Applications of electrophoresis for small enantiomeric drugs in real‐world samples: Recent trends and future perspectives

Separation and identification of chiral molecules is a topic widely discussed in the literature and of fundamental importance, especially in the pharmaceutical and food fields, both from industrial and laboratory points of view. Several techniques are used to carry out these analyses, but high‐performance liquid chromatography is often the “gold standard.” The high costs of chiral columns, necessary for this technique, led researchers to look for an alternative, and capillary electrophoresis (CE) is a technique capable of overcoming some of the disadvantages of liquid chromatography, often providing comparable results in terms of sensitivity and robustness. We addressed this topic, already widely discussed in the literature, providing an overview of the last 6 years of the most frequent and recent applications of CE. To make the manuscript more effective, we decided to divide it into paragraphs that represent the main field of application, from enantioseparation in complex matrices (pharmacokinetic studies or toxicological dosage of drugs, analysis of environmental pollutants, and analyses of foods) to quality control analyses on pharmaceutical formulas. About these, which are the fields of most meaningful use, we mentioned some of the most innovative and performing methods, with a look to the future on the application of new materials used, such as chiral selectors, that can make these types of analyses accessible to all, reducing cost, time, and excessive use of toxic solvents.


INTRODUCTION
Chirality is a property, that is, the basis of organic chemistry, and a molecule that has an atom bound to four different substituents is called chiral and is present in enantiomeric forms that are asymmetric and nonsuperimposable.These specular molecules have no chemical differences in atoms and bonds but differ in their arrangement in space [1].Over the years, this topic has drawn enormous attention because it was realized that molecules that present this feature and might be present within drugs, food, and all that surrounds us might have different behaviors.Much of the biochemical reactions of our organism are enantioselective; this means that the enzymes involved in these reactions or the receptors in our body can discriminate the enantiomers of the same molecule [2].Molecules that comprise the active ingredients of drugs with this characteristic can differ in activity, pharmacokinetic characteristics, and toxicity.As a result, it is inevitable to characterize the enantiomers as two separate species to assess the behavior of both and the possibility of administering the drug in the racemic form or the need to isolate the most active form called eutomer [3,4].
A tragic example was the use of Thalidomide, which was administered to pregnant women for the treatment of morning sickness, but this has led to an increase in births with malformations.Only after this was it understood that this molecule was chiral, and only the R enantiomer Thalidomide had pharmacological properties, whereas Sthalidomide was teratogenic.Initially, it was thought that the adverse effects were eliminated by isolating and administering only the R enantiomer.However, it was soon found that the R enantiomer could also racemize in its toxic Sform into the organism [5].Based on this event and the resulting studies, the importance of the characterization and separation of enantiomers after the synthesis process is understood, but at the same time, the importance of detecting the presence of enantiomers even within biological matrices.
Different analytical instruments have been adapted for the separation and characterization of chiral compounds, and among the most used certainly could be found high-performance liquid chromatography (HPLC), gas chromatography, thin-layer chromatography, and capillary electrophoresis (CE) [6].Based on our knowledge, articles in the literature examine the evolution of these techniques and the development of accurate chiral separation methods, and most of them refer to the use of liquid chromatography.On the contrary, this review highlights the advantages of using electrophoretic techniques to make the enantiomeric separation process more accurate and sensitive.Classical CE is based on the principle that different molecules can be separated, exploiting the different migration speeds when subjected to an electric potential (electrophoretic migration) having different sizes and charging densities [7].In the case of enantiomeric molecules, however, these differences are not clearly distinguished, and therefore it would not be possible to separate them.The challenge over the years has been to develop methods that provide for the presence of chiral selectors (CSs) in the stationary phase of the capillary.A lot of functional materials are studied as CSs, able to interact differently with the two enantiomers and obtain an accurate separation, for example, amino acids, sugar, nanoparticles (NPs), cyclodextrins (CD), porous organic materials or ionic liquids (ILs) [8][9][10][11], chiral crown ethers, proteins, cyclofructans, macrocyclic glycopeptide antibiotics, and most importantly polysaccharides [12,13].Studying these materials and the conditions of analysis has allowed us to obtain numerous advantages over liquid chromatography techniques, such as the highest separation efficiency, lower consumption of organic solvent, and the possibility to use small sample quantities [14].
All these features will be taken into consideration for the search of articles that allow having a broad vision of the use of this technique both in the field of quality control (QC) also to perform analysis from more complex matrices, highlighting established -and innovative-methods that can grow more and more the use of electrophoresis in enantiomeric characterization.
Resuming the main objectives of this review are: • Comparing CE with some other analytical tools; • Demonstrate the wide use of this technique; • Assuring wide versatility in different fields (from pharmaceutical to environmental analysis); • Highlighting possible future improvements following Green Analytical Chemistry.
As we previously told, we have considered the last 6 years to do Scopus research.As Figure 1 shows, we can note an endless (and relatively constant number of papers) interest in chiral CE (our main keywords).The data for 2023 are less than others, but we are sure that during the coming months, they will increase, giving continuity of interest.

APPLICATION OF ELECTROPHORETIC TECHNIQUE IN CHIRAL-BIOANALYSES
Nowadays, the main aim is to develop analytical methods that follow Green Analytical Principles to reduce the use of toxic solvents, costs, and analytical time.Electro-driven methods, such as CE or electrokinetic chromatography, allow to obtain the desired results, provide a valid alternative to chiral HPLC methods, which often involve very expensive and delicate stationary phases [15].Over the years, researchers tried to extend these chiral separation techniques on several fronts, such as pharmacokinetic and pharmacodynamic analyses, to evaluate the behavior and activity of these chiral molecules, particularly those that racemize during metabolism.At the same time, CE is also applied in food analyses to monitor the purity of protein amino acids, sugars, and organic acids or in environmental analyses to detect the presence of chiral pesticides in wastewater and soil [16][17][18][19].All these types of analysis are performed on complex matrices and often require specific CSs and particular sample preparation and pre-concentration treatments.

Dosage of enantiomeric drugs in human matrices
One of the significant applications of CE for enantiomeric separation takes place in the dosage of therapeutic drugs on patient samples or for the dosage of drugs of abuse  [20].As we have mentioned, the authors also stress the importance of the sample preparation method to obtain an accurate and sensitive result, as shown in Table 1 [20,21].
In their work, the authors validated a complex method that uses the ultrasound-assisted-low-density solvent dispersive liquid-liquid microextraction technique to optimize the extraction of the four enantiomers from plasma and urine.This extraction technique used field-amplified sample stacking related CE (FASS-CE) to detect the analyte, thus obtaining an excellent sensitivity compared to the other results presented in the literature (Table 1).The parameters optimized were the pH (2.5), phosphate buffer concentration (100 mM), and the concentration of the CS added (3 mM), which has explicitly been chosen, heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin.The instrumental setting was 50 cm capillary (40 cm effective length), hydrodynamic injection at 20 cm height for 10 s, 18 kV applied voltage, and 200 nm for UV detection [20].
On the same category of drugs, Andersen et al. developed a promising tool for enantiomeric determination of citalopram and its metabolite from the plasma of patients treated with a racemic antidepressant [22].The extraction, the clean-up, and the concentration on the sample were carried out using a liquid-phase microextraction (LPME) followed by CE using a fused-silica capillary and the mobile phases consisted of phosphate buffer (25 mM, pH 2.5) added of 1% of sulfated-β-cyclodextrin (S-β-CD), 12% of acetonitrile (ACN), and 0,1% of poly(vinyl alcohol).The analysis was performed in reverse polarity, with the anode at the end of the capillary; in this way, the CD used as the CS was negatively charged and interacted with analytes.In addition, the CS moved in a direction opposite respect to the analyte increasing the mobility difference between the complex and free analyte, optimizing the chiral resolution [22].Recently, Abolhasani et al. elaborated a new method to quantify enantiomers of citalopram in complex matrices, such as human urine [23].They used a new microextraction technique based on the hollow fiber-supported liquid phase to increase the effectiveness of extraction, limiting the presence of contaminants and avoiding the disadvantages of classic liquid-liquid and solid-liquid extraction.The novelty of this work is represented by the use of maltodextrin-modified CE, which improved the efficiency of chiral separation of a wide range of acidic and basic molecules.The change of conformation in the space, from the flexible coil to helix, when this CS interacts with chiral analytes is the bases of the separation.All experimental parameters optimized to obtain a LOQ = 30 ng/mL and a LOD = 10 ng/mL for both enantiomers are displayed in Table 2.
Wen et al. demonstrated in their study the possibility of obtaining the simultaneous enantioseparation of mirtazapine and its metabolite, detected with the capillary zone electrophoresis (CZE) aid of ACN-FASS in only 7 min [24].Based on validated methods, the authors have selected carboxymethyl-beta-cyclodextrin (CM-β-CD) as a CS, and to increase the limit of sensitivity, they have chosen the FASS (first used in 1979).In this technique there was a difference in the field intensity between samples.In the separation electrolyte, the analyte(s) migrates rapidly, accumulating and showing a sharp detection band, and in this way, the method's sensitivity is improved.They have chosen 80% ACN in distilled water for the sample solvent and a running buffer composed of 6.25 mM borate, 25 mM phosphate solution, and 5.5 mg/mL CM-β-CD with a pH of 20 mg/mL S-β-CD [26] Abbreviations: CE, capillary electrophoresis; CM-β-CD, carboxymethyl-beta-cyclodextrin; EBC, exhaled breath condensate; HP-α-CD, hydroxypropyl-alphacyclodextrin; LLE, liquid-liquid extraction; MECK-MS, micellar electrokinetic chromatography; NH 4 Ac, ammonium acetate; poly-l,l-SULA, poly-sodium N-undecanoyl-l,l-leucylalaninate; TEA, triethylamine; Tris-H 3 PO 4 , tris(hydroxymethyl)aminomethane-phosphoric acid.
2.8.Other optimized parameters were the applied voltage (16 kV) and the electrokinetic injection with the sample dissolved into 10% of running buffer, injection time 20 s, and 7.5 kV as injection voltage.
After optimizing the fundamental parameters, this method was applied to pharmacokinetic studies of healthy volunteers treated with racemic mirtazapine [24].The use of CE for dosing chiral molecules from biological matrices is increasingly used, and in recent years there have been numerous works published in the literature; through a critical look, we have selected the works that present an improvement of what are the analytical parameters of interest, and the most effective extraction techniques to make the optimal enantiomeric separation.3, investigated the differences between the use of HPLC with chiral column and CE to separate the enantiomers of pheniramine and chlorpheniramine from rat plasma [25,26].Considering the failure of their preliminary enantioseparation with HPLC, using five different columns, the authors developed a sensitive method for quantifying two molecules, applicable to pharmacokinetic studies.Two online pre-concentration methods were applied to enhance the sensitivity of separation, first large volume sample stacking and sweeping and then cation-selective exhaustive injection and sweeping followed by S-β-CD modified CE.The parameters of both methods were optimized, such as buffer pH and concentration, the concentration of CD, the voltage, and so on.Evaluating the data provided by the authors, both methods show the improvement of enhanced efficiency compared to simply CD-CE without online sweeping [25,26].Some recent applications of CE methods for enantioselective dosage of drugs in complex matrices are given in Table 3 [25][26][27][28][29][30][31][32][33][34].

Yao et al., in the two articles cited in Table
For the determination and quantification of methadone in cases of MTT, a treatment program for reducing opioid dependence symptoms, or in cases of abuse, Hamidi et al. validated a rapid method using CE.Exhaled breath condensate was selected as an alternative matrix, respect plasma, serum, or urine, to monitor the presence of the drug of interest.The excellent performance of electrophoresis was obtained after optimization of analysis parameters made it possible to achieve a sensitive and robust method and especially to avoid the operations of extraction and pre-concentration of the analyte(s) from the matrix of interest, minimizing the cost and time [27].The use of vancomycin as the CS for CE tandem mass spectrometry allowed Svidrnoch et al. to investigate the presence of the enantiomers of 2-hydroxyglutaric acid in human urine for diagnosis of hydroxy-glutaric acidurias [28].All the advantages of CE combined with the excellent enantioselectivity of vancomycin have guaranteed the desired results, reducing the pretreatment of the samples and the derivatization of the analytes [28].
The same topic was offered by Liu et al., highlighting the importance of CE versus the HPLC method to reduce the number of samples and the toxic solvents used.For this purpose, authors developed a CE-MS method for the simultaneous determination of enantiomers both of Venlafaxine and its metabolite, using a chiral polymeric surfactant, in particular polysodium N-indecency-l, l-leucyl-alanine (poly-l, l-SULA), they obtained higher performance compared to classical HPLC-MS methods [29].Electrophoretic techniques are increasingly frequent for the enantioseparation of drugs of abuse in the forensic field and so in the analysis of analytes present in postmortem samples.
The goal of the study presented by Bertaso et al. was to detect the presence of methorphan's enantiomers in blood collected from opiate overdose-related death; in this way, they could testify to the presence of this compound as impurities of clandestine Heroin.They validated the rapid and reliable method using HP-β-CD modified CE and the FASS sweeping to achieve the best results after an easy liquid-liquid extraction of analytes from cadaveric blood [31].
Porpiglia et al. demonstrated the advantages of using the CZE enantioselective method to determine and quantify ketamine and norketamine in hair samples.Using a simple liquid-liquid extraction and optimized analytical conditions, they obtained a low-cost method with excellent separation power in a short analysis time (10 min) [32].

Other applications of enantioseparation by CE
It is important to emphasize that CE is also applied in food and environmental analyses.Evaluating the presence and quantifying the molecules that occur in the enantiomeric form in food is essential information to monitor its quality and avoid the presence of toxic enantiomers already present inside or formed after storage.At the same time, it is essential to have an overview of environmental contamination, and also in this field, sensitive and rapid methods have been validated, thanks to the use of electrophoresis, to quantify the contaminants present enantiomerically, starting above all from water (lake, river, wastewater, and ground).Some developed methods in recent years, selecting from the literature those who proposed an innovative method applied to the biological matrices and the most exciting analytes were reported in Table 4 [33][34][35][36][37][38].
Aydoğan et al. described the application of chiral-ligand exchange separation using ad open tubular CE to determine the presence of an enantiomeric form of maleic acid in apple juice.This technique provides different materials for coating the capillary column, such as polymer, porous silica, and others, which can be further derived with other molecules that make the process more selective.In this work, the coating was carried out with the polymerization of 3-chloro-2-hydroxypropyl methacrylate, followed by derivatization with l-histidine used as a CS.The presence of this ligand allows the formation of complexes with the metal ions present in the buffer, in this case, Cu(II) ions, leading to selective interactions with the two enantiomers of the analyte(s) [35].Applying the same principle of separation, Kamencev et al. used an uncoated fused silica capillary for electrophoresis to detect tartaric and malic acids' enantiomers from wine.
The authors highlighted the possibility of applying HPLC methods with a chiral column for this type of analysis.However, the difficulty in the simultaneous separation of both acids is caused by strong matrix interferences.Ligand exchange CE is a useful tool to overcome these difficulties since simply CD-modified CE is inefficient in separating small hydrophilic acids.For this analysis, they used a bare-fused silica capillary and a background electrolyte composed as follows: • 20 mM acetate buffer • 100 mM d-quinic acid • 10 mM Cu(II) • 0.5 mM Al(III) The running buffer was added with different concentrations of hexadecyltrimethylammonium hydroxide, from 0.1 to 1 mM (optimized 0.5 mM) to achieve the anodic electroosmotic flow (EOF), because the cathodic EOF with uncoated capillary was much unstable.Thus, the separation was carried out at negative polarity, where both the EOF and the analytes run toward the anode [36].
The aim of the study presented by Fiori et al. was the enantioseparation and quantification of catechins present in green tea, which have potential bioactivity.For this type of analysis, high-performance capillary electrophoresis and the running buffer were composed of 25 mM borate-phosphate buffer at pH 2.5 to overcome the degradation of catechins during the run.Into the background electrolytes (BGEs) were also presented sodium dodecyl sulfate (SDS) 65 mM and DM-β-CD 28 mM.At the basis of this type of separation, there is the competition of analytes between micelles formed by SDS and cyclodextrin that can include some molecules based on their chemical affinity.The optimization of all these parameters has made it possible to obtain a robust method capable of offering quantitative data in just 5 min [37].
To monitor the presence of some pharmaceuticals in wastewater and, therefore, to control environmental pollution, Valimaña-Traverso et al. developed a sensitivity method to extract and concentrate this type of analytes from water and successively detect the enantiomeric forms of them by CE.Seven active substances of different categories of drugs were investigated thanks to the use of two novel periodic mesoporous silica materials, which have been developed to obtain a very selective solid-phase extraction prior to CE analysis.Separation was performed using an uncoated capillary and 25 mM phosphate buffer pH 3.0 as running buffer.After investigating eight different CD used as CSs, only S-γ-CD offered an excellent simultaneous separation of all compounds in only 16 min of analysis [38].
Using a similar method, Silva et al. investigated the presence of β-blockers drugs in groundwater and river.After optimization of new mesoporous silica functionalized with octadecyl groups, authors proposed the use of methylated-β-CD into 50 mM phosphate buffer pH 2.5 at the temperature of 30 • C to obtain a reasonable resolution of all enantiomers [39].
Another type of matrix to assess environmental pollution and, in this case, especially to monitor the quality of agricultural products is soil.Identifying traces of pollutants such as pesticides or herbicides in agricultural land has always been an essential aspect of environmental analytics.At the same time, assessing the formation of toxic metabolites over time and their persistence is crucial.In this context, we have shown in Table 4 one of the most recent applications of electrophoresis for this type of analysis; due to its simplicity and low-cost Jiménez-Jiménez et al. developed a method using CE to carry out the separation of prothioconazole and prothioconazole-death from soil samples, which until now had been analyzed only using HPLC.Soil samples were subjected to only extraction with water and centrifuge, and the analysis was conducted using 10 mM of S-γ-CD as a CS into a 75 mM of borate buffer pH 9.0 with a voltage +30 kV [40].

CAPILLARY ELECTROPHORESIS IN QUALITY CONTROL
ICH Q2 (R2) is the latest guideline regarding analytical procedures that includes identity, impurity (and purity), assay content, and potency [41].Concerning the safety and efficacy of new drugs, it is necessary to subject the final pharmaceutical formulation to various QCs, to control the presence of impurities, excipients, or other pharmacologically active ingredients, respecting regulatory systems [42].In this context, CE emerges, especially for the purification of enantiomeric drugs, chiral capillary electrophoresis (CCE).So it is fundamental to apply ICH Guidelines during the QC of a drug product.Separating the enantiomers is essential because, often, just one is biologically active.CE has become one of the most used separation techniques, preceded only by liquid chromatography, due to the low consumption of reagents and samples and brief analysis [14,43,44].The QC process needs high separation due to the presence of minimal parts of substances/impurities [42].

Dosage of enantiomeric drugs in pharmaceuticals
Varga et al. reported that some of CE's advantages could be short time methods, good screening, and the opportunity to reverse the migration's order, despite that CE continues to be not the first choice [45].
In industries, quality is a significant aim required, above all pharmaceutical industries, but also in scientific papers, this is an essential factor for disseminating an article [14].Regarding quality, the best approach to develop a robust CE method is quality by design (QbD), which includes analytical target profile (ATP), critical method parameters (CMP), and design space (DS).Acronym ATP identifies how to quickly determine the analyte and its impurity; CMP is all the parameters to consider when developing a method, such as a BGE, pH, temperature, and voltage.The last one, DS, defines all the experimental conditions used to identify the analyte(s) [14,46,47].
Among the factors to which attention must be paid, there are certainly concentrations of CSs.Each analyte has a specific concentration of CS that can be different between two enantiomers; for example, a concentration can enhance the resolution of just one of them and damage the other one.Therefore, CE aims to optimize a good separation of each analyte [47].
The significantly used approach in CCE is choosing a CS or plus of one, adding it directly into the medium, and, in this way, it is easier to adjust pH or concentration to have a good separation.Additionally, it is cheaper than LC [14].One of the most popular selectors used in CCE is CDs, which are (1,4)-linked α-d-glucopyranose units with a hydrophilic surface with a lipophilic cavity [48].In 1988, different articles were published using CD as a CS, but because of their insufficient aqueous solubility, they remained little used.Years later, CDs were synthesized with many different characteristics [48].
CDs are very familiar thanks to their stability and low cost, but their peculiarity is in optimally separating chiral compounds [42].Often CCE is combined with electrokinetic techniques so through a BGE added-in solution (Table 5 [49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65]).The separation is based on differences in the electrophoretic mobility of samples [14].Linking to the QbD, it is essential to report in the method all the tests carried out with the relevant findings because even using one CD instead of another can bring more resolution, as well as the molarity and type of BGE [42].
Interestingly, in 2018, Niedermeier et al. published an article about chiral separation of levomepromazine in HPLC [66].Thus, by comparing their two works mentioned in Table 5, where they used CE with aqueous and nonaqueous BGE, and their previous article with chiral HPLC, the most sensible method was obtained with CE with nonaqueous BGE because it permits to identify and quantify impurities until 0.01% [57,60,66].
Another exciting food for thought is the CS randomly methylated β-cyclodextrin (RAMEB) which is randomly methylated β-CD.Hancu et al. tried with different CSs, but they chose RAMEB because a more significant interaction between enantiomer and CS translates into slow migration of the first one [51].This approach, called one-variableat-a-time, systematically optimizes each parameter [42].They used this method on Amlodipine, a calcium channel blocker, because the S enantiomer is the only one with pharmacological activity.At the same time, the R enantiomer has significant collateral effects.The method was tried on tablets available on the market to confirm the validation of those mentioned above [51].
As mentioned previously, pH is an essential factor to analyze during CE.Harnisch et al. tried different pH in their study (7.12 and 12.2), observing that it is strictly related to the pKa and the type of groups in molecules (i.e., pindolol at pH 7.12 was protonated) [53].
Reminek et al. reported that following QdB principles, the most widely used approach is Monte Carlo simulation, which consists of optimizing electrophoretic conditions by response surface method.Another method used to determine impurities is Doehlert, as Orlandini et al. reported [42,49,53,54].Two aspects are fundamental in the QC of a drug product: good separation and robustness of the method.The first one comes from BGE and CS composition and modulation, as reported in each article mentioned above.The method's robustness, on the other hand, is synonymous with ensuring quality through the performance evaluation of finished products [14].
Another exciting aspect of CE is that by modifying pH, temperature, voltage, BGE, and CS, you can edit the migration order of enantiomers, which is an essential difference with chromatography [2].Using two CS is only possible, considering CE is divided into two passages: chiral recognition and separation from the first passage [2].Hancu et al. used a dual selector in their study because using one CS caused a partial or baseline separation; instead, the dual system peak shape was better [51].

CHIRAL RECOGNITION MECHANISM
Enantiomeric separation in CE involves using a CS, which is a molecule that interacts differently with the two enantiomers.The CS can be covalently attached to the capillary wall, added to the electrolyte solution, or used as a coating material for the capillary wall.When a sample containing enantiomers is introduced into the capillary, the electric field causes the enantiomers to migrate toward the electrode of the opposite charge.During migration, the enantiomers interact with the CS in a manner to form diastereomers that are specific to their molecular orientation.Due to their different physicochemical properties, the diastereomers migrate with different velocities in the capillary tubes, leading to enantiomeric separation [67].The optimization of the chiral separation is controlled by various factors, such as the properties of the CS, the composition and pH of the electrolyte solution, the temperature, the sample loading amount, and the applied electric field strength.By optimizing these parameters, it is possible to achieve high-resolution separation of enantiomers in CE.Overall, enantiomeric separation in CE involves using a CS to differentiate between the two enantiomers based on their differential interaction with the selector.A schematic representation of the enantiomeric separation in CE is given in Figure 2.

CHALLENGES AND FUTURE PERSPECTIVE
During the last 20 years, the use of CE for enantioseparation of chiral compounds is growing both in the of pharmaceuticals and in the dosage of chiral compounds present in biological matrices (human samples, animal samples, food samples, or environmental samples).As can be seen from the articles we mentioned and from a more general view of the articles in the literature, classical CSs, especially cyclodextrin, are dominant.Preference for using these types of CSs is due to the extensive knowledge of properties of CD, such as water solubility, UV transparency, and good stability in solution.In the years, it has been tried to improve their application, developing synthetic CD, with characteristics more appropriate to their use in CE, by applying short and easy synthesis protocols at low cost [68,69].
Even though the marked prevalence of CD among the CSs, research, and application of new materials is a desirable goal for researchers in this field, talking about new materials, we refer to deep-eutectic solvents (DESs), metal-organic frameworks (MOFs), covalent organic frameworks (COFs), other porous organic materials (including monolithic materials), NPs, and ILs, quoting only some of those that seem more advanced from the application point of view [11,[70][71][72][73][74].All these materials have different properties, which can be exploited to maximize the separation and the resolution of analytes of interest, they can be used both as a stationary phase, to coat the capillary, and both added into the running buffer, for example, to modify the EOF and to improve the peak shapes [75,76].The first use of ILs and DESs in chiral CE dates back to early 2000 and, until today, has continued to deepen and expand their use, finding applications in combination with the classic CSs such as the CD, widely demonstrated [74,[77][78][79][80].
In contrast to the ILs and DESs, NPs are often used only as carriers or adsorbents for CSs because most of these are achiral [81].Based on our knowledge of recent uses of NPs, we see composite NPs forming with appropriate CSs as pseudo-stationary phases in the running buffer [82][83][84].Still at the beginning, instead, is the research on the use of MOFs and COFs, which are porous materials capable of forming complex supramolecular structures with a large surface area, which seem suitable to be applied in chiral CE.Most studies concern the development of chiral stationary phases with these materials used for coating the capillary [85][86][87][88][89].It is essential to mention that costly CSs are always lost in CE.Besides, achieving the pure enantiomeric form is difficult after separating diastereomers.Therefore, there is a great demand for the chiral capillaries to separate the enantiomers without forming diastereomers.The other challenges may be summarized below: -Enhancing reproducibility: Capillary conditioning, buffer preparation, and sample preparation are a few variables that can impact the repeatability of chiral CE.
The approach would become more reliable and robust if repeatability were improved.-Producing more versatile CSs: The effectiveness of the CS utilized determines how well chiral CE performs.The selectivity and effectiveness of the separation could be increased by creating new, more adaptable CSs.-Detection sensitivity improvement: The ability of chiral CE to detect trim levels of chiral chemicals may be constrained by its current low detection sensitivity.Future studies could concentrate on creating more accurate detection techniques or enhancing existing ones.-Aggregating sample throughput: The time needed for each separation in chiral CE restricts the sample throughput.Researchers might look into strategies for cutting separation times without compromising separation effectiveness.-Increasing the range of analytes: Larger molecules like proteins and peptides and tiny molecules like chiral chemicals are increasingly being studied using chiral CE.The scope of the technique's uses would be significantly increased by developing techniques for separating and analyzing these more giant molecules.
Overcoming these obstacles would improve chiral CE's capabilities and broaden its applications, including those in biotechnology, pharmaceuticals, and environmental analysis.

CONCLUSION
CE has proven to be, in recent years, a good technique for enantioseparation of chiral molecules, a type of analysis that becomes increasingly important, especially in the pharmaceutical field.To avoid the presence of toxic enantiomers in pharmaceutical formulations or their formation in vivo due to metabolic processes, as already extensively explained in the previous sections.The use of this technique is possible to overcome more expensive techniques, such as enantioseparation by HPLC, and proves to be increasingly compatible with the needs of researchers to test new materials considered eco-friendly, or which may indirectly lead to an increase in the performance of analytical methods to limit the use of excessive volumes of toxic solvents.

A C K N O W L E D G M E N T S
The authors would like to thank the University "G.d'Annunzio" for the support in the literature survey.This review paper has not received any funding.Open access funding provided by University "G.d'Annunzio" of Chieti Pescara within the CRUI-CARE Agrement.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors have declared no conflict of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.

F
I G U R E 2 A schematic representation of the enantiomeric separation in capillary electrophoresis; 2a: racemic mixture was loaded; 2b: the enantiomers formed diastereomers; 2c: diastereomers started to separate; 2d: complete separation; 2e: elution of diastereomers.
General view of the methods reported in the literatures on enantiomeric separation of fluoxetine (Flx) and norfluoxetine (Nflx).
TA B L E 1 Capillary electrophoresis (CE) instrumentation and experimental parameters.Recent applications of capillary electrophoresis (CE) method to the analysis of enantiomeric drugs in biological matrices.
TA B L E 2Abbreviations: DAD, diode array detection; DE, dextrose equivalent; HCl, chloride acid; NaOH, sodium hydroxide.TA B L E 3 Enantioseparation of chiral molecules from food and environmental matrices.
Some examples of chiral capillary electrophoresis (CCE) used for quality control.
TA B L E 5