Liquid chromatographic enantiomer separation of 1-naphthylamides of chiral acids using several amylose- and cellulose-derived chiral stationary phases

The liquid chromatographic enantiomer separation of various chiral acids as 1-naphthylamides was performed using several chiral stationary phases (CSPs). The CSPs used in this study were six covalently bonded and four coated type CSPs derived from amylose and cellulose derivatives as chiral selectors. The degree of enantioseparation is affected by the structure of chiral acids and the CSPs used, which have different chiral selectors and types of immobilization. For the enantiomer resolution of chiral acids as 1-naphthylamide derivatives, the performance of the coated type Lux Cellulose-1 was superior to those of the other CSPs, except for 2-aryloxypropionic acid derivatives. Owing to the strong ultraviolet absorbance of the 1-naphthyl group, the convenient analytical method developed and validated in this study could be expected to be very useful for the enantiomer separation of various chiral acids as 1-naphthylamide derivatives using polysaccharide-derived CSPs.     

A convenient method for the enantiomeric separation of α-amino acid esters as benzophenone imine Schiff base derivatives

A convenient liquid chromatographic method for the separation of α-amino acid esters as benzophenone Schiff base derivatives on coated chiral stationary phases (CSPs) (Chiralcel OD-H, Chiralcel OD, Chiralpak AD-H, Chiralpak AD, and Chiralpak AS) or covalently immobilized CSPs (Chiralpak IA, Chiralpak IB, and Chiralpak IC) derived from polysaccharide derivatives is described. Benzophenone imine derivatives of α-amino acid esters were readily prepared by stirring benzophenone imine and the hydrochloride salts of α-amino acid esters in 2-propanol. The chromatographic separations were conducted at a flow rate 1.0 mL/min and a detection wavelength of 254 nm; 0.5% 2-propanol/hexane (v/v) was used on CSPs. In general, the resolution of Chiralpak IC was superior to those of the other CSPs. In addition, the resolutions of other arylimine derivatives of α-amino acid esters and the effects of different mobile phases on the enantiomeric separation of α-amino acid esters as benzophenone imine derivatives on Chiralpak IC were investigated.     

Immobilized proteins as chromatographic supports for chiral resolution.

This review examines the role of protein-bonded chiral stationary phases (CSPs) in enantiomeric separation and investigates the performance characteristics and desired properties of protein CSPs for separation and large-scale operation. The review also discusses the ability of protein-based CSPs to examine the stereochemistry of drug metabolism processes.     

Capillary electrophoretic separation of enantiomers of amino acids and amino acid derivatives using crown ether and cyclodextrin

The capillary zone electrophoresis using (+)-18-crown-6-tetracarbonic acid as a chiral selector was a suitable method for the enantiomeric separation of racemates of amino acids and of some amino acid derivatives (esters, dipeptides). The influence of the chemical structure of the compounds on the separation was investigated. After optimization of the separation conditions, baseline separations were obtained for most racemates. The addition of acetonitrile and TBAB yielded an improvement of the separation. Improved selectivity was further observed by the application of a cyclodextrin, HP-beta-CD, in combination with the crown ether.     

Comparison of three chiral stationary phases with respect to their enantio- and diastereoselectivity for cyclic beta-substituted alpha-amino acids.

Three chiral stationary phases were examined for the enantio- and diastereoseparation of cycloaliphatic beta-substituted alpha-quaternary alpha-amino acids. Resolution of diastereomeric analytes is feasible with a chiral crown ether based column, whereas the separation of enantiomers, except for one pair of amino acids, could not be achieved. The two chiral stationary phases with the glycopeptide antibiotic teicoplanin and with the copper(II)-D-penicillamine complex, respectively, are, however, both very potent in the separation of the enantiomeric, as well as of the diastereomeric amino acids. A baseline separation of all four stereoisomeric forms in one chromatographic run was possible with the exception of one type of amino acid. The results of the method development are presented in this paper.     

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.     

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.     

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.     

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.     

Enantiomeric separations of illicit drugs and controlled substances using cyclofructan‐based (LARIHC) and cyclobond I 2000 RSP HPLC chiral stationary phases

Recently a novel class of chiral stationary phases (CSPs) based on cyclofructan (CF) has been developed. Cyclofructans are cyclic oligosaccharides that possess a crown ether core and pendent fructofuranose moieties. Herein, we evaluate the applicability of these novel CSPs for the enantiomeric separation of chiral illicit drugs and controlled substances directly without any derivatization. A set of 20 racemic compounds were used to evaluate these columns including 8 primary amines, 5 secondary amines, and 7 tertiary amines. Of the new cyclofructan‐based LARIHC columns, 14 enantiomeric separations were obtained including 7 baseline and 7 partial separations. The LARIHC CF6‐P column proved to be the most useful in separating illicit drugs and controlled substances accounting for 11 of the 14 optimized separations. The polar organic mode containing small amounts of methanol in acetonitrile was the most useful solvent system for the LARIHC CF6‐P CSP. Furthermore, the LARIHC CF7‐DMP CSP proved to be valuable for the separation of the tested chiral drugs resulting in four of the optimized enantiomeric separations, whereas the CF6‐RN did not yield any optimum separations. The broad selectivity of the LARIHC CF7‐DMP CSP is evident as it separated primary, secondary and tertiary amine containing chiral drugs. The compounds that were partially or un‐separated using the cyclofructan based columns were screened with a Cyclobond I 2000 RSP column. This CSP provided three baseline and six partial separations. Copyright © 2013 John Wiley & Sons, Ltd.     

Analysis of amino acid enantiomers derived from antitumor antibiotics using chiral capillary electrophoresis

The chiral separation of enantiomeric forms of derivatized amino acids have been achieved based on a metal–chelate chiral capillary electrophoretic method and a cyclodextrin mediated host–guest interaction approach in micellar electrokinetic chromatography (MEKC) mode with laser-induced fluorescence detection. This approach has been applied to the determination of enantiomeric forms of amino acids derived from novel depsipeptide antitumor antibiotics, BMY-45012 and its analogs. Amino acids were analyzed by complete hydrolysis and the hydrolysate was derivatized with either dansyl chloride for UV absorbance detection or fluorescein isothiocyanate for laser based fluorescence detection. The presence of several amino acids, serine and β-hydroxyl-N-methy-valine in the proposed structure have been confirmed as d-serine and l-β-hydroxyl-N-methy-valine enantiomeric forms by both chiral capillary electrophoresis (chiral CE) and MEKC approaches. A non-chiral amino acid, sarcosine, was also confirmed. These methodologies provide a quick and sensitive approach for the determination of amino acids racemization of pharmaceutical natural products and have proven to be useful for structural elucidation refinement.     

Separation of neutral dihydropyridines and their enantiomers using electrokinetic chromatography

The separation and simultaneous enantiomeric separation of three neutral 1,4-dihydropyridine (DHP) derivatives (nimodipine, nisoldipine and nitrendipine) was studied using electrokinetic chromatography. Bile salts allowed the non-chiral separation of these DHP derivatives. With the taurine-conjugated bile salts a beginning of enantiomeric separation was observed for nimodipine and nisoldipine. Achiral micelles of sodium dodecyl sulphate mixed with neutral cyclodextrins did not allow enantioseparation. Baseline chiral separation of nisoldipine and nimodipine was obtained with carboxymethyl-beta-cyclodextrin at pH 5.0. The buffer type affected the chiral separation, especially in the case of nisoldipine. The addition of organic solvent decreased the enantioresolution of nimodipine. However, the resolution between the nisoldipine enantiomers was increased when methanol or ethanol were added to the background electrolyte. Varying the temperature had almost no effect on the enantioresolution of nisoldipine, whereas with nimodipine a clear improvement at lower temperatures was observed. Using the optimised method, the selectivity of this method was investigated for three possible impurities of nisoldipine.     

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.     

柱前衍生结合手性固定相高效液相色谱法拆分2种氨基酸对映体

目的建立柱前衍生结合手性固定相HPLC法拆分2种氨基酸对映体。方法衍生化试剂：4-氯-7-硝基-2,1,3-苯并噁二唑（NBD-Cl）;色谱柱：OA-2500S;流动相：甲醇（含5 mmol.L-1枸橼酸）∶-乙腈（50∶50,v/v）;检测波长：470 nm;流速：0.5 mL.min-1。结果在优化的色谱条件下,丝氨酸和缬氨酸对映体的分离度分别为2.4和2.5,分离因子分别为1.12和1.17。结论该方法简单、灵敏,可用于2种氨基酸对映体的拆分。     

Comparison of several polysaccharide-derived chiral stationary phases for the enantiomer separation of<Emphasis Type="Italic">N</Emphasis>-fluorenylmethoxy-carbonyl α-amino acids by hplc

The liquid chromatographic enantiomer separation of N-fluorenylmethoxycarbonyl (FMOC) protected alpha-amino acids was performed on nine polysaccharide-derived chiral stationary phases (CSPs). The cellulose derived coated CSPs, Chiralcel OD-H (separation factor = 1.09-2.70) and Chiralcel OD (separation factor = 1.08-2.55), had the best performance of all the CSPs for resolution of N-FMOC alpha-amino acids and therefore, all analyte enantiomers were base-line separated on Chiralcel OD-H and/or Chiralcel OD. Enantioseparation on cellulose tris(3,5-dimethylphenylcarbamate) derived CSPs (Chiralcel OD-H, Chiralcel OD and Chiralpak IB) is generally greater than that on amylose tris(3,5-dimethylphenylcarbamate) derived CSPs (Chiralpak AD-RH, Chiralpak AD and Chiralpak IA). Additionally, coated type CSPs (Chiralcel OD-H or Chiralcel OD, and Chiralpak AD) generally provided better enantioseparation for these analytes than the covalently bonded type CSPs (Chiralpak IB and Chiralpak IA) with the same chiral selector of cellulose tris(3,5-dimethylphenylcarbamate) and amylose tris(3,5-dimethylphenylcarbamate), respectively. However, Chiralpak IB and Chiralpak IA had an advantage over the coated type CSPs in that a broader range of solvents could be used due to its covalently bonded nature.     

Urethane-protected N-carboxyanhydrides (UNCAs) as unique reactants for the study of intrinsic racemization tendencies in peptide synthesis

We have established that urethane-protected N-carboxyanhydrides (UNCAs) are uniquely suited for the study of intrinsic racemization tendencies in peptide synthesis. The UNCA allows epimerization only by the direct enolization pathway (proton abstraction from the α-carbon) and does not decompose upon epimerization. A protocol employing the quantitative separation and analysis of enantiomeric N-protected amino acid derivatives by chiral HPLC has been developed to measure the intrinsic rate of racemization of UNCAs under widely varying reaction conditions. The influence of the tertiary amine structure, UNCA side chain structure, and solvent were studied. The same protocol was employed to study the intrinsic rate of racemization of N-protected activated amino acid intermediates generated via ‘onium-type’ activating reagents. We have shown that the trends influencing the intrinsic rate of racemization of UNCAs are maintained under the conditions of in situ activations, and are consistent with the trends found in classical studies in the literature. The results are relevant to peptide synthesis both in solution and on solid phase. The intrinsic rate of racemization for any type of activation with any tertiary amine can be measured by this protocol. © Munksgaard 1997.     

Application of chiral derivatizing agents in the high-performance liquid chromatographic separation of amino acid enantiomers: a review

The past 20 years has seen an explosive growth in the field of chirality, as illustrated by the rapid progress in the various facets of this intriguing field. The impetus for advances in chiral separation has been highest in the past decade and this still continues to be an area of high focus. This paper reviews indirect separation approaches, i.e. derivatization reactions aimed at creating the basis for the chromatographic resolution of biologically and pharmaceutically important enantiomers, with emphasis on the literature published in the last 12 years. The main aspects of the chiral derivatization of amino acids are discussed, i.e. derivatization on the amino group, transforming the molecules into covalently bonded diastereomeric derivatives through the use of homochiral derivatizing agents. The diastereomers formed (amides, urethanes, urea, thiourea derivatives, etc.) can be separated on achiral stationary phases. The applications are considered, and in some cases different derivatizing agents for the resolution of complex mixtures of proteinogenic d,l-amino acids, non-proteinogenic amino acids and peptides/amino acids from peptide syntheses or microorganisms are compared.     

Enantiomeric separation of optically active pyridazinone derivatives by chiral HPLC

Several 4,5-disubstituted 3(2H)-pyridazinone derivatives containing 2-hydroxymethylpyrrolidino moiety as a chiral building block were synthetized. Separation of enantiomers was carried out by chiral HPLC on Chiralcel OJ and OF columns. Mobile phases consisted of hexane, ethanol and 2-propanol. Chiralcel OJ column was capable of separating most of the enantiomeric pairs. For one type of compound. Chiralcel OF column was used for separation.     

Structural characterization and enantioseparation of the chiral compound praziquantel

In this study, we aimed to characterize the chiral compound type of a leading antischistosomal drug, praziquantel. The optically pure praziquantel enantiomers were recovered from the racemic mixture by enantiomeric separation, which was performed on preparative scale chromatography by using a novel beta-cyclodextrin type chiral column. The thermodynamic properties of praziquantel were determined from differential scanning calorimetry and the physical properties were studied by examining Fourier transform infrared spectroscopy and X-ray powder diffraction. Based on the differential scanning calorimetry data, a melting point binary phase diagram was constructed. A ternary solubility phase diagram of praziquantel in methanol was also determined at the temperature of 0 degrees C. All the experimental results support the conclusion that praziquantel is a racemic compound. The characterization of physical properties of praziquantel and the phase diagram are crucial for understanding the rationality for the successful resolution of praziquantel and also provide the basis for designing the strategy of separation and recovery of pure enantiomer.     

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。