*5.1. Capillary Electrophoresis*

CE, which belongs to electromigration separation techniques, possesses many advantages, such as low sample and reagen<sup>t</sup> consumption, high e fficiency, simplicity, short analysis time, automation and inexpensive cost of capillaries in comparison to HPLC columns. CE separations are also extremely effective and allow substances with similar structures to be separated. These advantages mean that this technique has become an interesting alternative analytical tool to other chromatographic methods. Generally, CE analysis is carried out on fused-silica capillaries with silanol groups on the inner surface which are normally negatively charged. This results in the formation of an electroosmotic flow (EOF) that moves compounds toward the cathode when a voltage is applied across a tube filled with an electrolyte solution. Contrary to this e ffect, electrophoretic mobility exists, which moves a molecule to its opposite electrode. Each ion possesses a specific electrophoretic mobility resulting in a charge-to-mass ratio. However, the e ffect of EOF is generally predominant in respect to electrophoretic mobility, causing all the molecules to be moved at di fferent speeds toward the cathode. A higher speed can be observed for cations, and neutral analytes take slightly longer to migrate, while negatively charged compounds take the longest to move because of their conflicting electrophoretic mobility. The fact that, simultaneously, both EOF and electrophoretic mobility occur, working on anions in opposite directions during electrophoretic separation, allows greater resolution to be obtained. The main parameters which can a ffect EOF mobility are the dielectric constant, the zeta potential value and the viscosity of the bu ffer. The values of these parameters can be regulated by the modification of the background electrolyte (BGE) and/or using di fferent bu ffer additives, as well as when the physicochemical properties of the wall of the capillary are changed. ILs are considered as good EOF modifiers because of their good electrical conductivity and they are slightly more viscous than organic solvents. In e ffect, low IL concentrations can be enough for a significant improvement in the electrophoretic separation. According to the literature data, ILs have been applied as the BGE, as additives to the BGE and/or as covalent coating reagents of the capillary. However, taking into account the costs of these modifications, ILs were mainly used as electrolytes or additives to electrolytes to modify the capillary wall. It should be highlighted that both cations and anions of ILs may change the migration behavior of analytes, although the activity of IL cations have a major impact on the resolution in CE. The IL cations, by the modification of the ionic strength of the BGE, can change the EOF, which influences the migration times of the analytes and may improve separation e fficiency. Other activity is related to the adsorption of IL cations on the capillary inner surface, which can reduce or even reverse the EOF as well as possibly correcting the peak tailing of some basic enantiomers. Both mechanisms mentioned above allow a significantly better resolution of analytes to be obtained [199,208].

For example, Qin et al. [209] used a 1-methylimidazolium-based IL for covalent bonding of the fused-silica capillary surface wall for reversing the EOF during the development of a CE-MS method for the determination of sildenafil (SL) and its metabolite UK-103,320 (UK) in human serum samples. The most e ffective separation was obtained with a BGE containing 10 mM of acetic acid (pH 4.5) and with a voltage of 25 kV. The sensitivity and resolution were significantly improved because this approach allowed the elimination of the adsorption of the compounds on the IL-coated capillary wall, which occurred on the bare fused-silica capillary wall. In e ffect, the analytes passed through the IL-coated capillary with a recovery of 98% and 100% for SL and UK, respectively. Moreover, the resolution between SL and UK was enhanced because of the modification of the EOF. The analytes were separated within 14 min with LODs of 14 and 17 ng/mL for SL and UK, respectively. El-Hady et al. [210] proposed a CE-UV method for the simultaneous determination of four anticancer drugs in human plasma and urine based on [C4MIM][Br] as a component of the BGE. During the study, the parameters of CE separation were optimized. The best results were obtained when the analysis was carried out on a BGE containing a 12.5 mmol/L phosphate bu ffer at pH 7.4 and 0.1 μmol/L of [C4MIM][Br] (IL), and 20 kV applied voltage. This approach allowed sensitivity to be increased 600 times over that observed

in CE performed without the IL. The developed CE-UV method for the quantification of methotrexate, vinblastine, chlorambucil and dacarbazine in human plasma and urine allowed the analytes to be monitored with the LODs in the range of 0.01 to 0.05 μg/mL.

It should also be noted that excellent separation is particularly required for the analysis of racemic mixtures, including various groups of pharmaceuticals the enantiomers of which can possess significant di fferent pharmacokinetic and pharmacodynamic properties and side e ffect profiles. The qualitative and quantitative analysis of the compounds in biological and environmental samples is necessary for better understanding the mechanism of their activity in live organisms and their influence on the environment. This issue was a predominant topic of many papers published in recent years in world scientific literature. In those studies, both achiral ILs and chiral ILs (CILs) were applied in combination with various types of chiral selectors (CS) like cyclodextrins (CDs) or their derivatives, antibiotics, polysaccharides or surfactants for the chiral separation of di fferent pharmaceuticals. Typical achiral ILs applied in CE enantioseparation were tetraalkylammonium ILs, alkylimidazolium ILs and alkylpyridinium ILs with inorganic anions such as [OH], [Cl], [Br], [BF4] and [PF6]. Among them, tetraalkylammonium-based ILs are considered as more e ffective because of their relatively more hydrophilic character, which decreases the likelihood of entering the hydrophobic cavity of the CS. Moreover, their relatively lower conductivity and UV transparency in the wavelength ranges applied for enantiomer detection allow them to be used in higher concentration levels. These data are in accordance with the study reported by Huang et al. [211] who tested alkylpyridinium, tetraalkylammonium and alkylimidazolium-based ILs along with β-CDs for the chiral separation of five β-agonists. The results confirmed that tetraalkylammonium-based ILs were more e ffective because they could be used at much higher levels than the other tested ILs. Poor resolution was achieved when the long-chain IL, [C8MPyrr][PF6], was used as the BGE modifier. Moreover, the presence of ILs was required for the full enantioseparation of salbutamol, cimaterol and formoterol, which were not resolved using the BGE containing only β-CD as the CS.

Jiang et al. [212] used [C2MIM][BF4] for the coating of a silica capillary during the enantioseparation of ibuprofen, fenoprofen, naproxen and ketoprofen. It enabled the EOF to be modified, which provided the e ffective resolution of the enantiomers. The tested IL not only a ffected the EOF but also acted as a discriminator. Moreover, the interaction between hydrogen at the C–2 carbon of the IL and the acid drugs played an important role in the separation. The same type of IL was selected for the enantiorecognition of nine tricyclic antidepressants in the study reported by Tsai et al. [213]. The optimal simultaneous separation of all the tested pairs of enantiomers was achieved with 50 mM of [C2MIM][BF4] as the sole BGE at pH 3. Zhao et al. [214] used three ILs and hydroxypropyl-β-cyclodextrin (HP-β-CD) as the components of the BGE for the enantioseparation of itraconazole, ketoconazole, econazole and miconazole. Compared with [C2MIM][L-lactate] or [C2MPyrr][BF4], [C12MAmm][Cl] was the most effective. When this reagen<sup>t</sup> was used along with HP-β-CD it allowed the resolutions of 3.8, 3.5, 2.8 and 2.5 for miconazole, econazole, ketoconazole and itraconazole, respectively, to be obtained.

In the paper published by Liu et al. [215], the e ffective chiral separation of racemic methyl-ephedrine hydrochloride, thebaine, codeine phosphate and acetylcodeine by capillary electrophoresis with electrochemical detection (CE-ECL) was observed when 0.6% [C4MIM][BF4] as the component of the BGE was applied (Figure 10).

**Figure 10.** Electropherograms of four standard samples: (**A**) without IL in electrophoretic buffer; (**B**) with the use of 0.6% BIMPF4 in the electrophoretic buffer. Peak: 1, 10 μmol/L methylephedrine hydrochloride; 2, 40 μmol/L of thebaine; 3, 25 μmol/L codeine phosphate; 4, 15 μmol/L acetylcodeine. Conditions: electrophoretic buffer, 14 mmol/L phosphate–borax at pH 7.4; electrokinetic injection, 10 s × 10 kV; separation voltage, 15 kV; detection potential, 1.2 V; ECL solution, 5 mmol/L Ru(bpy)3<sup>2</sup>+ with 50 mmol/L PBS at pH 8.2. Figure adopted from [215] with permission.

The developed method offered the quantification of four drug alkaloids in human urine samples with LODs from 1.4 × 10−<sup>7</sup> to 6.3 × 10−<sup>8</sup> mol/L. Jin et al. [216] reported the effective enantioseparation of propranolol, oxprenolol and pindolol by CE when a BGE containing the achiral IL—glycidyltrimethylammonium chloride ([GTMAmm][Cl]) as the modifier along with a dual CDs system based on 2,6-di-O-methyl-β-cyclodextrin (DM-β-CD) and 2,3,6-tri-O-methyl-β-cyclodextrin (TM-β-CD) was applied. The authors also used an on-line sample enrichment technique based on field-enhanced sample injection (FESI) for the improvement of sensitivity. The application of both approaches allowed the LODs of the enantiomers to be decreased from 0.10 to 0.65 nM. Finally, the developed CE method was successfully used for the analysis of spiked urine samples, with good recoveries.

Unfortunately, in many cases, the application of achiral ILs with a single chiral selector was not enough for the effective enantioseparation of the compounds of interest. An interesting alternative approach reported in the literature was using CILs which can possess either a chiral cation or achiral anion, or both. The application of these CILs in combination with traditional chiral selectors allows an extra "enantiorecognition" capability to be obtained while the capability of system modification is retained. In effect, a "synergistic system" occurs during electrophoretic separation, which can significantly improve the resolution of the analytes. The first paper reporting the use of this approach for the enantioseparation of pharmaceuticals was published by François et al. [217]. The authors developed and used two chiral choline-based ILs—ethylcholine bis(trifluoromethylsulfonyl)imide ([EtChol][Nf2T]) and phenylcholine bis(trifluoromethylsulfonyl)imide ([PhChol][Nf2T]) alone or in combination with DM-β-CD or TM-β-CD for the analysis of the anti-inflammatory drugs, 2-arylpropionic acids, as model compounds. The developed CILs were applied as BGE additives, chiral ligands and CSs. Moreover, the enantioseparation efficiency in respect to the type and concentrations of tested CILs and CDs, as well as the methanol addition to the BGE, were evaluated. The results indicated that the effective separation of the analytes was achieved only upon adding one of the CILs containing DM-β-CD or TM-β-CD and methanol to the BGE. Thus, the synergistic effect between the tested chiral choline-based ILs and CDs in the dual separation system was confirmed. In another study, two chiral synergistic systems based on tetramethylammonium-L-arginine (TMA-L-Arg)/glycogen and tetramethylammonium-L-aspartic acid

(TMA-L-Asp)/glycogen were compared with the system containing achiral tetramethylammonium hydroxide (TMA-OH)/glycogen for the chiral separation of nefopam, citalopram and duloxetine [218]. Each tested IL/glycogen synergistic system gave better resolutions of the tested enantiomers compared to those observed for the separation using glycogen alone. However, the addition of TMA-L-Arg to the BGE composition was more effective than TMA-L-Asp, while the TMA-OH/glycogen separation system gave poorer resolution. Zhang et al. [219] tested tetramethylammonium-L-arginine (TMA-L-Arg), tetramethyl-ammonium-L-hydroxyproline (TMA-L-Hyp) and tetramethylammonium-L-isoleucine (TMA-L-Ile) as BGE additives in combination with HP-β-CD for the enantioseparation of amlodipine, nefopam, duloxetine and propranolol. The highest signals of the tested analytes and the best resolution was achieved using a 40 mM Tris/H3PO4 buffer solution (pH 2.6) containing 20 mM of HP-β-CD and 30 mM of TMA-L-Arg (Figure 11). Zuo et al. [220] reported the enantioseparation of twelve pharmaceuticals using 1-ethyl-3-methylimidazolium-L-lactate ([C2MIM][L-lactate]) and 1-butyl-3-methylimidazolium-L-lactate ([C4MIM][L-lactate]) in combination with β-CD in a BGE. The resolution was better in a dual system based on one of the tested CILs and β-CD compared to the β-CD alone, although the addition of [C2MIM][L-lactate] was more effective. Finally, the BGE composed of 20 mM of [C2MIM][L-lactate] and 10 mM of β-CD at pH 2.5 was selected as optimal for the separation of most analytes.

Only the analysis of homatropine methylbromide was carried out on 30 mM of Tris-H3PO4 at pH 2.0 (more effective separation), while the enantiomers of venlafaxine and sibutramine were not baseline resolved. Kolobova et al. [221] confirmed that 1-butyl-3-methylimidazolium L-prolinate [C4MIM][L-Pro] as a CS in combination with 2-hydroxypropyl-β-cyclodextrin (2HP-β-CD) allowed a significant improvement in the chiral separation of carvedilol and propranolol.

**Figure 11.** Chiral separation of all drug enantiomers in the optimized HP-β-CD/TMA-L-Arg synergic system. Conditions: focused-silica capillary, 50 cm (41.5 cm effective length) × 50 μm i.d; applied voltage, 20 kV; capillary temperature, 15 ◦C; BGE, 40 mM Tris/H3PO4 buffer solution (Ph 2.6) containing 20 mM HP-β-CD/TMA-L-Arg. Figure adopted from [219] with copyright permission.

Zhang et al. [222] designed a lactobionic acid LA-based IL, namely tetramethylammoniumlactobionate (TMA-LA), which was applied for the chiral separation of atenolol, metoprolol, propranolol, nefopam and duloxetine. In the study, three combinations, namely a single LA system, β-TMA chloride (TMA-Cl) system and TMA-LA IL system, were tested. The best results were achieved when the IL TMA-LA as the CS was applied. Finally, the BGE containing 40 mM of borax bu ffer, pH 7.6, 40% *v*/*v* methanol, 200 mM of TMA-LA and 20 kV applied voltage was selected as the most e ffective. Zhang et al. [223] tested L-alanine tert-butyl ester bis (trifluoromethane) sulfonamide (L-AlaC4Nf2T) and L-valine *tert*-butyl ester bis (trifluoromethane) sulfonamide (L-ValC4Nf2T) as additives to the BGE in combination with M-β-CD, HP-β-CD and glucose-β-CDs (Glu-β-CD) for the enantioseparation of naproxen, pranoprofen and warfarin. Compared to CDs alone, significantly better chiral recognitions of all analytes were obtained, although the resolutions of these dual systems were di fferent. Moreover, the addition of organic modifiers to the BGE additionally improved selectivity. This was probably related to decreasing the EOF, which allowed interactions to be increased between AAILs, M-β-CD and the racemates. The best separations of the analytes were observed when 15 mM of CILs was introduced into the 30 mM sodium citrate/citric acid bu ffer solution at pH 5.0 containing 20 mM of M-β-CD and 20% ethanol as the organic modifier with a 20 kV applied voltage. The potential synergistic e ffects of L-AlaC4Nf2T and L-ValC4Nf2T were also checked in combination with vancomycin during the enantioseparation of naproxen, carprofen, ibuprofen, ketoprofen and pranoprofen [224]. Both dual synergic separation systems were also able more e ffectively to separate the enantiomers compared to the vancomycin-alone case. Xu et al. [225] applied tetramethylammonium-L-hydroxyproline (TMA-L-Hyp) with clindamycin phosphate (CP) for the separation of a racemic mixture of propranolol, nefopam, citalopram and chlorphenamine. The authors optimized the electrophoretic conditions in terms of the BGE composition, pH, voltage, temperature and UV parameters. The best results were obtained when the CE separation was carried out on an uncoated fused-silica capillary (50 cm total and 41.5 cm effective length × 50 μm i.d.) with a 40 mM borax bu ffer (pH 7.6) containing 80 mM of CP and 30 mM of TMA-L-Hyp and methanol (20% *v*/*v*). A voltage of 20 kV and a temperature of 20 ◦C were used. Nefopam, citalopram, chlorphenamine and propranolol were monitored at 289, 230, 265 and 237 nm, respectively. AAILs based on a tetramethylammonium cation were also tested with maltodextrin for the enantio-separation of pharmaceuticals belonging to di fferent classes. For example, Yang et al. [226] used tetramethylammonium-D-pantothenate (TMA-D-PAN) and tetramethylammonium-D-quinate (TMA-D-QUI) as additives to the maltodextrin-based synergistic systems in a CE method developed for the analysis of racemic mixtures of nefopam, ketoconazole, econazole and voriconazole. For both of the CIL/maltodextrin systems, significantly improved Rs were observed for all the tested enantiomers, although TMA-D-PAN o ffered better separation results. This synergistic e ffect was probably related to a decrease in the density of the negative charge as an e ffect of the adsorption of the CIL cations on the surface of the capillary. This caused increasing complexation between the racemates and the CIL, which improved the resolution for all analytes.

Chen et al. [227] used tetramethylammonium-L-arginine (TMA-L-Arg) and tetramethyl-ammonium-L-aspartic acid (TMA-L-Asp) in combination with maltodextrin for the enantioseparation of nefopam, citalopram, cetirizine, duloxetine and ketoconazole. The most effective chiral separation was observed when a BGE composed of 60 mM of TMA-L-Arg, 7.0% maltodextrin in 50 mM of Tris-H3PO4 (pH 3.0) and with a voltage of 18.0 kV was applied. Zhang et al. compared the separation systems based on 1-butyl-3-methylimidazolium (T-4)-bis[(2S)-2-(hydroxy-κO)-3-methylbutanoato-κO]borate ([C4MIM][BLHvB]) and 1-butyl-3- methylimidazolium (T-4)-bis[( αS)- α-(hydroxy-κO)-4-methylbenzeneacetato-κO]borate ([C4MIM][BSMB]) along with HP-β-CD [228] as well as dextrin [229] as the CS in CE enantioseparations. In both studies, the addition of the CIL enabled the synergistic e ffect to occur between them and the used CS, which allowed better resolutions to be obtained and higher peak e fficiencies compared to those calculated for the HP-β-CD or the dextrin alone. On the other hand, [C4MIM][BLHvB] was more e ffective than

[C4MIM][BSMB]. This was probably related to the structure of the [C4MIM][BSMB] anion whose aromatic ring substituent could disturb chiral recognition.

An interesting approach was presented by Zhang et al. [230] who employed IL-dispersed NPs as buffer modifiers for the chiral separation of laudanosine, propranolol, amlodipine, citalopram and nefopam in CE. In the study, [C4MIM]BF4], ([C4MIM][PF6]), 1-dodecyl-3-methylimidazolium chloride ([C12MIM][Cl]) and 1-aminoethyl-3-methylimidazolium bromide ([C2NH2MIM][Br]) ILs were dispersed in multi-walled carbon nanotubes (ILs-MWNTs) and applied as the BGE modifier in combination with chondroitin sulfate E (CSE), as the CS. The obtained results indicated that significantly better separation, selectivity and peak shapes were achieved in the ILs-MWNTs modified system compared to that observed in CSE alone. The parameters affecting the electrophoretic separation were also investigated and optimized. The best results were obtained when CE was carried out on a 20 mM Tris/H3PO4 buffer solution containing 2.5% CSE and 2.4 μg/mL of ILs-MWNTs at pH 2.8–3.4 and with 15 kV applied voltage.

It should be highlighted that most of the studies described above indicated that the application of CILs alone as BGE modifiers was not able to effectively to separate the enantiomers. However, in the literature there are also a few reports describing the synthesis of novel CIL structures the activity of which was enough to achieve full resolution of drug enantiomers. For example, Yu et al. [231] synthesized a β-CDs-based CIL, 6-O-2-hydroxypropyltrimethylammonium-β-cyclodextrin tetra-fluoroborate ([HPTMA-β-CD][BF4]), and used it as a CS for the enantioseparation of eight pairs of drug enantiomers. The novel CIL offered higher solubility of the analytes in the BGE and gave better stabilization of reversed EOF in CE compared to the parent β-CDs, which allowed a higher intensity of the signals and a more effective resolution to be obtained. The results confirmed that the enantiomers of chlorpheniramine, brompheniramine, promethazine, liarozole, tropicamide, warfarin, pheniramine and bifonazole were more effectively separated with [HPTMA-β-CD][BF4] as the CS than with β-CDs. Recently, a report describing the synthesis of mono-6-deoxy-6-(3-methylimidazolium)-β-cyclodextrin tosylate (β-CDMIMOTs) CIL was also published by Zhou et al. [63]. The authors applied this new CIL as a coating material to modify the EOF in the CE method for the enantioseparation of oxytetracycline, tetracycline, chlortetracycline and doxycycline in environmental samples. The researchers achieved good separation of the analytes due to the multiple functions of β-CD-IL, which enabled the tetracyclines to be entrapped to form an inclusion complex (Figure 12).

**Figure 12.** Mechanism of separation of four TCs using β-CD–IL as dynamic coating material. Figure adopted with permission from [63].

Compared to β-CD alone, β-CD-IL o ffered better solubility in an aqueous bu ffer. A stable suppressed EOF in the capillary was also generated as the e ffect of the occurrence of hydrogen bonding and the electrostatic interaction with the capillary inner wall. The authors selected the best CE conditions for tetracycline separation, which were achieved when a BGE composed of 10 mmol/L, a pH 7.2 phosphate bu ffer and 20 mmol/L of β-CD-IL and electrochemical detection at 1 V was used. The developed CE method allowed the compounds of interest to be monitored in environmental water samples with LODs from 0.33 to 0.67 μmol/L.

#### *5.2. Micellar Electrokinetic Chromatography*

Considered as a mode of CE, micellar electrokinetic chromatography (MEKC) allows both neutral and charged analytes to be separated. In MEKC, the surfactant monomers are added to the run separation bu ffer above the critical micelle concentration (CMC), which allows aggregates called micelles to form as a pseudostationary phase. The separation process is based on di fferences between the analytes partitioning in a micellar stationary phase, and is related to the electrophoretic mobility of the compounds. Therefore, the neutral and hydrophobic analytes incorporated into the micelles gain an apparent electrophoretic mobility and will move at the same velocity as the micelle under electrophoretic conditions. This allows the neutral and charged compounds with the same charge-to-mass ratio to be separated because the migration time in MEKC is dependent on the electrophoretic velocity of the micelle, the distribution ratio and the EOF velocity. The use of additional BGE modifiers can increase e fficiency and selectivity. ILs as BGE additions have become interesting alternatives because the long-chain part of the AAILs can act as a surfactant to form a micelle in the BGE when the level of ILs exceeds the CMC. Moreover, the electrostatic interaction between the acidic analyte and the cationic micelle (AAILs) o ffered a more e ffective enantiorecognition of the analytes. Higher concentrations of ILs may also be used compared to organic solvent surfactants because of higher conductivity, hydrophobicity and solvation, which decreases the risk of destroying the micellar system in MEKC. In the literature, there are a few papers reporting the use of ILs in MECK. For example, Wang et al. published two consecutive papers [232,233] demonstrating the combination of TM-β-CD with N-undecenoxycarbonyl-L-leucinol bromide (L-UCLB) CIL as a dual chiral selector for the enantiodiscrimination of fenoprofen, indoprofen, ketoprofen, suprofen and ibuprofen. In the study, di fferent levels of CILs and TM-β-CD were tested. The results indicated that TM-β-CD alone could not resolve the enantioseparation of the racemates, whereas the addition of L-UCLB at a concentration of 1.5 to 2.0 mM to the BGE with TM-β-CD provided an excellent resolution. This was related to the competitive inhibition of the interaction between the CIL and the capillary wall in the presence of TM-β-CD. Cui et al. [234] used L-ethyl-3-methylimidazolium-L-lactate, [C2MIM][L-lactate] and 1-ethyl-3-methylimidazolium-L-(β)-lactate [C2MIM][DL-lactate] alone or in combination with HP-β-CD for the chiral resolution of ten analytes belonging to di fferent classes of pharmaceuticals. The results confirmed that the best enantiorecognition was obtained when a BGE composed of 40 mM of HP-β-CD, 50 mM of NaH2PO4-H3PO4, pH 2.75, and 30 mM of [C2MIM][L-lactate] was used during the enantiomeric separation. Moreover, this e ffect was mainly correlated with the cationic activity of the IL, which played an important role in the increased resolution, whereas the anionic part of the CIL possessed a low influence on the chirality and nature of the enantioseparation. Su et al. [235] tested the addition of [C4MIM][Cl], [C4MIM][PF6], [C4MIM][Nf2T] and SDS as modifiers in the BGE during the optimization of MEKC conditions for the separation of seven benzodiazepines. The results confirmed that the BGE containing 170 mM of [C4MIM][Nf2T] and 10 mM of SDS o ffered the most effective selectivity and resolution of the compounds of interest. This was related to di fferent degrees of association of the tested analytes, which gave a more satisfactory separation compared to the results observed using the IL or SDS alone. The anionic moiety of [C4MIM][Nf2T] probably played a dominant function during the separation process as a heteroassociation site for the benzodiazepines, while the SDS improved the resolution. The developed MEKC method allowed the analytes to be detected in human urine samples with LODs in the range of 2.74 to 4.42 μg/mL.

#### *5.3. Non-Aqueous Capillary Electrophoresis*

In recent years, non-aqueous capillary electrophoresis (NACE) has become an interesting separation technique because it allows the detection of water-insoluble analytes which cannot be measured in traditional aqueous CE. Additionally, the analysis time in NACE can be shortened because of the lower viscosity of the buffer solution and the higher EOF as well as the reduction of the electrophoretic current. Moreover, the application of organic solvents allows the analytes to be detected online by MS. As it was earlier mentioned, ILs possess some advantages over conventional organic solvent modifiers, such as good conductivity. Hence, using ILs in NACE can give a better separation effect. These possibilities were confirmed by Ma et al. [236] who applied an ephedrine-based CIL as the CS for the enantiomeric resolution of omeprazole and rabeprazole by NACE. A reversed EOF (anodic flow), probably caused by the adsorption of the cations onto the capillary wall, was observed when (+)-N,N-dimethylephedrinium-bis(trifluoromethanesulfon)imidate ([DMP]<sup>+</sup>[Nf2T]−) was added to the BGE. The best resolution was achieved with the BGE containing an acetonitrile-methanol mixture (60:40, *v*/*v*) and 60 mM of [DMP]<sup>+</sup>[Nf2T]<sup>−</sup>. The authors found that the enantioseparation was related to ion-pair interactions dependent on equilibrium constants between the negatively charged enantiomers and DMP cations. Moreover, hydrogen-bonding between the hydroxyl group of DMP<sup>+</sup> and the sulfoxide group of the analytes as well as π–π and dipole–dipole interactions were responsible for the separation mechanism.

Summarizing, the application of ILs in electromigration techniques offers new opportunities to solve many analytical problems in the separation field. One of them is the chiral recognition of racemic mixtures of pharmaceuticals having different chemical structures and biological activity. The results of numerous studies based on drug standards confirmed the grea<sup>t</sup> potential of ILs in CE applications. On the other hand, there are relatively few reports describing the separations of drugs in real biological and environmental samples. This seems to be caused by the relatively low sensitivity of CE-based methods compared to LC and GC techniques, which may be not enough for many pharmaceutical, clinical and environmental applications. On the other hand, lower LOD values can be obtained in electromigration techniques supported by ILs, which allows a partial resolution for this analytical problem. Moreover, intensive progress is continuing systematically in developing new approaches for improving sensitivity in electromigration techniques based on techniques such as field-enhanced sample injection (FESI), field-amplified sample injection (FASI), field-amplified sample stacking (FASS) or a combination of simultaneous electrokinetic and hydrodynamic injection (SEHI) and field-enhanced sample injection in conjunction with a sweeping technique known as sequential stacking featuring sweeping (SSFS) [237,238]. Probably, when scientists apply both ILs and new technical resolutions in CE, it will allow the required sensitivity to be obtained for clinical and environmental studies. These studies are very important because both CE-based techniques and ILs are environmentally-friendly, so connecting them in one analytical tool could be an important factor supporting the protection of nature.

#### **6. Current Trends and Future Perspectives**

Pharmaceuticals possess high biological activity and they can take part in various types of interactions, which means that these substances have a huge influence on the functioning of both live organisms as well as whole ecosystems. Therefore, as it was mentioned in Section 1, it is very important to develop sensitive, selective, accurate and precise methods for reliable drug determination in biological and environmental samples. An interesting approach is the application of ILs during method development. According to the data presented in this review, there are several interesting trends in the application of ILs for the determination of pharmaceuticals. First of all, ILs are most often applied at the stage of sample preparation (Table 2). The vast majority of studies concerned the extraction (or actually microextraction) of biological and environmental samples. Moreover, the most common type of analyte extraction from both these matrices was DLLME. Researchers pay a lot of attention to improving these methods by introducing modifications using physical and chemical

factors. As a result, they promote the development of environmentally-friendly solutions in the field of analytical chemistry and the improvement of validation parameters. Unfortunately, it should be noted that despite the development of various IL-based methods, the majority of procedures are still supported by organic solvents. In DLLME, their basic function is the dispersion of ionic liquids. In turn, because of the high viscosity of ILs, sample detection is only possible after dissolving the sample in MeOH, ACN and others. Thus, the application of ILs leads to improved validation parameters, but the developed methods are not completely eco-friendly. The results prove that despite moving in the right direction, this area requires further development. Improving the results is possible not only by proper sample preparation, but also by the application of ILs in chromatographic and electrophoretic techniques. The addition of ILs to mobile phases is the main way of using them for the determination of pharmaceutical drugs by chromatographic techniques. As the results show, the suppression of the interaction of silanol by use of ILs is a huge advance in the problematic analysis of basic drugs (Table 3). The use of ILs in the BGE in electrophoretic techniques, which in many respects are compatible with green chemistry, although their sensitivity still remains a challenging task for the analyst, seems to be promising. It may be surprising that despite the existence of commercially available and described methods for the self-preparation of IL-based chromatographic columns and capillaries for electrophoresis, such methods of their use is very rare for pharmaceuticals. If the huge potential of ILs is to be discovered, it should also be noted that in addition to the above detection methods, researchers are trying to use them with other chromatographic techniques. Although such applications are not ye<sup>t</sup> widespread in the analysis of pharmaceuticals, their dynamic development may cause such experiments to be performed in the future. In addition to trends in the design of analytical methods, the qualification of ILs with similar structures to a specific stage of analysis is the constant rule. In many works, optimization concerns the selection of a specific IL from a large diverse group of IL molecules. However, according to the data presented in different reports, the final optimization effect leads to the selection of the same IL. For example, an IL with hydrophobic properties was sought for liquid-phase extraction and the best results were often achieved for the imidazolium cation and anion [PF6]. In turn, as an addition to the mobile phase, the selection of the IL [PF6] was not suitable due to too strong adsorption on the column and was replaced by [BF4]. It must be highlighted that these are trends for most, but not all papers (detailed in Tables 2 and 3). However, the fact is that despite access to a vast amount of ILs, only a few have been tested in experiments, and the final selection focuses on a small number. As mentioned, the samples are analyzed by various chromatographic and electrophoretic techniques, while a UV detector is almost always used for analyte detection, rarely FL and almost never MS/MS.

The above examples confirm that there are no ideal solutions in the design of analytical methods for the determination of pharmaceuticals in biological and environmental samples. However, in the case of ILs, their advantages over disadvantages and also the incomplete data on them prove the need for continuous interest and development in this area.
