit juices [124] and egg yolk [125]. In this sense, it is important to highlight that in certain cases, no additional solvents have been necessary to obtain a good dispersion of the HDES into the sample [18,86,[96][97][98]110]. Instead, the DLLME procedure has been assisted in different ways, including the vortex-assisted DLLME [86,97,99,100,104,106], ultrasound-assisted DLLME [16,85,87,101,113,116,123,124], air-assisted [109] and microwave-assisted DLLME [128]. However, other versions have also been used, such as DLLME based on the solidification of the floating organic drop (SFO) [18,115,122,126]; salting out-DLLME-back extraction or salt induced-homogenous liquid-liquid extraction-DLLME, in which a salt is added (e.g., NaCl, (NH 4 ) 2 SO 4 , Na 2 SO 4 or NH 4 Cl) to reduce the solubility of the analytes in water and to increase their distribution coefficients in the organic phase [108,114]; effervescence-assisted DLLME in which an effervescent reaction between a proton donor solvent (acetic acid, which has been previously mixed with HDES in a 3:1 (v/v) ratio) and an effervescent agent (sodium bicarbonate) is produced generating carbon dioxide that facilitates the dispersion of the extraction solvent (HDES, see Figure 2) [117]; or even the combination of DLLME with a previous extraction and clean-up stage, such as the QuEChERS method, in which the extracted supernatant was used as a dispersant in the following DLLME for further purification and preconcentration [112]. Another interesting modification of the LPME technique is the use of the above-mentioned ferrofluid as an extraction solvent [17,120]. On the other hand, in order to improve the sensitivity towards volatile compounds, headspace microextraction techniques have been used, such as headspace SDME, which is faster and cheaper than other extraction methods, and allows extracting a wide range of components with diverse physicochemical properties in matrices where no pre-treat- On the other hand, in order to improve the sensitivity towards volatile compounds, headspace microextraction techniques have been used, such as headspace SDME, which is faster and cheaper than other extraction methods, and allows extracting a wide range of components with diverse physicochemical properties in matrices where no pre-treatment is necessary. As an example, Triaux et al. [121] used a HDES (tetrabutylammonium bromide (N 4444 Br):dodecanol, 1:2 molar ratio) as extracting solvent for the extraction of 67 terpenes from six spices (cumin, cinnamon, clove, fennel, nutmeg and thyme) used as bought without additional grinding. The DES was introduced into the needle of a GC microsyringe, which was inserted into the headspace of the vial where the sample was located. The DES was pushed to form a drop at the tip of the needle and was left for 90 min at 80 • C for the absorption of the volatile analytes on the DES drop. The extracts were analysed by GC-MS obtaining limits of quantification (LOQs) between 0.47 and 86.40 µg/g.
As with hydrophilic DESs, hydrophobic NADESs can also be synthesized from natural compounds immiscible with water. As mentioned above, apart from all the inherent advantages of HDESs, they fully represent the GAC principles, since they are easily prepared, cost-effective and are not harmful to the environment [129]. In addition, as a result of their diverse compositions (among the components most used as HBA, menthol, thymol and camphor stand out, while carboxylic acids are usually used as HBDs), they have a wide range of polarity and physical properties [8]. Hydrophobic NADESs have also been used in food analysis, although they are still not very abundant compared to the use of "non-natural HDESs", as shown in Table 2. It is worth highlighting the work of Soltani and co-workers [100], who synthesized a hydrophobic NADES composed of thymol and vanillin (1:1, molar ratio in which a transparent yellow liquid remained). They calculated its solubility in water (0.005%, w/v) and its octanol/water distribution constant (log K OW = 4.30) to verify its hydrophobicity. Furthermore, they found that the thymol:vanillin (1:1) DES was stable for at least one week at ambient conditions. The authors used it as an extraction solvent in VA-LLME coupled with GC-µECD for the determination of 16 pesticides in olive oil samples (extra virgin, virgin and refined olive oils), complex food samples that showed a high matrix effect. Therefore, the authors developed a DES-based liquid-liquid solvent system (n-hexane/ACN/DES) to achieve cleaning the sample as much as possible and, thus, improve sensitivity and reduce the matrix effect. To do this, they mixed the sample with n-hexane, and then with ACN (used as extraction solvent) and a hydrophilic NADES composed of ChCl and urea. After shaking and centrifuging, a triphasic system was observed in which the medium layer was the ACN that contained the pesticide residues and was used for performing the preconcentration procedure worked up. The develop method provided high recovery percentages for the analytes (between 63.1-119.4%) with high precision (relative standard deviation values in the range 2-7%), and it was also simple and sensitive with LODs in the range 0.01-0.08 µg/kg.

Conclusions
Considering the current trends in the Analytical Chemistry field, DESs constitute a very interesting alternative to conventional solvents, not only because of their interesting physicochemical properties, but because they have made it possible to develop more sustainable analytical procedures, from an environmental point of view due to their low toxicity, and also from an economic point of view due to their general low cost and the simplicity of their synthesis. As with other solvents widely used in sample preparation techniques, the wide variety of HBD and HBA available make it possible to configure a large number of DESs, which has allowed their application for the extraction of a large number of organic and inorganic analytes from diverse food matrices. In this sense, the hydrophilic or hydrophobic nature of DES plays a fundamental role since it has a great influence on the extractive process. However, few studies have evaluated this aspect, as well as their toxicity. In fact, the greenness of many DESs currently used as green solvents has not been fully even evaluated, and many of them actually continue to present significant toxicity or pose a risk to the environment, in many cases due to synthesis procedures that use conventional solvents. In this context, the introduction of NADESs opens a window of hope for reducing the impact of this type of solvent on the environment.
It is also important to mention that, even with their previously mentioned great properties, there are still some aspects that limit the application of DESs to the analysis of food samples from an operational point of view, such as the miscibility of the hydrophilic ones with aqueous samples or the need of including organic solvents to provide a good dispersion of the DESs or to decrease their viscosity, which goes against the principles of developing sustainable analytical methodologies. Besides, the limited number of cheap, readily available and biodegradable components (especially in the case of hydrophobic NADESs) for the synthesis of HDESs also pose a limitation for their use in food analysis.
Despite the aforementioned problems that the use of DESs still poses today, this type of new solvents still has a wide margin for improvement and are proposed as an alternative for the future to be taken into account, not only at an analytical level, but also in a wide range of applications.