Update on chiral recognition mechanisms in separation science.

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.

Quality by design-guided development of a capillary electrophoresis method for the simultaneous chiral purity determination and impurity profiling of tamsulosin.

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 ß-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 ß-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.

Isothermal titration calorimetry and molecular modeling study of the complex formation of daclatasvir by γ-cyclodextrin and trimethyl-ß-cyclodextrin.

The complex formation between daclatasvir and γ-CD or heptakis(2,3,6-tri-O-methyl)-ß-CD (TM-ß-CD) was studied by isothermal titration calorimetry and molecular modeling. Both techniques supported the predominant formation of a 2:1 complex in case of γ-CD although a 1:1 complex may be formed to a much lower extent as well. In case of TM-ß-CD the stoichiometry of the complex was exclusively 1:1. Complex formation with γ-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-ß-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 γ-CD is enthalpy-driven, while the process is entropy-driven in case of TM-ß-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 γ-CD the thermodynamic stability might contribute to the overall slow dissociation.

Quality by design-guided development of a capillary electrophoresis method for the chiral purity determination of silodosin.

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-ß-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-ß-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.

Enantioseparation of chiral (benzylsulfinyl)benzamide sulfoxides by capillary electrophoresis using cyclodextrins as chiral selectors.

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, γ-CD derivatives displayed higher resolution ability toward the set of model compounds followed by ß-CD and α-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)-α-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.

Quality by design-assisted development of a capillary electrophoresis method for the enantiomeric purity determination of tenofovir.

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 ß-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 ß-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.

Structural characterization of methyl-ß-cyclodextrins by high-performance liquid chromatography and nuclear magnetic resonance spectroscopy and effect of their isomeric composition on the capillary electrophoresis enantioseparation of daclatasvir.

The separation of daclatasvir and its R,R,R,R-enantiomer was studied by capillary electrophoresis using various randomly methylated ß-CDs and the single isomer heptakis(2,6-di-O-methyl)-ß-CD (2,6-DM-ß-CD) as chiral selectors in an acidic background electrolyte. Opposite enantiomer migration order was observed for randomly substituted CDs compared to 2,6-DM-ß-CD as well as methylated ß-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-ß-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-ß-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.

Complexation of daclatasvir by single isomer methylated ß-cyclodextrins studied by capillary electrophoresis, NMR spectroscopy and mass spectrometry.

In capillary electrophoresis an enantioseparation of daclatasvir (DCV) was observed in case of heptakis(2,6-di-O-methyl)-ß-CD, heptakis(2-O-methyl)-ß-CD and ß-CD, while two peaks with a plateau were noted for heptakis(2,3,6-tri-O-methyl)-ß-CD and heptakis(2,3-di-O-methyl)-ß-CD indicating a slow equilibrium. Heptakis(6-O-methyl)-ß-CD and heptakis(3-O-methyl)-ß-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)-ß-CD, heptakis(2-O-methyl)-ß-CD and ß-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)-ß-CD and heptakis(2,3-di-O-methyl)-ß-CD yielded primarily 1:1 complexes. Higher order complexes between DCV and heptakis(2,6-di-O-methyl)-ß-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.

Effects of amino acid-derived chiral ionic liquids on cyclodextrin-mediated capillary electrophoresis enantioseparations of dipeptides.

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 ß-cyclodextrin and 2-hydroxypropyl-ß-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.

Combined Cardioprotective and Adipocyte Browning Effects Promoted by the Eutomer of Dual sEH/PPARγ Modulator.

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.

HPLC-MS identification of acid degradation products of dolutegravir.

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.

Advances of capillary electrophoresis enantioseparations in pharmaceutical analysis (2017-2020).

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.

Global testing of a consensus solubility assessment to enhance robustness of the WHO biopharmaceutical classification system.

The WHO Biopharmaceutical Classification System (BCS) is a practical tool to identify active pharmaceutical ingredients (APIs) that scientifically qualify for a waiver of in vivo bioequivalence studies. The focus of this study was to engage a global network of laboratories to experimentally quantify the pH-dependent solubility of the highest therapeutic dose of 16 APIs using a harmonized protocol. Intra-laboratory variability was ≤5 %, and no apparent association of inter-laboratory variability with API solubility was discovered. Final classification "low solubility" vs "high solubility" was consistent among laboratories. In comparison to the literature-based provisional 2006 WHO BCS classification, three compounds were re-classified from "high" to "low-solubility". To estimate the consequences of these experimental solubility results on BCS classification, dose-adjusted in silico predictions of the fraction absorbed in humans were performed using GastroPlus®. Further expansion of these experimental efforts to qualified APIs from the WHO Essential Medicines List is anticipated to empower regulatory authorities across the globe to issue scientifically-supported guidance regarding the necessity of performing in vivo bioequivalence studies. Ultimately, this will improve access to affordable generic products, which is a critical prerequisite to reach Universal Health Coverage.

12-Oxo-10-glutathionyl-5,8,14-eicosatrienoic acid (TOG10), a novel glutathione-containing eicosanoid generated via the 12-lipoxygenase pathway in human platelets.

Biologically active glutathione (GSH) conjugates of oxygenated fatty acids comprise a group of pro- and anti-inflammatory lipid mediators. While arachidonic acid (AA)-derived conjugates, as the cysteinyl leukotrienes (cys-LTs) and eoxins (EXs) have pro-inflammatory properties, conjugates in tissue regeneration (CTRs) biosynthesized from docosahexaenoic acid (DHA) exhibit pro-resolving activity. Human platelets express abundant amounts of platelet-type 12-lipoxygenase (pt12-LOX) and leukotriene C4 synthase (LTC4S). However, the only two described GSH conjugates formed by platelets are the AA-derived cys-LTs and the recently reported maresin CTRs (MCTRs). While cys-LTs are biosynthesized in a transcellular mechanism via the action of 5-LOX and LTC4S, MCTR1 is formed by 12-LOX and a yet unidentified GSH S-transferase (GST). Here, we present a novel GSH conjugate formed from AA via the 12-LOX pathway in human platelets. The 12-oxo-glutathione adduct, 12-oxo-10-glutathionyl-5,8,14-eicosatrienoic acid (TOG10), was identified by mass spectrometry using positive electrospray ionization. The structural proposal is supported by fragmentation data of the labeled metabolite obtained after incubation of deuterated AA (AA-d8). In platelets as well as in HEK293 cells stably expressing pt12-LOX, TOG10 biosynthesis was inhibited by the 12-LOX inhibitor ML-355 (5 µM), which confirms the involvement of pt12-LOX. Interestingly, TOG10 was formed independently of LTC4S in platelets. This is in accordance with the observation that the conjugate was also generated by AA-stimulated HEK_12-LOX cells in absence of LTC4S. Nevertheless, TOG10 can also be formed by LTC4S as the biosynthesis in HEK_12-LOX_LTC4S cells was reduced by the specific LTC4S inhibitor TK04a. In summary, TOG10 was identified as a new AA-derived GSH conjugate generated in human platelets via the action of pt12-LOX in combination with a GST.

Enantioseparation of alanyl-phenylalanine analogs by capillary electrophoresis using negatively charged cyclodextrins as chiral selectors.

The separation of the ll- and dd-enantiomers of the dipeptides Ala-Phe, Ala-phenylglycine (Phg), Ala-homoPhe, Ala-ß-Phe, Gly-Phe and ß-Ala-Phe was studied by capillary electrophoresis in the presence of negatively charged α-, ß- and γ-cyclodextrin (CD) derivatives. Analysis was performed under standardized conditions in fused-silica capillaries at pH 2.5, 3.5 and 5.3. All analyte enantiomers could be separated at pH 2.5 under at least one of the experimental conditions. Especially ß-CD derivatives proved to be effective chiral selectors. The enantiomer migration order depended on CD cavity size and substituent type, while peptide structure had only a minor effect. Upon increasing the pH from 2.5 to 5.3, reversal of the enantiomer migration order was observed frequently. Investigation of the apparent and, in the case of randomly substituted CDs, averaged complexation constants and mobilities of the diastereomeric peptide enantiomer-CD complexes indicated that in most cases the migration order in the presence of sulfated α-, ß- and γ-CD and heptakis(6-O-sulfo)-ß-CD could be explained by the stronger binding of the second migrating analyte by the CD at pH 2.5. However, in few cases the weaker bound enantiomer migrated second, which could be attributed to the higher mobility of the respective CD complexes. At pH 5.3, similar data were obtained for sulfated ß-CD and heptakis(6-O-sulfo)-ß-CD, i.e. the strength of the complexes determined the migration order for some peptides, while the migration sequence was based on the apparent (and averaged) mobility of the diastereomeric analyte-CD complexes in other cases.

Unusual complexation behavior between daclatasvir and γ-Cyclodextrin. A multiplatform study.

During a screening of cyclodextrins (CDs) as chiral selectors for the separation of daclatasvir (DCV) and its enantiomer by capillary electrophoresis (CE), an unusual phenomenon for CDs was observed, that is two peaks with a plateau in between using γ-CD as chiral selector. The same result was encountered when enantiopure DCV was injected or when analyzing a sample containing enantiopure DCV and γ-CD in a CD-free background electrolyte. Peak coalescence was observed at 45°C and at a pH above 3.5. Two peaks with a plateau were also observed for DCV stereoisomers as well as a structural analog. However, only a single peak was detected if one or both amino acid moieties of DCV were lacking. Nuclear magnetic resonance (NMR) experiments including Nuclear Overhauser effect-based methods showed that in solution DCV adopted a folded conformation in which the isopropyl side chain of the valine residues pointed toward the aromatic rings of DCV. Moreover, NMR unequivocally demonstrated the simultaneous formation of DCV-γ-CD inclusion complexes with 1:1 and 2:1 stoichiometry, which was corroborated by mass spectrometry. In both complexes, DCV also adopted a folded structure. The RSSR-diastereomer of DCV as well as an analog lacking one of the amino acid moieties also formed 1:1 and 2:1 complexes with γ-CD although a plateau was only observed in the case of the RSSR-diastereomer. As shown by CE-MS, both DCV-γ-CD complexes surprisingly comigrated as the first peak, while the second migrating peak represents non-complexed DCV.

Liquid chromatographic method for the simultaneous determination of achiral and chiral impurities of dapoxetine in approved and counterfeit products.

A reversed-phase high performance liquid chromatographic method was developed and validated for the simultaneous determination of the related substances of S-dapoxetine, including R-dapoxetine, (3S)-3-(dimethylamino-3-phenyl-1-propanol), S-3-amino-3-phenyl-1-propanol, 1-naphtol, 4-phenyl-2H,3H,4H-naphtho[1,2-b]pyran and 1-(2E)-Cinnamyloxynaphthalene. During the screening experiments seven different polysaccharide-type chiral stationary phases (amylose-based Lux-Amylose-1, Lux-i-Amylose-1 and Lux-Amylose-2, as well as cellulose-based Lux-Cellulose-1, Lux-Cellulose-2, Lux-Cellulose-3 and Lux-Cellulose-4) were tested in polar organic mode using a mobile phase consisting of 0.1% diethylamine in methanol, ethanol, 2-propanol and acetonitrile with 0.5 mL min-1 flow rate at 20 °C. Best results were obtained on Lux Cellulose-3 column with the ethanol-based mobile phase. To increase the retention factor of two, early-eluting impurities, water was added to the mobile phase. In order to counterbalance the increased total analysis time, higher column temperature (40 °C) and gradient elution, combined with flow-programming` was applied. Using the optimized conditions baseline separations were achieved for all compounds within 30 min. The method was validated according to the International Council on Harmonization guideline Q2(R1) and applied to the analysis of an approved, tablet formulation and dapoxetine-containing products sold on the internet. As expected, in the case of the pharmacy-acquired product, all of the monitored impurities were below 0.1%. However, interesting results were obtained when internet-acquired samples were analyzed. These tablets contained racemic dapoxetine and/or high concentration of R-dapoxetine impurity. Based on this work polysaccharide-based chiral stationary phases can be successfully applied for the simultaneous determination of achiral and chiral impurities in reversed-phase mode applying gradient elution and flow-rate programs. The study further underlines the importance of not only achiral, but also enantiomeric quality control, whenever counterfeiting of a single enantiomeric agent is suspected.

Enantioseparation of analogs of the dipeptide alanyl-phenylalanine by capillary electrophoresis using neutral cyclodextrins as chiral selectors.

In the present study, the enantioseparation of the ll- and dd-enantiomers of the dipeptides Ala-Phe, Ala-phenylglycine (Phg), Ala-homoPhe, Ala-ß-Phe, Gly-Phe and ß-Ala-Phe was studied by capillary electrophoresis in the presence of native α-, ß- and γ-cyclodextrin (CD) as well as their methyl and hydroxypropyl derivatives. Separations were performed under standardized conditions in fused-silica capillaries at pH 2.5, 3.5 and 9.5. All analyte enantiomers could be separated at acidic pH under at least one of the experimental conditions. ß-CDs proved to be more universal chiral selectors than α- and γ-CDs. Only few alkaline conditions led to an enantioseparation. For a given dipeptide, the enantiomer migration order depended on the type of CD with regard to cavity size and degree of substitution. Little effect was found with regard to the structure of the dipeptides. pH-dependent reversal of the enantiomer migration order upon increasing the pH from 2.5 to 3.5 was observed for all dipeptides with at least one of the ß-CD derivatives. In the case of ß-CD, analysis of the complexation constants and the apparent limiting mobilities of the diastereomeric peptide enantiomer-CD complexes revealed, that the enantiomer migration order of Ala-Phe, Ala-homoPhe and Ala-ß-Phe was determined by the stereoselective complexation by ß-CD at pH 2.5. At pH 3.5 opposite chiral recognition of the enantiomers by ß-CD was found for Ala-Phe and Ala-ß-Phe resulting in the reversed migration order. In contrast, chiral recognition did not change in the case of Ala-homoPhe, but reversal of the enantiomer migration order was based on the apparent mobility of the diastereomeric analyte-CD complexes.

Chiral separation of four phenothiazines by nonaqueous capillary electrophoresis and quality by design-based method development for quantification of dextromepromazine as chiral impurity of levomepromazine.

The separation of the enantiomers of mepromazine, promethazine, thioridazine and alimemazine was studied by nonaqueous capillary electrophoresis in the presence of cyclodextrins using 1 M acetic acid and 50 mM ammonium acetate in methanol as background electrolyte. Heptakis(2,3-di-O-acetyl-6-O-sulfo)-ß-cyclodextrin, heptakis(2,3-di-O-methyl-6-O-sulfo)-ß-cyclodextrin (HDMS-ß-CD) and octakis(2,3-di-O-methyl-6-O-sulfo)-γ-cyclodextrin were the most effective chiral selectors for mepromazine, promethazine and alimemazine. Subsequently, a method for the determination of dextromepromazine as chiral impurity of levomepromazine was developed employing quality by design principles. Using HDMS-ß-CD as selector, a fractional factorial resolution V+ design was employed for evaluating the knowledge space, while a central composite face centered design provided further method optimization and the basis for the computation of the design space by Monte Carlo simulations. The final experimental conditions included a 30/40.2 cm fused-silica capillary with 75 µm inner diameter and a background electrolyte composed of 0.75 M acetic acid and 55 mM ammonium acetate in methanol containing 27.5 mg/mL HDMS-ß-CD. The applied voltage was 22 kV and the capillary temperature was 15°C. Following method robustness testing via a Plackett-Burman design, the method was validated for dextromepromazine in the range of 0.01 to 3.0 % relative to a concentration of 0.74 mg/mL levomepromazine and applied to the analysis of reference standards of the European Pharmacopoeia and commercial tablets. The assay also allowed the detection of levomepromazine sulfoxide although the quantitation of the compound was hampered by the poor peak shape of the late migrating diastereomer.

Enantioselective resolution of biologically active dipeptide analogs by high-performance liquid chromatography applying Cinchona alkaloid-based ion-exchanger chiral stationary phases.

Sixteen pairs of enantiomeric dipeptides were separated on four chiral ion-exchanger-type stationary phases based on Cinchona alkaloids. Anion-exchangers (QN-AX, QD-AX) and zwitterionic phases [ZWIX(+)™ and ZWIX(-)™] were studied in a comparative manner. The effects of the nature and concentrations of the mobile phase solvent components and organic salt additives on analyte retention and enantioseparation were systematically studied in order to get a deeper insight into the enantiorecognition mechanism. Moreover, experiments were performed in the temperature range 10-50 °C to calculate thermodynamic parameters like changes in standard enthalpy, Δ(ΔH°), entropy, Δ(ΔS°), and free energy, Δ(ΔG°) on the basis of van't Hoff plots derived from the ln α vs. 1/T curves. Elution sequences of the dipeptides were determined in all cases and, with a few exceptions, they were found to be opposite on the pseudoenantiomeric stationary phases as of QN-AX/QD-AX and of ZWIX(+) and ZWIX(-). The stereoselective retention mechanism is based on electrostatically driven intermolecular interactions supported by additional interaction increments mainly determined by the absolute configuration of the chiral C8 and C9 atoms of the quinine and quinidine moieties.