Chiral separation of bupivacaine hydrochloride by capillary electrophoresis with high frequency conductivity detection and its application to rabbit serum and pharmaceutical injection

Conductivity detection was employed to detect the enantiomers of bupivacaine hydrochloride (Bup), which were separated by high performance capillary electrophoresis. A computer-aided technique was used to calculate the binding energies, and the interaction between Bup enantiomers and cyclodextrins (CDs) is preliminarily discussed. Factors affecting the separation efficiency such as the types and concentration of chiral selectors, running buffer, pH value, separation voltage and capillary inside diameter and length were studied. Under optimized conditions, a baseline separation of Bup enantiomers was achieved in less than 15 min in 4mM NH4Ac-NaAc-HAc (pH 4.00) -0.48mM sulfobutyl ether-beta-cyclodextrin running buffer at a separation voltage of 12 kV. The lowest detectable concentration was 0.052 microg/mL. The proposed method was applied to chiral separation of Bup enantiomers in rabbit serum and pharmaceutical injections.     

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.     

Chiral purity test of bevantolol by capillaryelectrophoresis and high performance liquid chromatography

Two methods for the chiral purity determination of bevantolol were developed, namely capillary electrophoresis (CE) using carboxymethyl-beta-cyclodextrin (CM-beta-CD) as a chiral selector and high-performance liquid chromatography (HPLC) using a chiral stationary phase. In the HPLC method, the separation of bevantolol enantiomers was performed on a Chiralpak AD-H column by isocratic elution with n-hexane-ethanol-diethylamine (10:90:0.1, v/v/v) as mobile phase. In the CE method, bevantolol enantiomers were separated on an uncoated fused silica capillary with 50 mM amonium phosphate dibasic adjusted to a pH 6.5 with phosphoric acid containing 15 mM CM-beta-CD as running buffer. Validation data such as linearity, recovery, detection limit, and precision of the two methods are presented. The detection limits of S-(-)-bevantolol were 0.1% and 0.05% for CE and HPLC method, respectively and R-(+)-bevantolol were 0.15% and 0.05% for CE and HPLC method, respectively. There was generally good agreement between the HPLC and CE results.     

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。     

Stereospecific determination of citalopram and desmethylcitalopram by capillary electrophoresis and liquid-phase microextraction

A chiral capillary electrophoresis (CE) system allowing simultaneous enantiomer determination of citalopram (CIT) and its pharmacologically active metabolite desmethylcitalopram (DCIT) was developed. Excellent chiral separation was obtained using 1% sulfated-beta-cyclodextrin (S-beta-CD) as chiral selector in combination with 12% ACN in 25 mM phosphate pH 2.5. Samples were prepared by liquid-phase microextraction (LPME) based on a rodlike porous polypropylene hollow fibre. CIT and DCIT were extracted from 1 ml plasma made alkaline with NaOH, into dodecyl acetate impregnated in the pores of a hollow fibre, and into 20 mM phosphate pH 2.75, inside the hollow fibre. The acceptor solution was directly compatible with the CE system. Efficient sample clean-up was seen, and the recoveries were 46 and 29% for the enantiomers of CIT and DCIT, respectively, corresponding to 31 and 19 times enrichment. The limit of quantification (S/N=10) was <11.2 ng/ml, intra-day precision was <12.8% RSD, and inter-day precision was <14.5% RSD, for all enantiomers. The validated method was successfully applied to simultaneous determination of enantiomer concentrations of CIT and DCIT in plasma samples from nine patients treated with racemic citalopram. The results confirm LPME-CE as a suitable and promising tool for enantiomeric determination of chiral drugs and metabolites in biological matrices.     

Comparison of chiral electrophoretic separation methods for phenethylamines and application on impurity analysis

A chiral microemulsion electrokinetic chromatography method has been developed for the separation of the enantiomers of the phenethylamines ephedrine, N-methylephedrine, norephedrine, pseudoephedrine, adrenaline (epinephrine), 2-amino-1-phenylethanol, diethylnorephedrine, and 2-(dibutylamino)-1-phenyl-1-propanol, respectively. The separations were achieved using an oil-in-water microemulsion consisting of the oil-component ethyl acetate, the surfactant sodium dodecylsulfate, the cosurfactant 1-butanol, the organic modifier propan-2-ol and 20 mM phosphate buffer pH 2.5 as aqueous phase. For enantioseparation sulfated β-cyclodextrin was added. The method was compared to an already described CZE method, which made use of heptakis(2,3-di-O-diacetyl-6-O-sulfo)-β-cyclodextrin (HDAS) as chiral selector. Additionally, the developed method was successfully applied to the related substances analysis of noradrenaline, adrenaline, dipivefrine, ephedrine and pseudoephedrine monographed in the European Pharmacopoeia 6.     

Enantioselective pharmacokinetics of Carvedilol in human volunteers

Carvedilol is administered as a racemic mixture of the R(+)- and S(-)-enantiomers, although they exhibit different pharmacological effects. To investigate the stereoselective pharmacokinetics, the enantiomeric separation of carvedilol in human plasma was undertaken using capillary electrophoresis (CE). Resolution of the enantiomers was achieved using 2-hydoxypropyl-beta-cyclodextrin as the chiral selector. Phosphate buffer (50 mM, pH 4.0) containing 10 mM of 2-hydoxypropropyl-beta-cyclodextrin was used as electrolytic buffer. Achiral separation was carried out with the same electrolytic buffer without chiral selector. Following a single oral administration of 25-mg carvedilol to 11 healthy, male volunteers, stereoselective pharmacokinetic analysis was undertaken. The maximum plasma concentrations (Cmax) were 48.9 and 21.6 ng/mL for (R)-carvedilol and (S)-carvedilol, respectively, determined by the chiral method. The profiles of the plasma concentration of (RS)-carvedilol showed Cmax of 71.5, 72.2, and 73.5 ng/mL, as determined by the CE, HPLC/FD methods and calculations from the data of the chiral method, respectively.     

Chiral separation and quantitation of pentazocine enantiomers in pharmaceuticals by capillary zone electrophoresis using maltodextrins

The chiral separation of pentazocine was achieved by capillary electrophoresis using oligosaccharides. Enantiomers were separated on 100 mM Tris/H3PO4 buffer (pH 2.5) with 5% maltodextrin as a chiral selector, and migration behavior was monitored at 200 nm. Under these conditions, (-)- and (+)-pentazocine and dextromethorphan (internal standard) migrated within 9 min, and the resolution of pentazocine enantiomers was 2.54. Linear calibration curves were obtained in the range 5-50 microg/ml(-1) for each enantiomer. The detection limit of pentazocine enantiomers was 29 pg, and the recoveries of(-)- and (+)-pentazocine were 98.9 (R.S.D., 3.4%) and 101.4% (R.S.D., 4.3%) with 10 microg/ml(-1), respectively.     

Capillary electrophoresis with (R)-(--)-N-(3,5-dinitrobenzoyl)-alpha-phenylglycine as chiral selector for separation of albendazole sulfoxide enantiomers and their analysis in human plasma.

The electrokinetic separation and analysis of the enantiomers of albendazole sulfoxide (ABZSO), a sulfoxide with a sulfur stereogenic center hepatically formed during therapy with the anthelmintic drug albendazole (ABZ), is reported. Using aqueous or nonaqueous alkaline background electrolytes, ABZSO enantiomers cannot be separated via single use of common neutral cyclodextrins and negatively charged carboxymethyl-beta-cyclodextrin. With the Pirkle-type (R)-(-)-N-(3,5-dinitrobenzoyl)-alpha-phenylglycine ((R)-DNBPG) chiral selector, however, ABZSO enantiomers do separate within a borate background electrolyte of pH 9.0-9.5 and can be detected by UV absorbance at 295 nm. Having untreated fused-silica capillaries and 50 mM (R)-DNBPG, enantiomeric resolution is dependent on capillary i.d., capillary length and operational temperature. Optimized separation is obtained for pH 9.25 and the lowest temperature setting. Preliminary data indicate that the same approach could be employed for analysis of the enantiomers of oxfenbendazole, a chiral anthelmintic sulfoxide employed in veterinary pharmacotherapy. Analysis of plasma extracts of patients under ABZ pharmacotherapy confirmed the known enantioselectivity in the sulfoxidation of ABZ with the (+)-ABZSO being the predominant enantiomer in blood. Commencing with 2 ml of plasma, enantiomers present at >1 microg/ml could be detected only, a limitation which is based upon the strong absorbance of the chiral selector. (R)-DNBPG and ABZSO are negatively charged at pH 9.0-9.5, which prevents the application of a partial filling technique. The mobility of (R)-DNBPG is significantly larger compared to that of ABZSO. A migrating plug-plug approach based upon a plug of (R)-DNBPG migrating across the sample plug in an electroosmosis free environment obtained via a dynamic coating produced by spermine is shown to provide chiral resolution but not increased sensitivity.     

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。     

High performance capillary electrophoresis for determination of the enantiomers of 2-arylpropionic acid derivatives in human serum. Pharmacokinetic studies of ketoprofen enantiomers following administration of standard and sustained release tablets

A stereospecific capillary zone electrophoresis (CZE) method for determination of the enantiomers of some 2-arylpropionic acid derivatives (2-APA, profens) in human serum has been developed. Racemic-ibuprofen (rac-IBP), racemic-flurbiprofen (rac-FBP), racemic-ketoprofen (rac-KTP) and (+)-S-naproxen ((+)-S-NPX--an internal standard) were chosen for these studies. The 2-APA enantiomers were extracted from acidified serum samples using methylene chloride separated in a fused silica capillary. The capillary was filled with a background electrolyte, which consisted of 0.05 M heptakis 2,3,6-tri-O-methyl-beta-cyclodextrin (TMbetaCD) (chiral selector) in 0.02 M triethanolamine-phosphate buffer of pH 5.0. Separation and resolution of the enantiomer mixture were obtained in one analytical run. The calculated electrophoretic parameters of the analytes were as follows: electrophoretic mobility, micorep(-)-R = -0.75 x 10(-4) to -0.30 x 10(-4) cm2/Vs; micorep(+)-S = -0.83 -(-0.38) cm2/Vs and electroosmotic mobility, microEOF = 2.35 x 10(-4) cm2/Vs, migration times, tmigr R = 12.55 - 16.07 min; tmigr s = 13.08 - 16.9 min, resolution factors, RS = 1.88 - 3.70 and chiral selectivity, alpha = 1.16 - 1.34. The method developed for the enantiomers was validated. The calibration curves were linear in the range of 0.5-50.0 microg/ml for FBP or KTP and of 1.0-50.0 microg/ml for IBP enantiomer concentrations. Recovery of the enantiomers from serum was about 90%. At the limit of quantification (LOQ) precision and accuracy were within 15%. The validated method was successfully applied to pharmacokinetic and bioavailability studies on KTP enantiomers in humans after administration of standard and sustained release tablets of rac-KTP. Significant differences in the pharmacokinetic parameters of both formulations were observed and the studied formulations were not bioequivalent.     

毛细管电泳法分离测定血浆样品中盐酸多奈哌齐对映体

目的分离、测定血浆样品中盐酸多奈哌齐对映体的含量。方法建立高效毛细管电泳法血浆中加入内标L-酒石酸布托菲诺后，碱化并以异丙醇-正己烷(3∶97)提取血浆样品，采用未涂层熔融石英毛细管柱(70 cm×50 μm ID)，以25 mmol·L^-1磷酸-三乙胺(pH 2.5)为运行缓冲液，2.5%磺化β-环糊精为手性添加剂，进样电压为-20 kV，进样时间为15 s，运行电压为-20 kV。对盐酸多奈哌齐对映体进行电泳分离，并进行家兔血浆样品中盐酸多奈哌齐对映体定量分析的方法验证。结果在上述电泳条件下，盐酸多奈哌齐对映体可达基线分离。测得血浆样品中R(-)-多奈哌齐的回归方程为：A_s/A_i=0.024 2+0.289 2C，r=0.999 2，S(+)-多奈哌齐的回归方程为：A_s/A_i=0.010 8+0.273 7C，r=0.999 7，线性范围为0.1～5 mg·L^-1，检测限为0.05 mg·L^-1。结论本法与手性柱HPLC法相比，有高效、快速和经济等优点。     

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.     

Enantioselective determination of chlorpheniramine in various formulations by HPLC using carboxymethyl-β-cyclodextrin as a chiral additive

A chiral mobile phase HPLC method is described for chiral separation and determination of chlorpheniramine (CP) enantiomers in various commercial preparations. Chromatographic separation was achieved on a conventional ODS column with a mixture of aqueous sodium phosphate (5 mM) containing 0.5 mM carboxymethyl-β-cyclodextrin, methanol and triethylamine (73:25:2, v/v/v, pH 4.3) as the mobile phase. The flow rate of isocratic elution was 0.24 mL/min and peaks were detected at 224 nm. The method was applied to nine commercial CP preparations in six dosage forms and CP enantiomers were well separated without any disturbance of other ingredients or impurities present. The results showed that only one preparation was d-CP and the others were dl-CP preparations. The contents of all the preparations were found to be in the range of 97%–104% of labeled contents. This method was economical and convenient, affording sufficient accuracy, precision and reproducibility, as well as sensitivity and selectivity.     

Use of teicoplanin stationary phase for the enantiomeric resolution of atenolol in human urine by nano-liquid chromatography-mass spectrometry

Nano-liquid chromatography (nano-LC) was used for the enantiomeric resolution of atenolol employing a teicoplanin modified silica stationary phase prepared in our laboratory. Experiments were carried out in a fused silica capillary of 75 microm i.d. packed with chiral modified silica particles of 5 microm diameter. Separated enantiomers were revealed by on-line UV detector at 205 nm or electrospray-ion-trap mass spectrometer (ESI-MS). Atenolol enantiomers were eluted utilizing a mobile phase with the following composition: 500 mM ammonium acetate pH 4.5/methanol/acetonitrile 1:60:39 (v/v/v) allowing to achieve good enantioresolution in a reasonable analysis time (about 8 min) with a flow rate of about 900 nL/min. After comparing the sensitivity of the nano-LC method using a conventional UV detector for capillary electrophoresis, a zeta cell (3 nL volume) employed in nano-LC and the ion-trap MS the method was validated with the MS detector offering the highest sensitivity (limit of detection (LOD)=50 ng/mL; limit of quantification (LOQ)=400 ng/mL for each atenolol enantiomer). (-)-Psi-Nor-ephedrine was used as the internal standard. The method was successfully applied to the analysis of atenolol enantiomers present in human urine samples of a patient under atenolol therapy.     

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.     

Chiral discrimination of sibutramine enantiomers by capillary electrophoresis and proton nuclear magnetic resonance spectroscopy

Capillary electrophoresis (CE) and proton nuclear magnetic resonance spectroscopy (¹H-NMR) have been used to discriminate the enantiomers of sibutramine using cyclodextrin derivatives. Possible correlation between CE and ¹H-NMR was examined. Good correlation between the ¹H-NMR shift non-equivalence data for sibutramine and the degree of enantioseparation in CE was observed. In CE study, a method of enantiomeric separation and quantitation of sibutramine was developed using enantiomeric standards. The method was based on the use of 50 mM of phosphate buffer of pH 3.0 with 10 mM of methyl-beta-cyclodextrin (M-β-CD). 0.05% of LOD, 0.2% of LOQ for S-sibutramine enantiomer was achieved, and the method was validated and applied to the quantitative determination of sibutramine enantiomers in commercial drugs. On a 600 MHz ¹H-NMR analysis, enantiomer signal separation of sibutramine was obtained by fast diastereomeric interaction with a chiral selector M-β-CD. For chiral separation and quantification, N-methyl proton peaks (at 2.18 ppm) were selected because of its being singlet and simple for understanding of diastereomeric interaction. Effects of temperature and concentration of chiral selector on enantiomer signal separation were investigated. The optimum condition was 0.5 mg/mL of sibutramine and 10 mg/mL of M-β-CD at 10°C. Distinguishment of 0.5% of S-sibutramine in R-sibutramine was found to be possible by ¹H-NMR with M-β-CD as chiral selector. Host-guest interaction between sibutramine and M-β-CD was confirmed by ¹H-NMR studies and CE studies. A Structure of the inclusion complex was proposed considering ¹H-NMR and 2D ROESY studies.     

毛细管电泳测定头孢曲松钠对映体含量

目的 :建立测定头孢曲松钠对映体含量的CD毛细管电泳方法。方法 :在不同电极性的条件下 ,运用 β-环糊精 (CD)手性添加剂毛细管电泳方法 ,同时考察了背景电解质pH值及 β-CD浓度对手性拆分的影响。结果 :拆分头孢曲松钠对映体的最佳条件 :缓冲液为50mmol/Lβ-CD ,3 0mmol/L三羟甲基氨基甲烷 ,分离电压为28kV ,pH为7 15。结论 :实验结果表明 ,在优化的实验条件下 ,头孢曲松钠对映体得到了基线分离。用该方法测定不同厂家的头孢曲松钠中两种对映体的含量 ,结果表明 ,不同厂家的头孢曲松钠中对映体的含量明显不同 ,故应建立控制头孢曲松钠对映体质量的方法。该方法可为药品质量控制及临床有效选择抗菌药物提供理论依据。     

HPLC enantioseparation of potential beta-blockers of the aryloxyaminopropanol type. Study of the mechanism of enantioseparation, Part VIII

This paper presents the results of HPLC enantioseparation of derivatives of aryloxyaminopropanols obtained using six chiral stationary phases [macrocyclic antibiotic (vancomycin, teicoplanin, teicoplanin aglycone, and methylated teicoplanin aglycone) and cyclodextrin (beta and gamma)] and a mixture of methanol/acetonitrile/acetic acid/triethylamine (45/55/0.3/0.2) as the mobile phase. No significant difference was observed in the separation of the enantiomers on the vancomycin and teicoplanin chiral stationary phases. Comparing the separation of enantiomers on teicoplanin-based columns the retention factors were increased in the order: native teicoplanin < teicoplanin aglycone < methylated teicoplanin aglycone. The highest values of resolution were obtained on the column containing carbohydrate moieties. The presence of saccharide moieties in the chiral stationary phase plays an important role, together with charge interactions and steric interactions, in the separation of enantiomers of derivatives of aryloxyaminopropanol.     

3种新型α1-受体阻断剂的手性流动相HPLC分离与制备

目的建立3种新型α₁-受体阻断剂的手性流动相HPLC分离与半制备方法。方法通过探讨有机相、手性添加剂、流动相的pH、扫尾剂、反相固定相对手性分离的影响,选择最佳的手性分离条件。结果基线分离了3种新型α₁-受体阻断剂对映体,并制备了毫克级样品。结论所建立的分离与制备方法可用于该类新药的手性研究与开发。