Cellulose tris(3,5-dichlorophenylcarbamate) immobilised on silica: a novel chiral stationary phase for resolution of enantiomers

CHIRALPAK IC is a new chiral stationary phase (CSP) made by immobilising cellulose tris(3,5-dichlorophenylcarbamate) on silica gel. The chiral selector is distinct from any other commercially available polysaccharide-based CSPs. Apart from its compatibility with the whole series of solvents; this CSP is able to operate under various chromatographic conditions and bring about new characteristics in enantiomeric recognition. It can afford many large and specific enantiomeric separations. It exhibits complementary properties with regard to the existing immobilised chiral packing materials of the same category.     

手性固定相HPLC法拆分苏氨酸和赖氨酸对映体

目的建立了柱前衍生结合手性固定相HPLC法拆分了苏氨酸和赖氨酸2种氨基酸对映体。方法利用4-氯-7-硝基-2,1,3-苯并噁二唑(NBD-Cl)柱前衍生,采用Sumichiral OA-2500S色谱柱,以甲醇-乙腈(60∶40,含5mmol.L-1柠檬酸)为流动相,检测波长为470nm。结果应用该法检查L-氨基酸原料药中的D-型异构体,D-苏氨酸和D-赖氨酸在2.0～16.0μg.mL-1质量浓度范围内线性关系良好,回收率均为101.0%,RSD分别为1.7%和0.37%。结论在最佳色谱条件下,2种氨基酸对映体均达到了基线分离。建立的对映体杂质检查方法灵敏度高,重复性好。     

A rational approach to the design of highly effective chiral stationary phases for the liquid chromatographic separation of enantiomers

The design and rationale of some novel chiral stationary phases (CSPs) are discussed with respect to methods for determining enantiomeric purity, absolute configuration and for obtaining enantiomerically pure materials by liquid chromatography. The commercially-available dinitrobenzoylamino CSP type 1 is discussed with respect to the chiral recognition mechanisms which may operate in the resolution of some polycyclic and heterocyclic aromatic molecules and some benzodiazepines. N-Acyl alpha-arylalkylamines are also employed as models to formulate mechanisms for the chiral properties of type 1 CSPs in terms of enantiomeric stacking of the most stable conformations in solution. The properties of new types of 'reciprocal' CSPs are discussed and illustrated by enantiomeric separation of some amino acid and amino phosphoric acid derivatives, and by the separation of the following enantiomeric drugs as their 3,5-dinitrobenzoyl derivatives: metoprolol, oxoprenolol, ephedrine and alprenolol.     

Enantiomeric separation of beta2-agonists on macrocyclic antibiotic chiral stationary phases in high performance liquid chromatography

The enantiomeric resolution of six beta2-agonists including bambuterol, clenbuterol, clenproperol, fumoterol, mabuterol and terbutaline, was studied on three macrocyclic antibiotic chiral stationary phases (CSPs): Chirobiotic V, T and R. By varying the mobile phase compositions for different CSPs, all the six compounds were successfully separated on Chirobiotic V and T CSPs. The thermodynamic parameter variation was calculated. The chiral recognition mechanism was discussed, the ionic interaction was confirmed to be the most dramatic interaction responsible for the chiral recognition. No enantiosepartion for all the beta2-agonists was found on Chirobiotic R CSP.     

Cyclodextrin chiral stationary phases for liquid chromatographic separations of drug stereoisomers

Many active drugs are racemic mixtures. Because the two enantiomers of a racemate often cause different pharmacological responses, the use of optically pure isomers is desirable and may be soon required. Cyclodextrin-bonded silica gel can be used as chiral stationary phase (CSP) in liquid chromatography. The enantiomers of 25 different racemic drugs were separated on such CSPs in the reversed-phase mode. The principal features of the cyclodextrin chiral recognition mechanism are recalled and some information on future trends for cyclodextrin CSPs is provided.     

Stereoselective formations of enantiomeric K-region epoxide and trans-dihydrodiols in dibenz[a,h]anthracene metabolism

Metabolism of dibenz[a,h]anthracene (DBA) to optically active epoxide and dihydrodiol products by rat liver microsomes was investigated. Enantiomeric separation of K-region 5,6-epoxide, trans- and cis-5,6-dihydrodiols, non-K-region trans-1,2- and trans-3, 4-dihydrodiols, and O-methyl ethers derived from methoxylation of racemic and enantiomeric K-region 5,6-epoxides was performed on HPLC columns packed with Pirkle chiral stationary-phase (CSP) (R)-N-(3,5-dinitrobenzoyl)phenylglycine (R-DNBPG) or (S)-N-(3,5-dinitrobenzoyl) leucine (S-DNBL), which was either ionically or covalently bonded to a silica gel support. Enantiomers of DBA 5,6-epoxide, trans-5,6-dihydrodiol, and its two isomeric O-methyl ethers were efficiently separated on the ionically bonded R-DNBPG column. Enantiomers of DBA cis-5, 6-dihydrodiol were resolved on both ionically and covalently bonded S-DNBL columns. Enantiomeric pairs of the non-K-region trans-1,2- and 3,4-dihydrodiols were poorly resolved on all CSPs tested. DBA was incubated with a NADPH-regenerating system and liver microsomes from untreated, phenobarbital- (PB) treated, 3-methylcholanthrene- (MC) treated, and polychlorinated biphenyl (PCB, Aroclor 1254) treated rats either in the absence or in the presence of an epoxide hydrolase inhibitor, 3,3,3-trichloropropylene 1,2-oxide (TCPO). Metabolites formed were analyzed by reversed-phase, normal-phase, and CSP HPLC. CD spectral and CSP-HPLC analyses of metabolically formed trans-dihydrodiols indicated that the dihydrodiols are highly enriched in the R,R-enantiomers.(ABSTRACT TRUNCATED AT 250 WORDS)     

羧甲基-β-环糊精为手性选择剂拆分托吡卡胺等4种手性药物对映体

目的以羧甲基-β-环糊精(CM-β-CD)为手性选择剂,建立毛细管区带电泳法分离托吡卡胺、文拉法辛、美托洛尔和瑞格列奈4种药物对映体。方法采用未涂壁熔融石英毛细管柱,磷酸盐缓冲液作为背景电解质溶液,分离电压为20 kV。考察了缓冲溶液的pH值、环糊精质量浓度、缓冲盐浓度对对映体分离的影响。结果在优化的分离条件下,4种手性药物均能达到完全分离。结论CM-β-CD适用于上述4种药物的对映体分离。     

Diastereomeric and enantiomeric high-performance liquid chromatographic separation of synthetic anisodamine

In order to investigate the enantiomeric pharmacokinetics and biotransformation of synthetic anisodamine (654-2), a cholinoceptor antagonist widely used in clinic in China, it has been preparatively separated into two racemates (I and II) by using ZORBAX Eclipse XDB-C18 column. The diastereo- and/or enantioseparations of 654-2, I and II were carried out by HPLC using CHIRALPAK AD-H as chiral stationary phase (CSP) and acetonitrile-2-propanol-DEA 97:3:0.1 (v/v/v) as mobile phase. The methods were optimized by studying mobile phase modifiers, concentration of modifier and column temperature. The HPLC method for the simultaneous separation of two pairs of enantiomers of 654-2 has been validated.     

利用Pirkle型手性固定相柱拆分对映体药物:用异氰酸-α萘酯作衍生化试剂拆分胺类、醇类药物

本文首次介绍了利用异氰酸-α萘酯(NI)作衍生化试剂,在Pirkle型DNBPG柱上对五种含胺基、醇基的药物对映体进行了拆分。其中拆分美西律(mexiletine)、氯胺酮(ketamine)、本芴醇(benflumelol)和苯丙醇(phenylpropanol)药物尚未见报道。结果表明,用异氰酸-α萘酯作衍生化试剂的方法,具体简便易行、分离度好、几乎没有衍生化带来的杂质、不需分离出未反应的异氰酸-α萘酯便可直接进行拆分实验等优点。可以预计,NI将可能成为一种很有发展前途的新衍生化试剂,而且为Pirkle型DNBPG柱开发了新的领域。     

色谱手性固定相在手性药物拆分中的应用

色谱手性固定相（chiral stationary phases，CSPs）在手性药物分析和制备拆分中具有很重要的作用。本研究对5种带有CSPs的手性色谱技术以及在高效液相色谱（high performance liquid chromatography，HPLC）和超临界流体色谱（supereritical fluid chromatography，SFC）中CSPs的4种流动相模式和6种类型进行了综述，提出了CSPs色谱柱的筛选策略，并讨论了手性药物拆分从分析到制备的转变。     

Enantiomeric separations and their application in pharmaceutical analysis using chiral eluents

The most important problems of enantiomeric separations using chiral eluents are discussed. The methods have been compared with respect to enantiomeric purity of reagents, reagent selection and separation variables. The most important considerations for methods based on inclusion-complexation with different CDs and on using chiral counter ions are summarised and compared. A new possibility for the separation of enantiomeric compounds with the aid of a combination of ion-pair and inclusion-complex formation has been introduced. As a consequence of this investigation, the selectivity of the separation can be significantly improved; those ionic isomers and enantiomers which cannot be separated or are unsatisfactorily separated by ion-pair chromatography or by inclusion-complex formation, can be separated by the combined technique. Comparison of methods applicable for enantiomeric separations is also given with respect to solving specific analytical tasks in the field of pharmaceutical analysis.     

The chiral chromatographic separation of beta-adrenoceptor blocking drugs.

Over 100 chromatographic procedures for the separation of beta-blocker enantiomers are reviewed including a large number for the analysis of biological samples. All the principal chiral chromatographic procedures have found use, namely Chiral Mobile Phase Additives (CMPA), Chiral Derivatization Agents (CDA) and Chiral Stationary Phases (CSP). Chiral Mobile Phase Additives are less frequently employed than the other two procedures and many of the earlier methods were based on the use of CDAs. However, the recent development of sophisticated custom-made CSPs has allowed the separation of native (underivatized) analytes and this approach appears to be gaining in popularity. The beta-blockers are an extensive group of drugs and stereoselective separations have been reported for 40 different structures.     

Monoliths with chiral surface functionalization for enantioselective capillary electrochromatography

The state-of-the-art in CEC enantiomer separations with monolithic capillary columns is comprehensively reviewed. The various types of monolithic columns comprising in situ organic polymer monoliths, molecularly imprinted polymer (MIP) monoliths, silica monoliths and monoliths made from particles are discussed with a focus on materials’ synthesis, chemistry and properties as well as column aspects. Monolithic MIP-type porous layer open-tubular (PLOT) columns are treated herein as well. From this survey of the literature, the authors come to the conclusion that monolithic silica capillaries appear to become the preferred column type for CEC enantiomer separations of low-molecular drugs and other chiral pharmaceuticals or chemicals.     

Comparison of liquid and supercritical fluid chromatography for the separation of enantiomers on chiral stationary phases

Comparisons of liquid (LC) and supercritical fluid chromatography (SFC) were conducted using commercially available chiral stationary phases (CSPs) bearing three different types of chiral selectors. Chiral compounds of pharmaceutical and agricultural interest were used to probe advantages or limitations of SFC relative to LC for enantiomeric separations. Column equilibration and parameter optimization were generally accomplished more rapidly in SFC than in LC. Although improved resolution was often observed in SFC, analysis times were not always lower in SFC than in LC. In some instances, SFC provided separation capabilities not readily accessible in LC.     

Chiral resolution of the enantiomers of 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide using high-performance liquid chromatography on cellulose-based chiral stationary phases

Analytical high-performance liquid chromatography (HPLC) methods using derivatized cellulose chiral stationary phases (CSPs) were developed for the separation of the enantiomers of 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide ((+/-) IDRA21). In previous studies, (+/-) IDRA21 has been found to have an interesting inhibitory effect on the desensitization of alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) receptor and improve cognition in animals. This compound possess one chiral carbon atom, but very little information has been reported on the stereoselectivity of his activity. Therefore resolution of the enantiomers of this compound and subsequent identification of stereospecificity in his pharmacological actions are clearly matters of interest. The resolution were made under normal- and reversed-phase conditions using a mobile phase consisting of n-hexane:2-propanol (70/30, v/v) and water:acetonitrile (60/40, v/v) respectively, and a CSP of silica-based cellulose tris-3,5-dimethyl-phenylcarbamate (Chiralcel OD and Chiracel OD-R). The enantiomeric nature of eluates was confirmed by circular dichroism (CD) spectra. A baseline separation (R(S) > 1.5) was obtained in both cases. Furthermore the isolation of optical isomers of (+/-) IDRA21 was performed using a semipreparative column packed with the same cellulose OD CSP.     

Comparative enantioseparation of selected chiral drugs on four different polysaccharide-type chiral stationary phases using polar organic mobile phases.

The enantiomers of randomly selected chiral drugs and drug analogs of various structural and pharmacological groups were resolved on four different polysaccharide-type chiral stationary phases (CSP) using pure methanol and acetonitrile as mobile phases. Polysaccharide phenylester type CSP, Chiralcel-OJ although resolving the enantiomers of some chiral drugs was less universal in the combination with methanol and acetonitrile as mobile phases. Among polysaccharide phenylcarabamates amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak-AD) was superior over the corresponding cellulose derivative, cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel-OD). However, another derivative of cellulose, namely, cellulose tris(3,5-dichlorophenylcarbamate) (CDCPC) exhibited higher chiral recognition ability compared to Chiralpak-AD material. This study confirms previous findings about the applicability of polysaccharide type CSPs in so called polar organic mode as well as shows high potential of CDCPC as a practically useful CSP for High performance liquid chromatography (HPLC) enantioseparations.     

Validation of HPLC and GC-MS systems for bisphenol-A leached from hemodialyzers on the basis of FUMI theory

Three different halogeno-phenylcarbamate derivatives of cellulose have been prepared and coated on silica gel. The coated materials have been immobilized and their chiral recognition ability as chiral stationary phase (CSP) has been evaluated with a set of reference racemates, including several drugs such as lormetazepam, glutethimide, and warfarin, using various mobile phase mixtures. The novel phases were found to exhibit unique enantioselective properties compared with more established polysaccharide-based CSPs. A good resolution of all racemates could be successfully achieved on at least one of the immobilized CSPs. Moreover, it has been pointed out that selectivity may considerably vary with the composition of the mobile phase.     

Coupled column chromatography in chiral separation of salmeterol

A coupled achiral-chiral high-performance liquid chromatographic system has been developed for the determination of the enantiomers of salmeterol, S-(+)-salmeterol and R-(-)-salmeterol in urine. The salmeterol was separated from the interfering components in urine and quantified on the silica column, and the enantiomeric composition was determined on a Sumichiral OA-4700 chiral stationary phase. The two columns were connected by a switching valve equipped with a silica precolumn. The precolumn was used to concentrate the salmeterol in the eluent from the achiral column before backflushing onto the chiral phase. The coupled system was validated.     

Enantiomeric separation of fluoxetine derivatives on polysaccharide-based chiral columns

A high performance liquid chromatography (HPLC) method employing amylose-based chiral columns (Chiralpak AD-RH and Chiralpak AD) and cellulose-based chiral columns (Chiralcel OD) as chiral stationary phases have been developed for the enantiomeric separation of fluoxetine (FLX) derivatives. The FLX was derivatized with 4-(N-chloroformylmethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole (NBD-COCl) and 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl), respectively. Influence of the mobile phase composition and column temperature on the enantioseparation was discussed during the separation. On the basis of separation of derivatized FLX enantiomers, the paper also discussed the separation mechanism on the chiral stationary phases used.     

Chiral separation of verapamil and some of its metabolites by HPLC and CE

HPLC and CE assays were developed for chiral separations of verapamil and its metabolites in serum samples. Three chiral HPLC columns (Chiralcel OJ, Chiralpak AD and Chiralcel OD-R) were tested in normal and reverse-phase modes. All HPLC analyses were performed with fluorescence detection at 276 and 310 nm. CE was realized using CM-beta-CD as a chiral selector for the enantiomeric analysis. The results of HPLC and CE studies were compared and the possibilities for the applications in therapeutic drug monitoring were discussed.