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.     

Stacking and separation of aspartic acid enantiomers under discontinuous system by capillary electrophoresis with light-emitting diode-induced fluorescence detection

We describe the stacking and separation of d- and l-aspartic acid (Asp) by capillary electrophoresis (CE) with light-emitting diode-induced fluorescence detection (LEDIF). In the presence of cyanide, d- and l-Asp were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) to form fluorescent derivatives prior to CE-LEDIF. The separation of NDA-derivatized d- and l-Asp was accomplished using a discontinuous system - buffer vials contained a solution of 0.6% poly(ethylene oxide) (PEO), 150 mM sodium dodecyl sulfate (SDS), and 60 mM hydroxypropyl-β-cyclodextrin (Hp-β-CD), while a capillary was filled with a solution of 150 mM SDS and 60 mM Hp-β-CD. The role of PEO, Hp-β-CD, and SDS is to act as a concentrating media, as a chiral selector, and as a pseudostationary phase, respectively. This discontinuous system could be employed for the stacking of 600 nL of NDA-derivatized d- and l-Asp without the loss of chiral resolution. The stacking mechanism is mainly based on the difference in viscosity between sample zone and PEO as well as SDS sweeping. The limits of detection at signal-to-noise of 3 for d- and l-Asp were down to 2.4 and 2.5 × 10⁻¹⁰ M, respectively. Compared to normal sample injection volume (25 nL), this stacking approach provided a 100- and 110-fold improvement in the sensitivity of d- and l-Asp, respectively. This method was further applied for determining d- and l-Asp in cerebrospinal fluid, soymilk, and beer.     

Enantiomeric analysis of methotrexate in pharmaceuticals by cyclodextrin-modified capillary electrophoresis

A capillary electrophoresis for the chiral separation of racemic methotrexate (rac-MTX) was developed and validated. The two enantiomers were separated by using fused-silica capillary and a running buffer containing phosphate and hydroxypropyl-β-cyclodextrin (HP-β-CD). Several parameters were studied, including concentration and pH of phosphate buffer, separation voltage, and type and concentration of CD. The quantitative ranges were 12.5-200.0 μM for each enantiomer. The intra- and inter-day relative standard deviations (R.S.D.) and relative errors (R.E.) (n=5) were all <5%. The detection limits were found to be about 4 μM (S/N=3, injection 5 s) at 280 nm. All recoveries were greater than 93%. This method was applied to the assay of l-MTX in pharmaceuticals.     

Electrophoretic separation strategy for chiral pharmaceuticals using highly-sulfated and neutral cyclodextrins based dual selector systems

The enantiomeric separation of chiral pharmaceuticals was investigated using dual systems with mixtures of cyclodextrin derivatives. The dual cyclodextrin systems, consisting of one highly-sulfated (α-, β-, and γ-HSCD) and one neutral cyclodextrin, i.e. either heptakis (2,3,6-tri-O-methyl)-β-CD (TMCD), heptakis (2,6-di-O-methyl)-β-CD (DMCD) or hydroxypropyl-β-CD (HPCD), are tested on 25 pharmaceutical compounds with different acid-basic properties (16 basics, 8 acids and 1 neutral). The influence on the separation of the type and concentration of neutral CD in highly-sulfated cyclodextrins-based dual selector systems, is investigated. For 11 of 16 basic compounds, a better separation is obtained with the CD mixtures compared to the use of only a highly-sulfated CD. Mixtures with TMCD give better results than those with DMCD and HPCD. Results showed that dual CD systems are useful to achieve and to optimise chiral separations of compounds not (sufficiently) separated with HSCDs alone. For example, ibuprofen was not resolved with α-, β- or γ-HSCD, but could be separated with the mixture 25 mM TMCD and 5% HS-β-CD. Based on the obtained results, a dual CD systems based separation strategy is defined.     

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.     

毛细管电泳法和高效液相色谱法拆分联萘二酚酸对映体

采用毛细管电泳法和高效液相色谱法直接拆分2,2′-二羟基-1,1′-联二萘-3,3′-二甲酸(HBNC)对映体.以四种不同的β-环糊精为手性添加剂,考察环糊精的种类与浓度、缓冲液pH值及浓度、分离电压、温度等因素对HBNC分离的影响.结果表明:采用10 mmol/L磺丁基醚-β-环糊精+20 mmol/L磷酸盐缓冲液(pH=7.0),20 kV分离电压,HBNC对映体在20 min内达到基线分离,分离度达到3.31.采用(S)-叔-亮氨酸基-(S)-1-(α-萘基)乙胺手性柱,正己烷-乙醇-三氟乙酸(97∶3∶0.2,V/V)流动相,HBNC对映体在40 min内也基本达到基线分离.     

Methoxypropylamino β-cyclodextrin clicked AC regioisomer for enantioseparations in capillary electrophoresis

In this work, a novel methoxypropylamino β-cyclodextrin (β-CD) clicked AC regioisomer, 6^A-4-hydroxyethyl-1,2,3-triazolyl-6^C-3-methoxypropylamino β-cyclodextrin (HETz-MPrAMCD), was synthesized via nucleophilic addition and click chemistry. The chiral separation ability of this AC regioisomer cationic CD was evaluated toward 7 ampholytic and 13 acidic racemates by capillary electrophoresis. Dependence of enantioselectivity and resolution on buffer pH (5.5–8.0) and chiral selector concentration (0.5–7.5 mM) was investigated. Enantioselectivities (α ≥ 1.05) could be achieved for most analytes under optimal conditions except dansyl-dl-noreleucine and dansyl-dl-serine. The highest resolutions for 2-chloromandelic acid p-hydroxymandelic acid were 15.6 and 9.7 respectively. The inclusion complexation between HETz-MPrAMCD and each 3-phenyllactic acid enantiomer was also revealed with nuclear magnetic resonance study.     

Analysis of enantiomers in biological matrices by charged cyclodextrin-mediated capillary zone electrophoresis in column-coupling arrangement with capillary isotachophoresis

The possibility to apply charged chiral selector as buffer additive in capillary zone electrophoresis (CZE) on-line coupled with capillary isotachophoresis (CITP) was studied. Enantioseparations and determinations of trace (ng/ml) antihistaminic drugs [pheniramine (PHM), dimethindene (DIM), dioxopromethazine (DIO)] present in samples of complex ionic matrices (urine) served as model examples. A negatively charged carboxyethyl-β-cyclodextrin (CE-β-CD) was used as a chiral selector in analytical CZE stage following upon a sample pretreatment by CITP (preconcentration of the analytes from 5 to 20-times diluted urine samples, partial sample clean up removing macroconstituents from the sample matrices). A high recognition capability of the oppositely charged CE-β-CD was demonstrated by enantioselective retardation of the drugs in presence of micro-and semi-macroconstituents migrating in CZE stage and detectable by UV detector. In this way, enantiomers of the drugs could be easily separated and determined. Due to lack of interferences between the drugs and sample-matrix constituents in presence of charged CE-β-CD, demands on both spacers in CITP step and multiple column-switching were minimized. CITP-CZE method with charged selector appeared to be a useful analytical approach for the trace enantiomers in complex ionic matrices as it combined enhanced separation selectivity and sample loadabitlity with high separation efficiency and provided favorable performance parameters including sensitivity, linearity, precision, accuracy/recovery and robustness with minimal demands on sample preparation. Analysis of urine sample taken from a patient treated by PHM, showing concentration profile of PHM enantiomers and their metabolites, illustrated potentialities of the method in clinical research.     

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.     

Computer-aided molecular modeling study of enantioseparation of iodiconazole and structurally related triadimenol analogues by capillary electrophoresis: Chiral recognition mechanism and mathematical model for predicting chiral separation

Chiral separation of iodiconazole, a new antifungal drug, and 12 new structurally related triadimenol analogues had been developed by capillary electrophoresis (CE) using hydroxypropyl-γ-cyclodextrin (HP-γ-CD) as the chiral selector. The effect of structural features of analytes on Δt and R_s was studied under the optimum separation conditions. Using molecular docking technique and binding energy calculations, the inclusion process between HP-γ-CD and enantiomers was investigated and chiral recognition mechanisms were discussed. The results suggest that hydrogen bonding between fluorine at position 4 of the phenyl group beside the chiral carbon and the hydroxyl group on the HP-γ-CD rim and face to face π–π interactions between two phenyl rings highly contributed to the enantiorecognition process between HP-γ-CD and iodiconazole. The N-methyl group beside chiral carbon also played an important role in enantiomeric separation. Additionally, the big difference in binding energy (ΔΔE) highly contributed to good separation in the presence of HP-γ-CD chiral selector, which may be a helpful initial guide for chiral selector selection and predicting the result of enantioseparation. Furthermore, the new mathematical equation established based on the results of molecular mechanics calculations exhibited good capability in predicting chiral separation of these triadimenol analogues using HP-γ-CD mediated CE.     

A novel capillary electrophoresis method for the determination of d-serine in neural samples

A capillary electrophoresis method has been developed for the determination of d-serine in neural samples. d/l-serine was tagged with naphthalene-2,3-dicarboxaldehyde (CBI-d/l-Ser), and the separation of CBI-d/l-Ser enantiomer was achieved by using a dual chiral selector system consisting of β-cyclodextrin (β-CD) and chiral micelles formed by sodium deoxycholate (SDC). No resolution was observed when either β-CD or SDC was used alone. Moreover, the combined use of β-CD with achiral micelles of sodium dodecylsulfate (SDS) exhibited no resolving effect. With laser induced fluorescence detection, the limit of detection was 3.0 × 10⁻⁸ M Ser. Under the separation conditions selected, no other amino acids co-eluted with l-/d-Ser enantiomers. Using the present method, d-Ser level in Aplysia ganglia homogenates was found to vary significantly from animal to animal. Interestingly, d-Ser was not detected in single neurons isolated from Aplysia ganglia.     

Fast enantiomeric separation of propranolol by affinity capillary electrophoresis using human serum albumin as chiral selector: application to quality control of pharmaceuticals

In the last years, capillary electrophoresis (CE) has gained considerable interest in pharmaceutical laboratories for controlling the chiral purity of drugs. This paper describes a simple and fast method for resolution of propranolol enantiomers by affinity capillary electrophoresis (ACE) using human serum albumin (HSA) as chiral selector. The effect of several experimental variables such as HSA concentration, temperature, chiral selector plug length and addition of organic modifiers, on the separation is evaluated. Complete enantioresolution of R- and S-propranolol was achieved in less than 5 min when the capillary was completely filled with 100 μM HSA solution and the electrophoresis was carried out with 67 mM phosphate buffer (pH 7.4) at 20 kV and 35 °C. Peaks were assigned to each propranolol enantiomer according to their relative affinities to HSA. The proposed method was applied to the analysis of pharmaceutical preparations containing propranolol. Resolution, accuracy, reproducibility, cost and sample throughput of the proposed method make it suitable for quality control of the enantiomeric composition of propranolol in pharmaceuticals.     

Fast determination of flavonoids in Hippophae rhamnoides and its medicinal preparation by capillary zone electrophoresis using dimethyl-beta-cyclodextrin as modifier

A fast capillary zone electrophoresis (CZE) method, using dimethyl-β-cyclodextrin (DM-β-CD) as modifier, has been developed for the determination of three flavonoids (quercetin (QU), kaempferol (KA) and isorhamnetin (IS)) in the Chinese herbal extract from Hippophae rhamnoides and its medicinal preparation (Sindacon tablet). Optimum separation was achieved with 20 mM borate buffer at pH 10.0 containing 5 mg ml⁻¹ of DM-β-CD. The applied voltage was 15 kV and the capillary temperature was kept constant at 25 °C. Regression equations revealed linear relationships (correlation coefficients: 0.9973, 0.9992 and 0.9996) between the peak area of each compound (QU, KA and IS) and its concentration. The relative standard deviations of migration times and peak areas were <1.53 and 4.14%, respectively. The effects of several CE parameters on the resolution were studied systematically. The contents of three flavonoids in H. rhamnoides were successfully determined with 4.5 min, with satisfactory repeatability and recovery. It was also tested that the possibilities of using this method for the determination of flavonoids in Chinese medicinal preparation.     

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.     

Hydroxyethylammonium monosubstituted cyclodextrin as chiral selector for capillary electrophoresis

A novel cationic cyclodextrin, mono-6^A-(2-hydroxyethyl-1-ammonium)-6^A-β-cyclodextrin chloride (HEtAMCD) has been successfully synthesized and applied as chiral selector in capillary electrophoresis. The NMR study revealed this chiral selector has three recognition sites: β-CD, ammonium cation and hydroxy group in the sidearm to contribute three corresponding driving forces including inclusion complexation, electrostatic interaction and hydrogen bonding. The effect of buffer pH and HEtAMCD concentration (2.5–10 mM) on enantioselectivity, chiral resolution as well as effective mobility of analytes was investigated. This elegantly designed CD exhibits outstanding enantioselectivities toward the studied hydroxyl acids and ampholytic racemates in CE with the aid of extra hydrogen bonding. Under optimum pH 6.0, chiral resolutions over 5 can be readily obtained for hydroxy acids with CD concentration below 5 mM. The comparison study between HEtAMCD and our earlier reported ammonium CDs indicates the hydroxyethylammonium group of HEtAMCD significantly increased the enantioselective capability.     

Enantioselective separation in capillary electrophoresis using a novel mono-6-propylammonium-β-cyclodextrin as chiral selector

The chiral resolving ability of a novel single-isomer cationic β-cyclodextrin (CD), mono-6^A-propylammonium-6^A-deoxy-β-cyclodextrin chloride (PrAMCD), as a chiral selector in capillary electrophoresis (CE) is reported in this work for the enantioseparation of hydroxy, carboxylic acids and amphoteric analytes. The effect of chiral selector concentration on the resolution was studied. Good resolutions were achieved for hydroxy acids. Optimum resolutions were obtained even at 3.5 mM CD concentration for carboxylic acids. The electrophoretic method showed good linearity and reproducibility in terms of migration times and peak areas, which should make it suitable for routine analysis. In addition, baseline chiral separation of a six-acid mixture was achieved within 20 min. PrAMCD proved to be an effective chiral selector for acidic analytes.     

Separation of chiral drugs with sulfobutylether-β-cyclodextrin bycapillary zone electrophoresis

IntroductionChaedcyclodextrlnswerefirstIntroducedby几rabelforchlralseparationofamlnoacids,andthechargedCDcommonlyusednowdaysarecarboxymethyl－p－CD（CM－p－CD）,p－CD－phosphate,Y－CD－phosphate,sulfobutylether－p－cyclodextrln（SBE－p－CD）etc．...     

Optimized Conditions of Enantioseparation of β-Blockers by CZE Using Carboxymethyl-β-Cyclodextrin as Chiral Selector

Capillary zone electrophoresis was used for the enantiomeric separation of six β-blocking drug substances with β-cyclodextrin (β-CD) and its derivatives as chiral selectors employing an uncoated capillary. The effects of pH value and composition of the background electrolyte (BGE), the capillary temperature and running voltage have been investigated. The results showed that β-CD type, concentration and pH value have a strong influence on the efficiency of the chiral separation. Carboxymethyl-β-cyclodextrin (CM-β-CD) gave a baseline enantiomeric separation for six β-blocking drug substances under optimal conditions, whereas the β-CD, hydroxypropyl-β-cyclodextrin (HP-β-CD) showed no chiral recognition. The potential and capillary temperature did not have a great effect on enantiomer resolution.     

Use of polystyrene nanoparticles to enhance enantiomeric separation of propranolol by capillary electrophoresis with Hp-beta-CD as chiral selector

We describe the use of polystyrene (PS) nanoparticles to manipulate chiral selectivity of propranolol analysis by capillary electrophoresis, by dispersing PS nanoparticles into the run buffer employing hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector. Distinct separational differences are observed between the buffer containing PS nanoparticles and buffer without, when changing separating conditions including PS nanoparticles concentration, pH, buffer concentration, HP-β-CD concentration and when adding an organic additive. Selectivity improvements are reflected by changes in the observed mobility as a result of interactions between the propranolol enantiomers and HP-β-CD governing the absorption process on the PS particles surface. The presence of PS nanoparticles increases the enantioseparation at low particle concentration in the presence of HP-β-CD as a chiral selector.     

Molecularly bonded chitosan prepared as chiral stationary phases in open-tubular capillary electrochromatography: comparison with chitosan nanoparticles bonded to the polyacrylamide phase

The chiral selector, chitosan (CS), was attached to the silanized capillary via a silane coupling agent, (3-glycidyloxypropyl)trimethoxysilane (GTS), to form the GTS-CS capillary, and results for this capillary were compared with those of a previous study on the copolymerization of CS with methacrylamide (MAA) (forming the MAA-CS capillary). The GTS-CS capillary did not exhibit enantioselectivity for d/l-tryptophan, whereas the GTS-BSA capillary, which was prepared by replacement of CS with bovine serum albumin (BSA), succeeded in the chiral separation with an Rs = 2.4 in Tris buffer (50 mM, pH 8.5). To increase CS attachment, the CS units were crosslinked by succinic acid, and the resulting GTS-CS-s capillary phase improved the resolution to 1.9. Alternatively, the SiH-CS-s capillary was constructed by CS attachment on the silicon hydride phase via stepwise silanization and hydrosilation reactions and crosslinking by succinic acid, but this approach could only achieve a resolution of 1.4 in Tris buffer (50 mM, pH 9.5). Although the GTS-CS-s and SiH-CS-s capillaries were still inferior to the MAA-CS capillary (Rs = 3.8), the enantioselectivities of the three capillaries were all in the range of 1.4-1.6. For the (±)-catechin sample, the plate heights of the GTS-CS-s and SiH-CS-s capillaries conditioned in pH 8.5 Tris buffer with 60% MeOH modifier were 0.9 cm ((−)-catechin) and 6.0 cm ((+)-catechin)) and 2.9 cm (−) and 3.2 cm (+), respectively, and these heights were comparable to the MAA-CS capillary (2.5 cm (−), 6.0 cm (+)) in pH 6.6 phosphate buffer with 80% MeOH. Finally, a racemate of ibuprofen, a weakly acidic anti-inflammatory drug, was successfully baseline resolved by the GTS-CS-s and SiH-CS-s capillaries in the borate buffers, which were 30 mM at pH 7.5 and 10 mM at pH 8.0, respectively.