Chiralpak AD Research Articles

Application and comparison of immobilized and coated amylose tris-(3,5-dimethylphenylcarbamate) chiral stationary phases for the enantioselective separation of β-blockers enantiomers by liquid chromatography

A direct liquid chromatographic enantioselective separation of a set of β-blocker enantiomers on the new immobilized and conventional coated amylose tris-(3,5-dimethylphenylcarbamate) chiral stationary phases (Chiralpak IA and Chiralpak AD, respectively) was studied using methanol as mobile phase and ethanolamine as an organic modifier (100:0.1, v/v). The separation, retention and elution order of the enantiomers on both columns under the same conditions were compared. The effect of the immobilization of the amylose tris-(3,5-dimethylphenylcarbamate) chiral stationary phase on silica (Chiralpak IA) on the chiral recognition ability was noted when compared to the coated phase (Chiralpak AD) which possesses a higher resolving power than the immobilized one (Chiralpak IA). A few racemates, which were not or poorly resolved on the immobilized Chiralpak IA were most efficiently resolved on the coated Chiralpak AD. However, the immobilized phase withstand solvents like dichloromethane when used as an eluent or as a dissolving agent for the analyte. The versatility of the immobilized Chiralpak IA in monitoring reactions performed in dichloromethane using direct analysis techniques without further purification, workup or removal of dichloromethane was studied on a representative example consisting of the lipase-catalyzed irreversible transesterification of a β-blocker using either vinylacetate or isopropenyl acetate as acyl donor in dichloromethane as organic solvent.

Evaluation of the performance of commercially available polysaccharide-based chiral stationary phases after multicycle operation in multimodal elution mode

The performance of four commercially available polysaccharide-based chiral stationary phases, Chiralcel OD, Chiralcel OJ, Chiralpak AD and Chiralpak AS was evaluated after several cycles of extended multimodal operations. Acetonitrile, methanol, ethanol and their mixtures were chosen for polar-organic mobile phases, ethanol– n-heptane mixture and n-heptane were selected for normal-phase mode. Retention factor ( k), selectivity ( α), resolution ( R s) and theoretical plate count ( N) were the chosen parameters to describe the column performance. One racemate for which all four columns have shown enantioselectivity was chosen as test compound. After 15 cycles of multimodal operations a slight decrease was seen in the retention factors ( k) however, column efficiency, selectivity ( α) and resolution ( R s) were maintained.

Use of enantioselective liquid chromatography for preparation of pure atenolol enantiomers

The study reported here shows a practicable preparation of pure atenolol enantiomers using enantioselective liquid chromatography. The successful separation of enantiomers of the final atenolol and the intermediate ester and the good peak shapes could not have been obtained without diethylamine as a component of the mobile phase. That makes difficult the recycling of the three-component mobile phase, an unavoidable step in simulated moving bed chromatography separation technology. The only suitable methodology for preparation of atenolol enantiomers proved to be synthesis from its N-benzyl-N-isopropyl precursor and the chiral stationary phase Chiralpak AD was found to be very convenient for preparative separation of these enantiomers. The enantiomeric purities and recovery of separated enantiomers of this N-benzyl-N-isopropyl precursor were very high, allowing high enantiomeric purities of the final products, ee's 99.3% for S- and 99.0% for R-atenolol. The chromatographic separation parameters, as well as solubility of racemate in the mobile phase, are good bases for the further examination of possible scale-up resolution of compound 6.

Reversal of elution order for profen acid enantiomers in packed‐column SFC on Chiralpak AD

Enantiomeric separations of four 2-substituted propionic acid drugs have been studied using packed-column supercritical fluid chromatography (SFC) with amylose tris(3,5-dimethylphenylcarbamate) coated on silica as support (Chiralpak AD). Under standard conditions (i.e., flow rate, 1.5 ml/min; column temperature, 30 degrees C; back-pressure, 150 bar), the order of elution could be reversed when the polar alcohol modifier methanol in carbon dioxide was replaced by 2-propanol for ibuprofen, ketoprofen, and naproxen. For flurbiprofen, with the highest selectivity factor, no reversal was observed, although selectivity was reduced significantly with higher alcohols. Naproxen and flurbiprofen were also investigated with 2-butanol and 2-pentanol. The former showed reversal of elution order but not the latter. For higher alcohol modifiers, including 2-propanol, the peak symmetry was poor but could be improved by addition of citric acid in the alcohol modifier. These results stress the importance to investigate enantiomer elution order during the development of enantioselective methods and when chromatographic conditions are optimized. Preliminary experiments with column temperatures over the range of -15 to 45 degrees C revealed that, in a few cases, reversal took place with a change in temperature only.

Direct resolution of chiral ‘pineno’ fused terpyridyl ligands on amylose based chiral stationary phase using long chain alcohol modifiers

Chiral separation of novel pineno fused terpyridyl ligands have been investigated on a silica-based amylose tris(3,5-dimethylphenyl carbamate) stationary phase (Chiralpak AD), using normal phase conditions. The chromatographic parameters investigated include retention factor ( k), separation factor ( α), column efficiency ( N) and resolution ( R S) of the analytes. In addition, the effect of alcohol modifiers on the enantioselectivity of the column has been discussed. The chiral resolution of the ligands was significantly improved by using 1-pentanol as alcohol modifier in the mobile phase. The reversal of the elution order achieved in this study allows a lower detection limit (<0.1%) of the minor enantiomer and the chromatographic system described is suitable for assessing the enantiomeric excess (ee) of the ctpy ≥ 99.9%. The method is selective and sensitive with a limit of detection (LOD) and quantification (LOQ) of 0.2 μg/ml and 0.6 μg/ml for ctpy, 0.6 μg/ml and 1.2 μg/ml for ctpy– x–ctpy, respectively. This study suggests that superior enantioseparation may be obtained for certain analytes using the same column by simply changing the type of alcohol modifier in the mobile phase instead of choosing another expensive chiral stationary phase for the same purpose.

Selectivity of amylose tris(3,5-dimethylphenyl-carbamate) chiral stationary phase as a function of its structure altered by changing concentration of ethanol or 2-propanol mobile-phase modifier

In a previous publication, solid-state NMR data showed that the structure of Chiralpak AD chiral stationary phase (CSP) was altered by changing the concentration of ethanol or 2-propanol modifier in the chromatographic mobile phase. This present paper reports the effect of the CSP structural change on chiral selectivity alpha. The enantiomers of a series of compounds were chromatographed using ethanol or 2-propanol in various concentrations as mobile-phase modifier and the alpha values were determined. Changes of alpha were observed for some enantiomeric pairs when ethanol and 2-propanol concentrations were varied. These data correlate with previous findings on the structural changes of the CSP. Not every enantiomeric pair showed changes in alpha as the alcohol concentration was varied, indicating that the chiral selectivity depends not only on the CSP's structure, but also on the structures of the analytes.

Development and validation of HPLC methods for the enantioselective analysis of bambuterol and albuterol

Suitable HPLC methods for the direct separation of bambuterol and albuterol enantiomers were developed. The enantioseparation was tested on numerous commercial chiral HPLC columns. For bambuterol the most convenient separation was determined on amylose Chiralpak AD column, and for albuterol on vancomycine Chirobiotic V column. The mobile phase compositions were systematically studied to obtain the optimal chromatographic methods. Validation of methods in selected conditions shows that the chosen methods are selective and precise with linear response of detector for both pairs of enantiomers.

Origin of enhanced chiral selectivity by acidic additives for a polysaccharide-based stationary phase

The effects of ethanesulfonic acid (ESA) and n-butylamine as additives were studied for a wide variety of chiral compounds using the polysaccharide chiral stationary phase (CSP), Chiralpak AD. The mobile phase consisted of hexane–ethanol (90:10, v/v). The additives typically had small effects, with one exception: the acidic additive had an enormous effect on the chiral selectivity of amino acid esters. The improved chiral selectivity was largely due to the longer retention of the later eluting enantiomer. Retention behavior of amines indicated that the higher selectivity for amino acid esters owes to increased hydrogen-bonding donation by the amine group of the analyte. Computation establishes the feasibility of a planar complex between the analyte and the cliral stationary phase, involving a pair of complementary hydrogen-bonding groups on each species, enabled by protonation of the analyte. Retention behaviors for a range of structures point to steric hindrance as the third interaction to comprise the requisite three interactions in chiral recognition.

New method for the chiral evaluation of mirtazapine in human plasma by liquid chromatography

A simple, rapid and sensitive high-performance liquid chromatography (HPLC) method was developed for the enantioselective analysis of the new antidepressant drug mirtazapine in human plasma. The procedure involved liquid–liquid extraction using toluene, followed by liquid chromatography coupled to UV detection at 292 nm. The chromatographic separation of the (+)-( S )- and (−)-( R )-enantiomers of mirtazapine was achieved on a Chiralpak AD column (250 mm × 4.6 mm, 10 μm particle size) protected with a CN guard column, using hexane–ethanol (98:2, v/v) plus 0.1% diethylamine as the isocratic mobile phase, at a flow rate of 1.2 ml/min. The total analysis time was less than 12 min per sample. The recoveries of (+)-( S )- and (−)-( R )-mirtazapine were in the 88–111% range with a linear response over the 6.25–625 ng/ml concentration range for both enantiomers. The quantification limit (LOQ) was 5 ng/ml. Within-day and between-day assay precision and accuracy were studied at three concentration levels (10, 50 and 250 ng/ml). For both mirtazapine enantiomers, the coefficients of variation (CV) and deviation from the theoretical value were lower than 15% at all concentration levels. The method proved to be suitable for pharmacokinetic studies.

Rapid method development for chiral separation in drug discovery using multi-column parallel screening and circular dichroism signal pooling

A novel strategy for rapid chiral method development has been developed using multi-column parallel screening and circular dichroism (CD) signal pooling. Described is the first use of a customized HPLC system that integrates an HPLC auto-sampler, one pump and five divided channels with five columns and five UV detectors to screen five chiral stationary phases (CSPs) simultaneously in parallel. A high-pressure semi-prep on-line pre-filter, a six-port manifold and five individually adjusted backpressure restrictors were installed in the system which allowed the sample and mobile phase to be evenly distributed over the five columns and UV detectors. The five CSPs, namely Chiralpak AD and AS, Chiralcel OJ and OD and Whelk-O1, were screened. The system guarantees a five-fold increase in speed for chiral column scouting compared with the widely used automated sequential column switching approach, and does not have the limitations of the coupled column screening approach for enantiomers whose elution order could be reversed on CSPs. Furthermore, the five channels after the UV detectors were recombined using a reversed flow splitter into a CD detector. The pooled CD signal from the five channels was recorded to track the elution order of the resolved enantiomers and to determine their sign, positive or negative. The signal pooling allows for the effective use of a single CD detector for multiple columns since unresolved racemate has little CD signal, and observing the sign of CD signal for one of the two enantiomer UV peaks is sufficient for tracking the enantiomeric elution order.

Rapid method development for chiral separation in drug discovery using multi-column parallel screening and circular dichroism signal pooling

A novel strategy for rapid chiral method development has been developed using multi-column parallel screening and circular dichroism (CD) signal pooling. Described is the first use of a customized HPLC system that integrates an HPLC auto-sampler, one pump and five divided channels with five columns and five UV detectors to screen five chiral stationary phases (CSPs) simultaneously in parallel. A high-pressure semi-prep on-line pre-filter, a six-port manifold and five individually adjusted backpressure restrictors were installed in the system which allowed the sample and mobile phase to be evenly distributed over the five columns and UV detectors. The five CSPs, namely Chiralpak AD and AS, Chiralcel OJ and OD and Whelk-O1, were screened. The system guarantees a five-fold increase in speed for chiral column scouting compared with the widely used automated sequential column switching approach, and does not have the limitations of the coupled column screening approach for enantiomers whose elution order could be reversed on CSPs. Furthermore, the five channels after the UV detectors were recombined using a reversed flow splitter into a CD detector. The pooled CD signal from the five channels was recorded to track the elution order of the resolved enantiomers and to determine their sign, positive or negative. The signal pooling allows for the effective use of a single CD detector for multiple columns since unresolved racemate has little CD signal, and observing the sign of CD signal for one of the two enantiomer UV peaks is sufficient for tracking the enantiomeric elution order.

Degradation and configurational changes of thioridazine 2-sulfoxide

Thioridazine (THD) is a phenothiazine neuroleptic drug used for the treatment of psychiatric disorders. After oral administration THD is extensively biotransformed to thioridazine 2-sulfone (THD 2-SO 2), thioridazine 5-sulfoxide (THD 5-SO) and thioridazine 2-sulfoxide (THD 2-SO). THD 2-SO and THD 5-SO have two chiral centres and therefore exist as two diastereoisomeric pairs. The degradation and epimerization of THD 2-SO in human plasma, buffer and methanolic solutions were studied using an enantioselective HPLC method. The samples were prepared by liquid–liquid extraction with diethyl ether and the chiral resolution of the enantiomers was carried out on a Chiralpak AD column using a mobile phase consisting of hexane:ethanol:2-propanol (90:7:3, v/v/v) containing 0.2% diethylamine. The method was validated and used to study the degradation and epimerization under different conditions of incubation. Our results showed that both enantiomers were stable at varying temperatures, pH and ionic strengths; however, solubility problems were observed, mainly at pH 8.5. The influence of light on stability was studied using methanolic solutions and degradation and epimerization of the THD 2-SO enantiomers were observed under UV light of 366 and 254 nm, respectively.

Chiral separation of some triazole pesticides by supercritical fluid chromatography☆

The enantiomeric separation of six triazole pesticides: cyproconazole, propiconazole, diniconazole, hexaconazole, tebuconazole and tetraconazole, using supercritical fluid chromatography and the Chiralpak AD column, is presented in this work. The effect of different organic modifiers such as methanol, ethanol and 2-propanol on the retention and resolution was studied as well as the presence of additives in the mobile phase. The results obtained were highly successful, all the compounds were enantiomerically separated and in most of the cases the analysis time was close to 10 min. The type of organic modifier that provided the best results depended on the compound.

New method for the chiral evaluation of mirtazapine in human plasma by liquid chromatography

A simple, rapid and sensitive high-performance liquid chromatography (HPLC) method was developed for the enantioselective analysis of the new antidepressant drug mirtazapine in human plasma. The procedure involved liquid–liquid extraction using toluene, followed by liquid chromatography coupled to UV detection at 292 nm. The chromatographic separation of the (+)-( S)- and (−)-( R)-enantiomers of mirtazapine was achieved on a Chiralpak AD column (250 mm × 4.6 mm, 10 μm particle size) protected with a CN guard column, using hexane–ethanol (98:2, v/v) plus 0.1% diethylamine as the isocratic mobile phase, at a flow rate of 1.2 ml/min. The total analysis time was less than 12 min per sample. The recoveries of (+)-( S)- and (−)-( R)-mirtazapine were in the 88–111% range with a linear response over the 6.25–625 ng/ml concentration range for both enantiomers. The quantification limit (LOQ) was 5 ng/ml. Within-day and between-day assay precision and accuracy were studied at three concentration levels (10, 50 and 250 ng/ml). For both mirtazapine enantiomers, the coefficients of variation (CV) and deviation from the theoretical value were lower than 15% at all concentration levels. The method proved to be suitable for pharmacokinetic studies.

Chiral separation of omeprazole and several related benzimidazoles using supercritical fluid chromatography.

A study of the enantiomeric separation of omeprazole and several related benzimidazoles, using supercritical fluid chromatography (SFC), on the amylose based column Chiralpak AD is presented in this work. The effect of the organic modifier as well as temperature on the retention and enantioresolution was investigated. Alcohol-type modifiers provided the best results, allowing the enantiomeric separation of all the compounds studied with resolutions that were in most cases higher than 2, and analysis times lower than 10 minutes. An investigation of the temperature effect revealed that the isoelution temperature was below the working temperature range in only two cases, and hence it was better to work at the highest temperature permitted.

Simultaneous determination of phenylglycidol enantiomers and cinnamyl alcohol in asymmetric epoxidation processes by chiral liquid chromatography

A high-performance liquid chromatographic method has been developed for the simultaneous determination of phenylglycidol enantiomers and cinnamyl alcohol (CA). Separations were achieved on an amylose tris(3, 5-dymethylphenylcarbamate) chiral stationary phase (Chiralpak AD). The effect of concentration of organic modifier (2-propanol and ethanol) in the mobile phase and flow-rate was studied. The mobile phase selected consisted of a mixture of n-hexane–ethanol (85:15, v/v) with a flow-rate of 1.2 ml/min. The UV–vis detector was set at 254 nm. Resolution for the phenylglycidol enantiomers in the suitable chromatographic conditions was 2.4 with an analysis time of 12 min. The method developed was validated and was found to be linear in the range from 5 × 10 −4 to 3 × 10 −2 M, for phenylglycidol enantiomers and in the range from 5 × 10 −5 to 1 × 10 −3 M, for CA ( r > 0.999 for the three compounds). Repeatability and intermediate precision for the three analytes at three different concentrations were below 3.6 and 2.8% R.S.D., respectively. This method has been applied to study the asymmetric epoxidation of CA with titanium(IV) alkoxide compounds as catalysts in order to evaluate their catalytic activity and stereoselectivity of the epoxidation processes.

Chiral separation of some triazole pesticides by supercritical fluid chromatography

The enantiomeric separation of six triazole pesticides: cyproconazole, propiconazole, diniconazole, hexaconazole, tebuconazole and tetraconazole, using supercritical fluid chromatography and the Chiralpak AD column, is presented in this work. The effect of different organic modifiers such as methanol, ethanol and 2-propanol on the retention and resolution was studied as well as the presence of additives in the mobile phase. The results obtained were highly successful, all the compounds were enantiomerically separated and in most of the cases the analysis time was close to 10 min. The type of organic modifier that provided the best results depended on the compound.

Enantiomers of New Synthetic Pyrrolylphenylethanoneamine Mono-Amino Oxidase Inhibitor Compounds: Analytical and Semipreparative HPLC Separations, and Chiroptical Characteristics

The polysaccharide chiral stationary phases (CSPs) Chiralcel OD and Chiralpak AD, and the brush-type (R,R)-Whelk-01 chiral stationary phases have been evaluated to separate new synthetic pyrrolylphenylethanoneamine racemic compounds, potentially monoamine oxidase (MAO) inhibitors, under various mobile phase compositions, using various temperatures. The enantioseparation was evaluated by comparing the (R,R)-Whelk-01 column performance with those of Chiralpak AD and Chiralcel OD. Significant differences were observed in their chiral recognition, as revealed from their retention, selectivity, resolution and elution order. Performances of the Chiralpak AD column were superior to those of the Chiralcel OD and (R,R)-Whelk-01 columns. Some of the racemic compounds were resolved by semipreparative chromatography on Chiralpak AD column in order to study the chiroptical proprieties of the single enantiomers.

Preparation and biological activities of optically active dehydroxymethylepoxyquinomicin, a novel NF-κB inhibitor

NF-κB is a transcription factor that induces inflammatory cytokines and anti-apoptotic proteins. We have designed a new NF-κB inhibitor based on the structure of the antibiotic epoxyquinomicin C. The designed compound, dehydroxymethylepoxyquinomicin (DHMEQ) was synthesized as a racemic form from 2,5-dimethoxyaniline through 5 steps. Application of racemic DHMEQ onto the chiral column (Chiralpak AD) directly gave enantiomeric DHMEQ after purification. (−)-DHMEQ was more potent than its enantiomer. (−)-DHMEQ was found to inhibit NF-κB activity and macrophage differentiation induced by 12- O-tetradecanoylphorbol-13-acetate (TPA) in human monocyte THP-1 cells.

Packed column supercritical fluid chromatography of a peroxysome proliferator-activating receptor agonist drug: Achiral and chiral purity of substance, formulation assay and its enantiomeric purity

This paper describes packed column supercritical fluid chromatography (SFC) for the analysis of a peroxysome proliferator-activating γ-receptor agonist that is a carboxylic acid. Evaluation of conditions for the separation of this candidate drug and related compounds in bulk substance is described. A Chiralcel OD column was used for this purpose due to its high selective retention of related substances and relative inertness, though the enantioselectivity was negligible, with methanol as polar modifier. A high enantioselectivity was obtained on Chiralpak AD and it was possible to determine the enantiomeric purity within 10 min on a 5 cm short column. Both the achiral and the chiral systems were run without acid additive in the mobile phase and the level of detection of impurities by area was about 0.1%. For the analysis of samples dissolved in water, without any isolation step, 2-propanol was used as modifier. Due to the column surface activity, evidently generated by injected water, citric acid 1 mM was included as additive in the 2-propanol in order to maintain symmetric and undistorted peak shape. The detection limit for the assay was 21 μg mL −1 (50 nmol mL −1) for 5 μL injected (R.S.D. 6.4 %, n = 8). A 5 cm short Chiralcel OD column was used. Determination of enantiomeric purity of the drug in aqueous samples required increased sensitivity. The sample was acidified and extracted into a small volume of 1-pentanol, out of which 25 μl was analyzed by SFC. The minor enantiomer at the 3% (w/w) level added could be confirmed. Its ratio remained constant during the procedure as measured relative to a reference solution in organic media.