Chiral Capillary Electrokinetic Chromatography

Chiral Capillary Electrokinetic Chromatography: Comparison

Please note this is a comparison between Version 1 by Sami El Deeb and Version 2 by Bruce Ren.

The separation principle of the technique is described and supported with simple graphical illustrations, showing migration under normal and reversed polarity modes of the separation voltage. The most relevant applications of the technique for enantioseparation of drugs and other enantiomeric molecules in different fields using chiral selectors in single, dual, or multiple systems are highlighted. Measures to improve the detection sensitivity of chiral capillary electrokinetic chromatography with UV detector are discussed, and the alternative aspects are explored, besides special emphases to hyphenation compatibility to mass spectrometry. Partial filling and counter migration techniques are described. Indirect identification of the separated enantiomers and the determination of enantiomeric migration order are mentioned. The application of Quality by Design principles to facilitate method development, optimization, and validation is presented. The elucidation and explanation of chiral recognition in molecular bases are discussed with special focus on the role of molecular modeling.

chiral capillary electrokinetic chromatography
enantioseparation
chiral selector
capillary electrophoresis
Quality by Design
enantiomers
capillary electrophoresis-mass spectrometry
enantiomeric impurity
molecular modeling
chiral recognition

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1. Introduction

Chirality plays a crucial role in life with a multidisciplinary interest—including, but not limited to, chemistry, biochemistry, food chemistry, agrochemistry, drug discovery, pharmacology, engineering, physics, and material science. Stemming from the chirality of natural amino acids, stereospecificity is a characteristic of many physiological reactions and pharmacological processes for all living systems ^[1][2][3][4][1,2,3,4].

Methods for enantioseparation, particularly on an analytical scale, are still gaining attraction, due to the continuous need for chiral separation and determination in different research and industrial fields. Enantiomers that have identical physical and chemical properties, might produce different pharmacological responses. Some counter enantiomers are recognized as being pharmacologically same active, less active, different active, inactive, antagonistic, or even toxic compared to their active enantiomeric pair. Distomer is considered the less potent, while the eutomer is more powerful for a particular action ^{[5][6][7][8][9][10][11]}[5,6,7,8,9,10,11].

Even when only considering the presence of inactive counter enantiomer of a drug in racemate, it constitutes an additional load on the body without benefit. Therefore, the number of drugs that are produced as enantiomers continually increases the expense of racemate drugs. Thus, there is usually a need for enantioselective methods to separate and quantitate enantiomers, but also to determine enantiomeric impurities with high sensitivity, which must be in accordance with the International Conference on Harmonization (ICH) regulations to quantify enantiomeric impurity of 0.1% relative to the main enantiomer. This has been established in the guidelines of ICH and the other regulatory authorities, such as the Food and Drug Administration (FDA) and European Medical Agency (EMA), for the validation of analytical methods [12].

Possible racemization of a single enantiomeric drug might occur during synthesis, storage, or in vivo metabolism. Possible in vivo inversion of a single enantiomeric drug (eutomer) to produce the unwanted enantiomer (distomer) in the biological system should be considered early during drug development. In vivo metabolism studies in rats are usually conducted using a proper enantioseparation method [13]. Furthermore, the determination of drug enantiomer, or its metabolite from biological body fluid and tissue samples, is highly valuable to study for its stereoselective pharmacokinetic and pharmacodynamic properties, to understand their enantioselective drug action in addition to stereoselective drug interaction and toxicity ^[6][14][6,14].

Enantioseparation methods are also important in food analysis of protein amino acids, organic acids, and sugars. For instance, it is necessary to check the absence of D-amino acids in food proteins, which might be formed due to racemization of natural L-amino acids during food production technology (e.g., treatment by alkaline conditions or heat). The presence of D-amino acids will decrease the susceptibility of the proteins to proteolytic enzymes ^[15][16][15,16].

Enantiomeric analytical methods are also required for agrochemical analysis of chiral pesticides and insecticides [17]. Environmental analysis of contaminated water (e.g., pharmaceutical wastewater), soil, and food by residues of chiral drug and/or their metabolites and chiral pesticides is an important field. Enantioselective separation is mandatory to explore the potential of these substances to exhibit stereoselective toxicity or degradation and its consequences on animals and humans ^[18][19][18,19]. Enantioseparation is also important in doping and forensic science.

Capillary electrophoresis (CE) and capillary electrochromatography (CEC) play more competitive and complementary roles to liquid chromatography (LC) than the other techniques for chiral separation, such as gas chromatography (GC) and supercritical fluid chromatography (SFC), and to less extent thin layer chromatography (TLC) ^{[6][20][21][22][23][24][25]}[6,20,21,22,23,24,25]. Microchips also contribute to the field of enantiomeric separation and detection with successful applications [26].

Compared to LC, the application of CE for chiral analysis involves lower sample and reagent consumption, making it a green alternative enantioseparation technique. CE techniques are also known to offer higher separation efficiency and resolution and mostly lower analysis time than HPLC. However, LC remains superior with regard to UV detection sensitivity and also mostly with regard to the repeatability of migration time and peak area. Moreover, the chiral separation principle in CE is different from that in HPLC, as described below. Therefore, CE is a good complementary technique to HPLC for chiral separation. It is also worth noting that most successful chiral selectors in HPLC can be transferred to CE except for aqueous buffer insoluble ones [27].

Among other CE modes, electrokinetic chromatography (EKC) is a widely used CE mode for enantioseparation ^[6][28][29][6,28,29]. EKC modes include micellar electrokinetic chromatography (MEKC), in which a micelle is used with or without chiral selector (CS) as a pesudostationary phase (PSP), and microemulsion electrokinetic chromatography (MEEKC), in which a microemulsion is used as carrier electrolyte. The present review draws attention to nonmicellar chiral capillary electrokinetic chromatography (CEKC) as a well-established technique for enantioseparation. The use of chiral MEKC is limited because uncharged enantiomeric compounds can also be separated by nonmicellar CEKC with the charged chiral selector. MEKC and MEEKC are out of the scope of this review and are described elsewhere ^{[30][31][32][33][34][35][36]}[30,31,32,33,34,35,36].

2. Chiral Capillary Electrokinetic Chromatography Principle and Selectors

2.1. Principle of CEKC

CEKC is a CE mode in which a CS is added directly to the background electrolyte (BGE). The separation of molecules will then result from the dynamic equilibrium with the CS inside the capillary and their different electrophoretic mobilities, if present, in a charged form in the BGE ^[37][38][37,38].

The added CS to the BGE will form noncovalent transient diastereomeric complexes with each of the two enantiomers of mostly the same drug with different complexation constants, and thus, different complex stability. In addition to the nonstereospecific binding forces, each enantiomer will form additional stereospecific binding with the chiral selector, mostly hydrogen bonding, but also forces like hydrophobic, electrostatic, van der Walls, and steric factors. In addition to possible inclusion forces, if cavity (or more) is/are available within the structure of the CS, then part of the enantiomer can pass inside. The two enantiomers will mostly have different affinity to the CS, i.e., the enantioseparation can be reached by different intermolecular forces and thermodynamic selectivity in the recognition mechanism ^[39][40][41][39,40,41].

It is proven, however, that it is possible in CE, and not in HPLC, that the enantioseparation selectivity can exceed the thermodynamic selectivity of the recognition. Therefore, CE can separate an enantiomeric pair of a compound even if the binding constants of its individual enantiomers with the CS are identical. That is to say, both or either a binding constant difference or analyte difference is/are required to achieve enantioseparation in CEKC ^[27][42][27,42].

Therefore, each enantiomer will migrate with a different speed (electrophoretic migration) because of the different interactions with the chiral selector, and thus, the two enantiomers will separate from one another. High flexibility is offered in choosing one (or more) of a variety of CS available for enantioseparation and simply adding it/them to the BGE in combination or not. This also offers the ability to easily adjusting the concentration of the CS to achieve the best equilibrium and to modify the electroosmotic flow (EOF) velocity through an effect on viscosity for best enantioseparation ^[43][44][43,44].

The CS can easily and cost-effectively be replaced by another in CEKC to identify the one with the best separation power for the intended enantiomeric molecules or to invert the migration order of the enantiomer peak pair. The concentration of the CS can also be modified for best selectivity ^[21][28][21,28].

CEKC can be conducted under normal or reversed polarity modes of the separation voltage. Enantiomers will migrate in a direction depending on its apparent mobility (µ_app), which is the sum of the electrophoretic mobility (µ_e) and the EOF mobility (µ_EOF), but also influenced by the µ_app of the CS to which it will complex. The normal polarity of the separation voltage (Figure 1) indicates having the anode (+) at the inlet and the cathode (−) at the outlet. Under the normal polarity mode of the separation voltage, the EOF is towards the cathode (detector/outlet). The normal polarity mode of the separation voltage is the standard mode in CE. In contrast, the separation can be conducted under a reversed polarity mode of the separation voltage. The reversed polarity mode of the separation voltage indicates having the cathode at the inlet and the anode at the outlet. Under reversed polarity of the separation voltage, the direction of the EOF will be mostly away from the detector, except if a zero-flow capillary or a low pH BGE is used. Negatively charged enantiomers with electrophoretic mobility greater than the EOF will pass the detector. Negatively charged cyclodextrins (CDs) have strong electrophoretic mobility toward the positive electrode (anode). Basic drugs are positively charged under low pH buffer and are more likely to interact with negatively charged CD, and then the CD drug complex will be attracted toward the anode at the outlet and pass the detection window (Figure 2). Therefore, the negatively charged CDs will act as a carrier of enantiomers by their self-mobility toward the anode at the outlet ^{[36][45][46][47][48]}[36,45,46,47,48].

Figure 1. Simple representative schematic diagram of CEKC normal polarity mode of the separation voltage separating a basic ionized enantiomeric racemate (R⁺ and S⁺) using CS in the BGE. Pink and green arrows show the apparent mobility (µ_app) of the enantiomeric pair and the mobility of the EOF (µ_EOF), respectively. The blue arrows show the difference in the speed of the migration of enantiomers upon interaction with the CS. µ_app of the CS would be according to its charge.

Figure 2. Simple representative schematic diagram of CEKC under reversed polarity mode of the separation voltage with a negatively charged chiral selector and a basic ionized enantiomeric pair (R⁺ and S⁺). Pink and green arrows show the apparent mobility (µ_app) of the enantiomeric pair and the mobility of the EOF (µ_EOF), respectively. The blue arrows show the difference in the speed of the migration of enantiomers upon interaction with the CS. Complexed enantiomers will migrate in the direction of the anode (here the outlet), due to interaction with the negatively charged CSs, which have strong µ_app in the direction of anode.

2.2. Types of Chiral Selectors in CEKC

Over the past years, many substances have been successfully used or unsuccessfully tried (e.g., without achieving the required resolution) as chiral BGE additives in nonmicellar CEKC to separate enantiomeric pairs. In principle, any substance with potential enantiomeric separation power, good stability in the BGE, and compatibility with the detection mode can be tried. Recently, new substances are showing good ability to separate enantiomers ^[49][50][49,50]. Additional special considerations should be taken, for example, when using EKC-mass spectrometry (MS), as described in Section 3.1.

In a published work from September 2020 about recent advances of novel chiral separation systems in CE, Qi [51] provided a circular statistical graphic (circle chart) divided into slices to illustrate the numerical proportion of the application of CSs in enantiomeric separation by CE, as shown in Figure 3.

Figure 3. The proportion of application of the various chiral selectors in CE in 2015–2019 (incomplete statistics). Reprinted with permission from reference [51].

A similar chart with a special focus on CSs in CEKC during 2017–2018 has before been published by Yu and Quirino [28]. Both authors show that derivatized CDs as a single selector are the most commonly used CSs and are responsible for about 50% of chiral separation methods.

Up-to-date derivatized CDs, which are formed through chemical substitutions with a stronger recognition ability and/or better solubility, are still pioneering the field of CSs in CEKC to separate enantiomers in either pharmaceutical formulations, biological samples, or other fields [52].

Uncharged derivatized CDs include substances as hydroxypropyl-α-CD, hydroxypropyl-β-CD, and hydroxypropyl-γ-CD. Negatively charged derivatized include high sulfated-α-CD, high sulfated-β-CD, high sulfated-γ-CD, sulfobutyl-ether-β-CD, carboxymethyl-β-CD, phosphated-α-CD, phosphated-β-CD, phosphated-γ-CD, and succinylated-β-CD. Positively charged derivatized CDs as a quaternary ammonium CD also showed successful applications. Among others, hydroxypropyl derivatized CDs are claimed to have the best enantioseparation power for a wider variety of chiral compounds ^{[6][52][53][54][55]}[6,52,53,54,55].

Negatively charged CDs (anionic CDs) are also especially important and widely applied for the enantioseparation of positively charged cationic compounds (basic chiral compounds) and neutral chiral compounds [56].

The single isomer CDs are preferred for chiral separation to obtain reproducible results, but also to study aspects of the selector-analyte interactions and to better understand the experimental design of chiral CE separations [53].

The CDs have been applied mostly as a single chiral selector, but also sometimes in a dual or multiple chiral system, as described below. Tuning of enantio-discrimination can be achieved by substitution of the single CD molecule, but also fine-tuning is possible by modifying the other separation conditions [53]. Cucinotta et al. [57] synthesized and characterized a new capped derivative of β-CD. The synthesized new lysin-bridged hemispherodextrin was characterized by electrospray ionization mass spectrometry (ESI-MS) and Nuclear magnetic resonance (NMR) spectroscopy. Circular dichroism and electron spin resonance spectroscopy were used to test its inclusion ability and its metal coordination ability, respectively. The new selector has been used to separate enantiomers with improved ability toward anionic and cationic guests compared to the single free CD. Recently, Salido et al. [58] reported the effect of eutectic solvents as choline chloride-ethylene glycol, choline chloride-urea, choline chloride-D-glucose, and choline chloride-D-sorbitol as additives in CD mediated CEKC to improve the enantioseparation of lacosamide enantiomers. The addition of choline chloride-D-sorbitol to the separation media containing succinyl-β-CD has been found to increase the resolution value from 1.5 to 2.8.

Room temperature ionic liquids (ILs) are the second most commonly used CSs in CEKC after CDs. ILs are responsible for about 17% of chiral separation. Chiral ILs (CILs) are organic salts that consist of organic cation and an organic or inorganic anion. Either the cation or the anion must be chiral to consider as CILs ^[28][59][28,59]. Recently, Nie et al. [60] reviewed and categorized ILs for enantioseparation and explored their resolution mechanism. Amino acid-based ILs have been proposed for potential use as sole CSs in CEKC [61]. Zhang et al. [62] reported the potential use of novel mono- and di-tetraalkylammonium L-tartrate ILs with different alkane chain lengths as sole CSs in CE. Successful enantioseparation of five model chiral analytes has been obtained with a high proportion of methanol. In many cases, ILs could not show enough enantiomeric discrimination power when used as sole CSs, and thus, have been usually used in combination with other CSs, mostly CDs, and particularly the derivatized ones. They act in synergism together to achieve better enantioseparation. It is suggested that the mechanism of separation is then based on the competition of the chiral IL and the enantiomer for inclusion into CD ^{[63][64][65][66][67]}[63,64,65,66,67]. Wahl and Holzgrabe prepared and used ILs combining tetrabutylammonium cations with chiral amino acid-based anions (tetrabutylammonium l-argininate) together with β-CD for the enantioseparation of ephedrine, pseudoephedrine, and methylephedrine isomers [68].

Polysaccharides, either the homo-polysaccharides as maltodextrin or the glycosaminoglycan as chondroitin sulfate and heparin, are contributing to about 5% of chiral separation and are much behind CDs in their enantiomeric discrimination power ^[69][70][71][69,70,71]. Sun et al. [72] described a CEKC approach using ethanediamine-bonded poly (glycidyl methacrylate) microspheres as CSs for enantioseparation with chondroitin sulfate E. Better enantioseparation results were obtained using the microsphere for separating basic model chiral compounds compared with using the single system of chondroitin sulfate E.

Macrocyclic antibiotics have molecular masses range between 600 and 2200. Their enantioseparation power is because they have multiple stereogenic centers. Most are accessible in charged forms, and thus, suit CEKC of uncharged enantiomeric drugs [73]. Examples include eremomycin, rifamycin B, vancomycin, thiostrepton, ristocetin A, fradiomycin, teicoplanin, azithromycin, erythromycin, and clarithromycin. Most of them have several chiral centers with or without cavities (e.g., teicoplanin has 23 chiral centers and 4 cavities), ensuring good enantioseparation power [74]. Post run washing of the capillary and the use of small volume samples have been recommended because of the strong ability of the glycopeptide antibiotics to adsorb to the inner wall of the capillary ^[75][76][75,76].

Recently, Ren et al. [77] reported using the second-generation macrolide antibiotic gamithromycin as a novel chiral selector for enantioseparation in CE. Gamithromycin showed particular enantioseparation power for chiral primary amines.

In another recently published research work, the steroid antibiotic fusidic acid has been proposed as a novel chiral selector for enantioseparation in CE. The authors suggested its special effective enantiodiscrimination power for chiral analytes containing rigid planar structures. The authors expected the finding to open the door for investigating other structurally similar steroids for possible enantioseparation power [78].

Chiral metal-ion complexes are small molecule chiral selectors for CEKC. Example include Cu(II)-L-ornithine, Cu(II)-D-phenylalanine, Cu(II)-l-lysine, Cu(II)-D-quinic acid, Zn(II)-l-arginine, Zn(II)-l-lysine, Co(II)-D-quinic acid and Ni(II)-d-quinic acid. They act through the formation of diastereomeric ternary metal-ligand-analyte complexes. Meta ligand complexes showed successful application for the enantioseparation of amino acids and dansylated amino acids, and glycyl peptides ^[79][80][79,80].

Among proteins, human or bovine serum albumins are the most commonly used chiral selectors in CEKC. A good washing protocol is required to minimize protein adsorption to the inner surface of the fused silica capillary. Proteins are still playing a particularly good role in investigating enantioselective protein interaction with enantiomeric drugs [81]. Ratih et al. [7] used human serum albumin as a chiral selector in 20 mM phosphate buffer pH 7.4 for enantioseparation and simultaneous binding constant determination of amlodipine and verapamil enantiomers. It is worth noting that microscale thermophoresis [82] also is useful in the study of enantioselective affinity of chiral drugs toward therapeutic target proteins [83].

Quintana et al. [84] synthesized carbosilane dendrimers functionalized with L-cysteine and N-acetyl-L-cysteine on their surface and used them as CS to separate razoxane by CEKC. Results showed partial resolution, but opens the door for more research in this regard to explore the use of dendrimers as CSs in CEKC.

New selectors are continuously proposed to enhance enantioselectivity. Meanwhile, modifications of existing selectors, for example, by derivatization, also takes place. However, random derivatization processes that cannot identically be repeated to produce the same product are not recommended, even if a good separation is obtained, because no result reproducibility can then be ensured.

Combination of two CSs in a dual chiral separation system or more in multiple chiral separation systems have been reported to improve the enantioseparation ^[85][86][85,86]. A dual chiral separation system is less complicated than the multiple ones, and is more commonly used. This has been achieved mostly by adding the two CSs mixed together to the BGE or sometimes by injecting them separately in two separated plugs without meeting each other in the capillary. Many examples showed an enhanced separation efficiency through the synergetic effect of dual CS systems of two mixed chiral selectors [87]. A dual CDs CS system of sulfobutyl-ether-β-CD and native γ-CD resulted in a good enantioseparation of lansoprazole and rabeprazole [88]. Nicolaou et al. [89] developed two dual CS systems to separate the enantiomers of warfarin, coumachlor, nefopam, and fexofenadine using either CD or cyclofructan with the CIL L-alanine tert butyl ester lactate. The addition of the CIL to the BGE was found to improve the resolution. Six basic racemic drugs (amlodipine, chlorphenamine, duloxetine, propranolol, nefopam, and citalopram) were separated using a dual chiral selector system of chondroitin sulfate D and carboxymethyl-β-CD [90]. Chen et al. [91] reported the synergetic action of the IL tetramethylammonium-L-arginine and maltodextrin in enantioseparation of the five studied drugs, including nefopam, duloxetine, ketoconazole, cetirizine, and citalopram. Better resolution values were obtained compared to the single maltodextrin system. Salido-Fortuna et al. [92] reported a CEKC method for the enantiomeric determination of econazole and sulconazole in pharmaceutical formulation using a dual chiral system consisting of hydroxypropyl-β-CD combined with ionic liquids. Best separation was obtained with hydroxypropyl-β-CD combined with tetrabutylammonium-L-lysine. High enantiomeric resolution values for econazole and for sulconazole have been achieved, as shown in Figure 4.

Figure 4. Electropherograms that correspond to the enantiomeric separation of a standard solution (40 mg/mL) and cream samples of econazole and sulconazole. CE conditions: 50 mM phosphate buffer pH 2.5 containing mixtures of (A) 5 mM hydroxypropyl-β-CD with 20 mM tetrabutylammonium-L-lysine at 25 °C; (B) 2 mM hydroxypropyl-β-CD with 25 mM tetrabutylammonium-L-lysine at 15 °C. Other conditions: Uncoated fused silica capillary, 50 μM i.d. × 48.5 cm (40 cm of effective length); UV detection at 200 nm; applied voltage, 30 kV; injection by pressure, 50 mbar for 10 s. Reprinted with permission from [92].

Some examples showed an enhanced separation efficiency through dual CS systems in which the two chiral selectors are introduced to the capillary in two separated plugs. Chalavi et al. [93] developed an enantioseparation method based on the partial filling technique with two chiral selector plugs. The first plug contains either hydroxypropyl-α or β-CD and the second plug contains maltodextrin. The two adjacent chiral plugs contain the same BGE. Each plug is supposed to separate the enantiomers independently. This approach is aimed to prevent the possible unwanted interaction between the two chiral selectors inside the capillary and is useful for chiral compounds that cannot be separated using either one of the two intended chiral selectors or a mixture of them. The method showed success in separating racemic drugs, including baclofen, carvedilol, cetirizine, chlorpheniramine, citalopram, fluoxetine, hydroxyzine, propranolol, tramadol, and trihexyphenidyl. No significant effect has been noted in selectivity factors (α) and migration times, and no significant differences between the resolutions when the order of the two chiral selector plugs in the capillary has been inverted. One should, however, note that in other cases (e.g., CDs with ILs), the interaction between the two chiral selectors is mostly advantageous and wanted to produce a synergetic effect.

Recently, La et al. [86] developed a CEKC method based on a multiple chiral separation system. The method has been used for separating dihydropyridone analog, a drug candidate proposed in type 2 diabetes treatment, which is a neutral hydrophobic molecule with multiple chiral centers. Its eight isomers have been successfully separated using a triple CD system consisting of 15mM sulfobutyl-ether-β-CD plus 15mM γ-CD and 40 mM hydroxypropyl-γ-CD in a 50 mM borate BGE at pH 10. Further relevant applications of CEKC dating between January 2016 and March 2021, other than those described in the text of this review, are summarized in Table 1.

Table 1. List of representative works published between 2016 and March 2021 dealing with the application of CEKC in enantiomeric separation and determination of enantiomeric compounds from different fields.

Analyte	Matrix	Separation Conditions	Detection	LOD/LOQ	Ref.
Cathinone derivatives	Human hair	Preconcentration by solid phase extraction, fused silica capillary of 50 µm i.d. and 80 cm total length, separation voltage of 35 kV, BGE of 80 mM disodium phosphate at pH 2.5, β-CD as CS	DAD at 200 nm	0.02 ng/mg 0.2 ng/mg	[94]
Proteinogenic amino acids	Cerebrospinal fluid	Derivatization by 9-fluorenylmethyl chloroformate fused silica capillary of 50 µm i.d. with a length of 70 cm to the detector and 80 cm of total length, separation voltage of 35 kV, BGE of 50 mM ammonium bicarbonate at pH 8 containing 15% (	v	/	v	) isopropanol and 10 mM β-CD, Sheath liquid interfacing of propan-2-ol:water: 1 M ammonium bicarbonate (50:50:1,	v	/	v	/	v	) at a flow rate of 3 L/min, and a nebulizer gas pressure of 2 psi	ESI-MS	0.9 µM -	[95]
Tedizolid	Pharmaceutical formulation	Fused silica capillary of 45 cm (effective length 35 cm) × 25 µm i.d., separation voltage of 12 kV, BGE of 37.5 mM heptakis-(2,3-diacetyl6-sulfo)-β-CD dissolved in 50 mM formic buffer pH 4.0 with the addition of acetonitrile (81.4:18.6,	v	/	v	)	DAD at 200 nm	- -	[96]
Radezolid	Pharmaceutical preparation	Fused silica capillary of 45 total length and 35 cm effective length and 25 µm i.d., separation voltage −28 kV, BGE of 40 mM heptakis(2,3-di-	O	-methyl-6-sulfo)-β-CD dissolved in 50 mM phosphate buffer pH 2.5	DAD at 265 nm	- -	[97]
Methadone	Exhaled breath condensate	Fused silica capillary of 50 cm length, 41.5 cm effective length, and 50 µm i.d., separation voltage of 25 kV, BGE of 150 mM phosphoric acid-tetraethylammonium at pH 2.5 containing 30% (	v	/	v	) methanol and 0.8% (	w	/	v	) carboxymethyl-β-CD	DAD at 200 nm	- 0.15 µg/mL	[98]
Colchicine	Pharmaceutical preparation	Fused silica capillary 58.5 cm (50 cm effective length) × 50 μm i.d., separation voltage of 20 kV, 50 mM or 25 mM borate buffer pH 9.0 using succinyl-γ-cyclodextrin or sulfated-γ-CD	UV at 243 nm	0.3 mg/mL 1 mg/mL	[99]
Praziquantel	Pharmaceutical preparation	Fused silica capillary of 50 µm i.d., 48.5 cm total and 40 cm effective length, separation voltage of 15 kV, BGE of 50 mM phosphate buffer pH 2.0, supplied with 15 mM sulfated-β-CD	DAD at 210 nm	0.75 µg/mL 2.0 µg/mL	[100]
Glycopyrrolate	Rat plasma	Online preconcentration by cation-selective exhaustive injection-sweeping, fused silica capillary (40.2 cm × 75 μm), separation voltage of −20 kV, BGE 30 mM phosphate solution at pH 2.0 containing 20 mg/mL sulfated-β-CD and 5% acetonitrile	DAD at 200 nm	2.0 ng/mL 0.0625 µg/mL	[101]
Brompheniramine	Rat plasma	Online preconcentration by cation-selective exhaustive injection and sweeping, fused silica capillary of the total length of 50 cm (effective length 40 cm) × 50 µm i.d., separation voltage of −20 kV, BGE in 50 mM phosphate buffer pH 3.5, containing 10% (	v	/	v	) acetonitrile and 30 mg/mL sulfated-β-CD	UV at 210 nm	- 0.01 µg/mL	[102]
Ivabradine	Pharmaceutical formulation	Fused silica capillary of 58.5 cm (50 cm to the detector window) × 50 µm i.d., separation voltage of −30 kV, BGE of 5 mM tetrabutylammonium-aspartic acid in 50 mM formate buffer pH 2.0 containing 4 mM sulfated-γ-CD	UV at 200 nm	0.22 and 0.28 µg/mL 0.73 and 0.93 µg/mL	[103]
Lansoprazole and rabeprazole	Pharmaceutical preparations	Fused silica capillary of 48 cm total, and 40 cm effective length and 50 μm i.d., separation voltage of +20 kV, BGE for lansoprazole: 25 mM phosphate buffer pH 7, 10 mM sulfobutyl-ether-β-CD/20mM γ-CD, +20 kV voltage; BGE for rabeprazole: 25 mM phosphate buffer pH 7, 15 mM sulfobutyl-ether-β-CD/30 mM γ-CD	UV at 210 nm	2 and 2 µg/mL 6 and 6 µg/mL	[88]
Pheniramine	Rat plasma	Online preconcentration by large volume sample stacking and sweeping, fussed silica capillary of a total length of 50 cm (effective length 40 cm) × 50 μm i.d., separation voltage of −20 kV, BGE of 30 mM phosphate buffer at pH 3.0 with 30 mg/mL sulfated-β-CD	UV at 262 nm	- 10 ng/mL	[104]
Phenothiazines	Urine sample	Preconcentration by solid phase extraction, fused silica capillary of 75 µm i.d. and 365 µm o.d., separation voltage from 10 to 12 kV, BGE of 75 mM phosphate buffer pH 3.0 and 0.9% poly (diallyldimethylammonium chloride), hydroxypropyl-γ-CD as a CS.	UV at 254 nm	2.1 to 6.3 nM-	[105]
Six phenoxy acid herbicides (Fenoprop 1, Fenoprop 2, Mecoprop 1, Mecoprop 2, Dichlorprop 1, Dichlorprop 2	Mixture of herbicides	Fused silica capillary of 58.5 cm total length and 50 cm length to the detector and 50 µm i.d., separation voltage of 25 kV, BGE of 50 mM phosphate buffer pH 7.0, dual CD (4 mM hydroxyl--β-CD and 16 mM heptakis(2,3,6-tri-	O	-methyl)-β-CD.	DAD at 200 nm for mecoprop, chlorprop, and 210 nm for fenoprop	- -	[106]
Homocysteine and cysteine	Stock standard solutions in borate buffer	Derivatization by 9-fluorenylmethyl chloroformate, fused silica capillary of 58.5 cm total length and 50 cm effective length and 50 µm i.d., separation voltage 20 kV, BGE for homocysteine: 2 mM γ-CD +5 mM L-Carnitine C1NTf2 in borate buffer pH 9.0, BGE for Cysteine: 2 mM γ-CD +5 mM L-CarnitineC1Lac in phosphate buffer pH 7.0	DAD at 210 nm	- -	[107]
Amlodipine	Pharmaceutical formulation	Fused silica capillary of 48 cm length (40 cm effective length) ×50 μm i.d., separation voltage of 25 kV, BGE of 25 mM phosphate buffer pH 9.0, 15 mM carboxymethyl-β-CD	UV at 230 nm	S 0.27 and R 0.32 µg/mL S 0.8 and R 0.96 µg/mL	[108]
L/D-Asp, L/D-Glu, and L/D-Ser	Bone cell lines (murine osteocytes and osteoblast	Derivatization by 4-fluoro-7-nitro-2,1,3-benzoxadiazole, capillary fused silica with 75 μm i.d. and a total length of 60 cm, separation voltage of 30 kV, BGE 137.5 mM borate buffer pH 10.25 and 12.5 mM β-CDs	LIF at λ	_ex	= 488 nm and λ	_em	= 522 nm	0.25 µmol/L	[109]
Venlafaxine	Pharmaceutical preparations	Fused silica capillary of 30 cm length (effective length 22 cm) × 50 µm, separation voltage of 25 kV, BGE of 25 mM phosphate buffer pH 2.5, 10 mM carboxymethyl-β-CD	UV at 230 nm	0.07 and 0.06 mg/mL 0.21 and 0.18 mg/mL	[110]
Methylparaben, ethylparaben, propylparaben, butylparaben, isobutylparaben, sorbic acid, benzoic acid, p-hydroxybenzoic acid	Pharmaceutical preparations	Online preconcentration by large volume sample stacking, fused silica capillary of 75 µm i.d. × 50 cm length, separation voltage 25 kV, BGE of 25 mM tetraborate pH 9.3 and α-CD	UV at 195 nm for ethylparaben, benzoic acid, and p-hydroxybenzoic acid, at 296 nm for methylparaben, propylparaben, butylparaben, and isobutylparaben, at 254 nm for sorbic acid at 254 nm.	0.8 to 5 ng/mL 3 to 16 ng/mL	[111]
L-Panthenol dexapanthenol	Pharmaceutical and cosmetic formulations	Fused silica capillary of a total length of 58.5 cm (50 cm effective length) and 50 µm i.d., separation voltage of 30 kV, BGE of 25 mM (2-carboxyethyl)-β-CD in 100 mM borate buffer pH 9.0	UV at 205 nm	1.0 and 4.0 mg/L 3.3 and 13.3 mg/L	[112]