Rapid determination of ambrisentan enantiomers by enantioselective liquid chromatography using cellulose-based chiral stationary phase in reverse phase mode

A sensitive, specific, and rapid high-performance liquid chromatography (HPLC) method for the determination of ambrisentan enantiomers has been developed and validated. Six chiral columns were tested in a reversed-phase system. Excellent enantioseparation with the resolution more than 2.5 was achieved on Chiralcel OZ-3R (cellulose 3-chloro-4-methylphenylcarbamate) using mixture of 20 mM sodium formate (pH 3.0) with acetonitrile (55:45; v/v). Validation of the HPLC method including linearity, limit of detection, limit of quantification, precision, accuracy, and selectivity was performed according to the International Conference on Harmonisation (ICH) guidelines. The method has an advantage of a very quick chromatographic separation (less than 6 min) and therefore is highly suitable for routine determination of (R)-ambrisentan in enantiopure active pharmaceutical ingredient (S)-ambrisentan.     

Separation of some chiral flavonoids by microbial cyclosophoraoses and their sulfated derivatives in micellar electrokinetic chromatography

Neutral cyclosophoraoses (Cys) and highly sulfated cyclosophoraoses (HS-Cys) were successfully applied as chiral selectors with SDS for the separation of some chiral flavonoids in MEKC. HS-Cys were synthesized by the chemical modification of a family of neutral Cys isolated from a soil microorganism, Rhizobium meliloti 2011. Chiral catechin was separated with a resolution (R(s)) of 0.754 by neutral Cys and SDS. In the case of isosakuranetin and neohesperidin, resolution (R(s)) values of 1.483 and 1.306 were obtained with HS-Cys and SDS, respectively.     

Enantiomeric separation of ketoconazole and terconazole antifungals by electrokinetic chromatography: Rapid quantitative analysis of ketoconazole in pharmaceutical formulations

EKC using a neutral CD as chiral selector was applied in this work to the development of a method enabling the enantiomeric separation of ketoconazole and terconazole antifungals. The influence of different experimental conditions such as temperature, CD concentration, pH, and nature and concentration of the buffer on the enantiomeric resolution of the compounds studied was investigated. The use of 10 mM heptakis-(2,3,6-tri-O-methyl)-beta-CD in a 100 mM phosphate buffer (pH 3.5) with a temperature of 15 degrees C allowed the separation of the enantiomers of ketoconazole and terconazole with high resolution (R(s) > 2.0). The rapid separation of ketoconazole enantiomers with an analysis time less than 3 min was carried out after fitting some experimental parameters. The developed method was applied to the determination of ketoconazole in different pharmaceutical formulations.     

Triticonazole enantiomers: Separation by supercritical fluid chromatography and the effect of the chromatographic conditions

Enantiomeric pairs of triticonazole have been successfully separated by supercritical fluid chromatography coupled with a tris(3,5‐dimethylphenylcarbamoyl) cellulose‐coated chiral stationary phase in this work. The effects of co‐solvent, dissolution solvent, flow rate, backpressure, and column temperature have been studied in detail with respect to retention, selectivity, and resolution of triticonazole. As indicated, the co‐solvents mostly affected the retention factors and resolution, due to the different molecular structure and polarity. In addition, the dissolution solvents, namely, chloromethanes and alcohols, have been also important for enantioseparation because of the different interaction with stationary phase. Higher flow rate and backpressure led to faster elution of the triticonazole molecules, and the change of column temperature showed slight effect on the resolution of triticonazole racemate. Moreover, a comparative separation experiment between supercritical fluid chromatography and high performance liquid chromatography revealed that chiral supercritical fluid chromatography gave the 3.5 times value of R_s/t_R2 than high performance liquid chromatography, which demonstrated that supercritical fluid chromatography had much higher separation efficiency.     

Direct chromatographic resolution of racemic aminoglutethimide and its acetylated metabolite using different cellulose based chiral stationary phases

Summary Two improved methods for the enantiomeric separation of racemic aminoglutethimide (±AG) and its acetylated metabolite (±AAG) have been developed. Direct liquid chromatographic resolution of the enantiomers of aminoglutethimide and its acetylated metabolite was accomplished using Chiralcel OD and Chiralcel OJ columns without any derivatization. Maximum resolution of 8.87 and 2.23 was obtained for the enantiomers of aminoglutethimide and its acetylated metabolite using a Chiralcel OD column, while maximum resolution of 10.34 and 7.01 was obtained for the enantiomers using a Chiralcel OJ column. Optimization of separation was obtained using different concentration of 2-propanol in hexane as a mobile phase.     

Validated chiral separation of M9, a Mannich ketone compound, by chiral ligand-exchange chromatography

Enantioseparation of the Mannich ketone M9, a potential antifungal compound, was examined using chiral ligand-exchange chromatography. The chiral mobile phase contained complexes of Cu(II) with the optically active selector L-aspartame (APM) and the organic modifier methanol. The separation was optimized with respect to the concentration of the Cu(II)-(L-APM) complexes, pH of mobile phase, methanol content, and column temperature. A baseline separation (R(s) = 3.08) was achieved for enantiomers of M9 under optimal conditions, and the analysis was accomplished in eleven minutes. The developed method was extensively validated. The sample stability, linearity, precision (method repeatability and intermediate precision) and accuracy, and the limits of detection and quantification of the developed method were studied. The proposed method was shown to be accurate and suitable for the quantitative determination of each enantiomer of M9.     

A Validated LC Method for Separation and Quantification of Voriconazole and Its Enantiomer

A simple liquid chromatographic method was developed for the separation and quantification of voriconazole and its enantiomer in drug substance. The separation was achieved on Chiral cel-OD (250 mm × 4.6 mm × 10 μm) using mobile phase consisting of n-hexane and ethanol in the ratio 9:1 (v/v) with a flow rate of 1.0 mL min⁻¹, at 27 °C column temperature and detection at 254 nm with an injection volume of 20 μL. Ethanol was used as diluent. The method is capable of detecting the (2S, 3R) enantiomer down to 0.0075% and can quantify down to 0.021% with respect to sample concentration. The method is rapid and the resolution achieved was about 3.0. This method can be employed for the quantification of (2S, 3R) enantiomer in voriconazole drug substance.     

A chiral porous organic cage for molecular recognition using gas chromatography

Molecular organic cages as shape-persistent organic molecules with permanent and accessible cavities have attracted a lot of interest because of their importance as host-guest systems. Herein, we report a chiral porous organic cage (POC) CC9 diluted with a polysiloxane OV-1701 to fabricate a CC9-coated capillary column, which was used for the high-resolution gas chromatographic separation of organic compounds, including positional isomers and racemates. On the CC9-coated capillary column, a large number of racemic compounds such as chiral alcohols, esters, ethers and epoxides can be resolved without derivatization. By comparing the chiral recognition ability of the CC9-coated column with the commercially available β-DEX 120 column and the POC CC3-R coated column recently reported by our group, the CC9-coated column offered good resolution during the separation of some racemates, that were not separated using the β-DEX 120 column or POC CC3-R coated column. Therefore, the CC9-coated column can be complementary to the β-DEX 120 column and CC3-R coated column. The results indicated that the CC9-coated column exhibited great potential for application in the separation of positional isomers and enantiomers with great selectivity, high resolution and good reproducibility.     

Past,present, and future developments in enantioselective analysis using capillary electromigration techniques

Enantioseparation of chiral products has become increasingly important in a large diversity of academic and industrial applications. The separation of chiral compounds is inherently challenging and thus requires a suitable analytical technique that can achieve high resolution and sensitivity. In this context, CE has shown remarkable results so far. Chiral CE offers an orthogonal enantioselectivity and is typically considered less costly than chromatographic techniques, since only minute amounts of chiral selectors are needed. Several CE approaches have been developed for chiral analysis, including chiral EKC and chiral CEC. Enantioseparations by EKC benefit from the wide variety of possible pseudostationary phases that can be employed. Chiral CEC, on the other hand, combines chromatographic separation principles with the bulk fluid movement of CE, benefitting from reduced band broadening as compared to pressure-driven systems. Although UV detection is conventionally used for these approaches, MS can also be considered. CE-MS represents a promising alternative due to the increased sensitivity and selectivity, enabling the chiral analysis of complex samples. The potential contamination of the MS ion source in EKC-MS can be overcome using partial-filling and counter-migration techniques. However, chiral analysis using monolithic and open-tubular CEC-MS awaits additional method validation and a dedicated commercial interface. Further efforts in chiral CE are expected toward the improvement of existing techniques, the development of novel pseudostationary phases, and establishing the use of chiral ionic liquids, molecular imprinted polymers, and metal-organic frameworks. These developments will certainly foster the adoption of CE(-MS) as a well-established technique in routine chiral analysis.     

合成高分子作为手性固定相在高效液相色谱中的应用

具有旋光活性的合成高分子基于它的手性结构而具有广泛的应用,其中最实际和广泛的应用是在高效液相色谱中作为手性固定相来拆分对映异构体,目前已成为合成化学、分析化学以及制药化学领域必不可少的分离材料.本文简要介绍了高效液相色谱手性固定相拆分法,综述了合成高分子,包括加聚物特别是聚甲基丙烯酸酯类和聚甲基丙烯酰胺类聚合物、聚酰胺...     

The effect of analyte lipophilicity on the resolution of α-amino acids on a HPLC chiral stationary phase based on crown ether

The effect of analyte lipophilicity on the resolution of α-amino acids on a chiral stationary phase based on chiral crown ether has been examined by the chromatographic resolution trends for the resolution of a homologous series of five α-amino acids with an alkyl group of different length at the chiral center. The retention factors (k₁ and k₂) for the two enantiomers and the separation factors (α) were found to depend on the lipophilicity of the α-amino acid. In general, the retention factors increased as the organic modifier content in the mobile phase increased, the degree of the enhancement of retention factors being dependent on the analyte lipophilicity. The separation factors also increased as the analyte lipophilicity and the organic modifier content in the mobile phase increased. Possible rationales for these behaviors have been proposed.     

Preparation of a new crown ether-based chiral stationary phase containing thioester linkage for the liquid chromatographic separation of enantiomers

A new chiral stationary phase (CSP) containing thioester linkages was prepared by bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to mercaptopropylsilica gel. The chiral recognition ability of the new CSP was found to be greater than that of the previously reported CSP containing amide linkages in the resolution of the various α-amino acids that were tested, except for that of Met, Ser and Thr. In the resolution of racemic amines and amino alcohols, the new CSP was always better than the one containing amide linkages in terms of the separation factors (α) and the resolutions (R_S). Given the identical elution orders on the two CSPs, it was concluded that the chiral recognition mechanism is not affected by the change of the linkage type. In addition, the new CSP was found to be quite stable under the acidic mobile phase conditions that were utilized, indicating that the thioester linkage is useful as a tethering group.     

Resolution of beta-amino acids on a high performance liquid chromatographic doubly tethered chiral stationary phase containing N-CH3 amide linkage based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid

A doubly tethered chiral stationary phase (CSP) containing N-CH(3 )amide linkage based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was applied to the resolution of various beta-amino acids. The chiral recognition behaviors for the resolution of beta-amino acids on the doubly tethered CSP were consistent with those on the corresponding singly tethered CSP while the chiral recognition ability of the doubly tethered CSP was generally greater in terms of both the separation (alpha) and the resolution factors (R(S)) than that of the corresponding singly tethered CSP. From these results, it was concluded that attaching the second tethering group to silica gel through a carbon atom of the first tethering group of the CSP improves the chiral recognition ability for the resolution of beta-amino acids without any change in the chiral recognition mode. The retention factors (k(1)) on the doubly tethered CSP were larger than those on the corresponding singly tethered CSP and these retention factors were found to be controllable with the variation of the type and the content of the organic and/or acidic modifier in the aqueous mobile phase without significant change in the separation and the resolution factors.     

Enantioseparation of phenylsuccinic acid by high speed counter-current chromatography using hydroxypropyl-β-cyclodextrin as chiral selector

High speed counter-current chromatography (HSCCC) was successfully applied to resolution of phenylsuccinic acid (PSA) with hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector (CS). The two-phase solvent system composed of n-hexane–methyl tert-butyl ether–0.1 mol L⁻¹ phosphate buffer solution with pH = 2.51 (0.5:1.5:2, v/v/v) was selected. Influence factors involved in the chiral separation were investigated, including the concentration of chiral selector, pH value of the aqueous phase, the separation temperature, and the thermodynamic parameters of inclusion complex were calculated. The complex formation constants were determined using analytical instrument. Two HSCCC elution modes were studied and peak resolution equation was discussed. Under optimum separation conditions, 712 mg of PSA racemate was separated using preparative apparatus. The purities of both of the fractions including (+)-PSA and (−)-PSA from the preparative CCC separation were over 98.5% determined by HPLC and enantiomeric excess of (+)-PSA and (−)-PSA reached 97.6% and 98.6%, respectively. Recovery for the target compounds from the CCC fractions reached 80–82% yielding 285 mg of (+)-PSA and 292 mg of (−)-PSA.     

Enantiomeric separation of (R, S)-naproxen by recycling high speed counter-current chromatography with hydroxypropyl-β-cyclodextrin as chiral selector

Recycling high speed counter-current chromatography (HSCCC) was successfully applied to resolution of (R, S)-naproxen (NAP) using hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector. The two-phase solvent system composed of n-hexane-ethyl acetate-0.1 mol L(-1) phosphate buffer solution with pH=2.67 (8:2:10, v/v/v) was selected. Influence factors for the chiral separation process were investigated, including concentration of HP-β-CD, equilibrium temperature and pH of aqueous phase. Suitable elution mode was selected for HSCCC enantioseparation of (R, S)-NAP. Under optimum separation conditions, 29 mg of (R, S)-NAP was separated using preparative recycling HSCCC with the molar ratio HP-β-CD/NAP racemate 83:1. Technical details for recycling elution mode were discussed as for chiral HSCCC separation. The purities of both (S)-NAP and (R)-NAP were over 99.5% as determined by HPLC. Enantiomeric excess of (S)-NAP and (R)-NAP reached 99.4%. Recovery for NAP enantiomers from HSCCC fractions was 82-89%, yielding 13 mg of (S)-NAP and 12 mg of (R)-NAP.     

Chromatographic separation and enantiomeric resolution of flurbiprofen and its major metabolites

Summary The chromatographic separation and resolution of the enantiomers of flurbiprofen and its two major metabolites, 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen was investigated using four different approaches: reversed-phase HPLC after pre-column derivatization with (R)-1-(naphthen-1-yl)ethylamine; reversed-phase HPLC using hydroxypropyl-β-cyclodextrin as a chiral mobile phase additive; chiral-phase HPLC using either an α₁-acid glycoprotein CSP (Chiral-AGP) or an amylose tris(3,5-dimethylphenylcarbamate) CSP (Chiralpak AD). Of all the approaches, only the direct method using the Chiralpak AD CSP demonstrated separation and enantiomeric resolution of all three analytes within an acceptable run time of 45 minutes. Enantiomeric resolution values of 1.67,3.67 and 3.44 were obtained for flurbiprofen, 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen respectively. Semi-preparative isolation of the individual enantiomers of both metabolites, followed by CD analysis, revealed that the elution order on the AD CSP wasR-beforeS-enantiomer for both metabolites and the same as that observed for flurbiprofen. The metabolite elution order was subsequently confirmed on the analysis of urine samples obtained from a healthy volunteer following oral administration of the individual drug enantiomers.     

Chiral separation of aminoalcohols by ion-pair chromatography

Summary A new chiral counter ion, N-benzoxycarbonyl-glycyl-L-proline (ZGP), added to the organic mobile phase (dichloromethane) has been used for separation of enantiomers of aminoalcohols with LiChrosorb DIOL as the solid phase. Separation factors of 1.2 to 1.4 for enantiomers of -adrenergic blocking agents (e.g., alprenolol, metoprolol and propranolol) were obtained. Some structural requirements in the solutes and the counter ion essential for chiral resolution were observed. The retention was regulated by the concentration of counter ion or by the addition of triethylamine to the mobile phase. The chiral counter ion was utilized to determine the enantiomeric impurity of less than 0.1% in S-alprenolol and for the analysis of propranolol enantiomers in plasma samples.Presented at the 9th International Symposium on Column Liquid Chromatography, Edinburgh, July, 1985.     

三七素手性拆分方法的研究

使用气相色谱法和高效液相色谱法分离了三七素对映体,并探讨了影响液相色谱法分离效果的因素。结果表明,HPLC法利用手性固定相进行直接拆分,无法实现对映体的完全分离;GC法和HPLC的手性试剂衍生化法均可对三七素对映体进行较好的分离。但GC法由于衍生化过程中副产物的存在,干扰了对映体的准确定量。手性试剂衍生化HPLC法,以邻苯二甲醛、N-酰化-L-半胱氨酸为衍生化试剂,反应得到的三七素对映体的衍生物在ODS柱上分离良好,且方法简单、快速。     

大分子键合型手性固定相的研究进展

高效液相色谱（HPLC）被广泛认为是分离制备光学纯单一对映体最有效的方法。在高效液相色谱手性拆分中,手性固定相的性能直接影响到色谱柱的手性分离能力。在众多手性固定相中,键合型手性固定相具有溶剂耐受性好,分离模式灵活等优点,是很重要的一大类手性固定相。本文主要针对大分子键合型手性固定相,包括多糖衍生物键合型手性固定相、蛋...