Simultaneous Determination of Enantiomeric Purity and Organic Impurities of Dexketoprofen Using Reversed-Phase Liquid Chromatography-Enhancing Enantioselectivity through Hysteretic Behavior and Temperature-Dependent Enantiomer Elution Order Reversal on Polysaccharide Chiral Stationary Phases.

A reversed-phase high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of the potential impurities of dexketoprofen, including the distomer R-ketoprofen. After screening the separation capability of four polysaccharide columns (Lux Amylose-1, Lux Amylose-2, Lux Cellulose-1 and Lux Cellulose-2) in polar organic and in reversed-phase modes, appropriate enantioseparation was observed only on the Lux Amylose-2 column in an acidified acetonitrile/water mixture. A detailed investigation of the mobile phase composition and temperature for enantio- and chemoselectivity showed many unexpected observations. It was observed that both the resolution and the enantiomer elution order can be fine-tuned by varying the temperature and mobile phase composition. Moreover, hysteresis of the retention times and enantioselectivity was also observed in reversed-phase mode using methanol/water mixtures on amylose-type columns. This could indicate that the three-dimensional structure of the amylose column can change by transitioning from a polar organic to a reversed-phase mode, which affects the enantioseparation process. Temperature-dependent enantiomer elution order and rare enthalpic/entropic controlled enantioseparation in the operative temperature range were also observed in reversed-phase mode. To find the best methodological conditions for the determination of dexketoprofen impurities, a full factorial optimization design was performed. Using the optimized parameters (Lux Amylose-2 column with water/acetonitrile/acetic acid 50/50/0.1 (v/v/v) at a 1 mL/min flow rate at 20 °C), baseline separations were achieved between all compounds within 15 min. Our newly developed HPLC method was validated according to the current guidelines, and its application was tested on commercially available pharmaceutical formulations. According to the authors' knowledge, this is the first study to report hysteretic behavior on polysaccharide columns in reversed-phase mode.

Arylnaphthalene Lignans with Anti-SARS-CoV-2 and Antiproliferative Activities from the Underground Organs of Linum austriacum and Linum perenne.

Diphyllin (1) and justicidin B (2) are arylnaphthalene lignans with antiviral and antiproliferative effects. Compound 1 is also known as an effective inhibitor of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). To evaluate the in vitro antiviral and cytotoxic potency of both lignans in SARS-CoV-2 -infected cells and various cancer cell lines, respectively, 1 and 2 were isolated from the underground organs of Linum austriacum and Linum perenne. Two previously undescribed arylnaphthalene lignans, denominated linadiacin A and B (3 and 4), were also isolated and identified. In acidic media, 3 was converted by a two-step reaction into 2 via the intermediate 4. Optimum acid treatment conditions were determined to isolate lignans by one-step preparative high-performance liquid chromatography (HPLC). The results of the conversion, HPLC-tandem mass spectrometry, nuclear magnetic resonance spectroscopy, and molecular modeling studies allowed complete structure analysis. Compounds 1 and 2 were the most effective against SARS-CoV-2 with a 3-log reduction in the viral copy number at a 12.5 µM concentration. Ten human cancer cell lines showed sensitivity to at least one of the isolated lignans.

Enantioselective Human Serum Albumin Binding of Apremilast: Liquid Chromatographic, Fluorescence and Molecular Docking Study.

The interaction between human serum albumin (HSA) and apremilast (APR), a novel antipsoriatic drug, was characterized by multimodal analytical techniques including high-performance liquid chromatography (HPLC), fluorescence spectroscopy and molecular docking for the first time. Using an HSA chiral stationary phase, the APR enantiomers were well separated, indicating enantioselective binding between the protein and the analytes. The influence of chromatographic parameters-type and concentration of the organic modifier, buffer type, pH, ionic strength of the mobile phase, flow rate and column temperature-on the chromatographic responses (retention factor and selectivity) was analyzed in detail. The results revealed that the eutomer S-APR bound to the protein to a greater extent than the antipode. The classical van 't Hoff method was applied for thermodynamic analysis, which indicated that the enantioseparation was enthalpy-controlled. The stability constants of the protein-enantiomer complexes, determined by fluorescence spectroscopy, were in accordance with the elution order observed in HPLC (KR-APR-HSA = 6.45 × 103 M-1, KS-APR-HSA = 1.04 × 104 M-1), showing that, indeed, the later-eluting S-APR displayed a stronger binding with HSA. Molecular docking was applied to study and analyze the interactions between HSA and the APR enantiomers at the atomic level. It was revealed that the most favored APR binding occurred at the border between domains I and II of HSA, and secondary interactions were responsible for the different binding strengths of the enantiomers.

The Applicability of Chromatographic Retention Modeling on Chiral Stationary Phases in Reverse-Phase Mode: A Case Study for Ezetimibe and Its Impurities.

Mechanistic modeling is useful for predicting and modulating selectivity even in early chromatographic method development. This approach is also in accordance with current analytical quality using design principles and is highly welcomed by the authorities. The aim of this study was to investigate the separation behavior of two different types of chiral stationary phases (CSPs) for the separation of ezetimibe and its related substances using the mechanistic retention modeling approach offered by the Drylab software (version 4.5) package. Based on the obtained results, both CSPs presented with chemoselectivity towards the impurities of ezetimibe. The cyclodextrin-based CSP displayed a higher separation capacity and was able to separate seven related substances from the active pharmaceutical ingredient, while the cellulose-based column enabled the baseline resolution of six impurities from ezetimibe. Generally, the accuracy of predicted retention times was lower for the polysaccharide CSP, which could indicate the presence of additional secondary interactions between the analytes and the CSP. It was also demonstrated that the combination of mechanistic modeling and an experimental design approach can be applied to method development on CSPs in reverse-phase mode. The applicability of the methods was tested on spiked artificial placebo samples, while intraday and long-term (2 years) method repeatability was also challenged through comparing the obtained retention times and resolution values. The results indicated the excellent robustness of the selected setpoints. Overall, our findings indicate that the chiral columns could offer orthogonal selectivity to traditional reverse-phase columns for the separation of structurally similar compounds.

Chiral Separation of Oxazolidinone Analogs by Capillary Electrophoresis Using Anionic Cyclodextrins as Chiral Selectors: Emphasis on Enantiomer Migration Order.

Comparative chiral separations of enantiomeric pairs of four oxazolidinone and two related thio-derivatives were performed by capillary electrophoresis, using cyclodextrins (CDs) as chiral selectors. Since the selected analytes are neutral, the enantiodiscrimination capabilities of nine anionic CD derivatives were determined, in 50 mM phosphate buffer pH = 6. Unanimously, the most successful chiral selector was the single isomeric heptakis-(6-sulfo)-ß-cyclodextrin (HS-ß-CD), which resulted in the highest enantioresolution values out of the CDs applied for five of the six enantiomeric pairs. The enantiomer migration order (EMO) was the same for two enantiomeric pairs, irrespective of the CD applied. However, several examples of EMO reversals were obtained in the other cases. Interestingly, changing from randomly substituted, multi-component mixtures of sulfated-ß-CD to the single isomeric chiral selector, enantiomer migration order reversal occurred for two enantiomeric pairs and similar observations were made when comparing heptakis-(2,3-di-O-methyl-6-O-sulfo)-ß-CD, (HDMS-ß-CD) with HS-ß-CD. In several cases, cavity-size-dependent, and substituent-dependent EMO reversals were also observed. Minute differences in the structure of the analytes were also responsible for several cases of EMO reversal. The present study offers a complex overview of the chiral separation of structurally related oxazolidinones, and thio-analogs, highlighting the importance of the adequate choice of chiral selector in this group of compounds, where enantiomeric purity is of utmost importance.

Chiral Separation of Apremilast by Capillary Electrophoresis Using Succinyl-ß-Cyclodextrin-Reversal of Enantiomer Elution Order by Cationic Capillary Coating.

A stereospecific capillary electrophoresis method was developed for the separation of the novel, antipsoriatic agent, apremilast (APR). Six anionic cyclodextrin (CD) derivatives were screened for their ability to discriminate between the uncharged enantiomers. Only succinyl-ß-CD (Succ-ß-CD) presented chiral interactions; however, the enantiomer migration order (EMO) was unfavorable, and the eutomer, S-APR, migrated faster. Despite the optimization of all possible parameters (pH, cyclodextrin concentration, temperature, and degree of substitution of CD), the method was unsuccessful for purity control due to the low resolution and the unfavorable enantiomer migration order. Changing the direction of electroosmotic flow (EOF) by the dynamic coating of the inner surface of the capillary with poly(diallyldimethylammonium) chloride or polybrene resulted in EMO reversal, and the developed method could be applied for the determination of R-APR as the enantiomeric purity. Thus, the application of the dynamic capillary coating offers a general opportunity for enantiomeric migration order reversal in particular cases when the chiral selector is a weak acid.

Simultaneous Determination of Escitalopram Impurities including the R-enantiomer on a Cellulose tris(3,5-Dimethylphenylcarbamate)-Based Chiral Column in Reversed-Phase Mode.

A high-performance liquid chromatographic method was developed for the simultaneous determination of the related substances-three potential synthesis-related chemical impurities and the distomer-of escitalopram. The separation capacity of seven different polysaccharide-type chiral columns, including three amylose-based (Lux Amylose-1, Lux i-Amylose-1, Lux Amylose-2) and four cellulose-based columns (Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3, and Lux Cellulose-4) were screened in the polar organic and reversed-phase modes. Lux Cellulose-1, based on cellulose tris(3,5-dimethylphenylcarbamate) as the chiral selector with an acetonitrile-water mixture containing 0.1% diethylamine was identified as the most promising separation system. Using the "one factor at a time" optimization approach, the effect of column temperature, flow rate, and mobile phase constituents on separation performance was evaluated, and the critical resolution values were determined. A U-shaped retention pattern was obtained when plotting the retention factors of the citalopram enantiomers versus the water content of the binary mobile phases on the Lux Cellulose-1 column. A thermodynamic analysis revealed enthalpy-driven enantioseparation in both the polar organic and reversed-phase modes. For further method optimizations, an L9 orthogonal array table was employed. Using the optimized parameters (Lux Cellulose-1 column with 0.1% (v/v) diethylamine in water/acetonitrile 55/45 (v/v); 0.8 mL/min flow rate at 25 °C), baseline separations were achieved between all compounds. Our newly developed HPLC method was validated according to the ICH guidelines and its application was tested with a commercially available pharmaceutical formulation. The method proved to be suitable for routine quality control of related substances and the enantiomeric purity of escitalopram.

Determination of Chiral Impurity of Naproxen in Different Pharmaceutical Formulations Using Polysaccharide-Based Stationary Phases in Reversed-Phased Mode.

A novel, validated, reversed-phase (RP), chiral high performance liquid chromatography (HPLC) method was developed for the enantiopurity control analysis of naproxen, a frequently used non-steroidal anti-inflammatory agent using polysaccharide-type chiral stationary phase (CSP). In the screening phase of method development, seven columns were tested in polar organic (PO) mode using mobile phases consisting of 0.1% acetic acid in methanol, ethanol, 2-propanol, and acetonitrile. Enantiorecognition was observed only in five cases. The best enantioseparation was observed on a Lux Amylose-1 column with 0.1% (v/v) acetic acid in ethanol with a resolution (Rs) of 1.24. The enantiomer elution order was unfavorable, as the distomer eluted after the eutomer. When the ethanolic mobile phase was supplemented with water, enantiomer elution order reversal was observed, indicating a difference in the enantiorecognition mechanism upon switching from PO to RP mode. Furthermore, by changing ethanol to methanol, not only lower backpressure, but also higher resolution was obtained. Subsequent method optimization was performed using a face-centered central composite design (FCCD) to achieve higher chiral resolution in a shorter analysis time. Optimized parameters offering baseline separation were as follows: Lux Amylose-1 stationary phase, thermostated at 40 °C, and a mobile phase consisting of methanol:water:acetic acid 85:15:0.1 (v/v/v), delivered with 0.65 mL/min flow rate. Using these optimized parameters, a Rs = 3.21 ± 0.03 was achieved within seven minutes. The optimized method was validated according to the ICH guidelines and successfully applied for the analysis of different pharmaceutical preparations, such as film-coated tablets and gel, as well as fixed-dose combination tablets, containing both naproxen and esomeprazole.

Chiral separation in the class of proton pump inhibitors by chromatographic and electromigration techniques: An overview.

Proton pump inhibitors (PPIs) are benzimidazole-derivative chiral sulfoxides, frequently used in the treatment of gastric hyperacidity-related disorders. Due to their stereoselective metabolism, the eutomeric forms of PPIs can present a more advantageous pharmacokinetic profile by comparison with the distomers or racemates. Moreover, two representatives of the class are used in therapy both as racemates and as pure enantiomers (esomeprazole, dexlansoprazole). A relatively large number of enantioseparation methods employed for the stereoselective determination of PPIs from pharmaceutical, biological, and environmental matrices were published in the past three decades. The purpose of the current overview is to provide a systematic survey of the available chiral separation methods published since the introduction of PPIs in the therapy up to the present. Analytical and bioanalytical methods using different chromatographic and electromigration techniques reported for the enantioseparation of omeprazole, lansoprazole, pantoprazole, rabeprazole, ilaprazole, and tenatoprazole are included. The analytical conditions of the presented methods are summarized in three comprehensive tables, while a critical discussion of the applied techniques, possible mechanism of enantiorecognition, and future perspectives on the topic are also presented.

Enantioseparation of solriamfetol and its major impurity phenylalaninol by capillary electrophoresis using sulfated gamma cyclodextrin.

R-solriamfetol is a recently approved drug used for the treatment of excessive sleepiness associated with narcolepsy and sleep apnea. Herein, a capillary electrophoretic method was developed, enabling the simultaneous analysis of the API and its S-enantiomer in addition to the enantiomers of its major impurity phenylalaninol. Twenty-nine different cyclodextrins (CDs), including native, neutral, and charged ones were screened as potential chiral selectors, and the best results were obtained with sulfated CDs. Randomly sulfated-ß-CD exhibited outstanding enantioresolution, the peaks of phenylalaninol enantiomers inserted between the two peaks of solriamfetol enantiomers, while sulfated-γ-CD (S-γ-CD) showed remarkable resolution values in a much shorter analysis time with the optimal enantiomer migration order. Among the single isomer sulfated CD derivatives, substituent dependent enantiomer migration order reversal could also be observed in the case of heptakis(6-O-sulfo)-ß-CD (HS-ß-CD) or heptakis(2,3-O-dimethyl-6-O-sulfo)-ß-CD (HDMS-ß-CD) with R-,S-solriamfetol, and heptakis(2,3-O-diacetyl-6-O-sulfo)-ß-CD (HDAS-ß-CD) resulting S-,R-solriamfetol migration order. The sulfated-γ-CD system was chosen for method optimization applying orthogonal experimental design. The optimized method (45 mM Tris-acetate buffer, pH 4.5, 4 mM S-γ-CD, 21°C, +19.5 kV) was capable for the baseline separation of solriamfetol and phenylalaninol enantiomers within 7 min. The optimized method was validated according to the ICH guidelines and successfully applied for the analysis of pharmaceutical preparation (Sunosi® 75 mg tablet), thus it may serve as a routine procedure for the laboratories of regulatory authorities as well as in Pharmacopoeias.

Synthesis of 3-O-Carboxyalkyl Morphine Derivatives and Characterization of Their Acid-Base Properties.

The C-3 phenolic hydroxy group containing morphine derivatives (morphine, oxymorphone, naloxone, naltrexone) are excellent candidates for the synthesis of 3-O-functionalized molecules. Achieving free carboxylic group containing derivatives gives the opportunity for further modification and conjugation that could be used for immunization and immunoassays. For this purpose ethyl bromo- and chloroacetate can be used as O-alkylating agents. Hydrolyzing the products affords the appropriate free carboxylic group containing 3-O-carboxyalkyl derivatives. As these molecules contain an acidic and a basic functional group the protonation macro- and microconstants were determined too, using pH-potentiometry and NMR-pH titration, beside fully characterizing their structure using IR, CD, NMR and HR-MS measurements.

Tissue-Specific Accumulation and Isomerization of Valuable Phenylethanoid Glycosides from Plantago and Forsythia Plants.

A comparative phytochemical study on the phenylethanoid glycoside (PhEG) composition of the underground organs of three Plantago species (P. lanceolata, P. major, and P. media) and that of the fruit wall and seed parts of Forsythia suspensa and F. europaea fruits was performed. The leaves of these Forsythia species and six cultivars of the hybrid F. × intermedia were also analyzed, demonstrating the tissue-specific accumulation and decomposition of PhEGs. Our analyses confirmed the significance of selected tissues as new and abundant sources of these valuable natural compounds. The optimized heat treatment of tissues containing high amounts of the PhEG plantamajoside (PM) or forsythoside A (FA), which was performed in distilled water, resulted in their characteristic isomerizations. In addition to PM and FA, high amounts of the isomerization products could also be isolated after heat treatment. The isomerization mechanisms were elucidated by molecular modeling, and the structures of PhEGs were identified by nuclear magnetic resonance spectroscopy (NMR) and high-resolution mass spectrometry (HR-MS) techniques, also confirming the possibility of discriminating regioisomeric PhEGs by tandem MS. The PhEGs showed no cytostatic activity in non-human primate Vero E6 cells, supporting their safe use as natural medicines and allowing their antiviral potency to be tested.

Comparative Chiral Separation of Thalidomide Class of Drugs Using Polysaccharide-Type Stationary Phases with Emphasis on Elution Order and Hysteresis in Polar Organic Mode.

The enantioseparation of four phthalimide derivatives (thalidomide, pomalidomide, lenalidomide and apremilast) was investigated on five different polysaccharide-type stationary phases (Chiralpak AD, Chiralpak AS, Lux Amylose-2, Chiralcel OD and Chiralcel OJ-H) using neat methanol (MeOH), ethanol (EtOH), 1-propanol (PROP), 2-propanol (IPA) and acetonitrile (ACN) as polar organic mobile phases and also in combination. Along with the separation capacity of the applied systems, our study also focuses on the elution sequences, the effect of mobile phase mixtures and the hysteresis of retention and selectivity. Although on several cases extremely high resolutions (Rs > 10) were observed for certain compounds, among the tested conditions only Chiralcel OJ-H column with MeOH was successful for baseline-separation of all investigated drugs. Chiral selector- and mobile-phase-dependent reversals of elution order were observed. Reversal of elution order and hysteresis of retention and enantioselectivity were further investigated using different eluent mixtures on Chiralpak AD, Chiralcel OD and Lux Amylose-2 column. In an IPA/MeOH mixture, enantiomer elution-order reversal was observed depending on the eluent composition. Furthermore, in eluent mixtures, enantioselectivity depends on the direction from which the composition of the eluent is approached, regardless of the eluent pair used on amylose-based columns. Using a mixture of polar alcohols not only the selectivities but the enantiomer elution order can also be fine-tuned on Chiralpak AD column, which opens up the possibility of a new type of chiral screening strategy.

The Use of Dual Cyclodextrin Chiral Selector Systems in the Enantioseparation of Pharmaceuticals by Capillary Electrophoresis: An Overview.

Cyclodextrin (CD) derivatives are the most efficient and frequently used chiral selectors (CSs) in capillary electrophoresis (CE). There are situations when the use of a single CD as CS is not enough to obtain efficient chiral discrimination of the enantiomers; in these cases, sometimes this problem can be resolved using a dual CD system. The use of dual CD systems can often dramatically enhance enantioseparation selectivity and can be applied for the separation of many analytes of pharmaceutical interest for which enantioseparation by CE with another CS systems can be problematic. Usually in a dual CD system an anionic CD is used together with a neutral one, but there are situations when the use of a cationic CD with a neutral one or the use of two neutral CDs or even two ionized CDs can be an efficient solution. In the current review we present general aspects of the use of dual CD systems in the analysis of pharmaceutical substances. Several examples of applications of the use of dual CD systems in the analysis of pharmaceuticals are selected and discussed. Theoretical aspects regarding the separation of enantiomers through simultaneous interaction with the two CSs are also explained. Finally, advantages, disadvantages, potential and new direction in this chiral analysis field are highlighted.

Groundwater, soil and compost, as possible sources of virulent and antibiotic-resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major public health concern all around the world. In the frame of this work, a set of diverse environmental P. aeruginosa isolates with various antibiotic resistance profiles were examined in a Galleria mellonella virulence model. Motility, serotypes, virulence factors and biofilm-forming ability were also examined. Molecular types were determined by pulsed-field gel electrophoresis (PFGE). Based on our results, the majority of environmental isolates were virulent in the G. mellonella test and twitching showed a positive correlation with mortality. Resistance against several antibiotic agents such as Imipenem correlated with a lower virulence in the applied G. mellonella model. PFGE revealed that five examined environmental isolates were closely related to clinically detected pulsed-field types. Our study demonstrated that industrial wastewater effluents, composts, and hydrocarbon-contaminated sites should be considered as hot spots of high-risk clones of P. aeruginosa.

Reversed-phase HPLC enantioseparation of pantoprazole using a teicoplanin aglycone stationary phase-Determination of the enantiomer elution order using HPLC-CD analyses.

A direct HPLC method was developed for the enantioseparation of pantoprazole using macrocyclic glycopeptide-based chiral stationary phases, along with various methods to determine the elution order without isolation of the individual enantiomers. In the preliminary screening, four macrocyclic glycopeptide-based chiral stationary phases containing vancomycin (Chirobiotic V), ristocetin A (Chirobiotic R), teicoplanin (Chirobiotic T), and teicoplanin-aglycone (Chirobiotic TAG) were screened in polar organic and reversed-phase mode. Best results were achieved by using Chirobiotic TAG column and a methanol-water mixture as mobile phase. Further method optimization was performed using a face-centered central composite design to achieve the highest chiral resolution. Optimized parameters, offering baseline separation (resolution = 1.91 ± 0.03) were as follows: Chirobiotic TAG stationary phase, thermostated at 10°C, mobile phase consisting of methanol/20mM ammonium acetate 60:40 v/v, and 0.6 mL/min flow rate. Enantiomer elution order was determined using HPLC hyphenated with circular dichroism (CD) spectroscopy detection. The online CD signals of the separated pantoprazole enantiomers at selected wavelengths were compared with the structurally analogous esomeprazole enantiomer. For further verification, the inline rapid, multiscan CD signals were compared with the quantum chemically calculated CD spectra. Furthermore, docking calculations were used to investigate the enantiorecognition at molecular level. The molecular docking shows that the R-enantiomer binds stronger to the chiral selector than its antipode, which is in accordance with the determined elution order on the column-S- followed by the R-isomer. Thus, combined methods, HPLC-CD and theoretical calculations, are highly efficient in predicting the elution order of enantiomers.

Planktonic and Benthic Bacterial Communities of the Largest Central European Shallow Lake, Lake Balaton and Its Main Inflow Zala River.

Lake Balaton is the largest European shallow lake, which underwent cultural eutrophication in the '70-80s. Therefore, strict pollution control measures were introduced and the water quality has become meso-eutrophic since the millennium. Due to the touristic significance and change in trophic levels of the lake, numerous ecological studies were carried out, but none of them was focused on both benthic and planktonic microbial communities at the same time. In our study, an attempt was made to reveal the spatial bacterial heterogeneity of the Lake Balaton and Zala River by 16S rDNA terminal restriction fragment length polymorphism fingerprinting and Illumina amplicon sequencing methods in the summer of 2017. According to the molecular biology results, mostly well-known freshwater microorganisms, adapted to nutrient-poor conditions were found in the pelagic water column. The LD12 subclade member Fonsibacter ubiquis, the cyanobacterial Synechococcus sp. and unknown Verrucomicrobia species were abundant in the less nutrient-dense basins, while the hgcI clade members showed various distribution. In the estuary and in the nutrient-dense western part of the lake, some eutrophic conditions preferring cyanobacteria (filamentous Anabaena and Aphanizomenon species) were also detectable. The benthic microbial community showed higher diversity, according to the observed appearance of microorganisms adapted to the deeper, less aerated layers (e.g. members of Desulfobacteraceae, Nitrosomonadaceae).

Synthesis of Potential Haptens with Morphine Skeleton and Determination of Protonation Constants.

Vaccination could be a promising alternative warfare against drug addiction and abuse. For this purpose, so-called haptens can be used. These molecules alone do not induce the activation of the immune system, this occurs only when they are attached to an immunogenic carrier protein. Hence obtaining a free amino or carboxylic group during the structural transformation is an important part of the synthesis. Namely, these groups can be used to form the requisite peptide bond between the hapten and the carrier protein. Focusing on this basic principle, six nor-morphine compounds were treated with ethyl acrylate and ethyl bromoacetate, while the prepared esters were hydrolyzed to obtain the N-carboxymethyl- and N-carboxyethyl-normorphine derivatives which are considered as potential haptens. The next step was the coupling phase with glycine ethyl ester, but the reactions did not work or the work-up process was not accomplishable. As an alternative route, the normorphine-compounds were N-alkylated with N-(chloroacetyl)glycine ethyl ester. These products were hydrolyzed in alkaline media and after the work-up process all of the derivatives contained the free carboxylic group of the glycine side chain. The acid-base properties of these molecules are characterized in detail. In the N-carboxyalkyl derivatives, the basicity of the amino and phenolate site is within an order of magnitude. In the glycine derivatives the basicity of the amino group is significantly decreased compared to the parent compounds (i.e., morphine, oxymorphone) because of the electron withdrawing amide group. The protonation state of the carboxylate group significantly influences the basicity of the amino group. All of the glycine ester and the glycine carboxylic acid derivatives are currently under biological tests.

Chiral separation of rasagiline using sulfobutylether-ß-cyclodextrin: capillary electrophoresis, NMR and molecular modeling study.

Pressure-assisted stereospecific capillary electrophoresis method was developed for the determination of enantiomeric purity of the antiparkinsonian agent (R)-rasagiline. The optimized method, 50 mM glycine-HCl buffer pH 2, supplied with 30 mM sulfobutylether-ß-cyclodextrin, at 35°C, applying 12 kV in reversed polarity, and -8 mbar pressure (vacuum), short-end injection with -25 mbar × 2 s, was successful for baseline separation of rasagiline enantiomers (Rs = 3.5 ± 0.1) in a short analysis time. The method was validated according to current guidelines and proved to be reliable, linear, precise and accurate for determination of 0.15% S-enantiomer as chiral impurity in R-rasagiline sample, as well as quantification of the eutomer. Method application was tested on a commercial tablet formulation. Determination of spatial structure of diastereomeric associates was based on 1 H and 2D ROESY NMR, indicating that the aromatic moiety of the molecule can enter the cyclodextrin cavity. NMR titration and molecular modeling revealed that S-rasagiline formed a more stable inclusion complex with sulfobutylether-ß-cyclodextrin, than its antipode, which is in agreement with electrophoretic results.

Chiral separation of lansoprazole and rabeprazole by capillary electrophoresis using dual cyclodextrin systems.

Novel capillary electrophoresis methods using CDs as chiral selectors were developed and validated for the chiral separation of lansoprazole and rabeprazole, two proton pump inhibitors. Fourteen different neutral and anionic CDs were screened at pH 4 and 7 in the preliminary analysis. Sulfobutyl-ether-ß-CD with a degree of substitution of 6.5 and 10 at neutral pH proved to be the most suitable chiral selector for both compounds. Various dual CD systems were also compared, and the possible mechanisms of enantiomer separation were investigated. A dual selector system containing sulfobutyl-ether-ß-CD degree of substitution 6.5 and native γ-CD proved to be the most adequate system for the separations. Method optimization was carried out using an experimental design approach, performing an initial fractional factorial screening design, followed by a central composite design to establish the optimal analytical conditions. The optimized methods (25 mM phosphate buffer, pH 7, 10 mM sulfobutyl-ether-ß-CD/20 mM γ-CD, +20 kV voltage; 17°C temperature; 50 mbar/3 s injection, detection at 210 nm for lansoprazole; 25 mM phosphate buffer, pH 7, 15 mM sulfobutyl-ether-ß-CD/30 mM γ-CD, +20 kV voltage; 18°C temperature; 50 mbar/3 s injection, detection at 210 nm for rabeprazole) provided baseline separation for lansoprazole (Rs = 2.91) and rabeprazole (Rs = 2.53) enantiomers with favorable migration order (in both cases the S-enantiomers migrates first). The optimized methods were validated according to current guidelines and proved to be reliable, linear, precise, and accurate for the determination of 0.15% distomer as chiral impurity in dexlansoprazole and dexrabeprazole samples.