CE, with Hydroxypropyl-β-Cyclodextrin as Chiral Selector, for Separation and Determination of the Enantiomers of Amlodipine in the Serum of Hypertension Patients

A capillary electrophoretic method for separation of the enantiomers of amlodipine in the serum of hypertension patients has been established and validated. The two enantiomers were separated in a fused-silica capillary with phosphate running buffer (75 mmol L^?1, pH 2.5) containing 15 mmol L^?1 hydroxypropyl-β-cyclodextrin (HP-β-CD). The effects on the separation of buffer pH and concentration, separation potential, and concentration of HP-β-CD were investigated. The range of quantitation for both enantiomers was 2.0–16.0 μg mL^?1. Intra-day and inter-day relative standard deviation (RSD; n = 5) was <10%. The limits of detection (LOD) and quantification (LOQ) of the amlodipine enantiomers, at 214 nm, were approximately 0.5 and 0.7 μg mL^?1, respectively (S/N = 3 and 10, respectively; 5-s injection). Recovery was always >85%. Results from enantiomer separation and quantification showed that concentrations of the enantiomers of amlodipine in serum from an elderly patient were higher than in serum from a young patient administered the same dose. The method was useful for determining the concentration of the enantiomers of amlodipine in hypertension patient serum and for monitoring the transition behavior of the enantiomers in humans. The method proved suitable for application to the separation of the enantiomers of amlodipine and analysis of clinical samples.     

Study on the Enantioselective Degradation of Imazethapyr in Soil by CE

A method was developed for the separation of imazethapyr enantiomers using capillary electrophoresis and large volume injection. Optimized buffer conditions were found using 6% hydroxypropyl-β-cyclodextrin as the chiral selector at pH 11 with an injection time of 20 s (0.5 psi). The limits of detection (LOD, S/N = 3) were 0.22 and 0.23 mg L^?1 for the two imazethapyr enantiomers (imazethapyr-I and imazethapyr-II) respectively. The relative standard deviation was less than 5%. This method was successfully used in the study of enantioselective degradation of this herbicide in field soil and the results suggested that imazethapyr-I degraded at a higher rate when compared with imazethapyr-II.     

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.     

Enantiomeric separation of racemic clenbuterol by capillary zone electrophoresis

A method for capillary electrophoretic enantiomeric separation of a racemic clenbuterol has been established with hydroxypropyl-β-cyclodextrin as the chiral selector. General equations and data analysis are presented to relate mobility to the equilibrium constants in simple binding equilibria and used to determine binding constants and thermodynamic parameters for host-guest complexation of clenbuterol enantiomers with hydroxypropyl-β-cyclodextrin as a selector. The effects of β-cyclodextrin type and concentration, buffer type, concentration and pH, as well as separation voltage and capillary temperature were investigated in detail. A maximal resolution of 6.78 was obtained. The binding constants of the host-guest complex of clenbuterol enantiomers with hydroxypropyl-β-cyclodextrin, K _R-CD and K _S-CD are 22.50 and 43.09 l mol^-1, respectively.     

Study on the Enantioselective Degradation of Imazethapyr in Soil by CE

A method was developed for the separation of imazethapyr enantiomers using capillary electrophoresis and large volume injection. Optimized buffer conditions were found using 6% hydroxypropyl-β-cyclodextrin as the chiral selector at pH 11 with an injection time of 20 s (0.5 psi). The limits of detection (LOD, S/N = 3) were 0.22 and 0.23 mg L⁻¹ for the two imazethapyr enantiomers (imazethapyr-I and imazethapyr-II) respectively. The relative standard deviation was less than 5%. This method was successfully used in the study of enantioselective degradation of this herbicide in field soil and the results suggested that imazethapyr-I degraded at a higher rate when compared with imazethapyr-II.

     

Enantiospecific HPLC and CE Methods for Separation and Determination of S-Darifenacin in Pharmaceutical Formulations

Two separation techniques were developed for the determination of S-(−)darifenacin (DAR) in the presence of its R-(+) isomer: The first method is high performance liquid chromatography (HPLC) and the second is capillary electrophoresis (CE). Chiral separation for chromatographic HPLC method development was carried out for S-DAR on Daicel CROWNPAK CR (+) (5 μm, 4.0 × 150 mm) column which contains (3,3-diphenyl-1,1-binaphthyl)-crown-6 coated onto a 5.5 μm silica support. The mobile phase system was aqueous acidic 70 % HClO₄ (pH 2.5): methanol in the proportion of 90:10 v/v. This current mobile phase was delivered at flow rate 0.8 mL min⁻¹ using UV detector adjusted at 286 nm. In CE method, the enantiomers were separated using 50 μm inner diameter fused-silica capillary cut to total lengths of 31.2 cm using 50 mM phosphate buffer as background electrolyte adjusted to pH 2.5 by triethanolamine. A wide range of cyclodextrins (CDs) were used such as highly sulfated α, γ CDs, hydroxyl propyl-β-CD and sulfobutyl ether-β-CD as chiral selectors. The effects of chiral additives regarding its concentration and content of organic modifier on the enantioseparation were investigated. Linear concentration ranges were from 2.5 to 50 and 40 to 300 μg mL⁻¹ with detection limits 0.67 and 12.28 μg mL⁻¹ for chromatographic HPLC and electrophoretic CE methods, respectively. The two methods were validated according to ICH guidelines with respect to linearity, accuracy, precision, LOQ, LOD and robustness. The suggested methods are suitable for separation and quantitation of S-DAR in tablets.

     

Simultaneous Chiral Separation of Geometry Isomers and Enantiomers of Nateglinide by Capillary Electrophoresis with Mixture of CD Derivations as Chiral Selector

Abstract

A capillary electrophoresis (CE) method for the simultaneous separation of geometry isomers and enantiomers of nateglinide was built. Several different dyclodextrin (CD) derivatives were tested for the chiral separation of nateglinide, and it was proved that ionic CDs [i.e., carboxymethy-β-CD (CM-β-CD) and sulphonic-β-CD (S-β-CD)] could show better chiral selectivity for both geometry isomers and enantiomers than the neutral CDs. The separation of geometry of both isomers and enantiomers of nateglinide was obtained by CE in a 75-µm i.d. × 60 cm (effective length 45 cm) fused-silica capillary at 11 kV voltage, while 30 mM phosphate (pH = 8.38) acted as running buffer and a mixture of 40 mM S-β-CD + 21 mM CM-β-CD served as chiral selector. The detective wavelength was set at 254 nm.     

Investigation of chondroitin sulfate D and chondroitin sulfate E as novel chiral selectors in capillary electrophoresis

Various chiral selectors have been utilized successfully in capillary electrophoresis (CE); however, the number of polysaccharides used as chiral selectors is still small and the mechanism of enantiorecognition has not been fully elucidated. Chondroitin sulfate D (CSD) and chondroitin sulfate E (CSE), belonging to the group of glycosaminoglycans, are linear, sulfated polysaccharides with large mass. In this paper, they were investigated for the first time for their potential as chiral selectors by CE. The effect of buffer composition and pH, chiral selector concentration, and applied voltage were systematically examined and optimized. A variety of drug enantiomers were resolved in the buffer pH range of 2.8–3.4 using 20 mM Tris/H₃PO₄ buffer with 5.0 % CSD or CSE and 20 kV applied voltage. A central composite design was used to validate the optimized separation parameters and satisfactory uniformity was obtained. As observed, CSE allowed satisfactory separation of the enantiomers of amlodipine, laudanosine, nefopam, sulconazole, and tryptophan methyl ester, as well as partial resolution of citalopram, duloxetine, and propranolol under the optimized conditions. CSD allowed partial or nearly baseline separation of amlodipine, laudanosine, nefopam, and sulconazole. The results indicated that CSE has a better enantiorecognition capability than CSD toward the tested drugs.

Enantioselective analysis of ofloxacin and ornidazole in pharmaceutical formulations by capillary electrophoresis using single chiral selector and computational calculation of their inclusion complexes

A capillary electrophoretic method for the separation of the enantiomers of both ofloxacin and ornidazole 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 mixtures was achieved in less than 16 min with resolution factors Rs = 5.45 and 6.28 for ofloxacin and ornidazole enantiomers, respectively. Separation was conducted using a bare fused-silica capillary and a background electrolyte (BGE) of 50 mM H₃PO₄-1 M tris solution; pH 1.85; containing 30 mg mL⁻¹ of sulfated-β-cyclodextrin (S-β-CD). The separation was carried out in reversed polarity mode at 25 °C, 18 kV, detection wavelength at 230 nm and using hydrodynamic injection for 15 s. Acceptable validation criteria for selectivity, linearity, precision, and accuracy were studied. The limits of detection (LOD) and limits of quantitation (LOQ) of the enantiomers (ofloxacin enantiomer 1 (OF-E1), ofloxacin enantiomer 2 (OF-E2), ornidazole enantiomer 1 (OR-E1) and ornidazole enantiomer 2 (OR-E2)) were (0.52, 0.46, 0.54, 0.89) and (1.59, 1.40, 3.07, 2.70) μg mL⁻¹, respectively. The proposed method was successfully applied to the assay of enantiomers of both ofloxacin and ornidazole in pharmaceutical formulations. The computational calculations for the enantiomeric inclusion complexes rationalized the reasons for the different migration times between the ofloxacin and ornidazole enantiomers.     

Comparison of enantioseparation of amino acid derivatives in aqueous and mixed aqueous-organic media by capillary zone electrophoresis

The chiral separation of dansyl-amino acids has been performed by capillary zone electrophoresis using ¶β-cyclodextrin as a chiral selector, urea as an additive and 2-propanol and methanol as organic modifiers. The enantiomeric separations of dansyl-amino acids were investigated in aqueous medium and compared with the separation in mixed aqueous-organic medium as background electrolytes. The separation conditions, (concentration of buffer, β-cyclodextrin, methanol, urea and the pH value of buffer) were optimized. In the absence of organic modifier, only five pairs of 8 separated dansyl-amino acids were resolved when run separately. A mixture of up to eight chiral amino acids can be baseline resolved in less than 19 min by β-cyclodextrin-modified capillary zone electrophoresis with a buffer of 60 mmol L^–1 H₃BO₃-KCl/40 mmol L^–1 NaOH (pH 9.0), 4 mol L^–1 urea, 100 mmol L^–1β-cyclodextrin and 10% (v/v) methanol.     

Study on the Effect of Moderate Exercise on Lactic Acid Content in Breast Milk by Indirect CE with Amperometric Detection

An indirect high-performance capillary electrophoresis with amperometric detection (CE-AD) method has been developed for determination of lactic acid (LA) in body fluids of lactating postpartum women. Several important factors such as the running buffer additive and concentration, the working electrode potential, the pH value and concentration of the running buffer, the separation voltage and injection time were investigated. Under the optimum conditions, LA could be well separated with co-existing interferences including uric acid (UA) in real samples in a 90-cm-length capillary at separation voltage of 12 kV in 4.0 × 10^?6 g mL^?1 3,4-dihydroxybenzylamine (DHBA)/40 mmol L^?1 H₃BO₃?CNa₂B₄O₇ buffer (pH 7.8). The linearity between peak current and concentration of LA was over three orders of magnitude with detection limit of 5.00 × 10^?7 g mL^?1 (S/N = 3). This proposed method has been successfully used to study the effects of moderate exercise on LA content in breast milk and urine samples of lactating postpartum women, and assay results showed that LA content in breast milk can return to normal level through 60 min rest without decreasing acceptance by breast-feeding infants, although the LA level did increase by 4?C6 times in both breast milk and urine samples at 10 min after moderate exercise.     

1-Hexadecyl-3-methylimidazolium Ionic Liquid as a New Cationic Surfactant for Separation of Phenolic Compounds by MEKC

The ionic liquid 1-hexadecyl-3-methylimidazolium bromide ([C₁₆MIm]Br) has been used as a novel cationic surfactant for separation of phenolic compounds, including quinol, phloroglucinol, resorcinol, phenol, p-cresol, and m-nitrophenol, by micellar electrokinetic capillary chromatography (MEKC). The effects of buffer concentration and pH, concentration of [C₁₆MIm]Br, and applied potential were studied. Use of the optimized buffer (25 mmol L^?1 NaH₂PO₄), 10 mmol L^?1 [C₁₆MIm]Br, and an applied potential of ?15 kV enables optimum separation with regard to resolution and migration time. The phenolic compounds were detected at 214 nm. The micelle of this long-alkyl-chain imidazolium ionic liquid acts as a pseudo-stationary phase in this MEKC separation.     

Enantioselective Separation of Basic Drugs by CE with Polygalacturonic Acid as a Novel Chiral Selector

Polygalacturonic acid, a linear high molecular weight homopolysaccharide was investigated as a chiral selector in capillary zone electrophoresis for the separation of enantiomers of basic drugs. The choices of running buffer pH and concentration of chiral selector were found to be important for the improvement of enantioselectivity. The effects of background electrolyte concentration and the capillary temperature on the separation were also examined. Enantioseparations were carried out in the acidic conditions using 1.5% polygalacturonic acid (w/v) in a 40 mM phosphate buffer under an applied voltage of 15 kV. The optimization of these separations was dependent on the nature of the analytes and could be achieved by the proper choice of experimental conditions. A brief mechanism of enantiorecognition by polygalacturonic acid was also given.

     

CZE Determination of Mismatched Double-Stranded Oligonucleotides (poly I:poly C12U) in Beagle Serum

A capillary zone electrophoretic (CZE) method with diode-array detection has been established for analysis of mismatched double-stranded oligonucleotides, poly I:poly C₁₂U, in beagle serum. The effects of sample pretreatment, buffer pH, buffer concentration, and applied voltage on the separation of inosine were optimized. Baseline separation was obtained for inosine within 11 min by use of 0.05 mol L^?1 borate running buffer, pH 9.60, and an applied voltage of 25 kV at 25 °C; the detection wavelength was 254 nm. To evaluate the performance of the method and demonstrate the utility of the approach, an experiment was designed in which poly I:poly C₁₂U were added to serum at different concentrations, artificially creating groups of samples. The proposed CZE method seemed a powerful technique for kinetic study of poly I:poly C₁₂U in serum.     

Chiral separation of ketoprofen on an achiral NH<Subscript>2</Subscript> column by HPLC using vancomycin as chiral mobile phase additive

A high-performance liquid chromatographic method for chiral separation of ketoprofen racemate was developed. (R)- and (S)-ketoprofen enantiomers were separated on a LiChrosorb NH₂ column (250 mm × 4.6 mm, i.d 5 µm) at 20 °C, using 2-propanol/potassium dihydrogen phosphate buffer (pH 6.0, 0.05 M) (50:50 v/v). Containing vancomycin as the mobile phase, at a flow rate of 0.8 ml min^?1 and detection wavelength of UV, the detector was set at 310 nm. Under these conditions, ketoprofen enantiomers could be separated with a selectivity factor (α) of 2.172 and a resolution (Rs) of 4.78 using extremely low concentrations of the vancomycin chiral additive.     

Kinetic study on reactive extraction for chiral separation of phenylsuccinic acid enantiomers

The kinetics of the extraction of phenylsuccinic acid (PSA) enantiomers by hydroxypropyl-β-cyclodextrin (HP-β-CD) in a modified Lewis cell was studied, in which HP-β-CD dissolved in 0.1 mol L^?1 NaH₂PO₄/H₃PO₄ buffer solution (pH = 2.5) was selected as the chiral extractant. PSA enantiomers were extracted from organic phase to aqueous phase in the extraction module. The theory of extraction accompanied by a chemical reaction has been used to obtain the intrinsic kinetics of this extraction module. The different parameters affecting the extraction rate such as agitation speed, interfacial area, initial concentration of PSA enantiomers in organic phase as well as HP-β-CD concentration in aqueous phase were separately studied. The experimental results demonstrate that the extraction reactions are fast. The reactions were found to be first order with respect to PSA and second order with respect to HP-β-CD with forward rate constants of 3.4 × 10^?2 m⁶ mol^?2 s^?1 for R-PSA and 9.96 × 10^?3 m⁶ mol^?2 s^?1 for S-PSA. These data will be useful in the design of extraction processes.     

α-萘基缩水甘油醚对映体的毛细管区带电泳手性拆分

建立了毛细管区带电泳手性拆分α-萘基缩水甘油醚对映体的方法.考察了不同手性拆分试剂对手性选择性的影响,实验结果表明,20 mmol/L H3PO4-三乙醇胺(pH 2.5)、2%(w/V)HS-β-CD、毛细管温度20 ℃、运行电压-18 kV为最佳分离条件,在该分离条件下α-萘基缩水甘油醚对映体实现基线分离.方法简便、准确,可用于α-萘基缩水甘油醚的手性拆分和对映体过量值(ee,%)测定.     

Enantiomeric separation of glycidyl tosylate by CE: Application to the study of catalytic asymmetric epoxidation of allyl alcohol

A CE method using CDs as chiral selectors was developed and validated to achieve the separation of glycidyl tosylate enantiomers originated by in situ derivatization of glycidol enantiomers obtained in asymmetric epoxidation of allyl alcohol with chiral titanium‐tartrate complexes as catalysts. The effects of the nature, pH and concentration of the buffer, the nature and concentration of chiral selector, the addition of SDS, methanol, ethanol or 2‐propanol, the capillary temperature, the effective capillary length and the applied voltage on the chiral resolution of glycidyl tosylate enantiomers were investigated. The best separation conditions were achieved using a Tris‐borate buffer mixture (50 and 25 mM, respectively) at pH=9.3 with a dual CD system consisting of 2.5% succinyl‐β‐CD and 1.0% β‐CD w/v at 15°C. A baseline separation (resolution～2.0) of the glycidyl tosylate enantiomers was obtained in a relatively short time (less than 12 min). Satisfactory results were obtained in terms of linearity (r>0.99) and intermediate precision (RSD below 8.5%). The LOD and LOQ were 3.0 and 10.0 mg/L, respectively, and the recoveries ranged from 99.8 to 108.8%. Finally, the method was applied to the determination of the enantiomeric excess and the yield obtained in the asymmetric epoxidation of allyl alcohol employing chiral titanium‐tartrate complexes as catalysts after an in situ derivatization of glycidol enantiomers to glycidyl tosylate.     

Capillary Electrophoresis Method for the Chiral Purity Determination of Pregabalin Derivatized with Dansyl Chloride

A capillary electrophoresis method for the determination of the chiral purity of pregabalin upon derivatization with dansyl chloride was developed using design of experiment methodologies. A D-optimal design was used for the identification of the critical process parameters, while a central composite face centered design and Monte Carlo simulations were employed for defining the design space of the method. Final working conditions consisted of a background electrolyte composed of a 100 mM sodium phosphate buffer, pH 2.5, containing 40 mg mL^?1 heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin, a separation voltage of 15 kV and a capillary temperature of 25 °C. The analyses were carried out in a 40/50.2 cm fused-silica capillary with an inner diameter of 50 µm. Upon testing the robustness using a Plackett–Burman design, the method was validated according to the International Council on Harmonization guideline Q2(R1). The method allowed the detection of the (R)-enantiomer at the 0.015% level with a limit of quantitation at the 0.05% level with regard to a sample containing 1.59 mg mL^?1 pregabalin. The method was subsequently applied to the determination of the stereochemical purity of the drug in commercial capsules.     

Enantioselective Separation of Basic Drugs by CE with Polygalacturonic Acid as a Novel Chiral Selector

Polygalacturonic acid, a linear high molecular weight homopolysaccharide was investigated as a chiral selector in capillary zone electrophoresis for the separation of enantiomers of basic drugs. The choices of running buffer pH and concentration of chiral selector were found to be important for the improvement of enantioselectivity. The effects of background electrolyte concentration and the capillary temperature on the separation were also examined. Enantioseparations were carried out in the acidic conditions using 1.5% polygalacturonic acid (w/v) in a 40 mM phosphate buffer under an applied voltage of 15 kV. The optimization of these separations was dependent on the nature of the analytes and could be achieved by the proper choice of experimental conditions. A brief mechanism of enantiorecognition by polygalacturonic acid was also given.