Amylose Chiral Stationary Phases Research Articles

HPLC Enantioseparation of Rigid Chiral Probes with Central, Axial, Helical, and Planar Stereogenicity on an Amylose (3,5-Dimethylphenylcarbamate) Chiral Stationary Phase

The chiral resolving ability of the commercially available amylose (3,5-dimethylphenylcarbamate)-based chiral stationary phase (CSP) toward four chiral probes representative of four kinds of stereogenicity (central, axial, helical, and planar) was investigated. Besides chirality, the evident structural feature of selectands is an extremely limited conformational freedom. The chiral rigid analytes were analyzed by using pure short alcohols as mobile phases at different column temperatures. The enantioselectivity was found to be suitable for all compounds investigated. This evidence confirms that the use of the amylose-based CSP in HPLC is an effective strategy for obtaining the resolution of chiral compounds containing any kind of stereogenic element. In addition, the experimental retention and enantioselectivity behavior, as well as the established enantiomer elution order of the investigated chiral analytes, may be used as key information to track essential details on the enantiorecognition mechanism of the amylose-based chiral stationary phase.

3-(Phenyl-4-oxy)-5-phenyl-4,5-dihydro-(1H)-pyrazole: A fascinating molecular framework to study the enantioseparation ability of the amylose (3,5-dimethylphenylcarbamate) chiral stationary phase. Part II. Solvophobic effects in enantiorecognition process

The enantiomers of five chiral compounds incorporating the 3-(phenyl-4-oxy)-5-phenyl-4,5-dihydro-(1H)-pyrazole scaffold and differing only in the linear alkyl chain (varying in length from 1 to 5 carbon atoms) linked to the oxygen atom were directly separated on the amylose (3,5-dimethylphenylcarbamate) based Chiralpak AD-3 chiral stationary phase. The effects of the mobile phase composition, the structure of the analytes and temperature on the retention and enantioselectivity were investigated. It was found that the enantiomeric separations were in all cases enthalpy-driven and disfavored by entropic term. U-shape curves obtained by plotting the chromatographic data versus the alcoholic percentage in n-pentane-methanol and n-hexane-ethanol mobile phases highlighted that, at higher alcohol concentrations, solvophobic interactions were operative in the retention mechanism. The unusual trend of such curves was linked to the nature of alkyl chain of the pyrazolines and it was indicative of the solvophobic contribution to the achievement of a high degree of enantioseparation.

Preparation and chromatographic evaluation of β-cyclodextrin derivative CSPs bearing substituted phenylcarbamate groups for HPLC

ABSTRACTFive β-cyclodextrin (β-CD) derivatives bearing substituted phenylcarbamate/3-(triethoxysilyl)propylcarbamate groups at the 2-, 3-, and 6-positions of glucose unit and another five derivatives containing benzoate at the 2-position and substituted phenylcarbamate/3-(triethoxysilyl)propylcarbamate groups at the 3- and 6-positions were synthesized using the regioselective esterification method. The obtained β-CD derivatives were efficiently immobilized onto the silica gel through the intermolecular polycondensation of a small amount of the triethoxysilyl groups, which were used as the chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC). The chiral separation properties of these CSPs were evaluated under the normal-phase HPLC. The effects of solvent polarity and the side chain structures of β-CD derivatives on the chiral recognition ability of the immobilized CSPs were investigated. Among these β-CD derivative CSPs, 2,3,6-tris(3,5-dichlorophenylcarbamate)-β-CD CSP showed a relatively high chiral recognition ability for the studied racemates. The regioselective esterification at the 2-position of glucose unit in the β-CD decreased the chiral recognition ability at the same conditions. For some racemates, the β-CD derivative CSPs showed chiral recognition abilities comparable or better to some chemical bonded β-CD derivative CSPs and 3,5-dichloro- and 3,5-dimethylphenylcarbamates of cellulose and amylose CSPs.

3-(Phenyl-4-oxy)-5-phenyl-4,5-dihydro-(1H)-pyrazole: A fascinating molecular framework to study the enantioseparation ability of the amylose (3,5-dimethylphenylcarbamate) chiral stationary phase. Part I. Structure-enantioselectivity relationships

Chiral stationary phases (CSPs) based on amylose (3,5-dimethylphenylcarbamate) (ADMPC) exhibit a wide-range of enantioselectivity in high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). Although this class of CSPs has been extensively used, chiral discriminations at receptorial level, which are useful to develop predictive molecular models, have been rarely reported in the literature.Herein, we describe the results obtained in the enantioselective HPLC of a set of six C5-chiral 4,5-dihydro-(1H)-pyrazole derivatives on the ADMPC-based Chiralpak AD-3 CSP (CSP) under normal-phase and polar organic conditions. Using pure methanol as a mobile phase the exceptional enantioseparation factor value of 50 at 25°C was found for one of the investigated analytes. To the best of our knowledge, the enantiomeric bias represents the most outstanding enantioseparation ever recorded on ADMPC-based CSPs.Systematic variations in chemical groups in specific positions of the 3-(phenyl-4-oxy)-5-phenyl-4,5-dihydro-(1H)-pyrazole molecular framework resulted in peculiar changes in retention and enantioselectivity. A careful analysis of the chromatographic data permitted to advance some hypotheses concerning the role played by the individual chemical groups in determining the exceptional enantioseparation.In particular, under methanol-rich mode, the prenyl moiety of the second eluted enantiomer of the better resolved analyte was recognized as a critical structural element to establish direct and favorable solvophobic interactions with apolar portions of selector.

Chiral separation and determination of enantiomeric purity of the pharmaceutical formulation of cefadroxil using coated and immobilized amylose-derived and cellulose-derived chiral stationary phases

Background and objectives In the present article, we describe the development of a simple, direct, and isocratic high-performance liquid-chromatographic method for chiral separation and the determination of the enantiomeric purity of cefadroxil. Cefadroxil has three chiral centers; the existence of eight different stereoisomers is possible. Only one of these isomers is currently under development as an antibiotic agent and, consequently, the other seven isomers are considered as unwanted chiral impurities.Materials and methods An analytical chiral separation was carried out to check its enantiomeric purity. The separation was carried out by exploiting the high efficiency of several coated/immobilized cellulose and amylose chiral stationary phases under normal-phase and polar-organic modes. The effects of type and concentration of the alcoholic modifiers, 2-propanol and ethanol, on the separation of stereoisomers were studied for optimum resolution.Results and conclusion Complete baseline separation of stereoisomers with good resolution was achieved within 40 min under normal-phase mode on Chiralpak IB column using hexane–2-propanol (60 : 40 v/v) as the mobile phase, without any organic additives, at a flow rate of 0.4 ml/min at 25°C and with the ultraviolet detection set at 268 nm. This process was found to be suitable for rapid enantiomeric purity analysis and a quality control of cefadroxil in pharmaceutical formulations without interference of the excipients. The chiral recognition mechanisms of separation were also discussed.

Enantiomeric Separations of N–H/N–Me Aziridines Utilizing GC and HPLC

High-performance liquid chromatographic (HPLC) and gas chromatographic (GC) methods were developed for the separation of enantiomers of 12 novel aziridines. The HPLC separations were performed on cyclodextrin, cyclofructan, amylose, cellulose and macrocyclic glycopeptide-based chiral stationary phases, whereas the GC separations were performed only on cyclodextrin-based chiral stationary phases. The amylose-based HPLC chiral stationary phases showed good selectivity toward the aziridines, while having low retention factors. It was shown that the HPLC generally provided better separations for most of the compounds as compared to GC. Effective enantiomeric separations for ten aziridines were developed.

Enantiomeric separation of 2-arylpropionic acid nonsteroidal anti-inflammatory drugs and β-blockers by RP-HPLC using an amylose chiral stationary phase for the enantioselective skin permeation study

The enantioselectivities of eight 2-arylpropionic acid nonsteroidal anti-inflammatory drugs (2-APA NSAIDs) and two β-blockers on an amylose-tris(3,5-dimethylphenylcarbamate)-based Chiralpak AD-RH (150 × 4.6 mm, 5 μm) column were studied in reverse phase (RP) elution mode. The influencing factors such as mobile phase composition, organic modifiers, and column temperature were investigated. For most of the selected NSAIDs, baseline enantioseparation was achieved using a mobile phase composition of acetonitrile–0.05% trifluoroacetic acid aqueous solution. For the β-blockers, baseline enantioseparation was achieved using a mobile phase composition of acetonitrile–borate buffer solution with 0.1% triethylamine (pH 9.0; 10 mM). Our study has demonstrated for the first time that the addition of an organic modifier, ethanol, in the mobile phase dramatically improves the resolution of the analytes such as flurbiprofen, suprofen, propranolol and metoprolol under reverse phase chromatographic conditions. The methods for the enantioselective determination of ibuprofen, flurbiprofen and propranolol were validated. The enantioselective skin permeation studies of propranolol hydrochloride presented here showed that there was no observable enantioselective permeation of propranolol across the excised rat skin, but L-linalool, a chiral enhancer, resulted in enhanced enantioselectivity, with a better permeation enhancing effect on R-propranolol than on S-propranolol and RS-propranolol across the excised rat skin.

Chiral Recognition Mechanisms of four β-Blockers by HPLC with Amylose Chiral Stationary Phase.

The high performance liquid chromatography (HPLC) enantioseparation of four β-blocking agents metoprolol, bisoprolol, propranolol and atenolol was performed on amylose tris-(3,5-dimethylphenylcarbamate) chiral stationary phase using n-hexane-ethanol-diethylamine (DEA) as the mobile phase and related chiral recognition mechanisms were discussed. Enantiomeric separation of the four β-blockers was a result of more than one type of interaction between solutes and CSP. Besides hydrogen bonding, there was another type interaction that was independent of solvent polarity and responsible for enantiomeric selectivity, such as - interactions. Both the groups close to the chiral centers and the substituent groups on the phenyl rings, which were far away from the chiral centers, could contribute to the good separation. The separations of the four β-blocker enantiomers were all enthalpy driven process. In the range of 293-308K (20-35 ℃), as the temperature increased, the retention as well as the resolution decreased. The molecular size rather than concentration of the alcohol modifiers affected the resolution and retention.

Development of a multi-residue enantiomeric analysis method for 9 pesticides in soil and water by chiral liquid chromatography/tandem mass spectrometry

A novel and sensitive chiral liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous measuring individual enantiomers of 9 pesticides including herbicides, insecticides, and fungicides in soil and water. The separation and determination were performed using reversed-phase chromatography on an amylose chiral stationary phase, a Chiralpak AD-RH column, under gradient elution using a mixture of ACN-2mM ammonium acetate in water as the mobile phase at 0.45mL/min flow rate. The effects of three cellulose-based columns and three amylose-based columns on the separation were also investigated. The QuEChERS (acronym for Quick, Easy, Cheap, Effective, Rugged and Safe) method and solid-phase extraction (SPE) were used for the extraction and clean-up of the soil and water samples, respectively. Parameters including the matrix effect, linearity, precision, accuracy and stability were undertaken. Under optimal conditions, the mean recoveries for all enantiomers from the soil and water samples were ranged from 77.8% to 106.2% with the relative standard deviations (RSD) less than 14.2%. Good linearity (at least R2≥0.9986) was obtained for all studied analytes in the soil and water matrix calibration curves over the range from 2.0 to 125μg/L. The limits of detection (LOD) for all enantiomers in the soil and water were less than 1.8μg/kg or μg/L, whereas the limit of quantification (LOQ) did not exceed 5.0μg/kg or μg/L. The results of the method validation confirm that this proposed method is convenient and reliable for the enantioselective determination of the enantiomers of 9 chiral pesticides in soil and water.

Preparative enantiomeric separation of new selective CB2 receptor agonists by liquid chromatography on polysaccharide-based chiral stationary phases: Determination of enantiomeric purity and assignment of absolute stereochemistry by X-ray structure analys

The development of high-performance liquid chromatography (HPLC) methods using derivatized amylose chiral stationary phases has permitted preparative enantioseparations of substituted 4-oxo-1,4-dihydroquinoline-3-carboxamide derivatives with satisfactory yields. These compounds constitute new potent selective agonists of the cannabinoid CB(2) receptor. Analytical enantioseparation methods using UV detection were validated to determine the enantiomeric purity of these compounds. Linear calibration curves in the range from 0.18 to 0.40 mM were obtained; repeatability, limits of detection (LOD), and quantification (LOQ) were determined: LOD varied, for the various solutes, from 0.5 to 1.2 μM. All the separated compounds were prepared with high enantiomeric purities superior to 99.3% Absolute configuration of the enantiomers was unequivocally established by single crystal X-ray diffraction method and correlated to the chiroptical properties of isolated enantiomers.

Kinetics and mechanism of racemization of Tic-hydantoins, potent sigma-1 agonists

The configurational stability of seven Tic-hydantoin sigma-1 agonists 1– 7 was studied in organic and aqueous media to mimic conditions encountered during either their synthesis or their evaluation as a drug candidate (physico chemical property determination and pharmacological study). This study was performed from the enantiomers directly obtained by asymmetric synthesis (hydantoins 1, 3– 7) or after semi-preparative separation of the racemic obtained according to the same asymmetric procedure (thiohydantoin 2), by chiral HPLC. The racemization phenomenon was followed using recently validated chiral HPLC and capillary electrophoresis separation methods using derivatized cellulose and amylose chiral stationary phases (Chiralcel OD-H and Chiralpak AD) and highly sulphated cyclodextrins [highly S-β-CD in the background electrolyte (BGE)], respectively. The kinetic parameters (rate constant, half-life and apparent free energy barriers) of racemization were calculated using a first-order reaction model. The influence of the acid–base content, pH in aqueous medium, concentration of the buffer, and temperature were investigated. The fastest racemization rates were observed under basic conditions. All hydantoins were shown to present the same magnitude of configurational stability, whereas thiohydantoin 2 was characterized by a high chiral instability, especially in ethanolic and aqueous media: its high instability in ethanol explains that a racemic mixture was obtained after asymmetric synthesis; its instantaneous racemization observed from the neutral pH makes the preparation of the enantiomers of 2 not relevant. Finally, the mechanism of racemization was elucidated using nuclear magnetic resonance (NMR): the comparison of the kinetics of the deuteration to the kinetics of the racemization suggests the involvement of a common reaction mechanism of S E1 type for hydantoin 1, while an S E2 type reaction seems to be involved for thiohydantoin 2.

Comparison of HPLC enantioseparation of substituted binaphthyls on CD‐, polysaccharide‐ and synthetic polymer‐based chiral stationary phases

Retention and enantioseparation behavior of ten 2,2'-disubstituted or 2,3,2'-trisubstituted 1,1'-binaphthyls and 8,3'-disubstituted 1,2'-binaphthyls, which are used as catalysts in asymmetric synthesis, was investigated on eight chiral stationary phases (CSPs) based on beta-CD, polysaccharides (tris(3,5-dimethylphenylcarbamate) cellulose or amylose CSPs) and new synthetic polymers (trans-1,2-diamino-cyclohexane, trans-1,2-diphenylethylenediamine and trans-9,10-dihydro-9,10-ethanoanthracene-(11S,12S)-11,12-dicarboxylic acid CSPs). Normal-, reversed-phase and polar-organic separation modes were employed. The effect of the mobile phase composition was examined. The enantiomeric separation of binaphthyl derivatives, which possess quite similar structures, was possible in different enantioselective environments. The substituents and their positions on the binaphthyl skeleton affect their properties and, as a consequence, the separation system suitable for their enantioseparation. In general, the presence of ionizable groups on the binaphthyl skeleton, substitution with non-identical groups and a chiral axis in the 1,2' position had the greatest impact on the enantiomeric discrimination. The 8,3'-disubstituted 1,2'-binaphthyl derivatives were the most easily separated compounds in several separation systems. From all the chiral stationary phases tested, cellulose-based columns were shown to be the most convenient for enantioseparation of the studied analytes. However, the polymeric CSPs with their complementary behavior provided good enantioselective environments for some derivatives that could be hardly separated in any other chromatographic system.

Separation of Tic-hydantoin enantiomers, potent sigma-1 agonists, by high performance liquid chromatography and capillary electrophoresis

Stereospecific separations of seven Tic-hydantoin sigma-1 agonists were performed by both HPLC method using derivatized cellulose and amylose chiral stationary phases and capillary electrophoresis (CE) method using neutral and anionic cyclodextrins added in the background electrolyte (BGE). An optimal baseline separation ( R s > 3.3 with analysis times < 25 min) was readily obtained with all silica-based celluloses and amyloses using a normal-phase methodology. CE was used as an alternative technique to HPLC for the Tic-hydantoin derivatives separation. The enantiomers were fully resolved with highly sulfated β-cyclodextrins at pH 2.5 ( R s > 1.5 with analysis times <11 min). Both methods were validated in terms of linearity, detection and quantification limits. They were used to check the enantiomeric purity of the enantiomers.

Separation of diastereoisomers of Ara-C phosphotriesters using solid phase extraction and HPLC for the study of their decomposition kinetic in cell extracts

Separations of the diastereoisomers of three nucleoside 5′-phosphotriester derivatives of Ara-C ( tBuSATE, hydroxy tBuSATE and bishydroxy tBuSATE phenylphosphotriester derivatives; pronucleotides) were performed by HPLC using derivatized cellulose and amylose chiral stationary phases. An optimal baseline separation ( Rs > 1.5) was readily obtained with an amylose based chiral column (AD-H) used in normal phase mode. This stereospecific HPLC method has been associated to a solid phase extraction step using a C18 cartridge and an internal standard for the quantification of one nucleoside 5′-phosphotriester derivative in cell extracts. After optimization, this method was validated in terms of specificity, recovery, linearity, precision and accuracy and detection limit. It was applied to the determination of the apparent rate constants of disappearance and half-lives of each diastereoisomer. This enabled us to conclude that the enzymatic activity involved in the first step of the decomposition pathway of the hydroxyl tBuSATE phenylphosphotriester of Ara-C is stereoselective and is related to the nature of the pyrimidic base.

Effects of solvent on chiral and enantiomeric separation of Koga bases using derivatized amylose chiral stationary phase

The separation of R,R-, S,S-, and meso-Koga bases on derivatized amylose chiral stationary phases (CSP) has been studied using different alcohol and alcohol-hexane mixtures as eluant. Straight-chain and branched alcohols with carbon numbers from one to four were investigated. The carbon number and geometry of the alcohol impacts the separation of Koga bases. The optimal separations were obtained using a mixture of methanol with linear or branched alcohol. Also, the elution order of meso- and R,R-Koga base was switched as content of branched alcohol increases in cosolvent. The study of acidic and basic additive effects demonstrated that maintaining analytes in the free base state is crucial in order to achieve retention and separation. TEA alone or TEA and TFA mixture were used in the studies.

Enantiomeric Resolution of Benzoxazolinonic Aminoalcohols, Potential Adrenergic Ligands, by LC and CE

Benzoxazolinone amino alcohols are chiral derivatives that carry one asymmetric center. Both capillary electrophoresis and high-performance liquid chromatography (LC) techniques were used to separate enantiomers with high resolution and thus permitted the determination of enantiomeric excess. Highly sulfated and anionic cyclodextrins were used as chiral selectors under CE conditions. Amylose and cellulose chiral stationary phases were used for LC for the enantioseparation of five racemic compounds. Separations were carried out on a Chiralpak AS column with various polar organic modifiers. The HPLC method appeared more to be more sensitive and was subsequently validated in terms of limits of detection and quantification. LC detection was based on UV and evaporating light scattering.

Enantioseparation of benzoxazolinone aminoalcohols and their aminoketone precursors, potential adrenergic ligands, by analytical and preparative liquid chromatography on amylose chiral stationary phases and characterization of the enantiomers

To obtain milligram amounts of the enantiomers of benzoxazolinone derivatives to be tested for binding to adrenergic sites, analytical HPLC methods using derivatized amylose chiral stationary phases were developed for the direct enantioseparation of benzoxazolinone aminoalcohols and their aminoketone precursors, derivatives with one or two chirals centers. The separations were made using normal phase methodology with a mobile phase of n-hexane-alcohol (ethanol, 1-propanol, or 2-propanol) in various proportions, and silica-based amylose (tris-3, 5-dimethylphenylcarbamate) Chiralpak AD and (tris-(S)-1-phenylethylcarbamate) Chiralpak AS columns. The effects of concentration of various aliphatic alcohols in the mobile phase were studied. The best separation was achieved on Chiralpak AS, so preparative HPLC was set up with this chiral stationary phase using a mobile phase consisting of n-hexane-alcohol using isocratic conditions and multiple repetitive injections. Physicochemicals properties of enantiomers were reported The effect of structural features of the solutes on discrimination between the enantiomers was examined. Limit of detection (LD) and limit of quantification (LQ) were determined using both ultra-violet (UV) and evaporative light-scattering detection (ELSD).

Chiral Separation of Carboprost Isomers by Normal Phase LC Using Amylose Chiral Stationary Phase

LC separation of carboprost diastereomers in bulk drug was developed and validated using normal-phase amylose stationary phase Chiralpak AD-H. The effect of the organic modifiers, namely 2-propanol and ethanol in the mobile phase was optimized in order to obtain the best separation. The retention time of (R)-carboprost and (S)-carboprost were 15.3 and 17.1 min, respectively. Calibration curves were linear over the range of 0.2–1.0%, with the regression coefficient (R2) of 0.9997. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.07 and 0.2%, respectively. The method was accurate, precise and suitable to use for the purpose of controlling unwanted (R)-isomer in the carboprost active pharmaceutical ingredient. This method can be successfully applied to the analysis of chiral purity of carboprost in pharmaceutical bulk drug samples.

Separation of nucleoside phosphoramidate diastereoisomers by high performance liquid chromatography and capillary electrophoresis

Separations of five diastereoisomers of nucleoside phosphoramidate derivatives (pronucleotides) were performed by both HPLC method using derivatized cellulose and amylose chiral stationary phases and CE method using anionic cyclodextrins added in the background electrolyte (BGE). An optimal baseline separation ( R s > 1.5) was readily obtained with all silica-based celluloses and amyloses using in a normal-phase methodology. Capillary electrophoresis was used as an alternative technique to HPLC for the separation of pronucleotides. The diastereoisomers were fully resolved with sulfated cyclodextrins at both BGE pH (2.5 and 6.2). Limits of detection and limits of quantification, calculated for both methods, are up to 200 times higher in CE separations than in HPLC separations. The analytical HPLC method was then applied in a preliminary study for the pronucleotide 1 quantification in cellular extract.

Column temperature programming in enantioseparation of dihydropyrimidinone compounds using derivatized cellulose and amylose chiral stationary phases

We report the application of column temperature programs as a tool to examine unusual temperature-induced behaviors of polysaccharide chiral stationary phases (CSPs). Using dihydropyrimidinone (DHP) compounds as probes we observed the heating (10-50 degrees C) and cooling (50-10 degrees C) van't Hoff plots of retention factors and/or selectivities of DHP compounds were not superimposable on AD, IA, and AS-H columns solvated with ethanol (EtOH)/n-hexane (n-Hex) mobile phases. The plots were not superimposable on AD, IB, and AS-H columns solvated with 2-propanol (2-PrOH)/n-Hex mobile phases. The thermally induced path-dependant behaviors were caused by slow equilibration as evidenced by the disappearance of the hysteresis in the second heating to cooling cycle and in a cooling to heating cycle. From the step-temperature program (10-50-10 degrees C), only EtOH solvated AD and AS-H phases showed the change of retention factors and/or selectivities with time while only 2-PrOH solvated AS-H phase showed similar behaviors.