Interinstrumental transfer of a fast short‐end injection capillary electrophoresis method: Application to the separation of niobium,tantalum, and their substituted ions

The interinstrumental transfer of a short‐end CE method was studied. A model separation of the hexameric forms of niobium, tantalum, and their substituted ions (Nb_6−xTa_x with 0 ≤ x  ≤ 6) was selected as test case. The method was first optimized on a Beckman instrument and in a second step transferred to an Agilent instrument. The transfer needed updated guidelines that tackled differences in effective capillary length, 8.5 (Agilent) versus 10 cm (Beckman), because of instrumental different capillary cartridges. Differences in effective length lead to migration time and separation efficiency inequalities, illustrated by a decrease in resolution between the substituted ions. The difference in effective length was overcome by adapting the lift offset parameter of the Agilent instrument. The lift offset default setting is 4 mm and by increasing this parameter both the inlet and outlet lifts are lowered and thus the detection window can be displaced and consequently the effective length was increased. The decrease in effective length difference and the effect on the separation efficiency was investigated and led finally to a restored separation of the substituted ions. The adaptation of the lift offset parameter during short‐end injection methods was added to earlier developed guidelines to facilitate interinstrumental method transfer of CE methods.     

Fast enantiomeric separation with vancomycin as chiral additive by co-electroosmotic flow capillary electrophoresis: increase of the detection sensitivity by the partial filling technique

A fast and sensitive method is described by using vancomycin as a chiral additive for enantiomeric separation by capillary electrophoresis (CE). In order to overcome disadvantages associated with use of vancomycin as chiral additive in CE, several strategies including the dynamic coating technique, the co-electroosmotic flow technique, and the partial filling technique were employed sequentially in this method. Using the polycationic polymer hexadimethrine bromide (HDB) as a buffer additive, the capillary wall was dynamically coated with a thin film formed by the adsorbed HDB. Consequently, the adsorption of vancomycin onto the capillary wall was minimized via electrostatic repulsion between the coating of the capillary wall and the vancomycin molecule. In addition, the reversed electroosmotic flow (from cathode to anode) produced by the positively charged capillary wall migrates in the same direction of negatively charged analytes (co-electroosmotic flow electrophoresis). Thereby the electrophoretic mobility of negatively charged analytes were drastically accelerated leading to a short separation time of less than 3.4 min. The separation time was further reduced by the use of a short-end-injection technique. For example, the analysis time was achieved by as short as 55 s for a baseline separation of dansyl-alpha-amino-n-butyric acid. Concurrently, the partial filling technique was used to avoid the loss of detection sensitivity caused by the presence of vancomycin in the running buffer. The effect of several parameters, such as HDB concentration, buffer pH, plug length of the chiral selector, concentration of the chiral selector and applied voltage, on enantioselectivity were investigated toward optimization. Besides the advantage of a very short separation time, the method is characterized by high detection sensitivity, high selectivity, and high efficiency.     

Determination of association constants between enantiomers of orciprenaline and methyl-beta-cyclodextrin as chiral selector by capillary zone electrophoresis using a partial filling technique

The principles for the determination of conditional association constants of enantiomers by capillary zone electrophoresis employing a partial filling technique (PFT) using methyl-beta-cyclodextrin as chiral selector is presented. Orciprenaline was used as a model compound. Partial filling is a separation technique, where different lengths of the chiral selector solution are introduced into the capillary to a final zone length shorter than the effective length of the capillary, prior to application of the solutes. Lengthening of the separation zone results in improving enantioresolution in addition to decreasing electrophoretic mobility of the enantiomers, because of longer interaction time between the solute and chiral selector. The degree of the reduction in electromobility depends on the affinity of the solute to the chiral selector, i.e. strength of the complex formed between the solute and cyclodextrin. The decrease in the electrophoretic mobility with increasing length of the separation zone is used for determination of the association constant. The association constants of the enantiomers of orciprenaline and the chiral selector were evaluated from the slope of the plot, observed electrophoretic mobility versus the ratio between the length of the separation zone and the effective length of the capillary. It was found that the association constants were independent of the chiral selector concentration. The mean values were 110 M(-1) and 160 M(-1) for respective enantiomer. Constants obtained by a conventional CE technique were in good agreement with those from the PFT experiments. The highest enantioselectivityy was obtained when about 50% of the solute was distributed to the selector phase.     

不依赖于统计模型的手性毛细管电泳分离多指标同时优化

考虑到手性毛细管电泳分离机理的复杂性，提出了不依赖于统计模型的二进制编码遗传算法多目标优化策略。根据实验参数的多少及其重要性，采用均匀设计安排完成初步实验。并用Derringer功效函数作为多指标同时优化的评价指标，以取得较高的分离度和较快分离速度；通过对实验参数给予编码，转化为二进制字符串，根据得到的总功效函数值大小进行遗传算法操作，产生新的一组实验条件。重复上述过程直到得到最佳分离条件为止。此方法成功地应用于华法令对映体的毛细管电泳分离条件优化。     

Chiral Capillary Electrokinetic Chromatography: Principle and Applications,Detection and Identification,Design of Experiment,and Exploration of Chiral Recognition Using Molecular Modeling

This work reviews the literature of chiral capillary electrokinetic chromatography from January 2016 to March 2021. This is done to explore the state-of-the-art approach and recent developments carried out in this field. The separation principle of the technique is described and supported with simple graphical illustrations, showing migration under normal and reversed polarity modes of the separation voltage. The most relevant applications of the technique for enantioseparation of drugs and other enantiomeric molecules in different fields using chiral selectors in single, dual, or multiple systems are highlighted. Measures to improve the detection sensitivity of chiral capillary electrokinetic chromatography with UV detector are discussed, and the alternative aspects are explored, besides special emphases to hyphenation compatibility to mass spectrometry. Partial filling and counter migration techniques are described. Indirect identification of the separated enantiomers and the determination of enantiomeric migration order are mentioned. The application of Quality by Design principles to facilitate method development, optimization, and validation is presented. The elucidation and explanation of chiral recognition in molecular bases are discussed with special focus on the role of molecular modeling.     

Chiral determination of amino acids by capillary electrophoresis and laser-induced fluorescence at picomolar concentrations

In this publication we present results on the determination of enantiomers of amino acids at very low concentrations. A fluoresceine-based chiral dye was synthesized to allow the separation of diastereoisomers of D- and L-amino acids. We used capillary electrophoresis with different non-ionic surfactants (Brij). The separation parameters were optimized and separations of D- and L-isovaline, an unusual terrestrial amino acid, were obtained. The sensitivity limits were also determined using a commercial laser-induced fluorescence detector. The quantitation of these amino acids is very important to understand the process of chiral selection on Earth.     

Enantioseparation of warfarin and its metabolites by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) method with direct ultraviolet (UV)-absorbance detection is presented for the simultaneous enantiomeric separation of warfarin and its main metabolites, including warfarin alcohols, 4'-, 6-, and 7-hydroxywarfarin, using highly sulfated beta-cyclodextrin (HS-beta-CD) as the chiral selector. This chiral separation method was optimized in terms of the electrophoretic parameters, which included the concentration of HS-beta-CD used, the type and composition of organic modifier added to the background electrolyte (BGE) buffer, and the BGE buffer pH. Chiral separation of warfarin and its major metabolites was achieved with high resolution, selectivity, efficiency, repeatability, and reproducibility. This optimized chiral analysis of warfarin along with its metabolites was completed within a satisfactory electrophoresis time of 20 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).     

On-line coupling of partial filling-capillary zone electrophoresis with mass spectrometry for the separation of clenbuterol enantiomers

The on-line coupling of capillary zone electrophoresis with mass spectrometry (CZE-MS) for the separation of enantiomers is hampered by the presence of nonvolatile chiral selectors such as cyclodextrins in the separation buffer. This problem can be overcome by use of the partial filling technique where only a part of the capillary is filled with the separation buffer containing chiral selectors. Since the electroosmotic flow is almost completely suppressed at acidic pH, that dimethyl-beta-cyclodextrin is neutral, no free cyclodextrin would reach the MS detector when using a partially filled capillary. By this method, clenbuterol enantiomers were successfully resolved and separated from salbutamol (internal standard) in aqueous solution and in plasma samples. A solid-phase extraction (SPE) was used for the preparation of plasma samples before analysis.     

Validation of capillary electrophoresis method for determination of N-methylpyrrolidine in cefepime for injection

The present study relates to a new capillary electrophoresis method for the determination of N-methylpyrrolidine, an impurity considered to be toxic and also potential degradation impurity in cefepime hydrochloride drug substance. The newly developed capillary electrophoresis method for determining the content of N-methylpyrrolidine in cefepime for injection has been validated as per International Conference on Harmonization (ICH) guidelines to prove the selectivity, sensitivity, suitability, robustness, and ruggedness of the method. This simple, efficient, and rapid methodology may be used by pharmaceutical industry for routine analysis as well as during stability studies. The newly developed capillary electrophoresis method to determine the content of N-methylpyrrolidine in cefepime for injection requires 10 min for data acquisition, and uses an indirect UV photometry method to detect the analyte signal at 240 nm against the reference signal at 210 nm. The electrophoretic system is optimized to get stable base line, higher signal to noise ratio and peaks with narrow peak width. The method employs bare fused silica capillary with extended light path, effective length of capillary is 56 cm and inner diameter of capillary is 50 μm, 5 mmole of imidazole buffer adjusted to pH 5.1 with 3 molar acetic acid solution is used as background electrolyte. The sample is introduced in hydrodynamic mode employing pressure of 50 mbar for 5 s, and the desired separation is achieved with constant applied voltage of 25 kV at ambient temperature (~25°C).     

Multi-purpose capillary electrophoresis system with concentration gradient detection

A robust, inexpensive and versatile capillary electrophoresis (CE) system for routine and rapid analysis is reported, which consists of a rugged cartridge holding a 20-mum i.d. 15-cm long capillary, and an inexpensive, universal and sensitive concentration gradient detector. The design of the cartridge simplifies the sample introduction process and makes it possible to perform many separation modes, including moving boundary capillary electrophoresis (MBCE), capillary zone electrophoresis (CZE), capillary isotachophoresis (CITP) and capillary isoelectric focusing (CIEF), on the same system. This arrangement provides more information about a sample's components since analytes can be separated by different modes performed on the same CE system. The detector only consists of a low-power HeNe laser, or laser diode, and a photodiode position sensor. Amino acids and proteins of 10(-6)-10(-3)M concentration can be separated by different capillary electrophoretic modes, and detected directly by the detector. The universal detector shows particularly good sensitivity when applied to CE separation modes having self-concentration and focusing effects. Femtomoles of proteins were separated and detected with CIEF. In addition, a short and narrow capillary allows use of high electrical fields which facilitate rapid separations. Four amino acids at millimolar concentrations were fully separated and detected in less than 80 sec by the MBCE mode when a high electric field was applied. The physical size of the whole system is much smaller than that of conventional CE instruments with UV absorbance or fluorescence detector.     

Determination of aluminum in serum by capillary zone electrophoresis with laser-induced fluorescence detection

Summary A novel method of separating and detecting trace aluminum by capillary zone electrophoresis is described. Aluminum is reacted with lumogallion [4-chloro-3-(2,4-dihydroxyphenylazo)-2-hydroxybenzen-1-sulphonic acid] so that the complex can be selectively and sensitively detected by a laser-induced fluorescence detector after capillary electrophoretic separation. Using the proposed method, limits of detection in the sub parts per billion range are achieved. The technique is applied to the determination of aluminum in human serum.     

Analytical potential of enzyme-coated capillary reactors in capillary zone electrophoresis

Enzymes immobilized on the inner surface of an electrophoretic capillary were used to increase sensitivity and resolution in capillary zone electrophoresis (CZE). Sensitivity is enhanced by inserting a piece of capillary containing the immobilized enzyme into the main capillary, located before the detector, in order to transform the analyte into a product with a higher absorptivity. This approach was used to determine ethanol. In order to improve resolution, capillary pieces containing immobilized enzymes were inserted at various strategic positions along the electrophoretic capillary. On reaching the enzyme, the analyte was converted into a product with a high electrophoretic mobility, the migration time for which was a function of the position of the enzyme reactor. This approach was applied to the separation and determination of acetaldehyde and pyruvate. Finally, the proposed method was validated with the determination of ethanol, acetaldehyde, and pyruvate in beer and wine samples.     

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.     

Evaluation of chiral purity for a substituted imidazole p38 MAP kinase inhibitor and its intermediates using a single chiral capillary electrophoresis method

The chiral separation of a substituted imidazole p38 MAP kinase inhibitor and its intermediates was investigated using capillary electrophoresis (CE) with various sulfated cyclodextrins. After initial screens, a single CE chiral method with a randomly sulfated beta-CD was selected for the evaluation of chiral purity for all three compounds. Operational parameters, such as the concentration of the chiral selectors, background electrolyte (or mobile phase) pH, organic modifiers, and temperature were varied in order to achieve an optimized method. The optimal method was validated in terms of linearity, sensitivity, precision, ruggedness, and specificity.     

Robustness testing of chiral separations by capillary electrophoresis using highly-sulfated cyclodextrins

The robustness of a generic method for chiral separation in capillary electrophoresis using highly-sulfated cyclodextrins in a low pH phosphate buffer and the "short-end injection technique" was studied. In this study, we focused on the robustness of the separations and not of the quantitative analysis of the enantiomers. The robustness was evaluated for the enantiomeric separation of a basic (propranolol), a neutral (praziquantel) and an acidic (warfarin) compound. The influence of eight factors which were believed to affect significantly the separations was studied using a 11-factor, 12-experiment Plackett-design. Statistical interpretation of the factor effects on different analytical responses (selectivity and resolution) was performed. The separations of the three compounds could be considered as rather robust as the factor effects were generally not significant (alpha = 0.05) and small.     

Evaluation of the interaction between sitagliptin and cyclodextrin derivatives by capillary electrophoresis and nuclear magnetic resonance spectroscopy

An aqueous capillary electrophoretic method was developed for chiral analysis of the novel anti-diabetic drug, sitagliptin. The acid-base profiling of the analyte was carried out using both capillary electrophoresis and nuclear magnetic resonance pH titrations. The apparent complex stability and chiral separation properties were investigated with 30 different cyclodextrins under acidic conditions. The effect of concentration and pH of the BGE, temperature of the capillary, and the type and concentration of the chiral selector on the enantiomer resolution were thoroughly investigated. The effects of dual cyclodextrin systems on separation were also extensively studied. Complete separation of racemic sitagliptin with good resolution (R(S)=2.24) was achieved within a short time (15 min) with optimized parameters (10°C, pH=4.4, 40 mM phosphate buffer) of a sulfobutylether-β-cyclodextrin (averaged degree of substitution ~4) and native β-cyclodextrin dual system. The averaged stoichiometry of the inclusion complex was determined using the Job plot method with both (1)H and (19)F NMR experiments and resulted in a 1:1 complex. The structure of the inclusion complex was elucidated using 2-D ROESY NMR experiments.     

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.

     

Enantiomeric determination, validation and robustness studies of racemic citalopram in pharmaceutical formulations by capillary electrophoresis

A chiral capillary electrophoresis (CE) method has been developed allowing the enantiomeric separation of racemic citalopram (R-(-) and S-(+) citalopram) using as chiral selector carboxymethyl-gamma-cyclodrextrin (CM-gamma-CD). The influence of chemical and instrumental parameters on the separation such as cyclodextrin (CD) and buffer concentrations, buffer pH, voltage, injection pressure, ..., was investigated. Good chiral separation of the racemic mixture was achieved in less than 4 min using a fused-silica capillary and as background electrolyte (BGE) a phosphate buffer solution (20 mM, pH 7) containing 0.15% (w/v) of CM-gamma-CD as chiral selector. The separation was driven in normal polarity mode at 15 degrees C, 30 kV and hydrodynamic injection. In order to validate the method, the stability of the solutions, precision (repeatability, reproducibility and F-Snedecor test), linearity (Lack of Fit and ANOVA tests) accuracy (98-101%), detection and quantitation limits (0.06 and 0.2 mg L(-1), respectively), on a selected analytical placebo, were examined. Besides, a robustness test was performed using the Plackett-Burman fractional factorial experimental design using a matrix of 15 experiments for seven factors (internal parameters) with a statistical treatment suggested by Youden and Steinner. The proposed method is fast, sensitive, inexpensive and, besides, it has been evaluated by means of an extensive validation study and an exhaustive robustness test. The scope of this validated and robust method has been proved in the analysis of four pharmaceutical formulations; two of them (recently available in Spain), which just contained S-(+)-citalopram (escitalopram) as active principle. Recoveries between 101 and 103%, with regard to their nominal contents were obtained. In the other two pharmaceutical ones, the method provided the separation and quantification of both chiral isomers in the existing racemic mixture.     

Enantioselective separations in capillary electrophoresis with dextran sulfate as the chiral selector

Dextran sulfate, a polyanionic polysaccharide, was evaluated as a chiral additive in capillary electrophoresis. Structurally related compounds having a variety of functional groups were utilized to probe the selectivity of the chiral selector. The effects of pH, chiral selector concentration, and chiral selector composition on resolution were also studied. At low pH, the reversed polarity mode was employed to achieve separation of the probe compounds. The electrophoretic results provided insight into the chiral recognition of dextran sulfate in capillary electrophoresis. Several factors, including hydrophobic, steric, and electrostatic interactions, appeared to play a role in the observed enantioseparations.