Derivatized cyclodextrins for the separation of enantiomers in capillary electrophoresis

The use of cyclodextrin derivatives for the efficient separation of enantiomeric drugs is described. Hydroxypropylation, methylation or carboxymethylation of the cyclodextrin not only result in a better solubility of the cyclodextrin in aqueous solutions, but also favor, via additional hydrogen bonding, the stabilization of one of the cyclodextrin-analyte complexes. The influence of the background electrolyte on peak shape is also described here. Carboxymethylated cyclodextrin can be used in similar manner to uncharged cyclodextrins at low pH values (below 4). At pH values above 5, however, its charge also allows the separation of uncharged enantiomers as in a micellar-like system.     

A further study on the chiral separation power of a soluble neutral β-cyclodextrin polymer

Enantiomeric separation of some basic compounds, namely selegiline, amphetamine, and clenbuterol, was studied by capillary electrophoresis using an uncharged β-cyclodextrin polymer added to the background electrolyte at pH 2.5. Both complexation and resolution were influenced by the concentration of the chiral polymer confirming our previous results obtained in our earlier work for a wide number of pharmaceutical compounds. In this further study, we examined the influence of different organic additives to the background electrolyte on the enantioselectivity of the chiral selector, also using an extended number of analytes. In most cases, the use of an organic additive resulted in a decrease of resolution. However opposite to that, in some cases, e.g. ephedrine, the organic solvent proved to be essential to achieve enantiomeric resolution. Furthermore the influence of the capillary temperature on the resolution of the analytes was evaluated. Increase of temperature had a deleterious effect on the resolution of the enantiomers. For ephedrine, however, relatively high temperature (50 °C) proved to be advantageous, for the resolution of the optical isomers.     

Analytical method development for directed enzyme evolution research: A high throughput high-performance liquid chromatography method for analysis of ribose and ribitol and a capillary electrophoresis method for the separation of ribose enantiomers

Analytical methods were developed for a directed enzyme evolution research programme, which pursued high performance enzymes to produce high quality l-ribose using large scale biocatalytic reaction. A high throughput HPLC method with evaporative light-scattering detection was developed to test ribose and ribitol in the enzymatic reaction, a β-cyclobond 2000 analytical column separated ribose and ribitol in 2.3 min, a C₁₈ guard column was used as an on-line filter to clean up the enzyme sample matrix and a short gradient was applied to wash the column, the enzymatic reaction solution can be directly injected after quenching. Total run time of each sample was approx. 4 min which provided capability of screening 4 × 96-well plates/day/instrument. Meanwhile, a capillary electrophoresis method was developed for the separation of ribose enantiomers, while 7-aminonaphthalene-1,3-disulfonic acid was used as derivatisation reagent and 25 mM tetraborate with 5 mM β-cyclodextrin was used as electrolyte. 0.35%of d-ribose in l-ribose can be detected which can be translated into 99.3% ee of l-ribose. Derivatisation reagent and sample matrix did not interfere with the measurement.     

Use of experimental design to optimize the analysis of volatile compounds by dynamic headspace extraction followed by cold trapping and capillary GC

A central composite design has been used to improve the extraction performance of a dynamic headspace method; extraction yield was evaluated on a homogeneous sample of cheese powder. The volatile compounds were stripped from the sample with nitrogen and adsorbed on a Tenax trap, analysis being performed with a thermal cold trapping injector connected directly to a capillary GC. Three variables (sample temperature, extraction time, and nitrogen flow rate) were investigated, and a quadratic model with interactions was postulated. Twenty experiments was performed, each producing five responses. It was shown that the best conditions for the extraction procedure were those characterized by the highest values of the three variables investigated.     

An experimental design based strategy to optimize a capillary electrophoresis method for the separation of 19 polycyclic aromatic hydrocarbons

Because of their high toxicity, international regulatory institutions recommend monitoring specific polycyclic aromatic hydrocarbons (PAHs) in environmental and food samples. A fast, selective and sensitive method is therefore required for their quantitation in such complex samples. This article deals with the optimization, based on an experimental design strategy, of a cyclodextrin (CD) modified capillary zone electrophoresis separation method for the simultaneous separation of 19 PAHs listed as priority pollutants. First, using a central composite design, the normalized peak-start and peak-end times were modelled as functions of the factors that most affect PAH electrophoretic behavior: the concentrations of the anionic sulfobutylether-β-CD and neutral methyl-β-CD, and the percentage of MeOH in the background electrolyte. Then, to circumvent computational difficulties resulting from the changes in migration order likely to occur while varying experimental conditions, an original approach based on the systematic evaluation of the time intervals between all the possible pairs of peaks was used. Finally, a desirability analysis based on the smallest time interval between two consecutive peaks and on the overall analysis time, allowed us to achieve, for the first time in CE, full resolution of all 19 PAHs in less than 18 min. Using this optimized capillary electrophoresis method, a vegetable oil was successfully analyzed, proving its suitability for real complex sample analysis.     

Borate-containing background electrolytes to improve CE separation in bare capillaries

The paper describes how borate-containing BGEs modify ζ-potential and so EOF in bare fused silica capillaries. This surface modification can be used to suppress EOF and improve the separation performance of CZE including capillary sieving electrophoresis (CSE). Boric acid forms complexes with polysaccharides used as sieving matrices in CSE and other compounds containing hydroxyl groups, including polyol bases such as Tris, triethanolamine, and Bis-Tris propane. High concentration of boric acid in BGEs leads to a strong interaction of boric acid with the silica surface of the capillary wall and this suppresses or even completely eliminates ζ-potential and EOF. Using a polyol base with several charge-carrying amino groups, such as Bis-Tris propane, can actually reverse EOF. We demonstrate the use of various borate-containing BGEs in bare fused silica capillaries for size-separation of DNA fragments, size-separation of proteins by SDS CSE, and also by CZE in the absence of any sieving matrix.     

Sweeping of alprenolol enantiomers with an organic solvent and sulfated β‐cyclodextrin in capillary electrophoresis

Sweeping, an on‐line sample concentration technique in CE, is the picking and accumulation of analytes by the pseudostationary phase or complexing additive. In the presence of an electric field, the analytes concentrated at the additive front that initially penetrated the sample zone. Here, we describe the sweeping of cationic alprenolol enantiomers using sulfated β‐CD and organic solvent. The separation solution contained the anionic additive while ACN was in the sample solution. With fused silica capillaries, positive polarity, and solutions buffered at pH 3, the direction of the enantiomers' effective electrophoretic mobility was the same as the electrophoretic mobility (or electrophoretic mobility without additive). When the amount of ACN in the sample was increased (i.e. 60%), the interaction between the analytes and additive became negligible. This caused the sweeping boundary to shift from the electrophoretically moving β‐CD front to the zone between the sample and separation solution. The equation that described the narrowing of injected sample zone was derived. The performance of sweeping with 60% ACN in the sample was then studied under different operating conditions (e.g. type of injection, injection time, and CD concentration). The low interaction between enantiomers and additive gave only moderate increases in sensitivity (approximately tenfold), but was improved when field enhancement was used during electrokinetic injection. With a conductivity difference (separation/sample solution) of 70 and a short injection time of 30 s at 20 kV, peak improvements of >100‐fold was easily achieved.     

Chemometric experimental design based optimization techniques in capillary electrophoresis: a critical review of modern applications

A critical review of recent developments in the use of chemometric experimental design based optimization techniques in capillary electrophoresis applications is presented. Current advances have led to enhanced separation capabilities of a wide range of analytes in such areas as biological, environmental, food technology, pharmaceutical, and medical analysis. Significant developments in design, detection methodology and applications from the last 5 years (2002–2007) are reported. Furthermore, future perspectives in the use of chemometric methodology in capillary electrophoresis are considered.     

Quantum dot-enhanced chemiluminescence detection for simultaneous determination of dopamine and epinephrine by capillary electrophoresis

A sensitive method based on quantum dot (QD)-enhanced capillary electrophoresis-chemiluminescence (CE-CL) detection was developed for simultaneous determination of dopamine (DA) and epinephrine (E). In this work, CdTe QD was added into the running buffer of CE to catalyze the post-column CL reaction between luminol and hydrogen peroxide, achieving higher CL emission. Negative peaks were produced due to the inhibitory effects on CL emission from DA and E eluted from the electrophoretic capillary. The decrease in CL intensity was proportional to the concentration of DA and E in the range of 8.0 × 10⁻⁸-5.0 × 10⁻⁶ M and 4.0 × 10⁻⁸-5.0 × 10⁻⁶ M, respectively. Detection limits for DA and E were 2.3 × 10⁻⁸ M and 9.3 × 10⁻⁹ M, respectively. Using this method, the levels of DA and E in human urine from healthy donors were determined.     

Optimizing the gas chromatographic separation and detection of polychlorinated biphenyls by use of electronic pressure programming and experimental design

The applicability of electronic pressure control and Taguchi L₂₇ experimental design to the optimization of the gas chromatographic separation and detection of polychlorinated biphenyls has been evaluated. The influence of several experimental variables, column temperature program, carrier gas pressure program, on-column injector temperature program, and make-up gas pressure program, was studied using analysis of variance. Simultaneous optimization of sample introduction, column efficiency, and detector performance could be achieved without compromising system performance. The relationships between system performance and experimental variables were established using regression analysis. Agreement between the simulated and experimental results obtained using suggested optimum conditions demonstrated the applicability of the technique developed in this study. The improvement achieved in the chromatographic separation of PCBs is presented.     

Application of experimental design and radial basis function neural network to the separation and determination of active components in traditional Chinese medicines by capillary electrophoresis

Orthogonal design has been used to the optimization of separation and determination of two active components in traditional Chinese medicines by capillary electrophoresis. The concentration of phosphate, applied voltage, organic modifier content and buffer pH were selected as variable parameters. Their different effects on peak resolution were studied by the experimental design method. Optimized separation conditions were obtained and successfully applied to the separation and determination of aconitine and hypaconitine in Aconitum medicinal herbs. Good separation was achieved within 7 min using a buffer system composed of 20 mmol L⁻¹ phosphate and 35% acetonitrile at pH 9.5. The applied voltage was 14 kV and the detection was set at 235 nm. In addition, a radial basis function neural network with a “4-18-1” structure was developed based on the experimental results of orthogonal design and uniform design, and was applied to the prediction of peak resolution of the two active components under the optimum separation conditions given by orthogonal design. The predicted results were in good agreement with the experimental values, indicating that radial basis function neural network is a potential way for the selection of separation conditions in capillary electrophoresis.     

The application of experimental design to the robustness testing of a method for the determination of drug-related impurities by capillary electrophoresis

Summary Robustness testing of capillary electrophoresis methods is an important part of method validation. Appropriate use of experimental designs can be employed in this robustness testing. In this study experimental designs were used in the assessment of a capillary electrophoresis method used to determine drug related impurities. Initially a fractional factorial screening design was used to identify the critical parameters which were found to be pH and voltage. A central composite design was then performed to evaluate the response surfaces for pH and voltage which showed operation at the optimum pH and voltage values.     

On the use of response surface strategy to elucidate the electrophoretic migration of carbohydrates and optimize their separation

This paper focuses on the optimization with a design of experiments of a new CE method for the simultaneous separation of four carbohydrates of interest (fructose, glucose, lactose, and sucrose) and five potentially interfering carbohydrates (ribose, xylose, maltose, mannose, and galactose) with a highly alkaline separation electrolyte for subsequent applications to food, beverage, forensic, or pharmaceutical samples. First, the factors that potentially affect the carbohydrate migration were identified: NaOH concentration in the separation electrolyte, separation temperature, and separation electrolyte conductivity. A central composite design was then carried out to determine and model the effects of these three factors on normalized migration times and separation efficiency. From the model, an optimization of the separation was carried out using a desirability analysis based on resolutions between adjacent peaks and analysis time. The optimum conditions obtained were a separation electrolyte composed of 98 mM NaOH and 120 mM NaCl to adjust the conductivity at 4.29 S/m and a separation temperature fixed at 26.5°C. Finally, these conditions were experimentally confirmed and the robustness of the obtained separation was checked.     

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.     

Experimental design methodologies to optimise the spectrofluorimetric determination of Losartan and Valsartan in human urine

A spectrofluorimetric method has been developed for the determination of two angiotensin II receptor antagonists (ARA II): Losartan and Valsartan. A fractional factorial design and a central composite design were used. The key factors considered in the optimization process were pH, temperature and emission slit width. Maximum fluorescent intensity was established as response for each experiment. The response surfaces confirmed the robustness of the method. A clean-up procedure was used for urine samples that consisted of a solid-phase extraction using C8 cartridges. The total analysis time was lower than 30 min. This method proved to be accurate (RE, 8%), precise (intra- and inter-day coefficients of variation were lower than 8% and sensitive enough (LOQ c.a. 0.5 μg ml⁻¹) to be applied to the determination of Losartan and Valsartan in urine samples.     

Chiral initiator-induces self-disproportionation of enantiomers via achiral chromatography: application to enantiomer separation of racemate

We report here the theoretical design and proof of principle of the first example of a conceptually new approach for the preparation of enantiomerically pure compounds from the racemates by chiral initiator-induced Self-Disproportionation of Enantiomers (SDE) via achiral chromatography.     

Separation of etodolac enantiomers by capillary electrophoresis. Validation and application of the chiral method to the analysis of commercial formulations

Separation of etodolac enantiomers, which exhibit different biological activity and pharmacokinetic profiles, has been achieved using the randomly substituted (2-hydroxy)propyl-beta-cyclodextrin (HP-beta-CD) as chiral selector in capillary electrophoresis. The selection of this CD was made after screening of different CD derivatives of neutral and anionic nature. The effect on the enantioresolution of the buffer concentration and of the degree of substitution (DS) and concentration of the CD as well as of instrumental parameters, such as the capillary temperature and the separation voltage, were studied. The highest resolution of etodolac enantiomers was around 2.5 using 100 mM phosphate buffer (pH 7.0) with 20 mM HP-beta-CD (DS approximately 4.2) and UV detection at 225 (10) nm with a reference wavelength at 360 (50) nm. Validation of the chiral method in terms of selectivity, linearity, precision (instrumental repeatability, method repeatability, intermediate precision), and the limits of detection and quantitation allowed to evaluate its quality to the analysis of etodolac enantiomers in different pharmaceutical preparations containing racemic etodolac.     

Mono‐6A‐(4‐methoxybutylamino)‐6A‐β‐cyclodextrin as a chiral selector for enantiomeric separation

A new member of the family of methoxylalkylamino monosubstituted β‐cyclodextrins, mono‐6^A‐(4‐methoxybutylamino)‐6^A‐β‐cyclodextrin, has been developed as a chiral selector for enantioseparation in capillary electrophoresis. This amino cyclodextrin exhibited good enantioselectivities for 16 model acidic racemates including three dansyl amino acids at an optimum pH of 6.0. Excellent chiral resolutions over six were obtained for α‐hydroxy acids and 2‐phenoxypropionic acids with 3.0 mM chiral selector. The good chiral recognition for α‐hydroxyl acids was attributed to inclusion complexation, electrostatic interactions, and hydrogen bonding. The hydrogen‐bonding‐enhanced chiral recognition was revealed by NMR spectroscopy. The chiral separation of acidic racemates was further improved with the addition of methanol (≤10 vol%) as an organic additive.     

Electrochemiluminescence quenching as an indirect method for detection of dopamine and epinephrine with capillary electrophoresis

An electrochemiluminescence (ECL) inhibition method was developed as an indirect detection method for the determination of dopamine and epinephrine separated by capillary electrophoresis (CE). When the concentration of Ru(bpy)(3) (2+) was 50 muM diluted by 50 mM phosphate (pH 8.5) in the cell and 0.5 M tripropylamine (TPA) was added to the running buffer (10 mM phosphate, pH 9.0), an inhibition of ECL of the Ru(bpy)(3) (2+)/TPA system by the analytes was observed. Under the optimized conditions, the relative standard deviations of migration time and negative peak area were less than 1% and 3%, respectively, for 1 microM dopamine or 1 microM epinephrine (n = 10). Linear ranges of 0.1-10 microM for both analytes and the detection limits (signal-to-noise ratio S/N = 3) of 10 nM for dopamine and 30 nM for epinephrine were obtained.     

Some design considerations in miniaturized electrokinetic separation systems

There is a great potential for miniaturized analytical separation systems, e.g. in process control, environmental monitoring, or clinical chemistry. Particularly, miniaturized electro-driven systems open many new possibilities. Most efforts in this area have been focused on cylindrical capillary columns. In the present paper, thin rectangular conduits are considered and comparisons with cylindrical tubes are made on the basis of theoretical models. A critical and limiting factor in electro-driven separations is the generation of heat. For a given cross-section, heat dissipation is more efficient in rectangular columns. Silicon is proposed as column material. Apart from the better thermal conductivity, monocrystalline silicon can be machined with an extraordinary precision when selective chemical etching procedures are employed. This precision is of central importance in the development of miniaturized high performance systems.