Enantiomeric separation of chiral theophylline derivatives by liquid chromatography on cellulose-based sorbents

The enantiomeric resolution of seven closely related theophylline racemates by high-performance liquid chromatography on cellulose-based sorbents, in particular Chiralcel-OD, -OC and -OJ, is described. Although all chiral stationary phases (CSPs) are suitable for the enantioseparation of all racemates investigated, it is obvious that method development is different for each stationary phase. The screenings with the above-mentioned CSP included variation of mobile phase and temperature. It turned out that Chiralcel-OD should be used with 2-propanol in n-hexane as the mobile phase at higher temperatures, whereas Chiralcel-OC performed best with methanol at ambient temperature. Improved enantioseparations were observed for Chiralcel-OJ with increased modifier concentrations in n-hexane at increased temperature.     

Enantiomeric resolution of new triazole compounds by high-performance liquid chromatography

Cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) was synthesized and coated on aminopropylsilica to prepare a chiral stationary phase (CSP). HPLC methods were developed for the direct enantioseparation of 12 chiral triazole compounds on the CSP. The separations were made using normal phase methodology with a mobile phase consisting of n-hexane-alcohol (ethanol, 1-propanol, 1-butanol, 2-propanol, and t-butanol) in various portions. The column temperatures were studied for the optimization of the resolutions. The effects of structural features of the solutes on the discrimination between the enantiomers were examined. Baseline separation was easily obtained in many cases.     

Separation of the enantiomers of substituted dihydrofurocoumarins by HPLC using macrocyclic glycopeptide chiral stationary phases

Enantiomer separations by HPLC using the macrocyclic glycopeptides teicoplanin (Chirobiotic T), teicoplanin aglycon (Chirobiotic TAG), and ristocetin A (Chirobiotic R) chiral stationary phases (CSP) have been achieved on a unique series of potentially biologically active racemic analogues of dihydrofurocoumarin. The macrocyclic glycopeptides have proven to be very selective for this class of compound. All of the 28 chiral analogues examined afforded baseline separation on at least one of the macrocyclic glycopeptide CSP. The teicoplanin CSP showed the broadest enantioselectivity with 24 of the compounds baseline separated. The TAG and the R CSP produced 23 and 14 baseline separations respectively. All three mobile phase modes, i.e. normal phase (NP), reversed phase (RP), and new polar organic modes (PO), have been evaluated. The NP mode proved to be most effective for the separation of chiral dihydrofurocoumarins on all CSP tested. In the reversed phase (RP) mode, all three CSP separated a similar number of compounds. It was observed that the structural characteristics of the analytes and steric effects are very important factors leading to chiral recognition. Hydrogen bonding was found to play a secondary role in chiral discrimination in the normal phase and polar organic modes. Hydrophobic interactions are important for chiral separation in the reversed-phase mode. Chromatographic retention data does not provide information on the absolute configuration of these chiral dihydrofurocoumarin derivatives. However, when coupled with circular dichroism using the exciton coupling chirality method, the enantiomer elution order and the absolute configuration of some chiral dihydrofurocoumarins were successfully determined.     

Advances in enantiomeric resolution on monolithic chiral stationary phases in liquid chromatography and electrochromatography

During the last decade, chiral monolithic stationary phases have been prepared and used for rapid enantioseparations in CEC and HPLC. Various chiral selectors are used to prepare these CSPs. The preparation, properties, and applications of these CSPs are discussed in this paper. Attempts have been made to describe optimization strategies and the chiral recognition mechanisms. A comparison of chiral separations in CEC and HPLC is described. Efforts have also been made to predict the future perspectives and challenges of chiral monolithic stationary phases. The most effective chiral selectors include polysaccharides, cyclodextrins, and macrocyclic glycopeptide antibiotics. These chiral phases produced acceptable analytical enantiomeric separation of a variety of racemates. However, the development of these CSPs for preparative‐scale separations is needed.     

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.     

Evaluation and comparison of a methylated teicoplanin aglycone to teicoplanin aglycone and natural teicoplanin chiral stationary phases

HPLC enantiomeric separations of a wide variety of racemic analytes was evaluated using chiral stationary phases (CSPs) based on the macrocyclic glycopeptides teicoplanin (T), teicoplanin aglycone (TAG), and methylated teicoplanin aglycone (Me-TAG) in two different mobile phase modes, i.e., the RP mode and the polar organic (PO) mode. Comparison of the enantiomeric separations using Chirobiotic T, Chirobiotic TAG, and the methylated form of TAG were conducted in order to gain a better understanding of the roles of the polar functional groups on the CSP. Substantial effects due to the cleavage of saccharides and/or methylation on chiral separations were observed in both separation modes. Improved separation efficiencies for many acidic analytes were obtained by methylating the H-bonding groups of TAG. These groups were believed to be a contributing factor to band broadening on TAG due to their negative effect on mass transfer between the stationary phase and mobile phase. Ionic/dipolar interactions between the carboxylate group of the analytes and the amine groups on T, TAG, or Me-TAG are important for chiral discrimination. Therefore, analytes possessing a carboxyl group are good candidates for successful separations on these CSPs. Hydrophobic interactions are important for enantiomeric separations in the RP mode where the H-bonding interactions between analytes and the chiral selectors are relatively weak. Me-TAG offers higher hydrophobicity, which can accentuate the interactions of analytes with hydrophobic moieties, but these interactions are not necessarily stereoselective. In the PO mobile phase, electrostatic/dipolar interactions between polar functional groups are the dominating interactions in chiral recognition. Another important factor is steric fit, which could be changed with every modification of the T structure. Therefore, substantial changes of enantioseparations were obtained within this studied group of CSPs. The PO mode was shown to be the most powerful mobile phase mode for enantiomeric separations on T-based stationary phases, mainly due to the improved efficiency. Methylation of the TAG proved to be a very useful tool for investigating the chiral recognition mechanism for this group of chiral selectors.     

Chiral Resolution of the Enantiomers of New Aromatase Inhibitors by Liquid Chromatography on Amylose Stationary Phases

Analytical HPLC methods for derivatized amylose chiral stationary phases were developed for the direct enantioseparation of substituted [1-(imidazo-1-yl)-1-phenylmethyl)] benzothiazolinone and benzoxazolinone derivatives with one stereogenic center. These analogues of fadrozole constitute new potent nonsteroidal inhibitors of aromatase (P450 arom.). The separations were made using normal phase methodology with mobile phase consisting of n-hexane-alcohol (ethanol, 1-propanol or 2-propanol) in various proportions, and a silica-based amylose tris-3,5-dimethylphenylcarbamate (Chiralpak AD), or tris-(S)-1-phenylethylcarbamate (Chiralpak AS). The effects of concentration of various aliphatic alcohols in the mobile phase were studied. Baseline separation (R_s > 1.5) was easily obtained in all cases, ethanol being often the more interesting modifier. The effects of structural features of the solutes along with the temperature of the column on the discrimination between the enantiomers were examined for different mobile phase compositions.     

Analytical and semipreparative chiral separation of cis‐itraconazole on cellulose stationary phases by high‐performance liquid chromatography

cis‐Itraconazole is a chiral antifungal drug administered as a racemate. The knowledge of properties of individual cis‐itraconazole stereoisomers is vital information for medicine and biosciences as different stereoisomers of cis‐itraconazole may possess different affinity to certain biological pathways in the human body. For this purpose, either chiral synthesis of enantiomers or chiral separation of racemate can be used. This paper presents a two‐step high‐performance liquid chromatography approach for the semipreparative isolation of four stereoisomers (two enantiomeric pairs) of itraconazole using polysaccharide stationary phases and volatile organic mobile phases without additives in isocratic mode. The approach used involves the separation of the racemate into three fractions (i.e. two pure stereoisomers and one mixed fraction containing the remaining two stereoisomers) in the first run and consequent separation of the collected mixed fraction in the second one. For this purpose, combination of cellulose tris‐(4‐methylbenzoate) and cellulose tris‐(3,5‐dimehylphenylcarbamate) columns with complementary selectivity for cis‐itraconazole provided full separation of all four stereoisomers (with purity of each isomer > 97%). The stereoisomers were collected, their optical rotation determined and their identity confirmed based on the results of a previously published study. Pure separated stereoisomers are subjected to further biological studies.     

Transforming chiral liquid chromatography methodologies into more sensitive liquid chromatography-electrospray ionization mass spectrometry without losing enantioselectivity

LC-electrospray ionization (ESI) MS conditions were optimized for the individual chiral separation of 19 compounds of pharmaceutical interest using the macrocyclic glycopeptide-based chiral stationary phases in both polar organic and reversed-phase modes (RPM). The influence of mobile phase composition and MS additive type on sensitivity was investigated for all classes of compounds tested. Compounds with amine or amide groups were efficiently separated, ionized, and detected with the addition of 0.1% (w/w) ammonium trifluoroacetate to the solvent system in either the reversed-phase or polar organic mode (POM). Macrocyclic glycopeptide coupled column technology was initially used to screen all chiral compounds analyzed. Baseline resolution of enantiomers was then achieved with relatively short retention times and high efficiencies on Chirobiotic T, Chirobiotic V or Chirobiotic R narrow bore chiral stationary phases. The polar organic mode offered better limits of detection (as low as 100 pg/ml) and sensitivity over reversed-phase methods. An optimum flow-rate range of 200-400 microl/min was necessary for sensitive chiral LC-ESI-MS analysis.     

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.     

Enantioseparation of vesamicol and novel vesamicol analogs by high-performance liquid chromatography on different chiral stationary phases

High-performance liquid chromatography enantioseparation of vesamicol and six novel azaspirovesamicols (amino alcohols) was accomplished on different chiral stationary phases (CSPs) by using an optical rotation based chiral detector for identification of the resolved enantiomers. The Pirkle-type column Reprosil Chiral-NR was found to be most suitable for chiral resolution in normal phase (NP) mode; all compounds could be enantioseparated successfully. Also the cellulose-based column Reprosil Chiral-OM showed appropriate separation properties by using NP conditions. The amylose-type column Reprosil Chiral-AM-RP was most suitable for enantioseparation in reversed phase (RP) mode; five out of seven compounds were resolved. This CSP showed a considerably higher capability for chiral recognition of vesamicol derivatives in RP mode than the corresponding cellulose-based column Reprosil Chiral-OM-RP. Enantioseparation with the teicoplanin aglycone-based column Reprosil Chiral-AA was successful under polar ionic mobile phase conditions.     

Enantiomeric separation of racemic sulphoxides on chiral stationary phases by gas and liquid chromatography

Summary The enantiomeric resolution of seven racemic sulphoxides on chiral stationary phases has been investigated by gas and liquid chromatography. In gas chromatography the separations were performed on octakis-(2,6-di-O-pentyl-3-O-butyryl)-γ-cyclodextrin (FS Lipodex-E) and heptakis-(2,6-di-O-methyl-3-O-pentyl)-β-cyclodextrin (DMP-β-CD). Both stationary phases were suitable for separation of the enantiomers of the sulphoxides. With one exception for each series all racemetes could be resolved on both stationary phases; FS Lipodex-E was more enantioselective than DMP-β-CD, whereas the latter seemed more generally applicable. Liquid chromatographic separations with Chiralcel-OB as stationary phase were significantly improved by optimization of mobile phase composition and temperature. Resolution factors up to R_s=6 were achieved indicating that the improved separations could now be easily used for preparative purposes.     

Hybrid polyacrylamide chiral stationary phases for HPLC prepared by surface-initiated photopolymerization

Two hybrid polyacrylamide chiral stationary phases (CSPs) for HPLC have been synthesized by a new surface-initiated photo-induced radical polymerization approach of enantiopure N,N'-diacryloyl derivatives of (1R,2R)-diaminocyclohexane (CSP1) and (1R,2R)-diphenylethylenediamine (CSP2). This system is based on the activation of mesoporous silica microparticles by chemically bonded trichloroacetyl groups and dimanganese decacarbonyl as catalyst. UV irradiation was performed using a lab-made quartz photochemical reactor, ad hoc designed for the photo-induced polymerization process on the surface of microparticles. The two phases were evaluated and compared as chromatographic supports for the enantioselective HPLC of model chiral compounds. Their physico-chemical properties and chromatographic performances were also evaluated in comparison with those exhibited by the homologue CSPs obtained by the grafting-from thermal-induced process (CSP3 and CSP4). The new photopolymerization approach yielded higher grafting density than the thermal-induced one, especially in the case of the less reactive monomer (the diacryloyl derivative of (1R,2R)-diphenylethylenediamine), good chromatographic efficiency and a broad application field under normal phase and polar organic mode conditions.     

Unusually high enantioselectivity in high-performance liquid chromatography using cellulose tris(4-methylbenzoate) as a chiral stationary phase

The cellulose tris(4-methylbenzoate) chiral stationary phase (CSP) (commercially known as Chiralcel OJ-H) exhibited an extremely high enantioselectivity when used in the HPLC resolution of N-thiocarbamoyl-3-(4′-prenyloxy)-phenyl-5-phenyl-4,5-dihydro-(1H) pyrazole (Compound 1), in both normal-phase and polar organic conditions. Using n-hexane–ethanol (80:20, v/v) as a mobile phase, an enantioseparation factor value of 138.5 was found. In order to modulate the elution time of the longer retained enantiomer, a simple HPLC procedure was developed. The optimized analytical protocol was based on the stopped flow technique and did not involve any change in mobile phase composition. The stronger interaction energy of the (S) enantiomer compared to that of the (R) enantiomer was mainly attributed to the formation of a hydrogen bonding between the amino group of the thiocarbamoyl moiety and the carbonyl oxygen of the CSP.     

Comparison of enantiomer separation on two chiral stationary phases derived from (+)-18-crown-6-2,3,11,12-tetracarboxylic acid of the same chiral selector

Liquid chromatographic comparisons for enantiomer resolution of α-amino acids and chiral primary amino compounds were made using chiral stationary phases (CSPs) prepared by covalently bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18-C-6-TA) of the same chiral selector. The resolution of all α-amino acids on CSP 1 developed in our group was found to be better than that on CSP 2 reported by Machida et al. All α-amino acids examined in this study were well enantioseparated on CSP 1 (α=1.22–2.47), while four analytes were not resolved or all the other analytes were poorly resolved on CSP 2 than on CSP 1. However, in resolving the primary amino compounds without a carbonyl group, CSP 1 was comparable with CSP 2. Although (+)-18-C-6-TA of the same chiral selector was used to prepare CSP 1 and CSP 2, this study showed that different connecting methods for the CSPs might influence their ability to resolve the analytes depending on their structures related to the chiral recognition mechanism.     

Preparation and evaluation of novel chiral stationary phases based on quinine derivatives comprising crown ether moieties

The C9‐position of quinine was modified by meta‐ or para‐substituted benzo‐18‐crown‐6, and immobilized on 3‐mercaptopropyl‐modified silica gel through the radical thiol‐ene addition reaction. These two chiral stationary phases were evaluated by chiral acids, amino acids, and chiral primary amines. The crown ether moiety on the quinine anion exchanger provided a ligand‐exchange site for primary amino groups, which played an important role in the retention and enantioselectivity for chiral compounds containing primary amine groups. These two stationary phases showed good selectivity for some amino acids. The complex interaction between crown ether and protonated primary amino group was investigated by the addition of inorganic salts such as LiCl, NH₄Cl, NaCl, and KCl to the mobile phase. The resolution results showed that the simultaneous interactions between two function moieties (quinine and crown ether) and amino acids were important for the chiral separation.     

纤维素衍生物手性固定相高效液相色谱法分离盐酸川丁特罗对映体

以纤维素三-(3,5-二甲基苯基氨基甲酸酯)为手性固定相(Lux Cellulose-1),建立了在正相色谱条件下直接分离盐酸川丁特罗对映体的高效液相色谱法。考察了乙醇、异丙醇等有机改性剂,三氟乙酸、二乙胺等流动相添加剂和柱温对对映体分离的影响。结果显示,酸性和碱性添加剂对对映体分离的影响最为显著: 添加二乙胺时两对映体无分离趋势;添加三氟乙酸时对映体保留强,且分离趋势明显;而同时添加三氟乙酸和二乙胺则两对映体分离显著改善,分离度可达4.0。优化后的色谱条件: 色谱柱为Lux Cellulose-1手性柱(250 mm×4.6 mm, 5 μm),流动相为正庚烷-乙醇-三氟乙酸-二乙胺(88:12:0.3:0.05, v/v/v/v),流速为1.0 mL/min,紫外检测波长为246 nm,柱温为25 ℃。该方法简便,快速,可用于左旋盐酸川丁特罗原料中右旋异构体杂质的检查。