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CE method for the baseline separation of structurally similar flavonolignans silybin A, silybin B, isosilybin A, isosilybin B, silychristin, silydianin, and their precursor taxifolin in silymarin complex has been developed and validated. The optimized background electrolyte was 100 mmol/L boric acid (pH 9.0) containing 5 mmol/L heptakis(2,3,6-tri-O-methyl)-β-CD and 10% (v/v) of methanol. The separation was carried out in an 80.5/72 cm (50 μm id) fused silica capillary at +25 kV with UV detection at 200 nm. Genistein (10 μg/mL) was used as internal standard. The resolution between the diastereomers of silybin and isosilybin was 1.73 and 2.59, respectively. The method was validated for each analyte in a concentration range of 2.5-50 μg/mL. The calibration curves were rectilinear with correlation coefficients ≥0.9972. The method was applied to determine flavonolignans in two dietary supplements containing Silybum marianum extract. The accuracy was evaluated by comparing the results of the CE analyses of the dietary supplements with those of the reference United States Pharmacopeial HPLC method. The unpaired t-test did not show a statistically significant difference between the results of both the proposed CE and the reference method (p > 0.05, n = 3).

Isomers and stereoisomers are always challenging to separate. Column coupling may provide improved chromatographic selectivity, necessary for the separation of the compounds with similar chemical and structural properties. The relatively low viscosity of supercritical fluids, used as mobile phases allows for the coupling of several columns in series in supercritical fluid chromatography (SFC), without exceeding the pressure limits of the system. The aim of this study is to propose reliable prediction of the retention behaviour of analytes on a coupled column system, based on a limited number of initial analyses. The chiral compounds atenolol, ephedrine, propranolol, mianserin, labetalol and nadolol, besides the diastereomers quinine and quinidine, and the structural isomers of aminophenol and aminocresol were used as model analytes. The retention behaviour of the analytes was determined on the individual chiral columns Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3, Lux Cellulose-4, Lux Amylose-2 and the achiral columns Luna NH2, Luna Silica, Synergi RP and FluoroSep RP. The mobile phase was composed of CO2 mixed with 20% (v/v) MeOH, which contained 0.1% (v/v) trifluoroacetic acid and 0.1% (v/v) isopropylamine. The retention factors of the analytes on coupled stationary phases were predicted, and subsequently compared to the experimentally obtained ones. Relative deviations of predicted and experimental retention factors were in range from 0.00% to 51.91%. Flow rate and back pressure of the screening conditions were adjusted to improve prediction precision on four column combinations, with varying success rates. The average relative deviations of retention factors were reduced to 2.84% - 6.59% by adjusting flow rate, and to 2.30% - 8.57% by adjusting back pressure. The most successful approach, flow rate adjustment, was then applied to select a column combination providing improved resolution of the structurally similar components of silymarin extract.

The stability of α-bromophenylacetic acid (BPAA) in 50% aqueous methanol solution has been tested. CE in different running buffers was used to separate BPAA from the decomposition reaction products α-hydroxyphenylacetic (mandelic) acid and α-methoxyphenylacetic acid. Suitable CE separation of all three compounds and other product, bromide, was achieved in 60 mmol/L formate buffer (pH 3.0) at -30 kV in 50 μm (i.d.) poly(vinyl alcohol)-coated fused silica capillary (30 cm/24.5 cm) with UV detection at 200 nm. The CE method was applied to determine the reaction order of the decomposition of BPAA (0.47 mmol/L) via nucleophilic substitution in 50% aqueous methanol. The first-order reaction kinetics was confirmed by linear and non-linear regression, giving the rate constants 1.52 × 10-4 ± 2.76 × 10-5 s-1 and 7.89 × 10-5 ± 5.02 × 10-6 s-1, respectively. Additionally, the degradation products were identified by CE coupled to mass spectrometric (MS) detection. The CE-MS experiments carried out in 60 mmol/L formate buffer (pH 3.0) and in 60 mmol/L acetate buffer (pH 5.0) confirmed the results obtained by CE-UV. Furthermore, the stability of BPAA in polar solvents was tested by 1H NMR experiments. Our results provide strong evidence of the instability and fast degradation of BPAA in 50% aqueous methanol indicating that BPAA is not suitable as the model analyte for chiral separations.

A micellar electrokinetic chromatography method for the determination of indomethacin impurities (4-chlorobenzoic acid, 5-methoxy-2-methyl-3-indoleacetic acid, and 3,4-dichloroindomethacin) has been developed. A 64.5/56-cm fused silica 50 μm id capillary with extended light path (150 μm id) detection window was used. Internal standard was 1-naphthylacetic acid. The analytes were separated at 30 kV with DAD detection at 224 nm. A central composite face-centered design was applied for the optimization of the separation conditions. The effect of SDS concentration, content of methanol, concentration of phosphate buffer, and pH of the buffer were studied at three levels. The optimized background electrolyte was 20 mmol/L phosphate buffer (pH 7.57) containing 58 mmol/L SDS and 0% MeOH. Sufficient resolution of all compounds with Rs ≥ 3.5 was achieved within 10 min. The method was validated for a range of 1.25-80 μg/mL of each impurity corresponding to 0.05-3.2% relative to the concentration of indomethacin (2.5 mg/mL). The calibration curves were rectilinear with correlation coefficients r2 exceeding 0.9994. The lower limit of quantification was 0.05% or 1.25 μg/mL that complies with the reporting limits regarding the ICH Q3A guideline. The method was applied to purity assay of indomethacin in both bulk drug and gel.

Due to their structural variability and their commercial availability, cyclodextrins are the most frequently used chiral selectors in capillary electrophoresis. A variety of migration modes can be realized depending on the characteristics of the cyclodextrins and the analytes. The basic considerations regarding the development of a chiral CE method employing cyclodextrins as chiral selectors are briefly discussed. The presented examples illustrate the separation modes of an acidic and a basic analyte with native and charged cyclodextrin derivatives as a function of the pH of the background electrolyte and the concentration of the cyclodextrin.

We present a simple and sensitive method for the determination of patulin at µg·kg−1 level in apple-based products. Our method relies on the application of an in-line molecularly imprinted polymer solid-phase extraction microcartridge in capillary electrophoresis coupled with mass spectrometry. Capillary zone electrophoresis method has been developed and parameters affecting the in-line process have been carefully optimized. Validation parameters were assessed for patulin, giving LOQ of 1 µg·kg−1 and linearity range 1–100 µg·kg−1 with R2 ≥ 0.997. The LOQ was below the maximum content of patulin requested by the European Union in this type of products. The precision of the peak area and the migration time were less than 14.9 and 1.6%, respectively. Patulin has been analyzed in the presence of 5-hydroxymethylfurfural, which is the main interference in this kind of matrix. The method was applied to assay patulin content in various apple-based products.

Due to their structural variability and their commercial availability, cyclodextrins are the most frequently used chiral selectors in capillary electrophoresis. A variety of migration modes can be realized depending on the characteristics of the cyclodextrins and the analytes. The basic considerations regarding the development of a chiral CE method employing cyclodextrins as chiral selectors are briefly discussed. The presented examples illustrate the separation modes of an acidic and a basic analyte with native and charged cyclodextrin derivatives as a function of the pH of the background electrolyte and the concentration of the cyclodextrin.

A new MEKC-MS/MS method was developed for the determination of four Sudan dyes in chili products. The separation and MS detection conditions were optimized to achieve fast, efficient, selective, and sensitive determination of Sudan I, Sudan II, Sudan III, and Sudan IV dyes. The target compounds were extracted from chili samples with acetonitrile and cleaned by freeze-out. This two-step sample preparation led to excellent extraction efficiency and minimal matrix effect. The analytical performance of the method was very good, with r2 ≥ 0.9914 and limits of quantification lower than 22 μg kg−1. The precision was below 15.7%. The recovery for spiked samples ranged from 84.4 to 99.6%, with relative standard deviations less than 8.0%. For all evaluated samples, the matrix effects did not exceed ± 10%. The applicability of the proposed method was demonstrated with 20 chili products, two of which were found to contain Sudan I and IV residues.

Hemicelluloses such as xylans play an increasing role as renewable raw materials for technological applications. The complex and variable composition of hemicelluloses requires powerful analytical techniques in order to assess their composition. In the present study, the neutral fraction of hydrothermally isolated xylan from beech wood was characterized by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) upon derivatization with 8-aminopyrene-1,3,6-trisulfonic acid. Reproducible separation of the xylo-oligosaccharides was achieved using a polyvinyl alcohol coated capillary and a 25 mM sodium acetate buffer, pH 4.75, as background electrolyte at an applied voltage of −30 kV. Intermediate precision expressed as relative standard deviation was below 2.0 % for migration times and below 10 % for relative peak areas except for the oligomers present at very low concentrations only. At the same time, derivatization conditions proved to be robust as well. Samples obtained by fractionation of the xylan were subsequently characterized by CE-LIF. In addition, capillary electrophoresis with mass spectrometry detection indicated the presence of small amounts of xylo-oligosaccharides containing additional sugar moieties such as 4-O-methylglucuronic acid. Moreover, minor components containing acetyl groups could be detected. The presence of these impurities was confirmed by nuclear magnetic resonance analysis of the fractions. In conclusion, although none of the techniques applied here gave a complete picture of the composition of the investigated xylan or its fractions, the combination provided insight into the complexity of the sample.

An electrophoretically mediated microanalysis assay (EMMA) for the determination of the stereoselective reduction of L-methionine sulfoxide diastereomers by methionine sulfoxide reductase enzymes was developed using fluorenylmethyloxycarbonyl (Fmoc)-L-methionine sulfoxide as substrate. The separation of the diastereomers of Fmoc-L-methionine sulfoxide and the product Fmoc-L-methionine was achieved in a successive multiple ionic-polymer layer-coated capillary using a 50 mM Tris buffer, pH 8.0, containing 30 mM sodium dodecyl sulfate as background electrolyte and an applied voltage of 25 kV. 4-Aminobenzoic acid was employed as internal standard. An injection sequence of incubation buffer, enzyme, substrate, enzyme, and incubation buffer was selected. The assay was optimized with regard to mixing time and mixing voltage and subsequently applied for the analysis of stereoselective reduction of Fmoc-L-methionine-(S)-sulfoxide by human methionine sulfoxide reductase A and of the Fmoc-L-methionine-(R)-sulfoxide by human methionine sulfoxide reductase B. The Michaelis-Menten constant, K m, and the maximum velocity, v max, were determined. Essentially identical data were determined by the electrophoretically mediated microanalysis assay and the analysis of the samples by CE upon offline incubation. Furthermore, it was shown for the first time that Fmoc-methionine-(R)-sulfoxide is a substrate of human methionine sulfoxide reductase B.

Capillary electromigration techniques have developed into significant analytical separation tools especially for enantioseparations. While CE can be considered a mature technique as documented by its wide applications, CEC is still in a developmental state despite many research efforts. The success of stereospecific CE separation methods is due to the high specificity and flexibility of the technique as well as the availability of many types of chiral selectors. Thus, numerous methods have been developed for the analysis of chiral compounds in chemical, biochemical, pharmaceutical, environmental, and forensic sciences. However, most reported applications deal with pharmaceuticals. The search for new chiral selectors also continued despite the fact that most applications were performed using cyclodextrins. Furthermore, CE has been combined with spectroscopic and molecular modeling studies in attempts to understand the interactions between chiral selectors and analytes. The present review focuses on recent examples of mechanistic aspects of capillary enantioseparations with regard to mathematical modeling of enantioseparations, investigations of the analyte-complex structures as well as new chiral selectors and applications of chiral analyses by CE and CEC. It covers the literature published between January 2011 and August 2012.

Capillary electrophoresis (CE) is a powerful separation method based on the migration of charged species under the influence of electric field. The main merits of CE are high separation efficiency, short analysis time and small consumption of solvents and samples. However, the main drawbacks of CE are generally lower sensitivity compared to classical column-chromatographic methods. Selectivity and/or sensitivity of CE separation can be improved by forming complexes between analytes and a complex-forming reagent present as an additive in the background electrolyte (BGE). We focus this review primarily on the application of inorganic complex-forming reagents added to the BGE to separate organic ligands. We briefly mention common CE separations of inorganic analytes (mainly metal ions) using BGEs with organic ligands (e.g., hydroxycarboxylic or aminopolycarboxylic acids) as selectors. The review involves brief theoretical consideration of the significance of the effect of complex formation on separation selectivity and/or sensitivity in CE, but the major topic is critical evaluation of different inorganic complex-forming reagents used recently in the CE analysis of organic compounds, including: (i) borate, tungstate and molybdate in separating organic compounds possessing vicinal -OH groups; (ii) ligand-exchange CE and capillary electrochromatography in chiral analysis; and, (iii) the role of metal ions as central ions employed for selectivity enhancement of CE separation of various classes of organic compounds, including biopolymers.

A novel capillary electrophoresis (CE) method with contactless conductivity detection suitable for the determination of glucosamine (GlAm) and K(+) in pharmaceuticals was devised. Under the optimum conditions (aqueous 30 mM acetate buffer of pH 5.2 as the background electrolyte; voltage 30 kV; 25 degrees C), GlAm (migrating as glucosaminium cation) was well separated from K(+) that could occur in the dosage forms as excipient. The CE analysis was performed in fused-silica capillaries (50 microm i.d., 75 cm total length, 27 cm to detector) and the separation took

A stereospecific capillary electrophoresis assay for the simultaneous determination of related substances and the enantiomeric purity of escitalopram was developed by a central composite face-centered factorial design and subsequently validated. Separations were carried out in a 50 microm, 47/40 cm fused-silica capillary. The optimized conditions included 20mM phosphate buffer, pH 2.5, containing 0.5mg/ml beta-cyclodextrin and 22 mg/ml sulfated beta-cyclodextrin as background electrolyte, an applied voltage of -20 kV and a temperature of 28 degrees C. Salicylic acid was used as internal standard. The assay was validated for the (R)-enantiomer of citalopram and the enantiomers of the impurity citadiol in the range of 2.5-150 microg/ml and 2.5-50 microg/ml, respectively. The limit of detection was 0.02% for all compounds, the limit of quantitation 0.05%, relative to a concentration of escitalopram of 5mg/ml. Intraday precision of migration time and peak area ratio were in the range of 0.17-0.44% and 1.64% and 6.25%, respectively. Relative standard deviations of interday precision ranged between 0.84% and 1.85% in the case of migration times and between 5.20% and 9.28% for peak area ratio. The assay was applied to the determination of the purity of escitalopram in bulk drug and tablets. (R)-Citalopram and (S)-citadiol were detected as impurities.

A micellar electrokinetic chromatography method for the analysis of the l-methionine sulfoxide diastereomers employing a successive multiple ionic-polymer layer coated fused-silica capillary was developed and validated in order to investigate the stereospecificity of methionine sulfoxide reductases. The capillary coating consisted of a first layer of hexadimethrine and a second layer of dextran sulfate providing a stable strong cathodic EOF and consequently highly repeatable analyte migration times. The methionine sulfoxide diastereomers, methionine as product as well as β-alanine as internal standard were derivatized by dabsyl chloride and separated using a 35 mM sodium phosphate buffer, pH 8.0, containing 25 mM SDS as BGE and a separation voltage of 25 kV. The method was validated in the range of 0.15-2.0 mM with respect to linearity and precision. The LODs of the analytes ranged between 0.04 and 0.10 mM. The assay was subsequently applied to determine the stereospecificity of methionine sulfoxide reductases as well as the enzyme kinetics of human methionine sulfoxide reductase A. Monitoring the decrease of the l-methionine-(S)-sulfoxide Km = 411.8 ± 33.8 μM and Vmax = 307.5 ± 10.8 μM/min were determined.

Due to their commercial availability, cyclodextrins are the most frequently used chiral selectors in capillary electrophoresis as documented by the numerous publications in the field. A variety of migration modes can be realized depending on the characteristics of the cyclodextrins and the analytes. The basic considerations regarding the development of a chiral CE method employing cyclodextrins as chiral selectors are briefly discussed. The presented examples illustrate the separation modes of an acidic and a basic analyte with native and charged cyclodextrin derivatives as a function of the pH of the background electrolyte and the cyclodextrin concentration.

Due to their commercial availability, cyclodextrins are the most frequently used chiral selectors in capillary electrophoresis as documented by the numerous publications in the field. A variety of migration modes can be realized depending on the characteristics of the cyclodextrins and the analytes. The basic considerations regarding the development of a chiral CE method employing cyclodextrins as chiral selectors are briefly discussed. The presented examples illustrate the separation modes of an acidic and a basic analyte with native and charged cyclodextrin derivatives as a function of the pH of the background electrolyte and the cyclodextrin concentration.

Capillary electromigration techniques have developed into significant analytical separation tools especially for enantioseparations. While CE can be considered a mature technique as documented by its wide applications, CEC is still in a developmental state despite many research efforts. The success of stereospecific CE separation methods is due to the high specificity and flexibility of the technique as well as the availability of many types of chiral selectors. Thus, numerous methods have been developed for the analysis of chiral compounds in chemical, biochemical, pharmaceutical, environmental, and forensic sciences. However, most reported applications deal with pharmaceuticals. The search for new chiral selectors also continued despite the fact that most applications were performed using cyclodextrins. Furthermore, CE has been combined with spectroscopic and molecular modeling studies in attempts to understand the interactions between chiral selectors and analytes. The present review focuses on recent examples of mechanistic aspects of capillary enantioseparations with regard to mathematical modeling of enantioseparations, investigations of the analyte-complex structures as well as new chiral selectors and applications of chiral analyses by CE and CEC. It covers the literature published between January 2011 and August 2012.

A novel capillary electrophoretic method for the separation of pancuronium (PM) and vecuronium (VM) ions utilizing capacitively coupled contactless conductivity detection was devised and validated. The separation was carried out in bare fused-silica capillaries (50 μm id, 75/45 cm) at 25°C. Optimal BGE was 50 mM borate buffer of pH 9.5 containing 12.5 mg/mL of (2-hydoxypropyl)-γ-CD. The samples were injected hydrodynamically at 1000 mbar for 3 s. Separation was performed at +30 kV. Under such conditions the PM and VM were base-line resolved and the separation took4 min. For quantification phenyltrimethylammonium iodide was used as internal standard. Calibration curves were linear for both pancuronium bromide (PMB) and vecuronium bromide (VMB) in the range 25-250 μg/mL with r0.9968. The limits of detection were 7 and 6 μg/mL for PMB and VMB, respectively. The accuracy tested by recovery experiment at three concentration levels of added PMB and VMB was satisfactory (95.7-102.7%, n =3, with RSD2.61%). The method was successfully applied to the assay of PMB and VMB in commercial injection solutions.

A novel electrophoretic BGE containing tungstate as complex-forming reagent is suitable for the separation of polyphenols. Similar to molybdate-containing BGE reported earlier (cf. M. Polasek, et al.., Talanta 2006, 69, 192) addition of tungstate to BGE affects significantly migration of compounds/ligands with vicinal -OH groups due to the formation of negatively charged complexes involving W(VI) as central ion. Baseline separation of mixtures of flavonoids (apigenin, luteolin, hyperoside, quercetin, and rutin) and phenolic acids (chlorogenic and p-coumaric acid) was achieved within 15 min with optimized BGE of pH 7.4 containing 50 mM N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES), 2.2 mM tungstate, and 25% v/v of methanol. The separation was performed in a 75 cm (effective length 42 cm)x 75 microm id uncoated fused-silica capillary at 30 kV with spectrophotometric detection at 275 nm. The calibration curves were rectilinear for 25-175 microg/mL of all analytes (cinnamic acid as the internal standard). The LODs ranged from 1.8 to 6 microg/mL for all analytes except for chlorogenic acid. Intraday precision (n = 6) of migration times (RSD < or = 1.2%) and peak areas (RSD < or = 5.6%) was evaluated. The tungstate-based BGEs can be alternatively utilized for the analysis of polyphenols at considerably lower pH than with conventional alkaline borate-based BGEs.

An overview mapping recent trends in the determination of polyphenols of natural origin (mostly flavonoids) and their synthetic derivatives by electromigration methods is presented. The overview (covering the period of the recent 5 years and comprising 61 references) is focused on capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) with various detection methods. Techniques comprising on-line pre-separation such as isotachophoresis (ITP)-CZE and flow-injection-CZE, chiral separations and CZE evaluation of antioxidation activity are also discussed.