Systematic screening approach for chiral separations of basic compounds by capillary electrophoresis with modified cyclodextrins

A simple, systematic method was developed for rapidly screening potential capillary electrophoresis (CE) separation conditions for small, amine-containing enantiomers. During method development, 39 pairs of enantiomers were investigated and partial or complete separation was achieved in every case. Baseline resolution was achieved by these initial screening conditions in over half of the cases. The screening strategy uses a bare fused silica capillary and a pH 2.5 amine-modified phosphate buffer containing one of the selected cyclodextrins (CD): dimethyl-beta-CD, hydroxypropyl-beta-CD, hydroxypropyl-alpha-CD, hydroxypropyl-gamma-CD and sulfated-beta-CD. An additional set of compounds have been screened by this approach to demonstrate the validity of the method. The paper outlines the experimental work carried out to develop the screen and describes how one might implement it for a new compound.     

Enantiomeric purity determination of tamsulosin by capillary electrophoresis using cyclodextrins and a polyacrylamide-coated capillary

The chiral separation of racemic tamsulosin hydrochloride (TH) was carried out using cyclodextrin (CD)-mediated capillary electrophoresis (CE) with DAD at 200 nm. The best separation of enantiomers of the studied compound was achieved at 20 kV with 30 cm x 50 microm I.D. polyacrylamide (PAA)-coated fused-silica capillary (effective length 20 cm) and running buffer with sulfated-beta-CD (S-beta-CD) as chiral selector. Other selected native or derivatized CDs were also tested: beta-CD (5, 15 mmol l(-1)), carboxymethyl-beta-CD (5, 30 mmol l(-1)), dimethyl-beta-CD (15 mmol l(-1)) and hydroxypropyl-beta-CD (5, 30 mmol l(-1)). Several parameters such as capillary pretreatment, buffer type and concentration, pH of background electrolyte, methanol content, separation temperature and voltage, were optimized. The excellent baseline separation of chiral TH was successfully achieved within 12 min using 100 mmol l(-1) phosphate buffer with pH 2.5 containing 1.7 mmol l(-1) S-beta-CD. Rectilinear calibration range was 50.0-500.0 mumol l(-1) of each enantiomer (r = 0.9993-0.9996). The method was applied to the assay of R-TH in Omnic, capsules (nominal content 0.4 mg per capsule) with R.S.D. 2.75% (n = 6), recovery 99.3-101.7% and it was suitable for the chiral purity control of the active enantiomer in the pharmaceutical.     

Enantioseparation of vesamicol in human serum by capillary electrophoresis with solid phase extraction and sulfated-beta-cyclodextrin

An enantioseparation of racemic vesamicol in human serum by capillary electrophoresis with solid phase extraction and sulfated B-cyclodextrin (S-B-CD) is presented The separation was achieved on an uncoated 72 cm x 50 microm id fused silica capillary maintained at 30 degrees C and + 15 kV applied voltage using a run buffer of 128 micro-B-CD in 50 mM phosphate buffer at pH 5. The detection wavelength was 260 nm. Bond Elut C18 solid phase extraction cartridges were used in the sample preparation of the vesamicol samples from serum. Among the CDs studied, the migration order of the enantiomers was reversed in CM-B-CD compared to S-B-CD. Increases in migration time and differences in time between enantiomers was observed with increasing concentrations of S-B-CD. Baseline separation was achieved in the 2-20 microg/ml range of enantiomer concentration (r > .996). A sample stacking technique was used to improve peak shape and LOD. LODs were 0.5 microg/ml for each enantiomer. Studies of various factors and CE conditions showed the effect of CD type, CD concentration, buffer type, buffer concentration and pH on stability and resolution.     

多聚乙酰神经氨糖酸毛细管电泳拆分新药马来酸氨氯地平对映体及手性分离机制

目的将多聚乙酰神经氨糖酸用作分离双氢吡啶类药物对映体的毛细管电泳手性选择剂，建立新药马来酸氨氯地平对映体的拆分方法。方法考察了手性选择剂浓度、运行缓冲液pH值、毛细管温度、工作电压和多糖分子量等因素对手性分离的影响。结果马来酸氨氯地平两对映体得到了完全分离，分离度为2.20，并研究了多聚乙酰神经氨糖酸对马来酸氨氯地平的手性识别机理。结论本法操作简便，可用于该新药对映体的分离分析。     

手性药物高效毛细管电泳拆分方法的研究进展

高效毛细管电泳法（high performance capillary electrophoresis,HPCE）在手性药物拆分领域得到了广泛的应用。目前,手性离子液体用于毛细管电泳拆分手性药物时,常与环糊精（CD）类手性选择剂构成二元体系产生协同作用,增强了手性离子液体潜在的手性拆分能力。依据对映体拆分的手性选择剂的种类及浓度、缓冲液的浓度和pH、电泳工作电压和温度,选出手性拆分的最佳条件。综述手性药物的发展以及应用HPCE拆分手性药物的文献资料,并对其研究的新进展作了分析。     

Chiral separation of oxprenolol by affinity electrokinetic chromatography-partial filling technique using human serum albumin as chiral selector

The intrinsic characteristics of capillary electrophoresis have made this technique a powerful tool in the chiral separation field. The present paper deals with the enantiomeric separation of oxprenolol enantiomers by affinity electrokinetic chromatography-partial filling technique using human serum albumin (HSA) as chiral selector. Several experimental conditions and variables affecting the separation such as pH, HSA concentration and plug length, background electrolyte concentration, temperature and voltage were studied. Baseline separation of oxprenolol enantiomers was obtained in less than 8 min under the following selected conditions: electrophoretic buffer composed of 50 mM Tris-(hydroximethyl)-aminomethane (Tris) at pH 8.5; 190 microM HSA solution applied at 50 mbar for 225 s as chiral selector; oxprenolol samples contained 190 microM HSA solution injected hydrodynamically at 30 mbar for 2s and the electrophoretic runs performed at 30 degrees C applying 15 kV voltage. The proposed methodology was applied for the analysis of two pharmaceutical preparations. Resolution, accuracy, reproducibility, speed and cost of the proposed method make it suitable for quality control of the enantiomeric composition of oxprenolol in drugs. The results show that a different affinity between oxprenolol enantiomers and HSA exists and can contribute to the pharmacokinetic differentiation of these enantiomers.     

Enantiomeric determination of ephedrine derivatives in unregulated drugs using capillary electrophoresis

Capillary electrophoresis (CE) was applied to enantiomeric separation of chiral ephedrine derivatives (d/l-ephedrine, d/l-methylephedrine, d/l-pseudoephedrine, and d/l-norephedrine) in unregulated drug products. Unregulated drugs, referred to as dietary supplements in U.S.A., have been used legally as tonic agents, but illegal substances such as ephedrine were often detected. Baseline separation of all enantiomers of ephedrine derivatives was achieved using an electrophoretic solution containing heptakis (2,6-di-O-methyl)-beta-cyclodextrin (DM-CD) as a chiral selector. The optimal conditions were established to be: capillary column of fused silica (50 microm i.d. x 56 cm); running buffer of 20 mM DM-CD with 50 mM potassium dihydrogenphosphate background electrolyte, pH 2.6; capillary temperature of 20 degrees C; applied voltage of 30 kV; on-column detection at 195 nm; and injection pressure of 50 mbar x 3 s. Under these conditions, all four pairs of enantiomers were sufficiently resolved, and eight peaks were observed with resolution factors of greater than 1.5. The calibration curves of all enantiomers showed good linearity over the concentration range of 2.5-10 microg/ml (r =0.999). The present method was used in a survey of marketed products. The resultant chiral contents were reported and the analytical data were also compared with those from HPLC. This method is useful in the simple and rapid analysis of ephedrine derivatives in marketed products.     

一叶萩碱的高效毛细管电泳手性分离及其大鼠体内立体选择性代谢研究

目的建立一叶萩碱(SE)的高效毛细管电泳手性分离方法。方法以羟丙基-β-环糊精(HP-β-CD)为手性选择剂,测定条件为:分离介质32 mmol·L^-1 HP-β-CD的Tris-H₃PO₄缓冲液(40 mmol·L^-1,H₃PO₄调至pH 6.0);分离电压15 kV,柱温16℃,压力进样6 s,检测波长254 nm;大鼠各生物样品碱化后乙酸乙酯萃取。结果测定条件下SE基本达到基线分离,大鼠生物样品测定不受内源及代谢物干扰。大鼠ip SE经胆汁、尿和粪排泄以D型为主,具有立体选择性。结论本法简便可靠,可适用于SE在大鼠体内立体选择性代谢研究。     

高效毛细管电泳法拆分氧氟沙星对映体的研究

用牛血清白蛋白为手性选择剂,异丙醇为修饰剂,应用毛细管电泳法拆分氧氟沙星对映体,实验时柱温为30℃,电泳电压15kV,采用50mg·ml^-1牛血清白蛋白—5%异丙醇—磷酸盐缓冲液(pH6.0)为电泳电解液,检测波长为293nm,得到了良好拆分结果。左、右旋成分迁移时间及峰面积的RSD分别为1.6%,1.8%和1.1%,2.8%。应用于氧氟沙星产品研究,其左、右旋成分峰面积比值为0.995～0.998,RSD为1.8%～2.0%。并研究了pH值、牛血清白蛋白浓度、异丙醇浓度、柱温、电泳电压对对映体迁移时间及分辨率的影响,方法简便快速。     

Chiral separation of β2-agonists by capillary electrophoresis using hydroxypropyl-α-cyclodextrin as a chiral selector

Enantiomers of five racemic beta2-agonists were investigated by capillary electrophoresis employing a hydroxypropyl-beta-cyclodextrin (HP-beta-CD). The effects of the concentration of HP-beta-CD added to the background electrolyte and of the pH of the buffer on the effective mobility and resolution of the studied compounds were examined. Very good resolution was achieved for terbutaline and clenbuterol; salbutamol and bambuterol was able to be partially resolved. Enantioselectivity and resolution were influenced by the concentration of the HP-beta-CD, buffer composition and pH.     

Quantitative determination of vasicine and vasicinone in Adhatoda vasica by high performance capillary electrophoresis

A new method of capillary electrophoresis was developed for the quantitative determination of vasicine and vasicinone from Adhatoda vasica (L.) Nees. The electrophoretic separation was performed using a 47 cm x 50 microm ID (38.5 cm effective length) fused silica capillary. The samples were injected by pressure for 3 s at 50 mbar and the running voltage was 19 kV at the injector end of the capillary. The capillary temperature was maintained at 40 degrees C. The separation of the two alkaloids has been achieved within 11 min with good repeatability. The method was validated in terms of reproducibility, linearity, accuracy and applied for the quantitative determination of vasicine and vasicinone in A. vasica plant samples/extracts. Parameters affecting the resolution such as pH, temperature, organic modifier, buffer concentration and capillary dimensions were reported.     

Optimisation, validation and application of a capillary electrophoretic method for the determination of zafirlukast in pharmaceutical formulations

Zafirlukast is a selective and competitive orally administered inhibitor of the cysteinyl leukotrienes and currently indicated for the prophylaxis and treatment chronic asthma. A simple, rapid, reliable capillary zone electrophoresis method for the determination of ZAF in pharmaceutical formulations was developed and validated. The influence of buffer concentration, buffer pH, organic modifier, capillary temperature, applied voltage and injection time was systemically investigated in a fused silica capillary (i.d. 50 microm, total length 80.5 cm and effective length 72.0 cm). Optimum results were obtained with 50mM borate buffer at pH 8.50, capillary temperature 25 degrees C and applied voltage 30 kV. The samples were injected hydrodynamically for 3s at 50 mbar. Detection wavelength was set at 240 nm. Meloxicam was used as internal standard. The method was suitably validated with respect to linearity, limit of detection and quantification, accuracy, precision, selectivity, robustness and ruggedness. The linear calibration range was 2.00-80.00 microg mL(-1) and the limits of detection and quantification were 0.75 and 2.00 microg mL(-1) with R.S.D. of 3.88 and 2.75%, respectively. The proposed method was applied for the determination of ZAF in its pharmaceutical formulations. The results obtained from developed method were compared with a HPLC method reported in the literature and no significant difference was found statistically.     

Quality control of pharmaceutical formulations containing cisplatin, carboplatin, and oxaliplatin by micellar and microemulsion electrokinetic chromatography (MEKC, MEEKC)

A micellar electrokinetic chromatography (MEKC) method was developed for the determination of cisplatin, carboplatin, and oxaliplatin in pharmaceutical formulations. The background electrolyte consisted of a phosphate buffer (pH 7.0; 25 mM) with sodium dodecyl sulfate (80 mM). The applied voltage was 30 kV and the sample injection was performed in the hydrodynamic mode. All analyses were carried out in a fused silica capillary with an internal diameter of 50 μm and a total length of 64.5 cm. The detection of target compounds was performed at 200 nm. Under these conditions, a complete separation of cisplatin, carboplatin and oxaliplatin was achieved in less than 10 min. The MEKC-UV method was validated and trueness values between 99.7% and 100.8% were obtained with repeatability and intermediate precision values of 0.7-1.4% and 1.1-1.7%, respectively for the three drugs. This method was found appropriate for controlling pharmaceutical formulations containing platinum complexes and successfully applied in quality control at the Geneva University Hospitals.     

Application of avidin-phospholipid vesicle complex as the stationary phase for chiral separation in open-tubular capillary electrochromatography

In the present study, we developed a novel open-tubular capillary electrochromatographic method using avidin-phospholipidvesicle complex as the stationary phase for chiral separation of mexiletine hydrochloride. The avidin immobilized on the phospholipid vesicle consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and L-α-phosphatidyl-L-serine (PS) (80:20, mg%) was coated in the capillary. The homogeneity and separation performance of the coating were evaluated in terms of phospholipid vesicle characterization and the resolution of D,L-Tryptophan. As for mexiletine hydrochloride, four vital parameters affecting the separation efficiency of coating capillary, including buffer type, buffer pH, buffer concentration and the applied voltage, were studied in detail. Under the optimum conditions, the enantiomers could be separated well with good resolution. All the satisfactory results indicated that this method using avidin-phospholipid vesicle complex as the stationary phase was suitable and feasible, which had great potential in pharmaceutical separation of enantiomers.     

磺丁基醚-β-环糊精的高效毛细管电泳-间接UV法分析

建立了高效毛细管电泳-间接UV法测定药用辅料磺丁基醚-B-环糊精(SBE-β-CD).采用未涂层熔融石英毛细管柱,以30 mmol/L苯甲酸-三羟甲基氨基甲烷(Tris)(pH 7.5)为运行缓冲液,分离电压30 kV,检测波长214 nm.考察了缓冲液类型与pH、检测波长、电压、温度和样品浓度对测定的影响,并评价了不同批次的SBE-β-CD.     

A new HPLC technique for the separation of methocarbamol enantiomers.

We have developed a stereoselective high-performance liquid chromatography technique for analytical separation of methocarbamol enantiomers. Precolumn derivatization was performed at room temperature using (-)-menthylchloroformate as a chiral reagent in the presence of pyridine as catalyst. The resulting diastereomers were separated on two Resolve C18 columns connected in series. The mobile phase was phosphate buffer (pH 7.5)-acetonitrile (50: 50, v/v) at a flow rate of 1 mL min(-1). UV detection was set at 274 nm. The optimum amount of reagent and the maximum peak intensity of the diastereomers were determined. The resolution of the diastereomers was satisfactory (alpha = 1.04) under the conditions used.     

苯磺酸氨氯地平对映体的高效毛细管电泳手性分离

目的：建立一种快速有效拆分苯磺酸氨氯地平对映体的分析方法。方法：采用高效毛细管电泳法。以羟乙基-β-环糊精为手性选择剂对苯磺酸氨氯地平对映体进行拆分,并考察了缓冲液的pH值、手性选择剂的浓度、缓冲液浓度、电压及柱温等对分离效果的影响。结果：确定了最佳分离条件：背景电解质为含30mmol·L^-1羟乙基-β-环糊精的100mmol·L^-1磷酸-三乙醇胺（pH4．0）体系,电压为28kV,柱温为20℃,进样压力为6kPa·s,检测波长为214nm。在此条件下,10分钟内完全分离了1．0g·L^-1苯磺酸氨氯地平外消旋体,分离度为4．5,并测得3批市售苯磺酸左旋氨氯地平片的光学纯度均不低于99．6％。结论：该法可快速有效地分离苯磺酸氨氯地平对映体,可用于其手性拆分及光学纯度测定。     

毛细管电泳法拆分盐酸安非他酮

目的:建立一种用毛细管电泳拆分盐酸安非他酮对映异构体的色谱方法。方法:以60 cm×75μm石英毛细管柱为分离通道,通过考察影响拆分的因素,如手性选择剂的种类、浓度,缓冲溶液的pH,分离电压和柱温,确定最佳拆分条件。结果:安非他酮拆分的最优条件为浓度15 mmol·L-1羟丙基-β-环糊精作手性添加剂,50 mmol·L-1的磷酸盐(pH 3.5)背景缓冲液,工作电压20 kV,柱温25℃。确定毛细管电泳中先出峰的是S-盐酸安非他酮。结论:该方法试样用量少、高效、快速、操作简单,环境污染少,可用于盐酸安非他酮的拆分,并为盐酸安非他酮的含量测定和单一对映体药理作用的研究奠定基础。     

Method development and validation for the analysis of meloxicam in tablets by CZE

A capillary zone electrophoresis assay for the analysis of meloxicam has been developed and validated. The influence of buffer concentration, buffer pH, methanol as organic modifier, capillary temperature, applied voltage and injection time was systemically investigated in a fused silica capillary (i.d. 50 microm, total length 44 cm and effective length 35.5 cm). Optimum results were obtained with a 100 mM borate buffer (pH 8.5) containing 5% methanol, capillary temperature 25 degrees C, applied voltage 20 kV and injection time 3 s hydrodynamic injection. The detection wavelength was set to 205 nm. Diflunisal was used as internal standard. The method showed good selectivity, accuracy, precision, linearity and sensitivity according to the evaluation of the validation parameters. The method was applied to the determination of six pharmaceutical preparations including two dosage forms. The relative standard deviation of 7 replicate analyses for each sample was less than 0.66%. The results were compared with a spectrophotometric method reported in literature and no significant difference was found statistically.     

Choice of capillary electrophoresis systems for the impurity profiling of drugs

In order to develop a strategy for the impurity profiling of drugs, the possibilities of some capillary electrophoresis systems were investigated. A mixture containing a drug and some of its possible impurities has been used as a model problem. The test compounds were investigated by capillary zone electrophoresis (CZE) and by micellar electrokinetic chromatography (MEKC). The pH of the CZE buffer was varied, but the two stereoisomers could not be separated. Moreover, CZE is not suitable for neutral compounds. In MEKC, two different types of surfactants, sodium dodecyl sulphate (SDS) and cetyltrimethylammonium bromide (CTAB), have been used and the effect of type and concentration modifier on the separation and the elution window was studied. In the SDS system, both the resolution and the elution window could be increased considerably by the addition of modifier. The use of two MEKC systems of different selectivity seems to be a combination with high potential for the impurity profiling of drugs.