Reversal Of Enantiomer Elution Order Research Articles

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.

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.

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.

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.

The effect of temperature on the separation of enantiomers with coated and covalently immobilized polysaccharide-based chiral stationary phases

This article describes our attempt to re-visit the role of temperature in the separation of enantiomers with polysaccharide-based chiral columns in high-performance liquid chromatography (HPLC). Rarely observed increased retention and separation factors with increasing temperature, as well as temperature dependent reversal of enantiomer elution order are reported for several arylpropionic acid derivatives. Chiral columns with coated and covalently immobilized chiral selectors were compared from the viewpoint of effect of temperature on analyte retention, enantiomer separation and enantiomer elution order. Thermodynamic parameters were calculated for analyte transfer from the liquid phase to the chiral stationary phase and the effect of temperature on chiral selectors was investigated by using differential scanning calorimetry (DSC). DSC results along with chromatographic studies indicate that polysaccharide-based chiral selectors undergo some kind of transition at elevated temperature that is not reversible in the thermodynamic sense of this term.

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: 0.6mL/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.

Separation of enantiomers of chiral weak acids with polysaccharide-based chiral columns and aqueous-organic mobile phases in high-performance liquid chromatography: Typical reversed-phase behavior?

When polysaccharide-based chiral columns are used in combination with aqueous-organic mobile phases for the separation of enantiomers in high-performance liquid chromatography the separation mode is commonly called “reversed-phase” in analogy to achiral separations. In several earlier and recent studies on neutral and basic chiral analytes it was shown by our and other groups that due to multiple type of interactions involved in selector-selectand binding and enantioselective recognition with polysaccharide derivatives, the above mentioned separation system may not always behave like a reversed-phase system. In the present study additional examples of non-reversed-phase behavior are described for the first time for weak acidic chiral analytes. In addition, the reversal of enantiomer elution order was observed again for the first time for several analytes based on water-content in the mobile phase.

Separation and elution order of the enantiomers of some β-agonists using polysaccharide-based chiral columns and normal phase eluents by high-performance liquid chromatography

In this study separation of enantiomers of 8 chiral β-agonists were studied on 6 polysaccharide-based chiral columns in polar-organic and alcohol-hydrocarbon mobile phases. No separation of enantiomers was observed on any column with polar-organic mobile phase eluents such as pure methanol, ethanol or acetonitrile. Most of the chiral analytes were resolved into enantiomers when alcohol-hydrocarbon type mobile phases were used. The most successful column was Lux Cellulose-2 on which all 8 chiral analytes were baseline resolved into enantiomers at least with one mobile phase used. The reversal of enantiomer elution order was observed dependent on the chemistry of the chiral selector and the composition of the mobile phase.

Role of substituents in cyclodextrin derivatives for enantioselective gas chromatographic separation of chiral terpenoids in the essential oils of Mentha spicata.

Enantioselective GC-FID and enantioselective GC-MS have been utilized under temperature gradient mode with differently substituted heptakis- and octakis-cyclodextrins to achieve the resolution of chiral terpenoids in the essential oil of indigenously grown cultivars of Mentha spicata. Modified cyclodextrins were derivatized in GC column for the separation of chiral terpenoids. A 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin doped into 14% cyanopropylphenyl/86%dimethylpolysiloxane (TBDE-β-CD) showed good enantioselectivity for all the studied chiral compounds excluding carvone. Carvone enantiomers were well resolved in 2,3-diacetoxy-6-tert-butyldimethylsilyl-β-cyclodextrin column (TBDA-β-CD) with enantioseparation (Es) of 1.006. A TBDE-β-CD provides maximum enantiomeric separation for β-pinene (Es 1.038), sabinene (Es 1.051), limonene (Es 1.045), isomenthone (Es 1.029) and α-terpineol (Es 1.014). Furthermore, enantiomer elution order reversal was observed for sabinene, menthone, terpinen-4-ol and menthol while changing from β- to γ-cyclodextrin phase. Carvone exhibits enantiomer elution order reversal by changing substituents i.e., methyl to acetyl at 2- & 3- position of the cyclodextrin derivative. Chiral constituents such as (+)-isomenthone, (-)-menthone, (1R,2S,5R)-(-)-menthol and (4S)-(+)-piperitone exist as a single enantiomer with >99% excess. Existence of (R)-(+)-limonene and (S)-(+)-carvone enantiomers has been proven first time in M. spicata essential oils and can be used as the marker for Indian origin.

HPLC Separation of Enantiomers of Some Chiral Carboxylic Acid Derivatives Using Polysaccharide-Based Chiral Columns and Polar Organic Mobile Phases

The separation of enantiomers of 14 chiral carboxylic acid derivatives was studied on six different polysaccharide-based chiral columns in high-performance liquid chromatography with methanol, ethanol and acetonitrile as mobile phases with emphasis on the elution order of enantiomers. Some interesting examples of enantiomer elution order reversal were observed as a function of the nature and composition of chiral selector and mobile phase. For instance, the enantiomer elution order for carprofen, ketorolac, naproxen, proglumide and surprofen reversed with changing the chemical structure of the chiral selector. Also, the enantiomer elution order for carprofen, ketorolac and naproxen changed by varying the composition of the mobile phase. In addition, the interesting effect of column temperature on the retention and separation of some analytes was observed. For instance, the enantiomers of surprofen were only partially resolved at lower temperatures but baseline resolved at higher temperature.

High‐performance liquid chromatography enantioseparation of polyhalogenated 4,4′‐bipyridines on polysaccharide‐based chiral stationary phases under multimodal elution

An investigation on the high-performance liquid chromatography enantioseparation of 12 polyhalogenated 4,4'-bipyridines on polysaccharide-based chiral stationary phases is described. The overall study was directed toward the generation of efficient separations in order to obtain pure atropisomers that will serve as ligands for building homochiral metal organic frameworks. Four coated columns--namely, Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-4, and Lux Amylose-2--and two immobilized columns--namely, Chiralpak IC and IA--were used under normal, polar organic, and reversed-phase elution modes. Moreover, Chiralcel OJ was considered under normal-phase and polar organic conditions. The effect of the chiral selector and mobile phase composition on the enantioseparation, the enantiomer elution order and the beneficial effect of nonstandard solvents were studied. The effect of water in the mobile phase on the enantioselectivity and retention was investigated and retention profiles typical of hydrophilic interaction liquid chromatography were observed. Interesting phenomena of solvent-induced enantiomer elution order reversal occurred under normal-phase mode. All the considered 4,4'-bipyridines were enantioseparated at the multimilligram level.

Preparation of a new immobilized cellulose-based chiral stationary phase and its enantioseparation behaviors

The immobilized polysaccharide-based chiral stationary phase has attracted considerable attention over the past decades due to its high chemical stability, good solvent resistance, great enantioseparation ability, etc. In this study, a new immobilized cellulose chiral stationary phase (denoted as ImCel) was prepared through the Staudinger reaction of 6-azido-6-deoxy-cellulose-3,5-dichlorophenylcarbamate and aminopropyl silica gel. The enantioseparation performance of the ImCel for 20 pairs of chiral analytes and the effect of non-standard solvents have been investigated by high performance liquid chromatography. Baseline separations of 17 pairs of enantiomers were achieved on the ImCel. The separation ability of the ImCel in the normal mode was much better than in reversed mode. In addition, the ImCel showed good chemical stability in the non-standard mobile phase due to the covalent bonds between the cellulose chiral selectors and silica support. Moreover, it exhibited complementarity with another immobilized-cellulose chiral stationary phase containing 3, 5-dimethylphenylcarbamate groups for the separation of a series of 9-fluorenylmethyloxycarbonyl (fmoc) -derived amino acids. The reversal of enantiomer elution order induced by the difference of the substituents in chiral stationary phase was observed under the same chromatographic conditions. In brief, a new immobilized cellulose chiral stationary phase with high stability and good separation performance was developed in this work.

Reversal of elution order of N‐(2,4‐dinitrophenyl)‐proline and N‐(2,4‐dinitrophenyl)‐serine in HPLC by BSA chiral stationary phase

N-(2,4-dinitrophenyl)-proline and N-(2,4-dinitrophenyl)-serine were enantiomerically resolved on the BSA chiral stationary phase by HPLC in reversed-phase mode. Effects of chromatographic conditions on enantioseparation and elution order have been investigated in detail. For these two samples, reversal of enantiomer elution order was observed by changing buffer pH, the content of acetonitrile, or alcohol modifiers in mobile phase, which is firstly reported in the BSA chiral stationary phase studies. More interestingly, combined effect between buffer pH and the content of acetonitrile was also observed. In addition, coelution range of enantiomers varied along with the content of acetonitrile in mobile phase.

Enantioseparation of Chiral Antimycotic Drugs by HPLC with Polysaccharide-Based Chiral Columns and Polar Organic Mobile Phases with Emphasis on Enantiomer Elution Order

The separation of enantiomers of 10 chiral antimycotic drugs was studied on polysaccharide-based chiral columns with polar organic mobile phases. The emphasis was placed on some interesting examples of enantiomer elution order reversal observed depending on the chemistry of the chiral selector, separation temperature, major component, as well as the minor additive in the mobile phase. In particular, it was found that the elution order of enantiomers of chiral drug terconazole was opposite on cellulose- and amylose-based columns with the same pendant group. The affinity pattern of enantiomers of another chiral drug bifonazole was opposite towards to two amylose-based chiral selectors with different pendant groups. The affinity pattern of terconazole enantiomers also changed on some columns when the alcohol-based mobile phase was replaced with acetonitrile. An interesting effect of the minor acidic (formic acid) additives to the mobile phase on the affinity pattern of terconazole enantiomers was observed on Cellulose-2 and Cellulose-4 columns. In addition, a reversal of elution order of bifonazole enantiomers was observed on Amylose-2 column by variation of a separation temperature.

Enantiomer elution order reversal of fluorenylmethoxycarbonyl-isoleucine in high-performance liquid chromatography by changing the mobile phase temperature and composition

In this paper the elution order reversal of enantiomers of fluorenylmethoxycarbonyl- or FMOC-isoleucine is described depending on the separation temperature and composition of the mobile phase when using the polysaccharide-based chiral column Lux Cellulose-1 in HPLC with normal-phase eluent. Reversal of the enantiomer elution order (EEO) in HPLC depending on the column temperature and content of the polar modifier in the mobile phase has been reported before in the literature. However, EEO reversal by changing the content of acidic modifier in the mobile phase seems to be described for the first time in the present work.

Parallel SFC/MS‐MUX screening to assess enantiomeric purity

Enantiomeric excess (ee) was evaluated for two internally synthesized compound libraries using a high-throughput automated, intelligent four-channel parallel supercritical fluid chromatography/mass spectrometry system equipped with a multiplexed ion source interface (SFC/MS-MUX). The two libraries contained compounds spanning a wide range of enantiomeric ratios with structurally diverse chemical scaffolds and stereogenic centers. The system analyzed each sample simultaneously against four chiral columns using up to six organic modifiers. Enhancements to our previously published parallel supercritical fluid chromatography/mass spectrometry system were implemented to address the challenges associated with automated trace enantiomer identification and quantitation. A reversal of enantiomer elution order was observed for several samples across multiple CSPs and modifiers. The relationship between elution order and % ee accuracy is presented for compounds exhibiting high, middle and low % ee values. Despite incidences in which the minor enantiomer eluted prior to the major enantiomer with less than baseline resolution, the overall % ee was in agreement with separations in which full baseline resolution was achieved. The methods presented here demonstrate the value and utility of high-throughput ee determinations to support drug discovery and development programs.

Chiral separations of transition metal complexes using capillary zone electrophoresis

Several buffer additives that may facilitate chiral separation for optically active transition metal (TM) systems are investigated using capillary zone electrophoresis. The TM complexes evaluated exhibit considerable heterogeneity with respect to total complex charge (0 to 4+), ligand type, and identity of the central metal including Ru 2+, Ni 2+, Cr 3+, and Co 3+. threo-D s[+]-Isocitrate, potassium antimonyl- d-tartrate and dibenzoyl- l-tartrate are identified as the most efficient chiral selectors. Interestingly, TM complexes exhibiting a (3+) total complex charge exhibit a reversal of enantiomer elution order versus all other complexes when separated using the tartrate additives. Operating parameters including pH, temperature, and capillary length are discussed, and chiral separations of complex mixtures are demonstrated.

Reversal of enantiomer elution order in capillary electrophoresis using charged and neutral cyclodextrins

Three different techniques for the reversal of the enantiomer elution order in capillary electrophoresis (CE) are reported in this paper. Intrinsic chiral recognition of the chiral selector remains the same in all cases and the reversal of enantiomer elution order is achieved by manipulation of the mobilities of the chiral selector and selectand. The mechanism of the reversal of the enantiomer elution order is described in detail for each particular case. In the first case a reversal of the enantiomer elution order is achieved by modification of the self-mobility of a chiral selector by introduction of an anionic ubstituent. In the second case the same effect was achieved by changing the pH of the separation buffer and in the third one by combination of the pH change and the reversal of polarity of the high-voltage supply.