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).     

Enantioseparation by CE with vancomycin as chiral selector: Improving the separation performance by dynamic coating of the capillary with poly(dimethylacrylamide)

An approach for improving the separation performance of the enantioseparation by CE with vancomycin as chiral selector is described. In the present method, a solution of poly(dimethylacrylamide) (PDMA) was used for dynamic coating of the capillary wall to minimize the adsorption of vancomycin onto the capillary wall, and to depress the EOF. Compared with the bare fused-silica capillaries and the capillaries coated with the polycationic polymer hexadimethrine bromide (HDB), the PDMA-coated capillary displayed the best separation performance. The resulting coating could withstand hundreds of runs without losing its function. Moreover, a partial filling technique was applied to avoid interference in detection caused by the presence of vancomycin in the buffer. The separation time was shortened when a short-end-injection technique was applied. Several parameters such as buffer pH, vancomycin concentration and plug length of the vancomycin solution for the separation were optimized. Under the optimal conditions, all tested enantiomers, including FMOC amino acids derivatives, ketoprofen and fenoprofen, were baseline-separated in less than 4.2 min.     

Enantioseparation of amino acid derivatives by capillary zone electrophoresis using vancomycin as chiral selector

The separation of racemic derivatized amino acids (N-acetyl) into their enantiomers was achieved using capillary zone electrophoresis employing vancomycin as a chiral selector. Due to the strong absorption properties of the chiral selector at the low wavelengths used, the partial-filling countercurrent method was adopted in order to improve method sensitivity. In the separation system studied, the chiral selector filled only a part of the capillary and, due to the appropriate selection of the pH, was moving in the opposite direction of the analytes keeping the detector free from absorbing compounds. The effect of several experimental parameters on the enantioresolution of analytes was studied, e.g., vancomycin concentration (0-5 mM), pH of the background electrolyte (pH 4-7), capillary temperature (15-35 degrees C), and the presence of an organic modifier in the run buffer (methanol or ethanol or n-propanol). N-Acetyl glutamic acid, serine, cystine, tyrosine, and proline were all baseline-resolved into their enantiomers and the enantioresolution factor (R(s)) was increased by raising the vancomycin concentration. pH 4 allowed the baseline resolution of the five studied analytes in the presence of 2.5 mM of chiral selector and an increase in pH caused a decrease of R(s).     

Separation of β-blocker and amino acid enantiomers on a mixed chiral sorbent modified with macrocyclic antibiotics eremomycin and vancomycin

A mixed chiral sorbent based on silica with immobilized macrocyclic antibiotics eremomycin and vancomycin was synthesized. A possibility of the separation of enantiomers of β-blockers (metoprolol, pindolol, alprenolol, oxprenolol, labetalol, and atenolol) and amino acids (tryptophan, phenylalanine, DOPA, methionine, and acetyl glutamic acid) on this chiral sorbent by HPLC was studied. The influence of the composition of the mobile phase (pH of buffer solution, its concentration, content of organic modifier, and its nature) on the retention times of β-blocker and amino acid enantiomers, selectivity, and resolution of peaks was studied. It was shown that the mixed chiral sorbent has enantioselectivity to both classes of compounds, while silica modified with vancomycin has no ability to the separation of enantiomers of non-derivatized amino acids, and silica modified with eremomycin has no ability to the separation of β-blocker enantiomers. High values of resolution for amino acids (max Rs > 4) and β-blockers (max Rs > 1) were obtained.     

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.     

Enantioselective separation of the novel antidepressant mirtazapine and its main metabolites by CEC

In this work, the simultaneous enantioseparation of the second-generation antidepressant drug mirtazapine and its main metabolites 8-hydroxymirtazapine and N-desmethylmirtazapine by chiral CEC is reported. The separation of all enantiomers under study was achieved employing a capillary column packed with a vancomycin-modified diol stationary phase. With the aim to optimize the separation of the three pairs of enantiomers in the same run, different experimental parameters were studied including the mobile phase composition (buffer concentration and pH, organic modifier type and ratio, and water content), stationary phase composition, and capillary temperature. A capillary column packed with vancomycin mixed with silica particles in the ratio (3:1) and a mobile phase composed of 100 mM ammonium acetate buffer (pH 6)/H(2)O/MeOH/ACN (5:15:30:50, by vol.) allowed the complete enantioresolution of each pair of enantiomers but not the simultaneous separation of all the studied compounds. For this purpose, a packing bed composed of vancomycin-CSP only was tested and the baseline resolution of the three couples of enantiomers was achieved in a single run in less than 30 min, setting the applied voltage and temperature at 25 kV and 20 degrees C, respectively. In order to show the potential applicability of the developed CEC method to biomedical analysis, a study concerning precision, sensitivity, and linearity was performed. The method was then applied to the separation of the enantiomers in a human urine sample spiked with the studied compounds after suitable SPE procedure with strong cation-exchange (SCX) cartridges.     

Chiral analysis of UV nonabsorbing compounds by capillary electrophoresis using macrocyclic antibiotics: 1. Separation of aspartic and glutamic acid enantiomers

Glycopeptide antibiotics, namely vancomycin or teicoplanin, were evaluated in capillary electrophoresis for the analysis of UV nonabsorbing compounds such as aspartic and glutamic acid enantiomers. Electrophoretic runs were performed in laboratory-made polyacrylamide-coated capillaries using the partial filling-counter current method in order to avoid the presence on the detector path of the absorbing chiral selector. The background electrolyte consisted of an aqueous or aqueous-organic buffer in the pH range of 4.5-6.5 of sorbic acid/histidine and the appropriate concentration of chiral selector. Several experimental parameters such as antibiotic concentration and type, buffer pH, organic modifier, type and concentration of absorbing co-ion (for the indirect UV detection) were studied in order to find the optimum conditions for the chiral resolution of the two underivatized amino acids in their enantiomers. Among the two investigated chiral selectors, vancomycin resulted to be the most useful chiral selector allowing relatively high chiral resolution of the studied compounds even at low concentration. The optimized method (10 mM sorbic acid/histidine, pH 5, and 10 mM of vancomycin) was used for the analysis of real samples such as teeth dentine and beer.     

Comparison of Separation Efficiency of Macrocyclic Glycopeptide-Based Chiral Stationary Phases for the LC Enantioseparation of β-Amino Acids

Direct reversed-phase high-performance liquid chromatographic methods were developed for the separation of enantiomers of eighteen unnatural β-amino acids, including several β-3-homo-amino acids. The direct separations of the underivatized analytes were performed on chiral stationary phases containing macrocyclic glycopeptide antibiotics such as teicoplanin (Chirobiotic T and T2), teicoplanin aglycone (Chirobiotic TAG), vancomycin (Chirobiotic V and V2), and ristocetin A (Chirobiotic R) as chiral selectors. The effects of the organic modifier, mobile phase composition and pH on the separations were investigated. A comparison of the separation performances of the macrocyclic glycopeptide stationary phases revealed that the Chirobiotic T2 column exhibited better selectivity than the Chirobiotic T column for the separation of β-3-homo-amino acid enantiomers; vancomycin or ristocetin A exhibited lower selectivity. The elution sequence was determined in some cases: the S enantiomers eluted before the R enantiomers, with the exception of the Chirobiotic R column, where the elution sequence R < S was observed.     

以万古霉素为流动相添加剂的高效液相色谱法手性分析系统的建立

以丹酰化苯甘氨酸、丹酰化苯丙氨酸、丹酰化丝氨酸、丹酯化丙氨酸为模型化合物，通过对色谱柱类型、缓冲液类型、pH和盐浓度、万古霉素用量、甲醇用量、柱温等色谱参数进行考察，建立了以万古霉素为手性流动相添加剂在普通色谱柱上进行手性分析的色谱系统，并在该系统中成功地拆分了酮洛芬对映体。     

Enantiomeric separation of ketoconazole and terconazole antifungals by electrokinetic chromatography: Rapid quantitative analysis of ketoconazole in pharmaceutical formulations

EKC using a neutral CD as chiral selector was applied in this work to the development of a method enabling the enantiomeric separation of ketoconazole and terconazole antifungals. The influence of different experimental conditions such as temperature, CD concentration, pH, and nature and concentration of the buffer on the enantiomeric resolution of the compounds studied was investigated. The use of 10 mM heptakis-(2,3,6-tri-O-methyl)-beta-CD in a 100 mM phosphate buffer (pH 3.5) with a temperature of 15 degrees C allowed the separation of the enantiomers of ketoconazole and terconazole with high resolution (R(s) > 2.0). The rapid separation of ketoconazole enantiomers with an analysis time less than 3 min was carried out after fitting some experimental parameters. The developed method was applied to the determination of ketoconazole in different pharmaceutical formulations.     

Optimization and Parameter Study for Chiral Separation by Capillary Electrophoresis

Orthogonal design and uniform design were used for the optimization of separation of enantiomers using 2,6-di-O-methyl-β-cyclodextrin (DM-β-CD) as a chiral selector by capillary zone electrophoresis. The concentration of DM-β-CD, buffer pH, running voltage, and capillary temperature were selected as variable parameters, their different effects on peak resolution were studied by the design methods. It was concluded that orthogonal design offers a rapid and efficient means for testing the importance of individual parameters and for determining the optimum operating conditions. However, for a large number of both factors and levels, uniform design is more efficient. The effect of addition of methanol and citric acid buffer on the separation of enantiomers was also examined.     

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.     

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.     

Sol‐gel technique for the preparation of β‐cyclodextrin gold nanoparticles as chiral stationary phase in open‐tubular capillary electrochromatography

A novel open‐tubular capillary electrochromatography column coated with β‐cyclodextrin was prepared using the sol‐gel technique. In the sol‐gel approach, owing to the three‐dimensional network of sol‐gel and the strong chemical bond between the stationary phase and the surface of capillary columns, good chromatographic characteristics and unique selectivity in separating enantiomers were shown. The influences of capillary inner diameter, coating time, organic modifier, buffer pH, and buffer concentration on separation were investigated. The sol‐gel‐coated β‐cyclodextrin column has shown improved enantioseparation efficiency of chlorphenamine, brompheniramine, pheniramine, zopiclone in comparison with the sol‐gel matrix capillary column. The migration time relative standard deviation of the separation of the enantiomers was less than 0.89% over five runs and 2.9% from column to column. This work confirmed that gold nanoparticles are promising electrochromatographic support to enhance the phase ratio of open‐tubular capillary electrochromatography column in capillary electrochromatography.     

Chiral Separation of Ketoprofen on a Chirobiotic T Column and Its Chiral Recognition Mechanisms

Purpose

In this study, direct separation of ketoprofen enantiomers was performed on a Chirobiotic T column.

Methods

The effects of the type and amount of the organic modifier, buffer concentration, pH value, temperature and flow rate on retention and selectivity were investigated. Experiments were carried out in the temperature range of 20–40 °C to study the effects of temperature. Thermodynamic parameters were calculated from plots of ln k or ln α versus 1/T. Molecular dynamics simulation was done to investigate interactions between ketoprofen enantiomers and the chiral selector—teicoplanin.

Results

It was observed that pH and flow rate had a large influence on resolution. Baseline separation of ketoprofen enantiomers could be achieved with low amounts of methanol, high temperature and high buffer concentrations.

Conclusions

Results from a thermodynamic study and molecular dynamics simulation show that steric hindrance effect, π–π complexation, hydrogen bonding and electrostatic forces are the main driving forces which cause chiral recognition of ketoprofen enantiomers.

     

Separation of enantiomers of <Emphasis Type="Italic">N</Emphasis>-derivatives of amino acids by capillary electrophoresis using macrocyclic antibiotics

The potential for the application of macrocyclic antibiotic eremomycin as a chiral selector for the separation of enantiomers of N-derivatives of amino acids in capillary electrophoresis was estimated. The influence of several factors (the composition and pH of the running electrolyte, the concentration of the chiral selector, capillary geometry, the value of the applied voltage) on the migration of the derivatives of amino acids and enantioselectivity in the presence of eremomycin was studied in order to choose the separation conditions. Using the example of the dansyl derivatives of amino acids the enantioselectivities of vancomycin and eremomycin in capillary electrophoresis were compared.     

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.     

Method development and validation for the separation and determination of omeprazole enantiomers in pharmaceutical preparations by capillary electrophoresis

A new capillary zone electrophoresis (CZE) method for the separation of omeprazole enantiomers has been developed. Methyl-β-cyclodextrin (methyl-β-CD) was chosen as the chiral selector, and several parameters, such as cyclodextrin structure and concentration, buffer concentration, pH, and capillary temperature were investigated in order to optimize separation and run times. Analysis times, shorter than 8 min were found using a background electrolyte solution consisting of 40 mM phosphate buffer adjusted to pH 2.2, 30 mM β-cyclodextrin and 5 mM sodium disulphide, hydrodynamic injection, and 15 kV separation voltage. Detection limits were evaluated on the basis of baseline noise and were established 0.31 mg/l for the omeprazole enantiomers. The proposed method was applied to five pharmaceutical preparations with recoveries between 84 and 104% of the labeled contents.     

Enantiomeric and isomeric separation of herbicides using cyclodextrin-modified capillary zone electrophoresis

Cyclodextrin-modified capillary zone electrophoresis (CD-CZE) was applied successfully to the enantiomeric and isomeric separation of three herbicides (imazaquin, diclofop and imazamethabenz). Commercially available cyclodextrins were evaluated for separation of the enantiomers and isomers of the three herbicides having varied molecular structures. The enantiomers of imazaquin and diclofop, and the isomers of imazamethabenz could be resolved with a resolution of ≥1.5. The resolution was found to depend on pH of the run buffer, cyclodextrin type and cyclodextrin concentration. By employing mixed cyclodextrins in the running buffer, the three herbicides were simultaneously separated in a single run. In addition, rapid (less than 3 min) enantiomeric separation is demonstrated using imazaquin as a model herbicide. The reported capillary electrophoresis (CE) methods are simple, rapid, efficient and reproducible and our results demonstrate that CE provides a powerful analytical tool for enantiomeric and isomeric separation of herbicides.     

Enatiomeric analysis of simendan by CE with beta-CD as chiral selector compared with CMPA-HPLC

The chiral separation of simendan enantiomers using capillary electrophoresis was studied with beta-cyclodextrin (beta-CD) as chiral selector. The influences of the concentration and pH of borate buffer solution, beta-CD concentration and methanol content in the background electrolyte were investigated. These factors were compared with those in an HPLC with beta-CD as chiral mobile phase additive (CMPA-HPLC). The quantification properties of the developed CE method were examined. A baseline separation of simendan enantiomers was achieved in the background electrolyte of 20 mmol/L borate buffer (pH 11.0) containing 12 mmol/L beta-CD-methanol (50:50 in volume ratio). The CE method is comparable with CMPA-HPLC in chiral resolution, although the optimal pH in CE (11.0) is much higher than that (6.0) in CMPA-HPLC. This chiral CE method is applicable to the quantitative ananlysis and enantiomeric excess value determination of L-simendan.