The use of NADESs as extraction solvents for the preparation and pretreatment of analytical samples, an essential step in almost every analytical procedure, is a fundamental and promising application [53,54,55]. Due to their high biocompatibility, stabilization, and solubilization abilities, NADESs have been used to extract bioactive compounds from natural products and different contaminants from food samples [30, 56,57,58,59,60,61,62]. The following pages will discuss NADES-based extraction methods of metals, pesticides, PAHs, antibiotics and mycotoxins from food samples, with some relevant examples reported in the literature.

Metal and metalloid analyses in food are an issue of global concern. Metals such as Zn, Cu, Mn, and Fe are essential in the human diet but can be toxic in high concentrations. Other heavy metals, such as As, Cd, Pb, and Se, are risky to human health even at trace levels. They are hazardous due to their toxicity, bioaccumulation, persistence, and non-biodegradable nature. The occurrence of these contaminants in the environment is due to natural sources (i.e., biogenic, terrestrial, marine, volcanic, forest fires, and erosion) and anthropogenic activities (i.e., industrialisation, urbanisation, inadequate waste handling, and energy generation) [63]. Almost 90% of human heavy metal intake comes from vegetable consumption. They can reach crop plants mainly through soil, water, and air. The content of plant carbohydrates, proteins, amino acids, fats, and fatty acids can be altered due to the presence of metals and metalloids, varying the nutritive properties of these foodstuffs [64]. Heavy metals can also be present in herbivorous animals and bioaccumulated in fish, so their consumption could also pose a human health risk.

There are numerous sample treatment approaches to extracting and analysing heavy metals in foodstuffs. The simplest is the direct analysis of heavy metals in liquid foods, but matrix interferences can occur in their detection. To overcome these possible interferences, diluting the sample in a suitable solvent is an alternative. However, sensitivity problems can be observed due to the decreased metal concentration. Organic solvents are usually employed to dilute the samples, but their toxic nature is well-known for humans and the environment [65]. Sample pretreatment by decomposition procedures is widely used for heavy metal analyses. They are based on the total or partial removal of the organic content. Dry ash and wet digestion are the most widespread methods for that purpose. Both have proven to be efficient but have several disadvantages. Decomposition by dry ash is very time-consuming and requires very high temperatures (500–600 ºC), which causes the loss of volatile elements. Wet digestion uses large inorganic acid volumes handled at elevated temperatures and pressure, posing a danger to the user and high costs [66]. Temperature and pressure working conditions can be significantly reduced using microwave and ultrasound-assisted extraction protocols [67,68,69], but large volumes of hazardous solvents are still used. Several organic solvents and ionic liquids have been widely used to extract heavy metals from foodstuffs in combination with different extraction methods [70,71,72,73,74,75,76,77,78]. However, it is well known that organic solvents are toxic to humans and harmful to the environment, and ILs, although a more environmentally friendly alternative, have poor biodegradability and synthesis and very varied toxicity and stability. NADESs are environment-friendly solvents and allow extraction processes to be carried out with reduced times and temperatures. Therefore, these green solvents are an excellent alternative for extracting heavy metals from food products, overcoming the abovementioned limitations [79]. Various authors have used NADESs as solvents for extracting heavy metals in different matrixes. Metal ions are extracted by forming ion-association complexes with other chelating agents. Before the NADES extraction procedure, a digestion step is needed to eliminate the organic matter and dissolve the ions. The selected NADES must be compatible with the chelating agent employed in these cases.

Nail Altunay et al. have recently studied the ability and possibilities of NADESs as extractants of different heavy metals in several foodstuffs, as shown in Table 1. They have applied the use of NADESs for the extraction of Cu, Pb, and Cd in honey [80], As and Sb in honey and rice [81], total Hg in fish [82], As and Se in rice [83] and Pb and Cd in vegetables [84] samples. In all cases, a standard methodology was successfully applied. Before extraction with NADESs, samples were conditioned and treated by wet digestion. This digestion was necessary to dissolve the metal ions and remove the organic matter. In the extraction stage using NADESs, the metal ions were chelated with different chelating agents under specific pH conditions that allowed the formation of the ion-association complex. After this step, the complexes were extracted with the appropriate NADES. Ultrasound- (UA) or vortex-assisted (VA) dispersive liquid–liquid microextraction (DLLME) was the extraction procedure in all cases. The many parameters for the extraction, such as the type, composition, and volume of the NADES, the type and concentration of the dispersant solvent, the volume of the pretreated sample, and the type and volume of the chelating agent, were optimised.

Specific physicochemical properties such as polarity, density, viscosity, and surface tension must be considered to select the optimal NADES for each application [82]. The constituent compounds of the solvent can be combined in different ratios and supplemented with a percentage of water that can modify the above parameters. All these possibilities demonstrated the versatility and tunability of NADESs. Therefore, different NADESs with different properties were used in each publication to determine the optimum in each case. The selection of the chelating agent and the pH of the solution were essential to ensure the selectivity of the extraction method. The appropriate chelating agent under optimal pH conditions can form the ion-association complex with the target metal ions and not with other common interfering cations and anions. Therefore, interference studies were successfully carried out, and tolerance limits were established for each interfering ion. All the NADES-based methods were validated using certified reference materials, with no significant differences from the certified values. The methodologies were successfully applied to various samples, obtaining recoveries close to 100% with a precision below 5% for all analytes in the analysed samples. Speciation is essential to determine the total content of an element reliably and not just in a particular oxidation state. In different works [81,82,83], a reduction stage is added in the pretreatment step, ensuring that all the metal content is in a single species to determine its total content.

Heavy metal detection was performed with simple and cheap atomic and molecular spectrometry techniques. Therefore, the detection limits must be highlighted because they are lower or at least comparable to those obtained by other authors with other extraction methodologies and even with more expensive and complex detection techniques (i.e., ICP-MS and ICP-OES). Even though the solvent extraction used is green, another critical part of the extraction process uses substances that are not environmentally friendly and dangerous for humans. In some cases, previous wet digestions still use hazardous acids at high temperatures. In addition, organic solvents are used in all DLLME as dispersant solvents (i.e., acetonitrile and tetrahydrofuran) and to reduce the viscosity of the NADES before the sample is analysed (i.e., methanol and ethanol). Nevertheless, using NADESs reduces the extraction time compared to conventional acid digestion. Moreover, in addition to separating the analyte from the matrix and avoiding possible interferences, the extraction helps to preconcentrate the analyte and improve the sensitivity of the analytical method.

A more exciting approach from the point of view of greener and simpler extraction procedures is to use NADESs as extracting and chelating agents of metal ions (Table 1). Thus, a chelating agent other than a natural deep eutectic solvent is unnecessary. In these cases, wet digestions were not required to remove organic matter and dissolve the ions, as the NADES could extract the metals from the matrix for both solid and liquid samples. The extractions were carried out in an acidic medium generated through buffer solutions from NADES with an acidic pH or by adding some acid, such as nitric acid. As there is no prior wet digestion, organic matter does not get removed, and the metal ions, complexed with proteins or other macromolecules, could not be dissolved and would remain with the residue. Protons released into the acidic medium compete with the metal ions (Lewis acids) for the electron-rich sites in the organic matter (Lewis bases). This mechanism is how the metal ions are released and can interact with NADESs (Fig. 5).

(Reproduced with permission from reference [93])

Potential mechanisms of metal removal from organic matter by NADESs.

Most of the NADESs contain carboxylic acids in their composition. It has been observed that carboxylic acids-based NADESs can dissolve metal oxides, which is a potential application for metal extraction [85]. In addition, the anions derived from carboxylic acids (i.e., tartrate, oxalate, citrate) lead to the formation of soluble complexes with different metal ions [86].

Sorouraddin et al. used a similar approach to extract Cd and Zn from fruit juices [87] and Cd, Cu, and Pb from milk samples [88]. A less-dense-than-water menthol:sorbitol:mandelic acid NADES was employed, and after the extraction procedure, fine drops of solidified NADES containing the metal ions were deposited on the top of the solution. The acidic medium was achieved with an acetate buffer at pH 4 and 5, respectively. Using the proposed NADES facilitates the isolation of the analyte-rich phase to be analysed. Although no prior digestion is required, with the consequent consumption of hazardous inorganic acids and energy, a large amount of organic solvent (methanol) is used to extract the metal ions from the milk (1.5 mL methanol per 100 μL NADES) to dissolve the solidified droplets before their introduction into the detection system. The proposed extraction method takes less than 5 min with a sensitivity comparable to traditional methods that use organic solvents as extractants.

A Choline chloride-oxalic acid NADES have been used to extract Cu, Fe, and Zn [89], Cu, Fe, Ni, and Zn [90], and Hg from marine fish samples [91], and Se and As from edible mushrooms [92]. Because each metal has a different affinity for food proteins and other macromolecules, nitric acid was added to release the metals, resulting in higher recovery rates. Thus, the addition of 7 mL HNO3 (2 M) increased the recoveries of Cu, Fe, Ni, and Zn from 87.0%–90.7% to values > 95% for all metals [90]. NADES-based microwave-assisted digestions in acidic media were possible in only 20 s, 100 times faster than it takes to dissolve samples in a conventional wet digestion [89, 91, 92]. Better or at least similar metal recovery and precision values were observed with this green methodology [90]. Although acid is still used to complete the extraction step, the following advantages over conventional wet digestions should be noted: 1) lower concentration of acid, 2) less time and energy consumption, 3) reduction of acid vapour emissions, 4) working conditions at atmospheric pressure and 5) safer extraction procedures.

Houng et al. [93] demonstrated the significant effect of the pH in the NADES-based extraction procedures. They investigated choline chloride- and glycerol-based NADESs as washing solvents in removing Cd from rice flour samples. ChCl-based NADESs demonstrated good Cd removal (51%–96%) due to the acidic pH values of the solvent, which release protons and help displace metal ions from organic matter (i.e., proteins and starch). In contrast, glycerol-based NADESs have weakly alkaline pH values, which hinders the release of Cd from organic matter, resulting in low Cd removal efficiencies (< 51%). The addition of a biodegradable surfactant, saponin, had a synergistic effect on Cd removal due to its ability to encapsulate Cd in micelles. Thus, adding 1% saponin to a ChCl:sorbose NADES produced a Cd removal increase from 56% to 72%. A high removal efficiency (> 99%) of Cd was observed by increasing the NADES volume and the rice flour amount ratio. The study showed the ability of this simple, inexpensive, and time-saving procedure to remove Cd from contaminated rice flour. Moreover, the washing process did not affect the main chemical components and the structure of rice flour.

Based on the principle of acidic NADES and the complexing capacity of carboxylic acids, Soylak et al. used a ChCl:lactic acid NADES to extract iron from sheep, bovine, and chicken livers [94] by an ultrasound-assited extraction procedure and a ChCl:tartaric/oxalic/citric acid NADES to extract Mn from vegetables [86] by a heat-assisted extraction procedure. In the case of iron extraction, accuracy similar to that reported by other authors using conventional wet digestion was found. In contrast, the three NADESs studied for the extraction of Mn were applied to different vegetable samples (i.e., spinach, dill, cucumber bark), with recoveries ranging from 80% to 112%.

NADESs were also used to extract heavy metals from edible oils. In this case, they should be more polar than the oil to separate the phases after extraction. However, at the same time, it should have some affinity with the oil phase to ensure the diffusion of the solvent into the sample and the successful extraction of the analytes. Thus, Soylak et al. tested four ChCl-based NADESs to separate and preconcentrate Pb, Co, Ni, and Mn from oil samples [95]. Although quantitative recoveries were obtained with the four NADESs studied, ChCl:urea was selected as an extraction solvent due to its simple preparation. Karimi et al. also showed that a ChCl:urea NADES was the optimal solvent to extract Cd and Pb from edible oils [96]. Amine-based NADESs have a medium polarity compared to NADESs based on carboxylic acids or alcohols [97], an advantage in extracting metal ions from edible oils. In addition, urea has two intermediate donor atoms of nitrogen that can interact with different elements [96]. The detection limits of both methods using atomic absorption spectroscopy agreed with the trace-level analysis of these metals. In the case of Pb, the limit of detection was 0.008 ng/g [96] and 2.4 ng/mL [95], far below the maximum permitted level of Pb in oils set at 100 ng/g.

One more notable example is from Santana et al. [98], who applied a constrained mixture design to estimate the optimal ratio of three NADESs consisting of two compounds and water, i.e., citric acid:xylitol:water (NADES 1), malic acid:xylitol:water (NADES 2) and citric acid:malic acid:water (NADES 3), to extract different metals (As, Cd, Hg, Pb, Se and V) from bovine liver and fish tissue and protein. The optimized proportion for each synthesised solvent was selected based on density and viscosity values. The lower the solvent viscosity and density, the higher the extraction efficiency due to lower diffusivity and higher mass transfer due to better interaction with the matrix. As the percentage of water in all mixtures increased, viscosities and densities decreased. In addition, solvent viscosity affects the efficiency of ICP-MS detection since the higher the viscosity, the lower the analyte transport efficiency. However, the properties of NADESs are altered with water percentages above 50%. Therefore, the following NADES proportions were estimated as optimal for achieving the lowest possible densities and viscosities: 42:13:45 (citric acid:xylitol:water) for NADES 1, 34:21:45 (malic acid:xylitol:water) for NADES 2 and 42:13:45 (citric acid:malic acid:water) for NADES 3. The three optimised mixtures were used as solvents to extract the metals from different samples using an ultrasound-assited (UAE) and microwave-assited (MAE) extraction procedure. Better recoveries were observed with NADES 1 combined with UAE and NADES 3 with MAE. Worse recoveries with NADES 2 may be due to the lower content of carboxylic acid, which has a high metal-complexing capacity. NADESs prove to be a very promising solvent for extracting inorganic elements in several types of samples, and it is highly compatible with plasma-based techniques, even improving their analytical performance. The results of the proposed methods were compared with those of microwave-assisted acid digestion, with extraction recoveries between 80% and 120% for most of the elements.

Pesticide is a generic term referring to those substances capable of controlling or eliminating pests in food and feed. Pesticides can be classified in many ways, for example, according to their purpose, chemical structure, or safety profile. According to the World Health Organization (WHO), pesticides are used in public health to kill vectors of potential disease, such as mosquitoes, and in agriculture to kill pests that damage crops. Pesticides are conceivably toxic to humans and other organisms, and therefore, they must be utilised safely and discarded appropriately. It is essential to monitor and analyse them thoroughly, as pesticides can accumulate in soil and water for years. Even though restrictions have increased and some pesticides have been banned, they are still used in many countries [111].

There have been many methods in the literature for detecting all kinds of pesticides in many different matrixes. Scientists have devised fancy ways to analyse pesticides in foodstuff samples, obtaining a detection limit in the femtomolar range [112]. However, in most cases, highly toxic and environmentally hazardous organic solvents are used to extract them from the original matrix. DESs as a greener alternative for extracting pesticides have attracted more attention recently. Due to the hydrophobic structure of pesticides, it is pretty challenging to use natural products as extractants. Several publications have been reported that can be used as a starting point for developing novel extraction methods for pesticides in foodstuffs. Table 2 presents the papers concerning determining pesticides in food samples using NADES-based extraction methods.

Recently, Jouyban et al. [113] developed a method to analyse pesticides in milk samples (Fig. 6). They combined a liquid phase extraction method and a NADES-based dispersive liquid–liquid microextraction method to extract different types of pesticides from the milk samples. A ChCl:decanoic acid-based DES and NaCl were dissolved into the milk samples to extract the analytes. After centrifuging, a menthol/decanoic acid mixture was added to the supernatant and dispersed in 5 mL of water. The mixture was cooled at 0 ºC, and the solid droplets were dissolved in acetonitrile. Finally, the solution was analysed by gas chromatography. Recoveries between 64% and 89% were obtained for the pesticides, with limits of detection ranging from 0.9 to 3.9 ng/mL. The results were not significantly different from similar extraction methods found in the literature for the same pesticides. Their precision and limit of quantification were the lowest of all the works compared, proving that the proposed method is suitable for analyzing those pesticides in milk samples.

(Adapted from reference [113]) [Created in BioRender. Benito-Peña, E. (2025) https://BioRender.com/z26a002]

Experimental procedure of a DES-based method for extracting pesticides from milk samples.

In some cases, miscibility plays a critical role when selecting the best solvent for extraction. Most NADESs are highly miscible in aqueous samples, so they did not make any aggregates with aprotic solvents. High miscibility sometimes makes it challenging to use NADESs, so non-natural and hydrophobic DESs are preferred due to their self-aggregation. This self-aggregation of DESs occurs due to the stronger interaction of the aprotic solvents with water than DES; therefore, DES molecules leave water molecules and aggregate into the aqueous sample. Thus, Heidari et al. [114] studied a method for extracting organophosphorus pesticides in juice samples using DESs. After performing the extraction with different DESs, including natural mixtures such as ChCl:urea and ChCl:ethylene glycol, a mixture of ChCl:phenol was finally used as a hydrophobic solvent to extract the pesticides from the juice samples because of the reason commented above. The parameters influencing extraction efficiency were studied and optimized using a central composite design. Using the optimised experimental parameters, a wide linear range, low detection limits, good precision, high enrichment factors, and good extraction recoveries were observed in determining organophosphorus pesticides in fruit juice samples.

It has also been observed that the efficiency of NADESs as extractants is sometimes limited, especially with hydrophobic analytes. Hence, these solvents must be combined with other materials or analytical procedures to enhance performance. For instance, Song et al. [115] recently described a NADES-based method to extract pyrethroid insecticides from different natural samples, such as beebread or Curcuma Wenyujin. They used NADES-derivatised graphene as an adsorbent of the target analytes in a dispersive micro-solid phase extraction (DMSPE) procedure. In this case, the role of the NADES was to avoid graphene agglomeration, preserve its high adsorption capacity, and improve the processing and storage of the material [116]. Unlike other materials commonly used for this purpose, such as diazonium salts or ILs, the ease of synthesis, low melting point, and lack of toxicity make the NADES an excellent alternative for graphene functionalisation. Even though the NADES was not the extractant, it actively participated in the extraction process, substituting the role of the IL or diazonium salts.

Nemati et al. [117] described using two NADESs combined with a stir-bar sorptive extraction (SBSE) method to extract different pesticides from tomato samples. First, a water-miscible NADES (ChCl:ethylene glycol) was used to elute the analytes from the stir bar. Afterwards, a derivatization agent (ethyl chloroformate) and a water-immiscible DES (ChCl:n-butyric acid) were added to the obtained eluant, and the obtained mixture was rapidly injected into deionised water placed in an ice bath, forming organic droplets that were frozen and separated from the aqueous solution. Once melted, the solution was ready for analysis. Under the optimum conditions, the proposed method showed a high enrichment factor, low limits of detection (7–14 ng/L) and quantification (23–47 ng/L), good linearity, and satisfactory precision (< 12%).

An ultrasound-assisted magnetic nanofluid-based liquid-phase microextraction (UA-MNF-LPME) was introduced by Shirani et al. [118] to determine pyrethroid insecticides. This nanofluid consisted of a mixture of magnetic multi-walled carbon nanotubes and a DES. The extraction method involved a combination of the MNF with the pretreated solution containing the analytes. The mixture was sonicated for 3 min, and the MNF was collected with a magnet, allowing the separation from the supernatant. The analytes are then collected by adding acetone to the MNF, mixing for 1 min, and separating the MNF with a magnet. The suggested method was simple, as the MNF could extract the analytes from the pretreated solution, and the separation of the two solvents was performed very quickly with a magnet. Other advantages of the proposed method were short extraction time, high enrichment factor, high sensitivity, high efficiency, and ability to be applied in complex matrixes.

While NADESs can be effectively employed in the extraction process for a range of pesticides, certain limitations persist regarding their use as these solvents may not always exhibit compatibility with the matrix. Additionally, superior results may occasionally be derived from the application of alternative non-natural solvents. The incorporation of NADESs into various materials, such as graphene or ferrofluid nanoparticles, presents an intriguing strategy for the extraction of these analytes.

PAHs are categorized as potential environmental contaminants that have significant and detrimental effects on human health. These compounds are predominantly toxic, with several recognized as carcinogenic. PAHs enter human organs through environmental pollution, arising from fuels utilized in various modes of transportation and industrial emissions; moreover, a notable incidence is observed among smokers. PAHs comprise a group of compounds characterized by various condensed benzene rings, typically containing between two and seven such rings. These compounds are primarily generated through the combustion of hydrocarbons at elevated temperatures (exceeding 500 ºC) in the presence of oxygen. They may also be produced from fossil fuels under high pressure and lower temperatures (around 200 ºC). Consequently, PAHs are primarily released from anthropogenic sources [130] and are continuously present in the environment. They can be distributed far from the emission point without significant degradation. These compounds were even detected in remote areas such as the interior regions of the Arctic and the Antarctic [131], and the Brazilian savanna [132]. Indirectly, food and feed are also contaminated with these compounds. Thus, a 2010 study revealed the detection of PAHs in different foodstuff samples [