Comprehensive Review on Chiral Stationary Phases in Single-Column Simultaneous Chiral-Achiral HPLC Separation Methods.

This comprehensive review explores the utilization of chiral stationary phases (CSPs) in the context of single-column simultaneous chiral-achiral high-performance liquid chromatography (HPLC) separation methods. While CSPs have traditionally been pivotal for enantioselective drug analysis, contemporary CSPs often exhibit notable chemoselective properties. Consequently, there is a discernible trend towards the development of methodologies that enable simultaneous enantio- and chemoselective separations utilizing a single CSP-based chromatographic column. This review provides an exhaustive overview of reported HPLC methods in this domain, with a focus on four major CSP types: cyclodextrin-, glycopeptide antibiotic-, protein-, and polysaccharide-based CSPs. This article delves into the diverse applications of CSPs, encompassing various chromatographic modes such as normal phase (NP), reverse phase (RP), and polar organic (PO). This review critically discusses method development, emphasizing the additional chemoselective separation mechanisms of CSPs. It also explores possibilities for method optimization and development, concluding with future perspectives on this evolving field. Despite the inherent challenges in understanding the retention mechanisms involved in chemoselective separations, this review highlights promising trends and anticipates a growing number of simultaneous enantio- and chemoselective methods in pharmaceutical analyses, pharmacokinetic studies, and environmental sample determinations.

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.

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.

Enantioseparation and molecular docking study of selected chiral pharmaceuticals on a commercialized phenylcarbamate-ß-cyclodextrin column using polar organic mode.

The chiral separation capability of Chiral-CD-Ph column, containing phenylcarbamate-ß-cyclodextrin as the chiral selector in polar organic mode was investigated. A total of twenty-five compounds with different structures and acid-base properties were evaluated, and twenty of them were separated using acetonitrile or methanol as eluent. The effects of various chromatographic parameters, such as the type and proportion of organic modifier, flow rate, and column temperature were analyzed in detail in relation to chromatographic performance. A U-shape retention curve was observed when a mixture of acetonitrile and methanol was used as the eluent, indicating different types of interactions in different solvent mixtures. Van 't Hoff analysis was used for calculation of thermodynamic parameters which revealed that the enantioseparation is mainly enthalpy controlled; however, entropic control was also observed. The enantiomer recognition ability at the atomic level was also investigated through a molecular docking study, which revealed surface binding in polar organic mode instead of inclusion complexation. Our work proves that the phenylcarbamate-ß-cyclodextrin-based chiral stationary phase can be effectively used in polar organic mode for the chiral separation of structurally diverse compounds. Furthermore, it is important to note that our study demonstrated that surface binding is responsible for the formation of supramolecular complexes in certain cyclodextrin derivatives.

Optimization and Production of Aceclofenac-Loaded Microfiber Solid Dispersion by Centrifugal Spinning.

Aceclofenac-loaded polyvinylpyrrolidone fiber-based amorphous solid dispersion was produced successfully by centrifugal spinning. The solution concentration and rotational speed were optimized to produce the fiber-based drug carrier system, with a determined production rate of 12.7 g/h dry solid fibers. The obtained fibers were bead-free and smooth-surfaced with an average diameter of 7.5 ± 2.5 µm. Gas chromatographic determinations revealed that ethanol, as a residual solvent, was well below the regulatory limit of 0.5%. Differential scanning calorimetric investigation and infrared spectroscopic measurements were used to track the physicochemical changes that intervene during fiber formation in the solid state. The results revealed that the rapid evaporation of the solvent was accompanied by a probable crystalline to amorphous transition of the active substance during centrifugal spinning. In vitro dissolution studies revealed an instantaneous disintegration of the fibrous structure and a rapid release of the active substance, with the microfibrous webs greatly outperforming the crystalline active substance, especially in the early time-points. This implies that centrifugal spinning offers a viable scale-up production process to prepare drug-loaded fiber-based solid dispersions.

Simultaneous determination of enantiomeric and organic impurities of vildagliptin on a cellulose tris(3-chloro-4-methylphenylcarbamate) column under revered-phase conditions.

A new, reversed-phase HPLC (RP-HPLC) method was developed for the simultaneous determination of the dipeptidyl-peptidase-IV-inhibitor antidiabetic drug vildagliptin (VIL) enantiomeric impurity and four other achiral related impurities. An initial screening was performed on five polysaccharide-type chiral stationary phases (Lux Amylose-1, Lux Amylose-2, Lux-Cellulose-1, Lux-Cellulose-2, Lux-Cellulose-3) in polar organic mode with methanol, ethanol, 2-propanol, or acetonitrile containing 0,1% diethylamine as mobile phase to identify the best conditions for the separation of VIL enantiomers. Lux-Cellulose-2 column was found to provide the best chiral resolution for VIL enantiomers. Further experiments were conducted using different aqueous-organic mobile phases to achieve the simultaneous chiral-achiral separation of the selected compounds. Experimental design-based optimization was performed by using a face-centered central composite design. The optimal separation conditions (Lux Cellulose-2 stationary phase, 45 °C, mobile phase consisting of methanol/water/diethylamine 80:20:0.2 (v/v/v), and 0.45 mL/min flow rate) provided baseline separation for all 6 compounds. The optimized method was validated according to the ICH guideline and proved to be reliable, specific, linear, precise, and accurate for the determination of at least 0.1% for all impurities in VIL samples. The validated method was applied for determinations from a commercially available drug formulation and proved to be suitable for routine quality control of both enantiomeric and organic impurities of VIL.

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.

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.

One-Step Preparation of Fiber-Based Chlorzoxazone Solid Dispersion by Centrifugal Spinning.

An amorphous fiber-based solid dispersion of chlorzoxazone was prepared for the first time by employing centrifugal spinning, using polyvinylpyrrolidone as the fiber-forming polymer. After optimization of the spinning parameters, the obtained fibers were characterized using a set of analytical techniques, both in a solid- and solution-state. Morphological characterization revealed a slightly aligned, defect-free fibrous structure with an average fiber diameter of d = 3.07 ± 1.32 µm. The differential scanning calorimetric results indicated a crystalline-to-amorphous transition of the active substance during the centrifugal spinning process, while gas chromatographic determinations revealed a residual ethanol content of 0.42 ± 0.04%. UV spectroscopy indicated the incorporation of chlorzoxazone in the fibrous structures, with an average active substance content of 15.91 ± 0.36 w/w%. During small-volume dissolution studies, the prepared fiber mats presented immediate disintegration upon contact with the dissolution media, followed by rapid dissolution of the active substance, with 84.8% dissolved at 1 min and 93.7% at 3 min, outperforming the micronized, pure chlorzoxazone. The obtained results indicate that centrifugal spinning is a low-cost, high-yield, viable alternative to the currently used methods to prepare fiber-based amorphous solid dispersions of poorly soluble drugs. The prepared chlorzoxazone-loaded microfibers could be used as a buccal dosage form for the systematic delivery of chlorzoxazone and could potentially lead to a rapid onset of action and longer efficacy of the muscle relaxant drug.

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.

Evaluation of Lapatinib-Loaded Microfibers Prepared by Centrifugal Spinning.

Lapatinib (Lap) is a lypophilic drug frequently used in cancer treatment; however, due to its limited solubility and permeability, achieving therapeutic dose through oral administration proves to be a challenge. There are various methods for enhancing the solubility of Lap and other similar drugs, one being the preparation of amorphous solid dispersions (ASD). In this study, a Lap-loaded polyvinylpyrrolidone (PVP) fiber mat was created with centrifugal spinning from a PVP/Lap solution in dimethyl formamide and ethanol. The production rate was 12.2 g/h dry fibers, and the fibers had an average thickness of 2.55 ± 0.92 µm. In the differential scanning calorimetry (DSC) thermogram of the fiber mat, the melting peak of the crystalline Lap was not visible, suggesting that Lap was in an amorphous state. A dissolution study was carried out in 0.2 M phosphate buffer saline solution at 37 °C. UV spectrophotometry data indicated that in the sample containing the fiber mat, the Lap concentration was 332 µg/mL (66%) in 10 min, decreasing to 227 µg/mL by 45 min. Meanwhile the crystalline Lap formed a 30-40 µg/mL (6-8%) solution in 5 min, maintaining that concentration. We conclude that centrifugal spinning can be an effective and easy method to produce ASDs.

New Trends in the Quality Control of Enantiomeric Drugs: Quality by Design-Compliant Development of Chiral Capillary Electrophoresis Methods.

Capillary electrophoresis (CE) is a potent method for analyzing chiral substances and is commonly used in the enantioseparation and chiral purity control of pharmaceuticals from different matrices. The adoption of Quality by Design (QbD) concepts in analytical method development, optimization and validation is a widespread trend observed in various analytical approaches including chiral CE. The application of Analytical QbD (AQbD) leads to the development of analytical methods based on sound science combined with risk management, and to a well understood process clarifying the influence of method parameters on the analytical output. The Design of Experiments (DoE) method employing chemometric tools is an essential part of QbD-based method development, allowing for the simultaneous evaluation of experimental parameters as well as their interaction. In 2022 the International Council for Harmonization (ICH) released two draft guidelines (ICH Q14 and ICH Q2(R2)) that are intended to encourage more robust analytical procedures. The ICH Q14 guideline intends to harmonize the scientific approaches for analytical procedures' development, while the Q2(R2) document covers the validation principles for the use of analytical procedures including the recent applications that require multivariate statistical analyses. The aim of this review is to provide an overview of the new prospects for chiral CE method development applied for the enantiomeric purity control of pharmaceuticals using AQbD principles. The review also provides an overview of recent research (2012-2022) on the applicability of CE methods in chiral drug impurity profiling.

Analytical quality by design-compliant retention modeling for exploring column interchangeabilities in separating ezetimibe and its related substances.

There are several potential advantages of using experimental design-based retention modeling for chromatographic method development. Most importantly, through the model-delivered systematic understanding (Design Spaces), users can benefit from increased method consistency, flexibility and robustness that can efficiently be achieved at lesser amount of development time. As a result, modeling tools have always been great supplementary assets and welcomed by both the pharmaceutical industry and the regulatory authorities. Most recently published chapters of ICH however - Q2(R2) and Q14 (both currently drafts) - evidence a further paradigm shift, specifying the elements of model-based development strategies in the so-called "enhanced approach". The main aim of this study was to investigate the impact of stationary phase chemistries on chromatographic method performance in the application example of ezetimibe and its related substances. A commercial modeling software package (DryLab®) was used to outline three-dimensional experimental design frameworks and acquire model Design Spaces (DSs) of 9 tested columns. This was done by performing 12 input calibration experiments per column, systematically changing critical method parameters (CMPs) as variables such as the gradient time (tG), temperature (T) and the ternary composition (tC) of the mobile phase. The constructed models allowed studying retention behaviors of selected analytes within each separation systems. In the first part of our work, we performed single optimizations for all nine stationary phases with substantially different surface modifications based on their highest achievable critical resolution values. For these optimum points in silico robustness testing was performed, clearly showing a change of CMPs, depending on the column, and specified optimum setpoint. In the second part of our work, we simultaneously compared the three-dimensional virtual separation models to identify all method parameter combinations that could provide at least baseline separation (Rs, crit.>1.50). These overlapping areas between the models described a common method operational design region (MODR) where columns were considered completely interchangeable - in terms of their baseline resolving capability - regardless of their exact physicochemical properties. A final optimized, column-independent working point within the common MODR was selected for verification. Indeed, experimental chromatograms showed excellent agreement with the model; all columns in the common condition were able to yield critical resolution values higher than 2.0, only their retentivity (elution window of peaks) was found different in some cases. Our results underline that a profound understanding of the separation process is of utmost importance andthat in some cases, adequate selectivity is achievable on various stationary phases.

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 of oxazolidinone analogues by liquid chromatography on polysaccharide stationary phases using polar organic mode.

The enantioseparation of four oxazolidinone and one biosimilar thiazolidine derivatives was performed on seven different polysaccharide-type chiral stationary phases (Lux Amylose-1, Lux i-Amylose-1, Lux Amylose-2, Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3, Lux Cellulose-4) differing in backbone (cellulose or amylose), substituent or the immobilization technologies (coated or immobilized). Polar organic mode was employed using neat methanol (MeOH), ethanol (EtOH), 2-propanol (IPA) and acetonitrile (ACN) either alone or in combinations as mobile phases. Amylose-based columns with ACN provided the highest enantioselectivities for the studied compounds. The replacement of an oxygen with a sulfur atom in the backbone of the studied analytes significantly alters the enantiomer recognition mechanism. Chiral selector-, mobile-phase-, and interestingly immobilization-dependent enantiomer elution order reversal was also observed. Reversal of elution order and hysteresis of retention and enantioselectivity was further investigated using different mixtures of IPA:MeOH and ACN:MeOH on amylose-type chiral stationary phases. Hysteresis of retention and enantioselectivity was observed on all investigated amylose-type columns and binary eluent mixtures, which can be further utilized for fine-tuning chiral separation performance of the studied columns.

Inclusion complexation of the anticancer drug pomalidomide with cyclodextrins: fast dissolution and improved solubility.

Pomalidomide (POM), a potent anticancer thalidomide analogue was characterized in terms of cyclodextrin complexation to improve its aqueous solubility and maintain its anti-angiogenic activity. The most promising cyclodextrin derivatives were selected by phase-solubility studies. From the investigated nine cyclodextrins - differing in cavity size, nature of substituents, degree of substitution and charge - the highest solubility increase was observed with sulfobutylether-ß-cyclodextrin (SBE-ß-CD). The inclusion complexation between POM and SBE-ß-CD was further characterized with a wide variety of state-of-the-art analytical techniques, such as nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR), circular dichroism spectroscopy, fluorescence spectroscopy as well as X-ray powder diffraction method (XRD). Job plot titration by NMR and the AL-type phase-solubility diagram indicated 1:1 stoichiometry in a liquid state. Complementary analytical methods were employed for the determination of the stability constant of the complex; the advantages and disadvantages of the different approaches are also discussed. Inclusion complex formation was also assessed by molecular modelling study. Solid state complexation in a 1:1 M ratio was carried out by lyophilization and investigated by IR and XRD. The complex exhibited fast-dissolution with immediate release of POM, when compared to the pure drug at acidic and neutral pH. Kinetic analysis of POM release from lyophilized complex shows that Korsmeyer-Peppas and Weibull model described the best the dissolution kinetics. The cytotoxicity of the complex was tested against the LP-1 human myeloma cell line which revealed that supramolecular interactions did not significantly affect the anti-cancer activity of the drug. Overall, our results suggest that the inclusion complexation of POM with SBE-ß-CD could be a promising approach for developing more effective POM formulations with increased solubility.

Computer-assisted UHPLC method development and optimization for the determination of albendazole and its related substances.

Computer-aided ultrahigh performance liquid chromatographic (UHPLC) method development and optimization was undertaken in order to replace an underperforming European Pharmacopoeia method for the determination of albendazole and its related substances. In the preliminary screening, a temperature-gradient time bidimensional model was chosen to aid selection of the proper stationary phase. Hereinafter temperature-gradient time-ternary composition and temperature-gradient time-pH tridimensional models were applied for the optimization of critical method parameters. The simulation and in silico robustness testing were realized using DryLab modeling software. The final method was validated for quantification of impurities and assay of the active substance according to the current ICH guidance. The validated methods were tested on a real, commercial tablet formulation. The experimental design-based and software-assisted method development proved to be a fast and reliable way of replacing a method with inadequate selectivity and long runtime with a robust UHPLC-based method, which offers baseline separation for all monitored impurities in 10 min. Results confirm that software-based chromatographic modelling can not only speed up the analytical method development process, but also improve the reliability of the developed method.

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.

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.