In the last four decades, computational techniques have become useful tools in liquid-phase enantioseparation for modeling analyte and chiral selector structures, as well as the mobile phase implicitly and explicitly, for exploring mechanisms underlying retention and enantioseparation, and for identifying noncovalent interactions that contribute to analyte-chiral selector binding and recognition. The present chapter briefly summarizes fundamentals and recent advances in the application of molecular modeling for understanding capillary electrophoresis enantioseparations promoted by cyclodextrin-based selectors. A practical example illustrates in detail the calculation and analysis of low-energy structures of single-isomer methylated cyclodextrins and their stereoelectronic properties.

CDs, cyclic oligosaccharides consisting of α-1,4 linked d-glucopyranose units, have been applied in many areas, such as food, cosmetics, environmental, agriculture, textile, pharmaceutical, and chemical industries, due to their ability to form guest-host complexes. Because CDs are chiral compounds, they have also been successfully used as chiral selectors in analytical enantioseparation techniques, especially CE. In the last decades, multidisciplinary approaches have been performed, combining CE with NMR spectroscopy, molecular modeling, and other techniques in order to understand aspects of chiral recognition underlying enantioseparations. The present review focuses on such mechanistic studies published between January 2023 and March 2026 in addition to the application of CD-mediated CE enantioseparations to the determination of the enantiomeric purity of drug substances as well as the analysis of pharmaceutical formulations.

Stereospecific recognition of chiral molecules plays an important role in nature as the basis of the interaction of chiral bioactive compounds with the chiral target structures. In separation sciences such as chromatographic and capillary electromigration techniques, interactions between chiral analytes and chiral selectors, i.e., the formation of transient diastereomeric complexes in thermodynamic equilibria, are the basis for chiral separations. Due to the large structural variety of chiral selectors, different structural features contribute to the overall chiral recognition process. This introductory chapter briefly summarizes the present understanding of the structural enantioselective recognition processes for various types of chiral selectors.

Cyclodextrins (CDs) are cyclic oligosaccharides composed of glucose units connected by α(1 → 4) glycosidic bonds. These hollow toroid structures are well known for their exceptional ability to form inclusion complexes. This makes them attractive in many fields, including separation science. CDs are especially popular as chiral selectors in capillary electromigration techniques. A brief overview of their physicochemical properties, structural variability of their derivatives, and basic principles of enantioseparation by means of capillary electrophoresis is provided, along with the discussion of separation modes and various strategies that may be used during method development employing CDs as chiral selectors. The presented examples show how to set up the experimental conditions to obtain enantioseparations of (i) a negatively charged analyte (1,1'-binaphthyl-2,2'-diyl hydrogen phosphate) using native β-CD and (ii) a positively charged analyte (ofloxacin) using a negatively charged derivative of CD (sulfated β-CD). The reversal of the enantiomer migration order as a function of (i) the pH of the background electrolyte and (ii) the concentration of sulfated β-CD is shown as well.

Multiple factors may impact enantiorecognition ability of CDs, and the validation of computational tools by using suitable experimental data is a critical point. The main advantage of using capillary electrophoresis (CE) for this purpose relies on its higher sensitivity to detect weak noncovalent intermolecular interactions compared to any other technique. In this study, we used CE enantioseparations of tetramisole with β-CD, heptakis(2,3-di-O-methyl)-β-CD, heptakis(2,3-di-O-acetyl)-β-CD and heptakis(2-O-methyl-3-O-acetyl)-β-CD as benchmark separation systems aiming at investigating the molecular bases of these processes by NMR spectroscopy and quantum mechanics (QM) methods. The aim of this study was explaining the subtle differences observed in enantiomer migration order, that means enantiomer affinity pattern of tetramisole toward the used CDs, migration times, and selectivity values. A good correlation between experimental and theoretical data was obtained along with noncovalent interactions patterns fully consistent with the CE outcomes. Significant differences in the enantioselective recognition ability were observed between β-CD and heptakis(2,3-di-O-acetyl)-β-CD in CE, which correlated very well with the recognition model derived from NMR spectroscopy and QM. Importantly, the introduction of dispersion corrections in the used QM model chemistry provided results in better agreement with the experimental observations for the complexes of tetramisole with the acetylated β-CDs.

A computational study was performed to unravel mechanisms underlying capillary electrophoresis enantioseparations of daclatasvir and its (R,R,R,R)-enantiomer with native and methylated β-cyclodextrins (β-CDs) as chiral selectors. Considering the enantioseparation results as benchmark, the structures of β-CD and seven methylated β-CDs were optimized by quantum mechanics, and their topography and computed molecular properties were compared. Furthermore, the electron charge density distribution of the macrocycles was also evaluated by calculating the molecular electrostatic potential of pivotal regions of native and methylated β-CDs. The function of hydrogen bonds in the complexation process of daclatasvir and the CDs was derived from quantum mechanics analysis and confirmed by molecular dynamics, as orthogonal computational techniques. The presence of a round-shaped cavity in the CDs used as chiral selector appeared as a necessary requirement for the enantioseparation of daclatasvir and its (R,R,R,R)-enantiomer. In this regard, it was confirmed that the round shape of the CDs is sustained by hydrogen bonds formed between adjacent glucopyranose units and blocking rotation of the linking glycosidic bonds. The presence of hydroxy groups at the 6-position of the glucopyranose units and the concurrent absence of hydroxy groups at the 2-position were evidenced as important factors for enantioseparation of daclatasvir and its enantiomer by methylated β-CDs.

The stereospecific analysis of chiral molecules is an important issue in many scientific fields. In separation sciences, this is achieved via the formation of transient diastereomeric complexes between a chiral selector and the selectand enantiomers driven by molecular interactions including electrostatic, ion-dipole, dipole-dipole, van der Waals or π-π interactions as well as hydrogen or halogen bonds depending on the nature of selector and selectand. Nuclear magnetic resonance spectroscopy and molecular modeling methods are currently the most frequently applied techniques to understand the selector-selectand interactions at a molecular level and to draw conclusions on the chiral separation mechanism. The present short review summarizes some of the recent achievements for the understanding of the chiral recognition of the most important chiral selectors combining separation techniques with molecular modeling and/or spectroscopic techniques dating between 2020 and early 2024. The selectors include polysaccharide derivatives, cyclodextrins, macrocyclic glycopeptides, proteins, donor-acceptor type selectors, ion-exchangers, crown ethers, and molecular micelles. The application of chiral ionic liquids and chiral deep eutectic solvents, as well as further selectors, are also briefly addressed. A compilation of all published literature on chiral selectors has not been attempted.

In the present review the potential of capillary electrophoresis for a better understanding of the fine structural mechanisms of noncovalent interactions and enantioselective intermolecular recognition is highlighted for cyclodextrins and their guests. The importance of applying additional experimental and molecular modeling tools is stressed. The concept is illustrated by results published in the recent literature. The critical opinion of the authors on some current tendencies is provided.

Analytical Quality by Design principles using the design of experiments were applied for the development of a capillary electrophoresis method for the determination of enantiomeric purity and chemically related impurities of tamsulosin. From initial scouting experiments, a dual cyclodextrin (CD) system composed of sulfated β-CD and carboxymethyl-α-CD was selected as the chiral selector. A fractional factorial resolution V+ design was used for the identification of the critical process parameters, while a face-centered central composite design and Monte Carlo simulations were employed for final optimization and defining the design space of the method. The experimental conditions of the working point were: 30 mM sodium phosphate buffer, pH 3.0, containing 40 mg/mL sulfated β-CD and 7 mg/mL carboxymethyl-α-CD, capillary temperature 18°C, applied voltage -23 kV. Following the assessment of robustness by applying a Plackett-Burman design, the method was validated according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use guideline Q2(R1). The method allowed the quantification of the chiral impurity and three other related impurities at the 0.1 % level with acceptable accuracy and precision.

The complex formation between daclatasvir and gamma-CD or heptakis(2,3,6-tri-O-methyl)-beta-CD (TM-beta-CD) was studied by isothermal titration calorimetry and molecular modeling. Both techniques supported the predominant formation of a 2:1 complex in case of gamma-CD although a 1:1 complex may be formed to a much lower extent as well. In case of TM-beta-CD the stoichiometry of the complex was exclusively 1:1. Complex formation with gamma-CD did not require dissociation of the daclatasvir dimer, which is present in solution, and resulted in a complex with a binding constant of 1.67.107 M-2. In contrast, formation of the weak TM-beta-CD complex (K = 371 M-1) required dissociation of the daclatasvir dimer. This is in line with the observation that the complex formation in case of gamma-CD is enthalpy-driven, while the process is entropy-driven in case of TM-beta-CD. It is concluded that the plateau observed in capillary electrophoresis is primarily based on the slow dissociation of the daclatasvir-CD complexes caused by steric constrains due to the folded terminal amino acid moieties of daclatasvir exerting a clip effect. In case gamma-CD the thermodynamic stability might contribute to the overall slow dissociation.

A CE method was developed and validated for the assessment of the chiral purity of the drug tenofovir applying a quality by design approach. Following selection of a quaternary ammonium β-CD as chiral selector, a fractional factorial resolution V+ design was employed for identification of the critical process parameters, while a central composite design served for method optimization. The final method used a 40/50.2 cm, 50 μm id fused-silica capillary, a BGE composed of a 100 mM sodium phosphate buffer, pH 6.4, containing 45 mg/mL quaternary ammonium β-CD, an applied voltage of 18 kV, and a capillary temperature of 22°C. Robustness was assessed by a Plackett-Burman design. The method was validated according to guideline Q2(R1) of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use and enabled the determination of the (S)-enantiomer of tenofovir at the 0.1% level.

Capillary electromigration, as well as liquid chromatography techniques, have been used for many years for the analysis of compounds of pharmaceutical interest, ranging from small molecules to macromolecules. The development and wide application of such methods is driven by industrial and academic research groups, which is documented in numerous publications including papers from the regulatory agencies. Furthermore, electromigration and chromatographic methods are indispensable in clinical and forensic laboratories. This Special Issue features the progress of electromigration and liquid chromatographic techniques in pharmaceutical analysis. This topic was already treated in the earlier Special Issues of Electrophoresis 2006, 27(12), 2261–2526, Electrophoresis 2008, 29(17), 3489–3714, Electrophoresis 2012, 33(11), 1493–1684, Electrophoresis 2015, 36(21–22), 2635–2850 and Electrophoresis 2018, 39(20), 2485–2626. Even if there were already impressive applications in earlier volumes, especially capillary electrophoresis has substantially improved over the years. Today, it is certainly one of the methods of choice for enantioseparations, as well as for the analysis of macromolecules and biopharmaceuticals in particular. In the area of biopharmaceuticals, besides the analysis of the therapeutics, we also observe a slow increase in applications for other analyses, such as medium components and clearance of process additives. In addition, with the large body of experience that has accumulated over the years and the identification of best practice, the successful development and validation of methods have become straightforward. Recently, capillary electrophoresis and derived techniques, as well as liquid chromatography, proved their particular value as quality-indicating tools for vaccines during the pandemic. This Special Issue features the general improvement of various techniques including sensitivity enhancements and featuring rational method development. Four articles focus on biopharmaceuticals, and in addition, as mentioned, four papers relate to vaccines, mRNA and lipid nanoparticles (LNPs) as a particular highlight. Another key topic is the analysis from body fluids and cell cultures with all the matrix-related issues, which is discussed in five articles. The editors would like to thank all authors for their important contributions and all reviewers for their efforts and valuable comments on the manuscripts. Furthermore, we acknowledge the conference series which supported us with synergy for this Special Issue over the years, CE Pharm and ITP in particular. We certainly missed some of the exciting and fruitful discussions of the live conferences in the last two years, even though every organizer took a lot of effort to allow for some interactions.However, nowwe are looking forward to stimulate this live and lively discussion again, and to meet you all in presence very soon!

Silodosin is a single isomer selective α1-adrenoreceptor antagonist used for the treatment of benign prostatic hyperplasia. In order to control the enantiomeric purity of the drug a capillary electrophoresis method was developed that is applicable to the analysis of drug substance as well as pharmaceutical formulations. Method development followed a quality by design strategy. After selection of carboxymethyl-β-cyclodextrin as suitable chiral selector and the starting conditions in the scouting phase, a two-level full factorial design was applied to identify the critical process parameters. The final method optimization was performed using a face-centered central composite design resulting in the conditions 100 mM sodium phosphate buffer, pH 2.9, containing 40 mg/mL car-boxymethyl-β-cyclodextrin, a capillary temperature of 17 °C and an applied voltage of 28 kV. Robustness testing employing a Plackett-Burman design revealed the importance of careful pH adjustment in order to achieve suitable peak shape and resolution. The method was validated according to the guideline Q2(R1) of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use and applied to the analysis of a commercial capsule formulation.

Sulfur as a stereogenic center can be found in synthetic compounds and natural products. The current study evaluated the enantioseparation of 16 chiral (benzylsulfinyl)benzamide compounds by capillary electrophoresis using charged cyclodextrins (CDs) as chiral selectors in 50 mM sodium acetate buffer, pH 5.5. The sulfoxides varied in the type and position of the substituent of the benzyl moiety as well as the position and methylation of the amide group. Typically, randomly substituted CDs separated the majority of the model analytes in contrast to single isomer CDs. In case of random substitution, gamma-CD derivatives displayed higher resolution ability toward the set of model compounds followed by beta-CD and alpha-CD derivatives. Except for a few examples, the ( + )-enantiomer of the analytes migrated before the (-)-isomer irrespective of the type of the CD so that the chiral recognition appeared to be also mostly independent on the structure of the sulfoxides. Evaluation of complexation constants and complex mobilities of selected CD-analyte pairs revealed that the separations were based on the stereoselective complexation by the CD expressed as complexation constants but examples for complex mobilities as the determining factor for the enantiomer migration order were also found. In case of 2-(4-bromobenzylsulfinyl)-N-methyl benzamide in the presence of heptakis(2,3-di-O-methyl-6-O-sulfo)-alpha-CD reversal of the enantiomer migration order as a function of the CD concentration was observed. Using neutral CD derivatives in the presence of sodium dodecyl sulfate-based micelles at pH 9.0 only few sulfoxides could be enantioseparated. (C)& nbsp;2022 Elsevier B.V. All rights reserved.

The metabolic syndrome (MetS) is a constellation of cardiovascular and metabolic symptoms involving insulin resistance, steatohepatitis, obesity, hypertension, and heart disease, and patients suffering from MetS often require polypharmaceutical treatment. PPARγ agonists are highly effective oral antidiabetics with great potential in MetS, which promote adipocyte browning and insulin sensitization. However, the application of PPARγ agonists in clinics is restricted by potential cardiovascular adverse events. We have previously demonstrated that the racemic dual sEH/PPARγ modulator RB394 (3) simultaneously improves all risk factors of MetS in vivo. In this study, we identify and characterize the eutomer of 3. We provide structural rationale for molecular recognition of the eutomer. Furthermore, we could show that the dual sEH/PPARγ modulator is able to promote adipocyte browning and simultaneously exhibits cardioprotective activity which underlines its exciting potential in treatment of MetS.

In capillary electrophoresis an enantioseparation of daclatasvir (DCV) was observed in case of heptakis(2,6-di-O-methyl)-β-CD, heptakis(2-O-methyl)-β-CD and β-CD, while two peaks with a plateau were noted for heptakis(2,3,6-tri-O-methyl)-β-CD and heptakis(2,3-di-O-methyl)-β-CD indicating a slow equilibrium. Heptakis(6-O-methyl)-β-CD and heptakis(3-O-methyl)-β-CD yielded broad peaks. Nuclear magnetic resonance experiments including nuclear Overhauser effect-based techniques revealed inclusion complex formation for all CDs with the biphenyl ring of DCV within the cavity and the valine-pyrrolidine moieties protruding from the torus. However, in case of heptakis(2,6-di-O-methyl)-β-CD, heptakis(2-O-methyl)-β-CD and β-CD higher order structures with 1:3 stoichiometry were concluded, where the valine moieties enter additional CD molecules via the secondary side. Heptakis(2,3,6-tri-O-methyl)-β-CD and heptakis(2,3-di-O-methyl)-β-CD yielded primarily 1:1 complexes. Higher order complexes between DCV and heptakis(2,6-di-O-methyl)-β-CD were corroborated by mass spectrometry. Complex stoichiometry was not the reason for the slow equilibrium yielding the plateau observed in capillary electrophoresis, but structural characteristics of the CDs especially complete methylation of the secondary rim.

Capillary electrophoresis is a powerful technique for the analysis of polar chiral compounds and has been widely accepted for analytical enantioseparations of drug compounds in pharmaceuticals and biological media. In addition, many mechanistic studies have been conducted in an attempt to rationalize enantioseparations in combination with spectroscopic and computational techniques. The present review will focus on recent examples of mechanistic aspects and summarize recent applications of stereoselective pharmaceutical and biomedical analysis published between January 2017 and November 2020. Various separation modes including electrokinetic chromatography in combination with several detection modes including laser-induced fluorescence, mass spectrometry and contactless conductivity detection will be discussed. A general trend also observed in other analytical techniques is the application of quality by design principles in method development and optimization.

Dolutegravir is an integrase strand transfer inhibitor used for the treatment of human immuno-deficiency virus infections. The present study was conducted in order to identify degradation products formed in acidic solution upon heating. The structures were assigned based on low resolution collision-induced dissociation tandem mass spectra as well as high resolution higher-energy collisional dissociation tandem mass spectra. The major degradation products resulted from hydrolytic opening of the oxepine ring leading to bis-hydroxy diastereomers (DP2 and DP3) as well as a mono-hydroxy derivative (DP1) as the result of dehydration of the diastereomers. Furthermore, two carboxylic acid derivatives (DP4 and DP5) could be identified, which can be explained as the result of the hydrolysis of the exocyclic amide bond of dolutegravir and DP1, respectively. During the fragmentation process of dolutegravir and its degradation products DP1 to DP3 a formal addition of oxygen resulting in the respective carboxylic acid fragments was detected. This could be evidenced based on high resolution masses of the fragments as well as the comparison of the MS/MS spectra of the fragments with the spectra of the carboxylic acids DP4 and DP5.

The separation of daclatasvir and its R,R,R,R-enantiomer was studied by capillary electrophoresis using various randomly methylated β-CDs and the single isomer heptakis(2,6-di-O-methyl)-β-CD (2,6-DM-β-CD) as chiral selectors in an acidic background electrolyte. Opposite enantiomer migration order was observed for randomly substituted CDs compared to 2,6-DM-β-CD as well as methylated β-CDs with different composition according to the specifications of the manufacturers. HPLC and NMR analyses confirmed that the presence of a high 2,6-DM-β-CD content in the CDs enables to achieve the migration order R,R,R,R-enantiomer > daclatasvir. In contrast, products with low 2,6-DM-β-CD isomer content and/or the presence of a large amount of methylated CD isomers, in which d-glucopyranose moieties are not substituted in either position 2 or 6, displayed the opposite enantiomer migration order daclatasvir > R,R,R,R-enantiomer. The study indicated the importance of the type and composition of derivatized CDs on chiral separations in capillary electrophoresis as well as the importance of proper quality control for cyclodextrin manufacturers. Moreover, the observed migration order could be rationalized based on the composition and substitution pattern of the CDs.

The synergistic effect of chiral ionic liquids composed of tetraalkylammonium ions and the amino acids Asn, Asp or Pro on the enantioseparations of dipeptides mediated by β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin in capillary electrophoresis was studied. Addition of a chiral ionic liquid resulted in a concentration-dependent increase in the enantioresolutions compared to the sole presence of a cyclodextrin in the background electrolyte. The extent varied with the tetraalkylammonium cation (tetramethylammonium versus tetrabutylammonium) as well as the amino acid component of the ionic liquid. The presence of a chiral ionic liquid did not counteract the pH-dependent reversal of the enantiomer migration order of the dipeptides Ala-Phe, Ala-Tyr and Phe-Phe when increasing the pH of the background electrolyte from 2.5 to 3.5. Comparing the effect of a chiral ionic liquid based on Asp with the addition of equimolar concentrations of the individual components of the ionic liquid, a diverse picture was observed. In some cases, higher resolution values were obtained with the chiral ionic liquid, while for other cases superior enantioseparations were obtained upon separate addition of the amino acid component and a tetraalkylammonium chloride. With regard to the stereochemistry of the amino acid, a superior effect was typically observed using the l-configured amino acid, but in some cases higher resolution values were found in the presence of d-Asp. The rationale for the diverse observations is not obvious and may be due to the zwitterionic nature of analytes as well as the amino acid component of the chiral ionic liquid.