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.

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.

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

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

Simultaneous determination of chiral and achiral impurities of ivabradine on a cellulose tris(3-chloro-4-methylphenylcarbamate) chiral column using polar organic mode.

A high performance liquid chromatographic method was developed for the simultaneous determination of the related substances (R-ivabradine, dehydro-S-ivabradine, N-demethyl-S-ivabradine, ((S)-3,4-dimethoxy-bicyclo[4.2.0]octa-1,3,5-triene-7-yl-methyl)-methyl-amine) and 1-(7,8-dimethoxy-1,3,4,5-tetrahydro-2H-3-benzazepine-2-on-3-yl)-3-chloro-propane) of the heart-rate lowering drug, ivabradine. The separation capability of seven different polysaccharide-type chiral columns (Lux Amylose-1, Lux i-Amylose-1, Lux Amylose-2, Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3 and Lux Cellulose-4) was investigated with a mobile phase consisting of 0.1% diethylamine in methanol, 2-propanol and acetonitrile. During the screnning experiments the best results were obtained on Lux Cellulose-2 (based on cellulose tris(3-chloro-4-methylphenylcarbamate) column with methanol with an ideal case, where all the impurities eluted before the S-ivabradine peak. Chromatographic parameters (flow rate, temperature and mobile phase constituents) were optimized by a full factorial screening design. Using optimized parameters (Lux Cellulose-2 column with 0.06% (v/v) diethylamine in methanol/acetonitrile 98/2 (v/v) with 0.45 mL/min flow rate at 12 °C) baseline separations were achieved between all compounds. The optimized method was validated according to the International Council on Harmonization Q2(R1) guideline and proved to be reliable, linear, precise and accurate for determination of at least 0.05% for all impurities in S-ivabradine samples. Method application was tested on a commercial tablet formulation and proved to be suitable for routine quality control of both chiral and achiral related substances of S-ivabradine.

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.

Enantiomeric quality control of R-Tofisopam by HPLC using polysaccharide-type chiral stationary phases in polar organic mode.

A novel, fast and economic chiral HPLC method was developed and validated for the resolution of the four isomers of tofisopam. The separation capacity of eleven different chiral columns: six polysaccharide-type including three amylose-based (Chiralpak AD, Chiralpak AD-RH and Chiralpak AS) and three cellulose-based (Chiralcel OD, Chiralcel OJ and Lux Cellulose-4); three cyclodextrin- (Quest-BC, Quest-C2 and Quest-CM) and two macrocyclic glycopeptide antibiotic-type (Chirobiotic T and Chirobiotic TAG) were screened using polar organic or reversed-phase mode. Chiralpak AD, based on amylose tris(3,5-dimethylphenylcarbamate) as chiral selector with neat methanol was identified as the most promising system. In order to improve resolution, an orthogonal experimental design was employed, altering the concentration of 2-propanol, column temperature, and flow rate in a multivariate manner. Using the optimized method (85/15 v/v methanol/2-propanol, 40°C, flow rate: 0.7 mL/min) we were not only able to separate the four isomers but also detect 0.1% S-enantiomer as chiral impurity in R-tofisopam. This is important since the latter is under development as a single enantiomeric agent. Thermodynamic investigation revealed an unusual entropy and enthalpy-entropy co-driven controlled enantioseparation on Chiralcel OJ and on Chiralpak AD column, respectively. Our newly developed HPLC method was validated according to the ICH guidelines and its application was tested on a pharmaceutical formulation containing the racemic mixture of the drug. As a further novelty, a separate circular dichroism method was applied for the investigation of the interconversion kinetics of tofisopam conformers, which proved to be crucial for sample preparation and method validation.

Enantioseparation of racecadotril using polysaccharide-type chiral stationary phases in polar organic mode.

Enantioseparation of the antidiarrheal drug, racecadotril, was investigated by liquid chromatography using polysaccharide-type chiral stationary phases in polar organic mode. The enantiodiscrimininating properties of 4 different chiral columns (Chiralpak AD, Chiralcel OD, Chiralpak AS, Chiralcel OJ) with 5 different solvents (methanol, ethanol, 1-propanol, 2-propanol, and acetonitrile) at 5 different temperatures (5-40 °C) were investigated. Apart from Chiralpak AS column the other 3 columns showed significant enantioseparation capabilities. Among the tested mobile phases, alcohol type solvents were superior over acetonitrile, and significant differences in enantioselective performance of the selector were observed depending on the type of alcohol employed. Van't Hoff analysis was used for calculation of thermodynamic parameters which revealed that enantioseparation is mainly enthalpy controlled; however, enthropic control was also observed. Enantiopure standard was used to determine the enantiomer elution order, revealing chiral selector-and mobile-phase dependent reversal of enantiomer elution order. Using the optimized method (Chiralcel OJ stationary phase, thermostated at 10 °C, 100% methanol, flow rate: 0.6 mL/min) baseline separation of racecadotril enantiomers (resolution = 3.00 ± 0.02) was achieved, with the R-enantiomer eluting first. The method was validated according to the ICH guidelines, and its application was tested on capsule and granules containing the racemic mixture of the drug.

Chiral separation of lenalidomide by liquid chromatography on polysaccharide-type stationary phases and by capillary electrophoresis using cyclodextrin selectors.

Complementary techniques were applied for the investigation of the chiral recognition and enantiomeric resolution of lenalidomide using various cyclodextrins and polysaccharides as chiral selectors. The high-performance liquid chromatography enantioseparation of the anticancer drug was achieved using polysaccharide-type chiral stationary phases in polar organic mode. Elution order and absolute configuration were elucidated by combined circular dichroism spectroscopy and time-dependent density functional theory calculations after the isolation of pure enantiomers. Chiral selector dependent and mobile-phase dependent reversal of the enantiomer elution order was observed, and the nonracemic nature of the lenalidomide sample was also demonstrated. Eight anionic cyclodextrins were screened for their ability to discriminate between the uncharged enantiomers by using capillary electrophoresis. Only two derivatives presented chiral interactions, these cases being interpreted in terms of apparent stability constants and complex mobilities. The best results were delivered by sulfobutylether-ß-cyclodextrin, where quasi-equal stability constants were recorded and the enantiodiscrimination process was mainly driven by different mobilities of the transient diastereomeric complexes. The optimized high-performance liquid chromatography (Chiralcel OJ column, pure ethanol with 0.6 mL/min flow rate, 40°C) and capillary electrophoresis methods (30 mM sulfobutylether-ß-cyclodextrin, 30 mM phosphate pH 6.5, 12 kV applied voltage, 10°C) were validated for the determination of 0.1% (R)-lenalidomide as a chiral impurity, which could be important if a racemic switch is achieved.