新型含苯并噻唑α-氨基膦酸酯类化合物在淀粉类手性固定相上的液相色谱分离

本文运用涂敷型(Chiralpak AD-H)和键合型(Chiralpak IA)两种淀粉类手性固定相高效液相色谱法,进行了新型含苯并噻唑α-氨基膦酸酯类化合物的手性分离。从色谱分离的保留因子(k)、分离系数(α)和分离度(Rs)三个方面考察了两种类型色谱柱的分离性能,上述化合物在Chiralpak IA柱上能够得到较好的基线分离。同时,讨论了温度、流动相极性和目标分析物的结构等因素对Chiralpak IA柱分离性能的影响。由于键合型固定相较稳定的性能,使某些非常规的溶剂(如THF)成功地应用于手性α-氨基膦酸酯类化合物的分离。     

Optimization of the HPLC enantioseparation of 3,3′‐dibromo‐5,5′‐disubstituted‐4,4′‐bipyridines using immobilized polysaccharide‐based chiral stationary phases

The HPLC enantioseparation of nine atropisomeric 3,3′,5,5′‐tetrasubstituted‐4,4′‐bipyridines was performed in normal and polar organic (PO) phase modes using two immobilized polysaccharide‐based chiral columns, namely, Chiralpak IA and Chiralpak IC. The separation of all racemic analytes, the effect of the chiral selector, and mobile phase (MP) composition on enantioseparation and the enantiomer elution order (EEO) were studied. The beneficial effect of nonstandard solvents, such as tetrahydrofuran (THF), dichloromethane (DCM), and methyl t‐butyl ether on enantioseparation was investigated. All selected 4,4′‐bipyridines were successfully enantioseparated on Chiralpak IA under normal or PO MPs with separation factors from 1.14 to 1.70 and resolutions from 1.3 to 6.5. Two bipyridines were enantioseparated at the multimilligram level on Chiralpak IA. Differently, Chiralpak IC was less versatile toward the considered class of compounds and only five bipyridines out of nine could be efficiently separated. In particular, on these columns, the ternary mixture n‐heptane/THF/DCM (90:5:5) as MP had a positive effect on enantioseparation. An interesting phenomenon of reversal of the EEO depending on the composition of the MP for the 3,3′‐dibromo‐5,5′‐bis‐(E)‐phenylethenyl‐4,4′‐bipyridine along with an exceptional enantioseparation for the 3,3′‐dibromo‐5,5′‐bis‐ferrocenylethynyl‐4,4′‐bipyridine (α = 8.33, R_s = 30.6) were observed on Chiralpak IC.     

Enantiomeric analysis of simendan on polysaccharide‐based stationary phases by polar organic solvent chromatography

Herein, the enantiomeric separation of simendan by high‐performance liquid chromatography with ultraviolet detection using polysaccharide‐based chiral stationary phases in polar organic mode is described. Three chiral columns (Chiralpak AD‐H, Chiralcel OD‐H, and Chiralpak AS) were screened using pure methanol and acetonitrile without additives under isocratic conditions. A reversed elution order was observed on the Chiralpak AD‐H column when the methanol content in the mobile phase (methanol–acetonitrile mixtures) was above 10%, whereby levosimendan eluted prior to dextrosimendan. Further, it was found that increasing temperature effectively improved the enantioresolution on the Chiralpak AD‐H column. Van't Hoff analysis was performed to evaluate the contribution of enthalpy and entropy to the chiral discrimination process. The best enantioseparation (α = 3.00, Rs = 12.85) was obtained on the Chiralpak AD‐H column with methanol as the mobile phase at 40°C. Thus, a quantitative method for the resolution of dextrosimendan was established and validated, which could be used as a reference for the determination of dextrosimendan in levosimendan products.     

Characterization of Column Hold-Up Volume with Static and Dynamic Methods on an Immobilized Polysaccharide-Based Chiral Stationary Phase

Polysaccharide-based chiral stationary phases (CSPs) are efficient for enantioseparation of many chiral compounds. Immobilized polysaccharide CSP, as used in the Chiralpak IA column, is a new configuration that was recently introduced for application in chiral separation. As shown in several previous studies, the characteristics of Chiralpak IA columns cannot be simply extrapolated from the coated version. In this study, hold-up volume of a Chiralpak IA column was evaluated by static and dynamic methods. The static pyconometry method gave similar hold-up volumes either as an average value from a range of solvents or a direct measurement from the carbon tetrachloride-isopropanol (IPA) solvent pair. The dynamic method with 1,3,5-tri-tert-butylbenzene (TTBB) was influenced by the ratio of n-hexane and 2-propanol in the mobile phase but not by the dissolving solvent of TTBB. The two methods resulted in the same hold-up volume of ∼3.0 mL. TTBB showed weaker retention on the IA column after correction of isobaric thermal expansion of the mobile phase. During temperature variations in the range of 15–50 °C, the hold-up volume of TTBB was highly reproducible. Results of this study improve our understanding of the chromatographic features of the immobilized polysaccharide IA column.     

Liquid chromatographic separation and thermodynamic investigation of stereoisomers of darunavir on Chiralpak AD‐Hcolumn

Liquid chromatographic separation of stereoisomers of darunavir on Chiralpak AD‐H, a column containing the stationary phase coated with amylose tris(3,5‐dimethylphenylcarbamate) as a chiral selector, was studied under normal‐phase conditions at different temperatures between 20 and 50°C. The effect of quality and quantity of different polar organic modifiers viz: methanol, ethanol, 1‐propanol, and 2‐propanol in the mobile phase as well as column temperature on retention, separation, and resolution was investigated and optimized. The optimum separation was accomplished using a mobile phase composed of n‐hexane/ethanol/diethyl amine (80:20:0.1 v/v/v) at 40°C. Apparent thermodynamic parameters ΔH⁰ and ΔS* were derived from the Van't Hoff plots (lnk′ versus 1/T) and used to explain the strength of interactions between the stereoisomers and amylose tris(3,5‐dimethylphenylcarbamate) coated chiral stationary phase.     

Chiral separations of piperidine-2,6-dione analogues on Chiralpak IA and Chiralpak IB columns by using HPLC

Recently, two new immobilized polysaccharides based CSPs, namely tris-(3,5-dimethylphenylcarbamate) derivatives of amylose and cellulose known as Chiralpak IA and Chiralpak IB were introduced, which may be used with a wide range of solvents including standard and prohibited ones. Several racemic piperidine-2,6-dione analogues [aminoglutethimide, p-nitro-glutethimide, p-nitro-5-aminoglutethimide, cyclohexylaminoglutethimide, phenglutarimide and thalidomide] have been resolved on Chiralpak IA and Chiralpak IB columns (25 cm × 0.46 cm). The non-conventional mobile phases used were methyl-tert-butyl ether-THF (90:10, v/v) [I], 100% dichloromethane [II] and 100% acetonitrile [III] separately at a flow rate of 1.0 mL/min using a UV detector at 254 nm. The resolution factors for Chiralpak IA and Chiralpak IB columns were 1.00-5.33 and 0.33-0.67, respectively. Chiralpak IA column gave better results than Chiralpak IB column for the reported molecules using the developed HPLC conditions. Experimental conditions and the possible chiral recognition mechanisms have been discussed.     

Analytical and semi-preparative HPLC enantioseparation of novel pyridazin-3(2H)-one derivatives with α-aminophosphonate moiety using immobilized polysaccharide chiral stationary phases

The direct HPLC enantioseparation of a novel series of chiral pyridazin-3(2H)-one derivatives with α-aminophosphonate moiety was performed on two immobilized polysaccharide chiral stationary phases (Chiralpak IA, Chiralpak IC) using n-hexane (n-Hex)/dichloromethane (DCM) mobile phase with 5% alcohol additive. Good baseline separation of the enantiomers was achieved using amylose tris-(3,5-dimethylphenylcarbamate) chiral stationary phases (Chiralpak IA) on analytical scale. The analytical method was further scaled up to semi-preparative loading to obtain small amounts of both the enantiomers of pyridazin-3(2H)-one derivative. The semi-preparative resolution of all compounds was successfully achieved with n-hexane/dichloromethane/ethanol (EtOH) as mobile phase using a semi-preparative Chiralpak IA column. The first fractions were isolated with purities of >99.9% (enantiomeric excess (e.e.), and the second fractions were obtained with purities of >98.2% (enantiomeric excess). The assignment of the absolute configuration was established for the F1 fraction of compound a-2 by single-crystal X-ray diffraction method.     

Enantiomeric separation of indoxacarb on an amylose‐based chiral stationary phase and its application in study of indoxacarb degradation in water

Direct semipreparative enantioseparation of indoxacarb was performed on a semipreparative Chiralpak IA column using normal‐phase high‐performance liquid chromatography (HPLC) with n‐hexane–isopropanol–ethyl acetate (70:20:10) mixture as mobile phase. Degradation of indoxacarb (2.33S + 1R) and its two enantiopure isoforms in three aqueous buffer solutions and four water samples collected from natural water sources was then elucidated by HPLC analysis on Chiralpak IA column. Degradation of all three indoxacarbs complied with first‐order kinetics and demonstrated linearity with regression coefficients R² > 0.88. Indoxacarb (2.33S + 1R) underwent enantioselective degradation in river water, rain water, and buffer solution of pH 7.0. Enantiopure S‐(+)‐indoxacarb and R‐(?)‐indoxacarb were both found to be configurationally stable in water. Copyright © 2014 John Wiley & Sons, Ltd.     

Validated LC Method for Determination of Enantiomeric Purity of Apremilast Using Polysaccharide-Type Stationary Phases in Polar Organic Mode

Enantioseparation of an anti-psoriatic agent, apremilast (APR), was performed by HPLC using polysaccharide-type chiral stationary phases in polar organic mode for the first time. The separation capability of six different chiral columns (Chiralpak AD, Chiralpak IA, Chiralpak AS, Lux Amylose-2, Chiralcel OD and Chiralcel OJ-H) was investigated using neat MeOH and ACN. During the preliminary experiments the best results were obtained on Chiralpak IA column with ACN (R_s?=?5.4). The effects of binary mobile phases on the resolutions and retention factors were also investigated containing different percentages of MeOH:ACN. U-shaped retention pattern was obtained when plotting the retention factors of the APR enantiomers versus the MeOH content of the binary mobile phases on Chiralpak IA column. For further method optimizations an L25 orthogonal array table was employed altering the concentration of MeOH in ACN, column temperature, and flow rate. The best result was achieved on Chiralpak IA column with 80/20 (v/v%) MeOH/ACN with 0.7 mL min^?1 flow rate at 25 °C (R_s?=?5.4, t₂?=?7.45 min). Thermodynamic analysis revealed an enthalpy-driven enantioseparation. The developed HPLC method was validated according to the ICH guideline Q2(R1) and proved to be reliable, linear, precise and accurate for the determination of 0.1% R-enantiomer as chiral impurity in S-APR as well as quantification of the S-enantiomer.

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Enantiomeric separation of precursors of rho-kinase inhibitors by HPLC on polysaccharide-based chiral stationary phases

Summary The separation of enantiomers of substituted cyclohexanecarboxamides, benzamides and chemical precursors of Rho-kinase inhibitors was achieved using derivatized polysaccharide-based chiral stationary phases. Separations were by normal phase HPLC with a mobile phase ofn-hexane-alcohol (methanol, ethanol or 2-propanol) in various proportions, and a silica-based cellulose tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H), tris-methylbenzoate (Chiralcel OJ), a silica-based amylose tris-(S)-1-phenylethylcarbamate (Chiralpak AS), or tris-3,5-dimethylphenylcarbamate (Chiralpak AD). The effects of cencentration of various aliphatic alcohols in the mobile phase were investigated. The effect of structural features on the discrimination between the enantiomers was examined. The isolation of milligram amounts of enantiomers of two derivatives was performed on an analytical column by multiple repetitive injections under overload conditions.     

Application and comparison of immobilized and coated amylose tris-(3,5-dimethylphenylcarbamate) chiral stationary phases for the enantioselective separation of beta-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.     

Enantiomeric separation of six chiral pesticides that contain chiral sulfur/phosphorus atoms by supercritical fluid chromatography

Six chiral pesticides containing chiral sulfur/phosphorus atoms were separated by supercritical fluid chromatography with supercritical CO₂ as the main mobile phase component. The effect of the chiral stationary phase, different type and concentration of modifiers, column temperature, and backpressure on the separation efficiency was investigated to obtain the appropriate separation condition. Five chiral pesticides (isofenphos‐methyl, isocarbophos, flufiprole, fipronil, and ethiprole) were baseline separated under experimental conditions, while isofenphos only obtained partial separation. The Chiralpak AD‐3 column showed a better chiral separation ability than others for chiral pesticides containing chiral sulfur/phosphorus atoms. When different modifiers at the same concentration were used, the retention factor of pesticides except flufiprole decreased in the order of isopropanol, ethanol, methanol; meanwhile, the retention factor of flufiprole increased in the order of isopropanol, ethanol, methanol. For a given modifier, the retention factor and resolution decreased on the whole with the increase of its concentration. The enantiomer separation of five chiral pesticides was an “enthalpy‐driven” process, and the separation factor decreased as the temperature increased. The backpressure of the mobile phase had little effect on the separation factor and resolution.     

HPLC with polysaccharide chiral stationary phase in polar‐organic phase mode: Application to the asymmetric epoxidation of allylic alcohols

A simple and rapid HPLC method using a polysaccharide‐based chiral stationary phase (Chiralpak AD‐H) in polar‐organic phase mode has been developed for direct resolution of glycidyl nitrobenzoate (GNB) and 2‐methyl glycidyl nitrobenzoate (MGNB) enantiomers. ACN and methanol were used as mobile phase and the effects of the addition of ethanol and 2‐propanol as organic modifier in the mobile phase, flow rate and the column temperature were tested. The optimized conditions were: methanol/ethanol (80:20) at a flow rate of 0.9 mL/min and 40°C. Analysis time was ?13 min and the chiral resolution was ?2. The method was validated and resulted to be selective, precise and accurate. The method was found to be linear in 2–300 μg/mL range (R² >0.999) with an LOD nearly 0.5 μg/mL for four enantiomers. GNB and MGNB enantiomers were obtained by asymmetric epoxidation of allyl alcohol and 2‐methyl allyl alcohol, respectively, using chiral titanium–tartrate complexes as catalyst and dichloromethane as solvent after in situ derivatization of the intermediate glycidols derivatives. The quite simple and rapid validated method was applied successfully for direct determination of the enantiomeric excess (?90%) and yield obtained in real samples of asymmetric epoxidation of allylic alcohols without further purification, workup or solvent removal. The method provides a useful and value‐added tool for controlling the enantiomeric purity of the synthesized epoxides.     

Efficient preparative separation of 6‐(4‐aminophenyl)‐5‐methyl‐4, 5‐dihydro‐3(2H)‐pyridazinone enantiomers on polysaccharide‐based stationary phases in polar organic solvent chromatography and supercritical fluid chromatography

6‐(4‐Aminophenyl)‐5‐methyl‐4,5‐dihydro‐3(2H)‐pyridazinone is a key synthetic intermediate for cardiotonic agent levosimendan. Very few studies address the use of chiral stationary phases in chromatography for the enantioseparation of this intermediate. This study presents two efficient preparative methods for the isolation of (R)(?)‐6‐(4‐aminophenyl)‐5‐methyl‐4,5‐dihydro‐3(2H)‐pyridazinone in polar organic solvent chromatography and supercritical fluid chromatography using polysaccharide‐based chiral stationary phases and volatile organic mobile phases without additives in isocratic mode. Under optimum conditions, Chiralcel OJ column showed the best performance (α = 1.71, Rs = 5.47) in polar organic solvent chromatography, while Chiralpak AS column exhibited remarkable separations (α = 1.81 and Rs = 6.51) in supercritical fluid chromatography with an opposite enantiomer elution order. Considering the sample solubility, runtime and solvent cost, the preparations were carried out on Chiralcel OJ column and Chiralpak AS column (250 × 20 mm i.d.; 10 µm) in polar organic mode and supercritical fluid chromatography mode with methanol and CO₂/methanol as mobile phases, respectively. By utilizing the advantages of chromatographic techniques and polysaccharide‐based chiral stationary phases, this work provides two methods for the fast and economic preparation of (R)(?)‐6‐(4‐aminophenyl)‐5‐methyl‐4,5‐dihydro‐3(2H)‐pyridazinone, which are suitable for the pharmaceutical industry.     

Chiral separation of disease biomarkers with 2‐hydroxycarboxylic acid structure

Chiral 2‐hydroxycarboxylic acids are compounds that have been linked to particular diseases and are putative biomarkers with some diagnostic potential. The importance of identifying whether a particular enantiomer is related to certain diseases has been encouraged recently. However, in many cases it has not yet been elucidated whether there are stereochemical implications with respect to these biomarkers and whether their enantioselective analysis provides new insights and diagnostic potential. In this study 13 disease‐related chiral 2‐hydrocarboxylic acids were studied for their chiral separation by high‐performance liquid chromatography on three cinchona alkaloid‐derived chiral stationary phases. From a subgroup of eight 2‐hydroxymonocarboxylic acids, baseline resolution could be achieved and inversion of elution order by exchanging tert‐butylcarbamoyl quinidine chiral stationary phase (Chiralpak QD‐AX) for the corresponding quinine analogue (Chiralpak QN‐AX) is shown for seven of them. Furthermore, conditions for chiral separation of the 2‐hydroxydicarboxylic acids, citramalic acid, 2‐isopropylmalic acid, and 2‐hydroxyadipic acid are reported and compared to the previous reported conditions for 2‐hydroxyglutaric acid and malic acid.     

Immobilized versus coated amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phases for the enantioselective separation of cyclopropane derivatives by liquid chromatography

The solvent versatility of Chiralpak IA, a new chiral stationary phase (CSP) containing amylose tris(3,5-dimethylphenylcarabamate) immobilized onto silica gel, is investigated for the enantioselective separation of a set of cyclopropane derivatives using ethyl acetate or dichloromethane (DCM) as non-standard mobile phase eluent and diluent, respectively in high-performance liquid chromatography (HPLC). A comparison of the separation of cyclopropanes on both immobilized and coated amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phases (Chiralpak IA and Chiralpak AD, respectively) in HPLC using a mixture of n-hexane/2-propanol (90/10 and 99/1, v/v) as mobile phase with a flow rate of 0.5 ml/min and UV detection at 254 nm, is demonstrated. The optimized method of separation is used for an online HPLC monitoring for the Rh(II)-catalyzed asymmetric intermolecular cyclopropanations in dichloromethane. Direct analysis techniques without further purification, workup or removal of dichloromethane were summarized. The method provides an easy and direct determination of the enantiomeric excess of the cyclopropanes and selectivity of the catalyst used without any further work up.     

Enantioseparation of kavain on Chiralpak IA under normal-phase, polar organic and reversed-phase conditions

First baseline HPLC enantioseparation of kavain is described. Complete enantiodiscrimination was achieved on the immobilised-type Chiralpak IA chiral stationary phase (CSP) using pure methanol and simple methanol-water and ethanol-water mixtures as eluents. A water-dependent enantioselectivity was clearly demonstrated. Performance of the Chiralpak IA CSP in polar organic and RP conditions was compared with that of five coated polysaccharide-derived CSPs used in normal-phase mode.     

Chiral separation of isoxanthohumol and 8‐prenylnaringenin in beer,hop pellets and hops by HPLC with chiral columns

Xanthohumol, isoxanthohumol, and 8‐prenylnaringenin in beer, hop and hop pellet samples were analyzed by HPLC using an InertSustain phenyl column and the mobile phase containing 40% methanol and 12% 2‐propanol. Fractions of isoxanthohumol and 8‐prenylnaringenin obtained by the above HPLC were separately collected. Isoxanthohumol and 8‐prenylnaringenin were enantioseparated by HPLC using a Chiralcel OD‐H column with a mobile phase composed of hexane–ethanol (90:10, v/v) and a Chiralpak AD‐RH column with a mobile phase composed of methanol–2‐propanol–water (40:20:40, v/v/v), respectively. Isoxanthohumol and 8‐prenylnaringenin from beer, hop and hop pellet samples were found to be present in a racemic mixture. This can be explained by the fact that the two analytes were produced by a nonenzymatic process. The effects of boiling conditions on the conversion of xanthohumol into isoxanthohumol were also studied. A higher concentration of ethanol in heating solvent resulted in a decrease in the conversion ratio and the conversion was stopped by addition of ethanol at >50% (v/v). The isomerization was significantly affected pH (2−10) and the boiling medium at pH 5 was minimum for the conversion. Therefore, it was suggested that xanthohumol was relatively difficult to convert to isoxanthohumol in wort (pH 5−5.5) during boiling.     

Enantioselective separation of twelve pairs of enantiomers on polysaccharide‐based chiral stationary phases and thermodynamic analysis of separation mechanism

The enantioseparation of twelve pairs of structurally related 1‐aryl‐1‐indanone derivatives was studied in the normal‐phase mode using three different polysaccharide‐type chiral stationary phases, namely Chiralpak IB, Chiralpak IC, and Chiralpak ID. n‐Hexane/2‐propanol and n‐hexane/ethanol were employed as mobile phases. Among all the investigated chiral columns, Chiralpak IC exhibited the most universal and the best enantioseparation ability toward all the racemates, particularly with the mobile phase composed of n‐hexane/2‐propanol (90/10, v/v). Then the effects of column temperature on retention and enantioselectivity were examined in the range of 25–40°C. Satisfactory enantioseparation was obtained at ambient temperature. The natural logarithm of retention and separation factors (ln k and ln α) versus the reciprocal of absolute temperature (1/T) (Van't Hoff plots) were found to be linear for all racemates, indicating that the retention and separation mechanisms were independent of temperature in the range investigated. Then, the thermodynamic parameters (ΔΔH°, ΔΔS°, and ΔΔG°) were calculated from Van't Hoff plots. These values indicated that the solute transfer from the mobile to stationary phase was enthalpically favorable, and the process of enantioseparation was mainly enthalpy controlled. At last, the impact of small changes in molecular structures of the tested 1‐indanone derivatives on enantioseparation was also discussed.     

Comparative studies of immobilized chiral stationary phases based on polysaccharide derivatives for enantiomeric separation of 15 azole compounds

Immobilized polysaccharide‐based columns showed excellent enantioselectivity in normal phase separation mode. In this work, enantioseparation abilities of four immobilized polysaccharide‐derived chiral stationary phases (Chiralpak IA, Chiralpak IB, Chiralpak IC, and Chiralpak ID) toward 15 azole compounds were evaluated. Separation was carried out using n‐hexane as mobile phase with ethanol, 1‐propanol, 1‐butanol, and 2‐propanol as modifiers. And twelve compounds have achieved baseline separation with the resolutions ranging between 2.05 and 21.73. The enantioseparation on the four polysaccharide‐based chiral columns using different alcohol modifiers was compared. In general, the best separation performance was identified as Chiralpak IC, which was able to resolve 11 compounds to baseline and two partially under the screening conditions. Separation on Chiralpak IB was not satisfactory, because only four compounds were baseline separated.