Investigation of stereochemical stability of 2-, 3- and 4-chloromethcathinones in various biological matrixes.

The stereochemical stability of the popular drugs of abuse 2-, 3- and 4-chloromethcathinone was studied in the mobile phase used for the isolation of their enantiomers by high-performance liquid chromatography, as well as in various biological matrixes such as whole blood, saliva and urine. For 2-, 3-, and 4-chloromethcathinones the rate constants and half-lives of their first order racemization reaction was assessed. It was found that at 25 °C the racemization rate constant decreases in the order 2-CMC > 3-CMC > 4-CMC while their stereochemical stability in biological matrixes decreases in the order urine > saliva > whole blood. This information must be considered for the adequate storage of purified enantiomers in the collected fractions, as well as in the studies focused on their enantioselective transformation in the human body.

Separation of undeuterated and partially deuterated enantioisotopologues of some amphetamine derivatives on achiral and polysaccharide-based chiral columns in high-performance liquid chromatography.

The different behavior of enantiomers of chiral compounds in non-isotropic environments (among them in living organism) is well known. On the other hand, the importance of a kinetic isotope effect in the biomedical field has become evident during past few decades. Thus, separation of both, enantiomers and isotopologues is now critical. Only very few published studies have attempted the simultaneous separation of enantioisotopologues. In this article we report baseline separation of partially deuterated isotopologues of a few amphetamine derivatives in high-performance liquid chromatography (HPLC) using achiral columns. In addition, the simultaneous separations of enantiomers and isotopologues (i.e. enantioisotopologues) were attempted on polysaccharide-based chiral columns. For several compounds the isotope effect was tunable and could be switched from a "normal" to "inverse" by making changes to the mobile-phase composition. A stronger isotope effect was observed in acetonitrile-containing mobile phases compared to methanol-containing ones with both chiral and achiral columns. In a separation system where both "normal" and "inverse" isotope effects were observed the "normal" isotope effect was favored in polar organic solvents while increasing content of the aqueous component in the reversed-phase (RP) mobile phase favored an "inverse" isotope effect. This observation indicates that polar, hydrogen bonding-type noncovalent interactions are involved in the "normal" isotope effect, while apolar hydrophobic-type interactions are mostly responsible for the "inverse" isotope effect.

Analytical and preparative chiral supercritical fluid chromatography resolutions using crown ether-derived column.

In this study, crown ether-derived column Crownpak® CR-I (+) was evaluated under SFC conditions using 12 primary amines, and the chromatographic results were compared against eight immobilized polysaccharide-based columns. Crownpak® CR-I (+) achieved a significantly higher success rate. It was found that the addition of 5% water to the modifier dramatically improved the peak shape for chiral separation of primary amines on Crownpak® CR-I (+). The first reported preparative SFC separations on Crownpak® CR-I (+) are shown, offering a new approach for the preparative resolution of primary amines. The case studies demonstrate that Crownpak® CR-I (+) is a very useful column in the chiral separation of challenging compounds that contain a primary amine group in the pharmaceutical industry.

Enantioseparation of selected chiral agrochemicals by using nano-liquid chromatography and capillary electrochromatography with amylose tris(3­chloro-5-methylphenylcarbamate) covalently immobilized onto silica.

In this paper enantiomers of selected chiral agrochemicals representing various structural classes were separated by using nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC) employing a capillary column packed with silica particles containing immobilized amylose tris(3­chloro-5-methylphenylcarbamate) (i-ADMPC) as a chiral selector (CS). Special attention was paid to peak dispersion in nano-LC and CEC instruments used in order to make comparison between these two techniques more reliable. Enantioseparations were studied utilizing methanol (MeOH) or acetonitrile-water (ACNH2O), both containing 5 mM of ammonium acetate as the mobile phases (MPs). The tested chiral stationary phase (CSP), containing 20% (w/w) of the neutral CS onto native silica, allowed the generation of sufficiently strong electroosmotic flow (EOF) to observe separation of enantiomers of studied agrochemicals in a reasonable time also in CEC mode. Modestly higher efficiencies and enantioresolutions were obtained in CEC than in nano-LC. Just a moderate preference of CEC over nano-LC in this particular study can be explained with a significant mass transfer resistance through the CSP that is caused due to high content of the CS in CSP.

Our research cooperation with Professor Yoshio Okamoto.

This article summarizes our cooperation with the research group of Prof. Yoshio Okamoto at Nagoya University during the period of time between 1992 and 2005. Although the text deals entirely with enantioseparations in high-performance liquid chromatography, capillary electrophoresis, and capillary electrochromatography, this is not a detailed review in any of these areas. The text highlights selected aspects of these techniques, which have been the subject of our joint research and in part their reflection in follow-up research by our and other research groups. Together with more systematically studied topics, aspects such as ultrafast separation of enantiomers, uncommonly high separation factor of enantiomers and other related issues are also addressed.

History, advancement, bottlenecks, and future of chiral capillary electrochromatography.

Capillary electrochromatography (CEC) represents a technique with less than 30 years of intense development and in this period this technique has seen huge promise, fast development, stagnation, and significant decline of innovative activity. The major goal of the present overview is not to present an extensive review of the literature on chiral CEC but to analyze the reasons for this dramatic development and attempting to answer questions such as: 1) Was the potential of CEC reasonably evaluated in 1990s before starting the explosive development in this field? 2) Did the development of this technique take the right track? 3) What other developments and competitive trends led to stagnation in the advancement of CEC? 4) Why is the activity in this field currently decreasing? 5) What are the current challenges and promises and what is the future of chiral CEC?

Potential and current limitations of superficially porous silica as a carrier for polysaccharide-based chiral selectors in separation of enantiomers in high-performance liquid chromatography.

In this study superficially porous silica particles with a nominal pore size of 450 Å and average particle size of 2.6 micrometers was compared to fully porous silica particles with nominal particle size 3 micrometers and nominal pore size 1000 A as carriers for a polysaccharide based chiral selector for the separation of enantiomers in high-performance liquid chromatography. In addition, the effects of chiral selector loading onto the silica support and of column internal dimeter in the case of both, superficially porous and totally porous silica, as well as of the pore size of superficially porous silica on column performance were studied. The dependence of plate height on mobile phase flow rate was also studied and attempts were made for shortening analysis time. The baseline separation of enantiomers of some chiral sulfoxides was obtained within 2.0-4.5 s.

Separation of enantiomers of chiral basic drugs with amylose- and cellulose- phenylcarbamate-based chiral columns in acetonitrile and aqueous-acetonitrile in high-performance liquid chromatography with a focus on substituent electron-donor and electron-acceptor effects.

In this study, amylose- and cellulose-phenylcarbamate-based chiral columns with different chiral-selector (CS) chemistries were compared to each other for the separation of enantiomers of basic chiral analytes in acetonitrile and aqueous-acetonitrile mobile phases in HPLC. For two chemistries the amylose-based columns with coated and immobilized CSs were also compared. The comparison of CSs containing only electron-donating or electron-withdrawing substituents with those containing both electron-donating and electron-withdrawing substituents showed opposite results for the studied set of chiral analytes in the case of amylose and cellulose derivatives. Along with the chemistry of CS the focus was on the behavior of polysaccharide phenylcarbamates in acetonitrile versus aqueous acetonitrile as eluents. In agreement with earlier results, it was found that in contrast to the commonly accepted view, polysaccharide phenylcarbamates do not behave as typical reversed-phase materials for basic analytes either. In the range of water content in the mobile phase of up to 20-30% v/v the behavior of these CSs is similar to hydrophilic interaction liquid chromatography (HILIC)-type adsorbents. This means that with increasing water content in the mobile phase up to 20-30% v/v, the retention of analytes mostly decreases. The important finding of this study is that the separation efficiency improves for most analytes when switching from pure acetonitrile to aqueous acetonitrile. Therefore, in spite of reduced retention, the separation of enantiomers improves and thus, the HILIC-range of mobile phase composition, offering shorter analysis time and better peak resolution, is advantageous over pure polar-organic solvent mode. Interesting examples of enantiomer elution order (EEO) reversal were observed for some analytes based on the content of water in the mobile phase on Lux Cellulose-1 and Lux Amylose-2 columns.

Further study on enantiomer resolving ability of amylose tris(3-chloro-5-methylphenylcarbamate) covalently immobilized onto silica in nano-liquid chromatography and capillary electrochromatography.

In the present study separation of enantiomers of some chiral neutral, basic and weakly acidic analytes was investigated on the chiral stationary phase (CSP) made by covalent immobilization of amylose tris(3-chloro-5-methylphenylcarbamate) onto aminopropylsilanized (APS) silica in nano-liquid chromatography (nano-LC) in aqueous methanol or acetonitrile mixtures. It has been shown that similar to high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) this chiral selector is useful for separation of enantiomers of neutral, basic and acidic analytes also in nano-LC. In comparison to our previous research, in which the chiral selector (CS) was bonded on native silica, in this study, the CS was immobilized on APS silica in order to improve chromatographic performance towards basic analytes. In fact, some improvement was observed and surprisingly not only for basic but also for neutral and acidic analytes. Again, quite unexpectedly almost no electroosmotic flow (EOF) was observed in capillaries packed with ca. 20% (w/w) amylose tris(3-chloro-5-methylphenylcarbamate) immobilized onto APS silica although the same APS silica before attachment of chiral selector exhibited significant EOF. In order to generate EOF in the capillaries with the CSP and enable capillary electrochromatographic (CEC) experiment on it, the short segment of the capillary column was packed with APS silica without chiral selector. The EOF in such capillary enabled CEC experiment and some preliminary results are reported here.

Chiral separation of underivatized amino acids in supercritical fluid chromatography with chiral crown ether derived column.

The supercritical fluid chromatographic separation of underivatized amino acids was explored using immobilized chiral crown ether column CROWNPAK CR-I (+) and mass spectrometric detection. The type of modifier, acidic additives, and the role of water were investigated. Enantioseparation was achieved for all 18 amino acids investigated with short retention times (less than 3 minutes) and average resolution of greater than 5.0. Analysis of enantiomerically pure standards demonstrated the D enantiomer eluted first for all amino acids using a CROWNPAK CR-I (+) column.

Defining a system suitability limit to decide on column deterioration and to facilitate column transfers in chiral supercritical fluid chromatography.

The separation of enantiomers is an important requirement during the entire drug life cycle in the pharmaceutical industry. High-performance liquid chromatography and supercritical fluid chromatography (SFC) are the main chromatographic techniques used to separate enantiomers. Since chiral stationary phases are often extensively used once a method has been developed, columns will age and must be replaced after a certain period. However, no practical guidelines exist to determine when a column is deteriorated or to decide whether a transfer to another column (with the same chiral selector) is successful. In this study, a system suitability limit for resolution was defined, based on an intermediate (time-different) precision study in SFC on four immobilized polysaccharide-based columns that only differed in manufacturer or particle size. This system suitability limit could be used to decide on column deterioration or as a requirement to evaluate whether a separation transfer was successful. Some method adaptations may be necessary to obtain successful transfers. An approach was proposed, which helped the analyst to make successful transfers. Graphical abstract.

Comparative study on enantiomer resolving ability of amylose tris(3-chloro-5-methylphenylcarbamate) covalently immobilized onto silica in nano-liquid chromatography and capillary electrochromatography.

In the present study separation of enantiomers of some chiral neutral and weakly acidic analytes was investigated on the chiral stationary phase (CSP) made by covalent immobilization of amylose tris(3-chloro-5-methylphenylcarbamate) onto silica in nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC) in acetonitrile and aqueous acetonitrile. Few comparisons were made also between the enantioseparations in nano-LC and high-performance liquid chromatography (HPLC) with the chiral column of 4.6 × 250 mm dimension. Slightly better separation of enantiomers was observed in HPLC mode compared to nano-LC mode. It was shown that in the capillary columns packed with the CSP containing about 20% (w/w) of a covalently immobilized neutral chiral selector, amylose tris(3-chloro-5-methylphenylcarbamate), sufficient electroosmotic flow has been generated and enantioseparations with reasonable analysis time were performed also in CEC mode. It was shown once again that CEC offers a clear advantage over nano-LC from the viewpoint of plate numbers and peak resolution.

Polysaccharide-Based Chiral Stationary Phases for Enantioseparations by High-Performance Liquid Chromatography: An Overview.

This chapter summarizes the application of polysaccharide-based chiral stationary phases (CSPs) for separation of enantiomers in high-performance liquid chromatography (HPLC). Since this book contains dedicated chapters on enantioseparations using supercritical fluid chromatography (SFC), or capillary electrochromatography (CEC), the application of polysaccharide-based materials in these modes of liquid-phase separation techniques is touched just superficially. Special emphasis is directed toward a discussion of the optimization of polysaccharide-based chiral selectors, their attachment onto the carrier, and the optimization of the support. The optimization of the separation of enantiomers based on various parameters such as mobile phase composition and temperature is discussed.

HPLC Enantioseparations with Polysaccharide-Based Chiral Stationary Phases in HILIC Conditions.

In contrast to achiral hydrophilic interaction liquid chromatography (HILIC), which is a popular and largely applied technique to analyze polar compounds such as pharmaceuticals, metabolites, proteins, peptides, amino acids, oligonucleotides, and carbohydrates, the introduction of the HILIC concept in enantioselective chromatography has been relatively recent and scarcely debated. In this chapter, the HILIC enantioseparations carried out on polysaccharide-based chiral stationary phases are grouped and discussed. Another objective of this chapter is to provide a comprehensive overview and insight into the experimental conditions needed to operate under HILIC mode. Finally, to stimulate and facilitate the application of this chromatographic technique, a detailed experimental protocol of a chiral resolution on a chlorinated cellulose-based chiral stationary phase under HILIC conditions is described.

Separation of Bruton's tyrosine kinase inhibitor atropisomers by supercritical fluid chromatography.

Bruton's tyrosine kinase (BTK) plays an essential role in multiple cell types responsible for numerous autoimmune diseases, thus inhibition of BTK is anticipated to provide an effective strategy for the clinical treatment of autoimmune diseases. Preparative-scale super/subcritical fluid chromatography (SFC) separation methods for four groups of highly potent and selective BTK inhibitor atropisomers were successfully developed. Depending on the rotation barrier around the chiral axis, the compounds were prepared as a single stereochemically stable atropisomer or as an atropisomeric mixture. Among the four, compound 2 with one rotationally stable atropisomeric center (carbazole/quinazolinedione based) was resolved as a mixture of two atropisomers, while compound 3 (carbazole-chlorine/quinazolinedione based) and 4 (tetrahydrocarbazole-fluorine/quinazolinedione based) with two rotationally stable atropisomeric centers were resolved into a single stable atropisomer. This article discusses the challenges and strategies in preparing large quantities of these atropisomeric active pharmaceutical ingredients (APIs) in support of the BTK program discovery efforts.

The Way to Ultrafast, High-Throughput Enantioseparations of Bioactive Compounds in Liquid and Supercritical Fluid Chromatography.

Until less than 10 years ago, chiral separations were carried out with columns packed with 5 or 3 µ m fully porous particles (FPPs). Times to resolve enantiomeric mixtures were easily larger than 30 min, or so. Pushed especially by stringent requirements from medicinal and pharmaceutical industries, during the last years the field of chiral separations by liquid chromatography has undergone what can be defined a "true revolution". With the purpose of developing ever faster and efficient method of separations, indeed, very efficient particle formats, such as superficially porous particles (SPPs) or.

On our way to sub-second separations of enantiomers in high-performance liquid chromatography.

In this study our preliminary attempt for obtaining fast and highly efficient separations of enantiomers in high-performance liquid chromatography with slightly modified state-of-the-art commercial instrumentation is described. In order to reach this goal after careful selection of chiral analytes, the preparation of chiral stationary phase (CSP), mobile phase composition and column dimensions were optimized. The concept of segmented chiral-achiral column was introduced. As the result of these optimizations baseline separation of enantiomers was achieved with the analysis time between 1-2 s.

Separation of terbutaline enantiomers in capillary electrophoresis with cyclodextrin-type chiral selectors and investigation of structure of selector-selectand complexes.

The affinity pattern of terbutaline enantiomers towards various cyclodextrins was studied using capillary electrophoresis. The affinity pattern of terbutaline enantiomers was the same towards all studied cyclodextrins except heptakis(2-O-methyl-3,6-di-O-sulfo)-ß-CD. Nuclear magnetic resonance spectroscopy was used for understanding of fine structural mechanisms of interactions of ß-cyclodextrin and its two sulfated derivatives with the enantiomers of terbutaline. The structure of terbutaline complexes with all 3 cyclodextrins studied was different from each other. In confirmation with our earlier studies it was shown again that capillary electrophoresis represents very sensitive technique for studies of affinity patterns in cyclodextrin complexes with chiral guests. Other instrumental (e.g. NMR spectroscopy and X-ray diffraction analysis) and theoretical techniques, although very useful for obtaining the information regarding the stoichiometry, binding constants and structure of intermolecular complexes, as well as about the forces involved in selector-selectand binding and chiral recognition, may sometimes fail to properly sense those fine differences in the affinity patterns. Therefore, it is recommended to use capillary electrophoresis in order to examine correctness of affinity pattern determined for intermolecular complexes of cyclodextrins with guest molecules by other instrumental or computation techniques.

Application of cellulose 3,5-dichlorophenylcarbamate covalently immobilized on superficially porous silica for the separation of enantiomers in high-performance liquid chromatography.

Our earlier studies have demonstrated the applicability of polysaccharide-based chiral selectors in combination with superficially porous (or core-shell) silica (SPS) particles for the preparation of highly efficient chiral stationary phases (CSP). In earlier studies, CSPs were prepared by coating (adsorption) of the chiral selector onto the surface of silica. In this study we report for the first time the CSP obtained by covalent immobilization of a chiral selector onto the surface of SPS particles. The applicability of this CSP for the separation of enantiomers in pure methanol and acetonitrile, as well as in n-hexane/2-propanol mobile phases is shown. The effect of the injected sample amount, mobile phase flow rate and detection frequency on separation performance were studied, as well as high efficiency separation of enantiomers with the analysis time less than 30 s was attempted.

Contemporary theory of enantioseparations in capillary electrophoresis.

The first separation of enantiomers in capillary electrophoresis (CE) counts slightly longer than three decades. Fast development of the practice and theory of chiral CE occurred in the past 30 years and today one can consider this technology to have a solid and mature theoretical background. The goal of the present review is not only to summarize the history and contemporary theory of enantioseparations by using CE but also to present the authors personal view where shall we head to with this attractive technology not only from the viewpoint of separation of enantiomers but also for better understanding the mechanisms of non-covalent (enantioselective) interactions in chemistry, biology, medicine and related disciplines.