Iva Valášková

Direct quantitative determination of amlodipine enantiomers in urine samples for pharmacokinetic study using on-line coupled isotachophoresis-capillary zone electrophoresis separation method with diode array detection.

The present work illustrates possibilities of column-coupling capillary electrophoresis (CE-CE) combined with chiral selector (2-hydroxypropyl-beta-cyclodextrin, HP-beta-CD) and fiber-based diode array detection (DAD) for the direct quantitative enantioselective determination of trace drug (amlodipine, AML) in biological multicomponent ionic matrices (human urine). Capillary isotachophoresis (ITP) served as an ideal injection technique in CE-CE. Moreover, the ITP provided an effective on-line sample pretreatment prior to the capillary zone electrophoresis (CZE) separation. Enhanced separation selectivity due to the combination of different separation mechanisms (ITP vs. CZE-HP-beta-CD) enabled to obtain pure zones of the analytes, suitable for their detection and quantitation. The DAD, unlike single wavelength UV detection, enabled to characterize the purity (i.e. spectral homogeneity) of the analytes zones. A processing of the raw DAD spectra (the background correction and smoothing procedure) was essential when a trace analyte signal was evaluated. Obtained results indicated pure (i.e. spectrally homogeneous) zones of interest confirming effective ITP-CZE separation process. The proposed ITP-CZE-DAD method was characterized by favorable performance parameters (sensitivity, linearity, precision, recovery, accuracy, robustness, selectivity) and successfully applied to an enantioselective pharmacokinetic study of AML.

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-beta-cyclodextrin (CE-beta-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-beta-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-beta-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.

Isotachophoretic separation of alkali and alkaline earth metal cations in water—polyethylene glycol mixtures

Abstract The influence of polyethylene glycol (PEG) on the effective mobilities of alkali and alkaline earth metal cations was investigated. It is shown that appropriate proportions of water and PEG in the leading electrolyte enable a complete separation of this group of cations to be achieved by capillary isotachophoresis (ITP). The high dynamic concentration ranges of the ITP determinations achieved in the operational systems based on water—PEG mixtures are illustrated by practical examples (water samples, urine, aqueous extract of apple flesh).

Determination of fexofenadine in tablets by capillary electrophoresis in free solution and in solution with cyclodextrins as analyte carriers.

ABSTRACT Capillary electrophoresis (CE) methods for the determination of fexofenadine (FEX) in commercial pharmaceuticals were developed. It was demonstrated that FEX could be effectively analyzed in free solution cationic CE at low pH. Another analytical approach studied was based on cyclodextrin (CD) modified CE where highly charged CD derivatives served as analyte carriers. In this way, the separation range was spread to physiological pH region and a CE analysis of FEX, present actually in its zwitterionic form, could be accomplished. Several parameters affecting the separations were studied, including the type and concentration of carrier ion, counterion, analyte carrier, and pH of the buffer. The methods based on the free solution CE and CD-modified CE were compared each other, validated, and applied for the determination of FEX in tablets.

Determination of terbinafine in pharmaceuticals and dialyzates by capillary electrophoresis

A capillary electrophoresis method has been developed for the separation and determination of terbinafine (TER) in various pharmaceutically relevant matrices. Capillary zone electrophoresis (CZE) separation and UV absorbance photometric detection were carried out in a 160mm capillary tube with a 300mum i.d., hydrodynamically (membrane) closed. The influences of pH, carrier cation and counterion on migration parameters of TER were studied and the following conditions were selected: a 20mmoll(-1) glycine running buffer adjusted to pH 2.7 with acetic acid, 0.2% (w/v) methylhydroxyethylcellulose (m-HEC) as an electro-osmotic flow (EOF) suppressor, a 250muA driving current, and 20 degrees C. The optimized separation conditions were convenient for the determination of TER in commercial tablets and spray and in dialyzates. Here, the dialysis was used to investigate in vitro permeation of TER through the skin from the gel. The samples of dialyzates were examined with and without simple extraction procedure and the results were compared. A permeation profile of the drug present in the gel of given composition was obtained analyzing pretreated samples. The proposed electrophoretic method was successfully validated. It was suitable for the simple, sensitive, rapid and highly reproducible assay of TER. CZE analysis was completed within 5.5min. The detection limit of TER was 1.73mumoll(-1) at a 224nm detection wavelength. The intra- and inter-laboratory precisions over the concentration range 6.0-60.0mumoll(-1) were between 0.32-0.69% and 1.04-1.44% including R.S.D. of migration times and peak areas, respectively. The mean absolute recoveries of drugs from samples were found to be 98.34 (tablets) and 99.47% (spray). It is suggested that there are potentialities to determine TER present in unpretreated complex samples, as CZE in a hydrodynamically closed separation system may be easily on-line combinable with purification and preconcentration CE modes (e.g., isotachophoresis, ITP).

Chiral separation of dioxopromethazine in eye drops by CZE with charged cyclodextrin.

Capillary zone electrophoresis (CZE) with carboxyethyl-beta-cyclodextrin (CE-beta-CD) dissolved in the operating buffer was used for the separation and determination of enantiomers of phenothiazine antihistaminic, dioxopromethazine, in commercial pharmaceutical preparation, eye drops. This chiral selector, negatively charged under given separating conditions (20 mmol/l epsilon -aminocaproic acid, acetic acid, pH 4.5), was effective in enantioresolution of the antihistamine even at its low concentrations (3-6 mg/ml) in the buffer solution. CZE identification and quantitation of the relevant constituents present in the preparation (dioxopromethazine enantiomers, phenylephrine) were based on the response of photometric absorbency detector, operating at a 275 nm detection wavelength. Changes in pH, type and concentration of chiral selector were studied in relation to chiral resolution. Acceptable validation criteria for sensitivity, precision, linearity and repeatability are included.

Isotachophoretic analysis of inorganic ions

Capillary isotachophoresis is frequently used in analytical practice for the determination of inorganic ions in various matrices. Proper selection of the separation conditions enables one to attain the maximum ratio of effective mobilities of the individual components of a mixture under analysis, and thus their mutual separation. This article examines possibilities of how to differentiate the effective mobilities of inorganic ions along with their use for the analysis of real samples.

Chiral separation of alkylamine antihistamines in pharmaceuticals by capillary isotachophoresis with charged cyclodextrin.

Cyclodextrin-mediated capillary isotachophoresis (ITP) in cationic regime of the separation was developed for the separation and quantitation of alkylamine antihistamine dimethindene (DIM) and pheniramine (PHM) enantiomers in various pharmaceutical preparations (capsules, oral drops, gel, granulated powder). Several electrolyte systems of different compositions and pH were examined. The optimized chiral ITP electrolyte system was consisted of 10 mmol/L potassium acetate adjusted to pH 4.8 with acetic acid, containing 4 mmol/L negatively charged CE-β−CD (chiral selector) as the leading electrolyte with electroosmotic flow (EOF) suppressing additive, 0.2% (w/v) methylhydroxyethylcellulose (m-HEC), and 5 mmol/L β-alanine as the terminating electrolyte. The proposed electrophoretic method was successfully validated. It was convenient for the sensitive, simple, rapid, and highly reproducible assay of these antihistamine enantiomers. The calibration graphs relating the ITP zone length to the concentration of DIM and PHM enantiomers were rectilinear (r = 0.999) in the range 40.0–200.0 mg/L of each enantiomer. The relative standard deviations (RSD) were 0.75% for DIM(1), 0.63% for DIM(2), 1.05% for PHM(1), and 0.83% for PHM(2) (n = 6) when determining 100 mg/L DIM and PHM, respectively, standard solutions. According to the validation procedure based on the standard addition technique the recoveries were 97.66–98.34%. Good quantitation was obtained in short analysis time (a single analysis took about 12 min). The minimal sample pretreatment and low running costs make the proposed ITP method a good alternative to commonly used analytical methods (CZE, HPLC). The obtained results suggest that the proposed method is suitable for routine assay of dimethindene and pheniramine enantiomers in various pharmaceuticals.

Monitoring of lithium levels in human serum after therapy with lithium preparations by capillary isotachophoresis

An isotachophoretic method for the evaluation of the level of lithium salts in serum samples was optimized. Use of operating systems containing polyethylene glycol permitted the separation of cationically migrating components from Li (i.e., Na, K and Ca). The pretreatment of serum samples involves only appropriate dilution with demineralized water depending on the concentration of the major components such as sodium. The lithium levels were studied both in model samples and serum from patients treated with lithium preparations.

Direct determination of celiprolol in human urine using on-line coupled ITP-CZE method with fiber-based DAD.

The present work illustrated possibilities of column‐coupling electrophoresis combined with DAD for the direct quantitative determination of trace drug (celiprolol, CEL) in clinical human urine samples. ITP, on‐line coupled with CZE, served as an ideal injection technique (high sample load capacity, narrow and sharp drug zone). Moreover, the ITP provided an effective on‐line sample pretreatment (preseparation, purification and preconcentration of the drug) producing analyte zone suitable for its direct detection and quantitation in CZE stage. Spectral DAD in comparison with single wavelength ultraviolet detection enhanced value of analytical information (i) verifying purity (i.e., spectral homogeneity) of drug zone (according to differences in spectrum profiles when compared tested and reference drug spectra) and (ii) indicating zones/peaks with spectra similar to the drug spectrum (potential structurally related metabolites). The characterization of trace analyte signals superposed on the baseline noise was more definite thanks to the application of background correction and smoothing procedure to the raw DAD spectra (producing relevant spectral response). The proposed ITP‐CZE‐DAD method was characterized by favorable performance parameters for CEL in urine matrices {e.g., the lower limit of quantification was 9.7 ng/mL, RSD and relative error of the determinations were lower than 3% (precision) and 1% (accuracy), respectively, analyte peak exhibited spectral homogeneity (reflecting separation selectivity), separation efficiency was 84 500 theoretical plates} and successfully applied in a trial pharmacokinetic study of CEL.