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.

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.

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.

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.

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.

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.