Chiral separation of highly negatively charged enantiomers by capillary electrophoresis

The separation of two highly negatively charged enantiomeric organic disulfates containing two chiral centers was investigated by capillary electrophoresis using cyclodextrin based chiral selectors added to the run buffer. The optimum separation for the enantiomers was achieved in less than 3 min at 25 degrees C with a run buffer of 10 mM glycine pH 2.4 and 5 mM QA-beta-CD, which is a positively charged quaternary ammonium beta-cyclodextrin derivative. The method resulted in baseline resolution, excellent linearity, and highly reproducible migration times allowing facile evaluation of the enantiomeric purity of the individual isomers. Detection limits for the enantiomeric pair were determined to be 0.3 ng/microl (S/N = 3). The nature of the selector-enantiomer interaction and a quantitative measurement of the apparent stability constants that governed chiral discrimination of the enantiomers with QA-beta-CD were also investigated by UV-Vis spectroscopy and electrospray ionization mass spectrometry.     

手性化合物的毛细管胶束电动色谱分离研究

以4种不同的N-长链烷酰-L-氨基酸胶束为手性选择剂,对3种不同性质的手性化合物(α-氯代丙酰替苯胺,2-氨基-3-对硝基苯基-1,2-丙二醇和华法林)的毛细管胶束电动色谱分离进行研究.结果表明,手性表面活性剂中不同的氨基酸残基和烷基链的长度对分离影响较大;随手性表面活性剂浓度增加,溶质保留时间增大,分离度增加,不同溶质的最佳分离浓度在100～150mmol/L之间;pH对电中性手性化合物分离影响不大,但对酸性或碱性手性化合物的分离影响较大.在选定的条件下,3种样品均在20min内完全分离,分离柱效达1×10⁵理论板数/m.     

Use of multivariate analysis for optimization of separation parameters and prediction of migration time, resolution, and resolution per unit time in micellar electrokinetic chromatography

The optimization of separation parameters in chromatography for better separation and resolution of analytes continues to be a labor intensive procedure usually performed by a trial and error method. A multivariate analysis in the form of multilinear regression (MLR) is used to optimize separation parameters and predict the migration behavior, resolution, and resolution per unit time of achiral (4-chlorophenol, pentachlorophenol, clonazepam, and diazepam) and chiral (1,1'-binaphthyl 2,2'-dihydrogen phosphate (BNP), and 1,1'-bi-2-naphthol (BOH)) compounds in MEKC. Separations of achiral and chiral analytes were performed using an achiral (poly(sodium N-undecylenic sulfate)) molecular micelle and chiral (poly(sodium N-undecanoyl-L-leucylvalinate) or poly(sodium N-undecanoyl-L-isoleucylvalinate)) molecular micelle, respectively, at various operating temperatures, applied voltages, pH values, and molecular micelle concentrations in the BGE. The separation parameters were subsequently used as input variables for MLR models. The models were validated with independent samples. The root-mean-square percent relative error (RMS%RE) is used as a figure of merit for characterizing the performance of the migration time, resolution, and resolution per unit time models. The RMS%RE obtained for predicted migrated times, resolutions, and resolution per unit time of 4-chlorophenol, pentachlorophenol, clonazepam, diazepam, BNP, and BOH ranged between 8 and 19%. The same experimental procedure was used to optimize the separation parameters of six other chiral analytes of different compound class. The predicted migration times, resolutions, and resolution per unit time of the chiral as well as the achiral analytes compare favorably with the experimental migration times and resolutions, indicating versatility and wide applicability of the technique in MEKC.     

Use of vancomycin as chiral selector in capillary electrophoresis. Optimization and quantitation of loxiglumide enantiomers in pharmaceuticals

Vancomycin has been used as chiral selector for the enantiomers separation of D, L-loxiglumide, a new drug proposed for the treatment of gastrointestinal pathology. The chiral selector, dissolved at very low concentration in the running buffer, filled only part of the capillary (polyacrylamide coated) and allowed chiral resolution in less than 12 min using a 50 mM phosphate buffer at pH 6. The partial separation technique allowed to obtain a detection limit of 0.5 μg/ml for each enantiomer avoiding the drop in sensitivity due to the strong UV absorption of vancomycin when present in the detector path. The effects of vancomycin concentration and buffer pH on enantiomers resolution have been studied in order to find the optimum experimental conditions for the chiral purity control of drug. The optimized method, using the internal standard, showed good reproducibility for both migration times and normalized peak area ratio and for linearity. Under the studied operating conditions it was possible to detect 0.2 % (w/w) of L-loxiglumide as a chiral impurity. Analysis of pharmaceutical preparations of D-loxiglumide did not reveal the presence of the impurity (L-isomer).     

Experimental design methodologies to optimize the CE separation of epinephrine enantiomers

Summary A capillary electrophoretic method using a chiral selector was optimized by experimental design for the enantioresolution of epinephrine enantiomers. Two β-cyclodextrins derivatives, namely heptakis-2,6-di-O-methyl-β-cyclodextrin and carboxy-methyl-β-cyclodextrin, respectively neutral and charged, were used as chiral selectors employing an uncoated capillary. By using a statistical experimental design in which all factors are varied at the same time, it was possible to optimize the method with regard to the resolution between peaks and the two migration times. A fractional factorial design and a central composite design were used. A compromise between conflicting goals, such as maximization of resolution and minimization of analysis time, was found by means of a desirability function D. Balancing these goals against each other, the most acceptable solution to the problem was found and the optimized method gave a fast separation with complete resolution between the adrenaline enantiomers. The response surfaces obtained confirmed the robustness of the method.     

Resolution of the enantiomers of oxamniquine by capillary electrophoresis and high-performance liquid chromatography with cyclodextrins and heparin as chiral selectors

The methods of separation of the enantiomers of the chiral drug oxamniquine are compared, between HPLC with either cyclodextrins and their related derivatives as chiral selectors in the mobile phase or immobilisedin a chiral stationary phase (as Cyclobond I and II) and between capillary zone electrophoresis (CZE) where the cyclodextrins are added to the buffer solution. The HPLC experiments, which included structured method optimisation were largely unsuccessful in resolving the enantiomers, with the exception of when a Chiral-AGP protein stationary phase was introduced into the programme. However although this chiral stationary phase provided baseline resolution of the enantiomers the stability of the method was suspect to small changes in the pH (0.2 units). In contrast the CZE method developed for both cyclodextrins and their derivatives gave good resolution of the enantiomers and method stability (R.S.D. <1%, N = 10 on precision). The basis of the interaction mechanism between selector and selectand was shown as a 1:2 relationship of cyclodextrin to analyte by NMR. In addition the polysaccharide, heparin was investigated as a chiral additive and excellent resolution of the oxaminiquine was achieved with 3 mM heparin in 50 mM sodium dihydrogenphosphate (pH 3.0) as buffer in CZE, which also gave a stable procedure. This method allowed the detection of each of the enantiomers in the presence of the other down to 0.23% (m/m). The overall composition of the heparin material from different sources can however be slightly variable and this can result in small differences in resolution capability.     

Separation of basic drug enantiomers by capillary electrophoresis using ovoglycoprotein as a chiral selector: comparison of chiral resolution ability of ovoglycoprotein and completely deglycosylated ovoglycoprotein

Separations of basic drug enantiomers by capillary electrophoresis (CE) using ovoglycoprotein (OGCHI) as a chiral selector are described. The effects of running buffer pH and 2-propanol content on the migration times and resolution of basic drug enantiomers were examined using a linear polyacrylamide-coated capillary. High resolution of basic drug enantiomers was attained using a mixture of 50 mM sodium phosphate buffer (pH 4.5-6.0) and 2-propanol (5-30%) including 50 microM OGCHI. It was found that ionic and hydrophobic interactions could work for the recognition of basic drug enantiomers. Further, we compared the chiral resolution ability of OGCHI with that of completely deglycosylated OGCHI (cd-OGCHI) using them as chiral selectors in CE. OGCHI showed higher resolution for basic drug enantiomers tested than cd-OGCHI. The results suggest that the chiral recognition site(s) for OGCHI exists on the protein domain of OGCHI.     

A comparison of vancomycin and sulfated beta-cyclodextrin as chiral selectors for enantiomeric separations of selenoamino acids using capillary electrophoresis with UV absorbance detection

The enantiomeric separation of three selenoamino acids, D,L-selenomethionine, D,L-selenoethionine and D,L-selenocystine is described. Both sulfated beta-cyclodextrin and vancomycin have been successfully used to separate all enantiomers of the compounds with UV detection. Reproducible separations, in terms of peak area and migration time were obtained using sulfated beta-cyclodextrin with reversed polarity and UV detection. With vancomycin as a chiral selector, reversed polarity was found to be more reproducible than positive polarity in terms of peak migration times.     

Enantioselective determination of modafinil in pharmaceutical formulations by capillary electrophoresis, and computational calculation of their inclusion complexes

A fast capillary electrophoretic method is described for the separation and determination of the enantiomers of the novel wake-promoting agent, modafinil. Several parameters affecting the separation were studied, including the type and concentration of chiral selector, buffer pH, buffer concentration, voltage and temperature. Good chiral separation of the racemic mixture was achieved in less than 5 min with resolution factor Rs?=?2.51, using a bare fused-silica capillary and a background electrolyte (BGE) of 25 mM H₃PO₄?1 M tris solution; pH 8.0; containing 30 mg mL^?1 of sulfated-β-cyclodextrin (S-β-CD). The separation was carried out in normal polarity mode at 25 ^?C, 18 kV and using hydrostatic injection. Acceptable validation criteria for selectivity, linearity, precision, and accuracy were included. The developed method was successfully applied to the assay of enantiomers of modafinil in pharmaceutical formulations. The computational calculations for the enantiomeric inclusion complexes rationalized the reasons for the different migration times between the modafinil enantiomers.     

酰胺型手性固定相直接拆分克仑特罗对映体

 将酰胺型手性固定相用于正相高效液相直接拆分 β2 受体兴奋剂克仑特罗。讨论了三元流动相中正己烷、1,2 二氯乙烷和甲醇含量的变化以及柱温和流速对分离的影响 ;优化了实验条件 :流动相为V(正己烷 )∶V(二氯乙烷 )∶V(甲醇 ) =5 4∶38∶8,柱温为 17℃ ,流速为 1 0mL/min ;并对拆分的机理加以探讨。方法简单、快速。     

Aspergiolides C and D: spirocyclic aromatic polyketides with potent protein kinase c-Met inhibitory effects

Variation of the cultivation conditions for Aspergillus glaucus led to the discovery of two novel spirocyclic aromatic polyketides, aspergiolides C (3) and D (4). Their constitutions were elucidated by a combination of spectroscopic methods and isotope-labeling experiments. Aspergiolides C (3) and D (4) occur as racemic mixtures, the resolution of which was succeeded by HPLC on a chiral phase. The absolute configurations of their enantiomers were assigned online, from the peaks in the chromatogram, by a combination of HPLC-CD and quantum chemical CD calculations. Both compounds were found to inhibit the kinase activities of the receptor tyrosine kinases (RTKs) c-Met, Ron, and c-Src with low-micromolar IC(50)s. The enantiomers of 3 were resolved by HPLC on a chiral phase. Both enantiomers showed a comparable inhibition of the HGF-induced autophosphorylation of c-Met and of subsequent cell migration.     

Chiral high-performance liquid chromatography of aromatic cyclic dipeptides using cyclodextrin stationary phases

A series of enantiomers of cyclic and linear dipeptides containing aromatic amino acids was prepared and chromatographed on beta- and gamma-cyclodextrin (CD) columns. The retention times, separation factor alpha and resolution values were calculated. The relevance of the distance of the chiral center from the phenyl ring for chiral resolution was studied. A model was developed using X-ray crystallographic data for an inclusion complex of beta-CD and the enantiomers of cyclic (Phe-Gly).     

吸附固定相电色谱和动态改性电色谱的手性分离

对动态改性电色谱手性分离进行了研究。电色谱柱填充强阴离子交换固定相（SAX0,添加在流动相中的磺化β－环糊精（S－CD）动态地吸陵于SAX填料表面,形成一层准手性固定相。色氨酸、阿托品和异博定对映体在本体系获得了很好的分离,它们的分离分别为2．06,10．1和1．96,对映体峰的柱效价于85,000塔板数／米和412,000塔板数／米之间。连续运行17次,死时间和色氨酸对映体的电色谱保留因子的相对标准偏差分别为0．53％,0．62％和0．69％。此外,以吸附于SAX填料的牛血清白蛋白和S－CD为手性固定相进行了电色谱手性分离的研究。在这两种体系下分离色氨酸对映体的分离度分别为3．86和2．97。吸附S－CD柱电色谱和动态改性电谱的重现性进行子比较,发现动态改性电色谱有更好的重现性。     

万古霉素键合手性固定相高效液相色谱法直接分离泰妥拉唑对映体

利用反相高效液相色谱法在大环抗生素类手性固定相万古霉素键合手性固定相(Chirobiotic V)上直接分离了泰妥拉唑对映体。考察了缓冲溶液的种类、浓度和pH值,有机改性剂的种类和浓度,柱长和柱温等对手性分离的影响。优化后的色谱条件为:Chirobiotic V色谱柱(150 mm×4.6 mm,5 μm),流动相为0.02 mol/L 醋酸铵缓冲液(pH 6.0)-四氢呋喃(体积比为93∶7),流速为0.5 mL/min,柱温为20 ℃,检测波长为306 nm。在此条件下泰妥拉唑对映体达到了基线分离,分离度达1.68;对映体保留时间的相对标准偏差分别为0.48%和0.49%(n=6),峰面积的相对标准偏差分别为0.45%和0.55%(n=6)。所建立的手性分离方法具有简便快速及重复性好等优点。     

Determination of aminoglutethimide enantiomers in pharmaceutical formulations by capillary electrophoresis using methylated-β-cyclodextrin as a chiral selector and computational calculation for their respective inclusion complexes

A capillary electrophoretic method for the separation of the aminoglutethimide (AGT) enantiomers using methylated-β-cyclodextrin (M-β-CD) as chiral selector is described. Several parameters affecting the separation were studied, including the type and concentration of chiral selector, buffer pH, voltage and temperature. Good chiral separation of the racemic mixture was achieved in less than 9 min with resolution factor Rs = 2.1, using a fused-silica capillary and a background electrolyte (BGE) of tris-phosphate buffer solution (50 mmol L⁻¹, pH 3.0) containing 30 mg mL⁻¹ of M-β-CD. The separation was carried out in normal polarity mode at 25 °C, 16 kV and using hydrostatic injection. Acceptable validation criteria for selectivity, linearity, precision, and accuracy/recovery were included. The proposed method was successfully applied to the assay of AGT enantiomers in pharmaceutical formulations. The computational calculations for the inclusion complexes of the R- and S-AGT-M-β-CD rationalized the reasons for the different migration times between the AGT enantiomers.     

Separation of basic drug enantiomers by capillary zone electrophoresis using glucuronyl glucosyl beta-cyclodextrin as a chiral selector.

Separations of basic drug enantiomers have been investigated using glucuronyl glucosyl beta-cyclodextrin (GUG beta-CD) as a chiral selector in the background electrolyte by capillary zone electrophoresis. The effects of GUG beta-CD concentration and running buffer pH on the migration times and resolution of 16 basic drug enantiomers were precisely examined using a linear polyacrylamide-coated capillary. High resolution of 16 basic drug enantiomers was generally attained with a running buffer pH 2.5 or 3.5 containing 10 mM GUG beta-CD. Next, we compared the chiral resolution abilities of GUG beta-CD with those of beta-CD and maltosyl beta-CD (G2 beta-CD). GUG beta-CD showed higher resolution for basic drug enantiomers tested than beta-CD and G2 beta-CD. This could be due to that hydrogen bonding or ionic interactions of uncharged and charged glucuronyl glucosyl groups of GUG beta-CD with an analyte could stabilize the inclusion complex.     

薄层色谱在有机物顺反及对映异构体分离中的应用

体异构体和光学异构体分离技术的发展已有较综合的论述ｌ‘”‘］。利用ＴＬＣ对光学异构体进行拆分的方法主要有两种。１）使用手性同定相ｌ’,‘］;２）利用手性流动相在非手性固定相上进行拆分ｒ‘。本文对立体异构体的分离采用一般的ＴＬＣ法,对对映异构体的分离选用ｐ－ＣＤ手性流动相,＊一奎宁手性流动相和手性固定相纤维素板及非手性Ｃ;ｓ反相板对三哇及氨基酸进行了拆分,并初步探讨了方形板及楔形板展开对分离效果的影响。１实验部分１．１试剂及材料二哩类化合物及标样均为自制,标样均通过元素分析;Ｄ,Ｌ一色氨酸,Ｄ一色…     

High-resolution GC separation of alkyl lactate enantiomers and the determination of the enantiomeric excess of the optical isomers of alkyl lactates

Summary In this paper, the recently reported chiral stationary phase [1,2] was applied to the chiral resolution of alkyl lactates in the investigation of the effect of a new asymmetric heterogeneous catalyst system (cinchonidine-modified finely dispersed polyvinylpyrrolidone-stabilized platinum clusters) in the enantioselective hydrogenation of α-ketoesters. The enantiomers of methyl, ethyl, isopropyl, n-propyl and n-butyl lactates were successfully resolved while the enantiomers of isobutyl lactate could not be resolved, at all. The separation of the enantiomers of isopropyl, n-propyl and n-butyl lactates are reported for the first time. The results show that the new asymmetric heterogeneous catalyst system is effective in the enantioselective hydrogenation of α-ketoesters.     

Determination of fenfluramine enantiomers in pharmaceutical formulations by capillary zone electrophoresis

Summary Capillary zone electrophoresis has been used for the enantiomeric separation of racemic ortho-fenfluramine and meta-fenfluramine employing a phosphate buffer at pH 2.5 added with cyclodextrins. The cyclodextrin type and concentration strongly influenced the chiral resolution. The uncharged β-cyclodextrin polymer gave enantiomeric resolution of both ortho and meta isomers, while γ-cyclodextrin was a good chiral selector for only ortho-fenfluramine; heptakis-2,3,6-tri-O-methyl-β-cyclodextrin permitted base line separation of meta-fenfluramine enantiomers but only partial resolution of racemic ortho-fenfluramine. The optimized electrophoretic method was applied to the quantitative analysis of 1-meta-fenfluramine (minor component in the mixture) and d-meta-fenfluramine in a commercial pharmaceutical formulation. Good reproducibility for migration time and corrected peak areas (R.S.D. <0.8% and <1.2%, respectively) was achieved and the presence of the minor component of the mixture was found to be in accord to previous determinations performed by other analytical methods.     

Enantiomeric separation of asymmetric triacylglycerol by recycle high-performance liquid chromatography with chiral column

In our previous studies, we employed recycle HPLC for the separation of triacylglycerol (TAG)-positional isomers (PIs). In this study, a recycle HPLC system equipped with a polysaccharide-based chiral column was applied to the enantiomeric separation of some asymmetric TAGs having straight-chain C16-C18 acyl residues. As a result, 1,2-dipalmitoyl-3-oleoyl-rac-glycerol (rac-PPO), 1,2-dioleoyl-3-palmitoyl-rac-glycerol (rac-OOP), and 1,2-dipalmitoyl-3-linoleoyl-rac-glycerol (rac-PPL) were resolved into their respective enantiomers. However, neither 1,2-dioleoyl-3-linoleoyl-rac-glycerol (rac-OOL), consisting of only unsaturated fatty acids, nor 1,2-dipalmitoyl-3-stearoyl-rac-glycerol (rac-PPS), consisting of only saturated fatty acids, was resolved. These results suggest that the asymmetric TAGs, used in this study, having both a palmitic acid moiety and an oleic acid (or a linoleic acid) moiety at the sn-1 or sn-3 positions are resolved by the chiral column. This new chiral separation method can be used in combination with atmospheric pressure chemical ionization mass spectrometry to determine the sn-OOP/sn-POO ratio in palm oil. This method is applicable for the chiral separation of asymmetric TAGs in palm oil.