Determination of polyphenol components in herbal medicines by micellar electrokinetic capillary chromatography with Tween 20

A simple and convenient method of micellar electrokinetic capillary chromatography (MEKC) using polyoxyethylene sorbitan monolaurate (Tween 20) to form single micelle and methanol as a buffer additive was introduced for the simultaneous determination of five polyphenols, including scopoletin, rutin, esculetin, chlorogenic acid and caffeic acid. A running buffer solution of pH 9.3, 20 mmol/L sodium tetraborate containing 64 mmol/L Tween 20 and 9% (v/v) methanol was adopted in the separation. Because rutin and esculetin were difficult to be separated by capillary zone electrophoresis (CZE) and SDS-based MEKC, Tween 20-based MEKC was adopted and the polyphenols were separated satisfactorily. The proposed method was used to determine the polyphenol components in the herbal medicine of Cortex fraxini. The separation mechanism of Tween 20-based MEKC for the polyphenols was discussed preliminarily.     

Capillary electrophoresis determination of six bioactive constituents in San-huang-hsieh-hsin-tang

A capillary electrophoretic method for simultaneous determination of six bioactive ingredients (berberine, palmatine, baicalin, sennoside B, emodin, and sennoside A) in the Chinse herbal formula San-huang-hsieh-hsin-tang was established. A carrier composed of aqueous buffer solution (50 mM sodium cholate, 15 mM sodium dihydrogen phosphate, and 4.25 mM sodium borate)-acetonitrile (3:2) was found to be the most suitable electrolyte for this separation. Contents of these constituents in a non-pretreated methanol-water extract of San-huang-hsieh-hsin-tang sample could be easily determined within 20 min. The effects of borate, cholate, and organic modifier (acetonitrile) concentration of the carrier on the migration behavior of the solutes were also studied.     

Profiling and screening analysis of 27 aromatic amino acids by capillary electrophoresis in dual modes

An efficient capillary electrophoretic (CE) profiling and screening system based on dual modes of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) was developed for the simultaneous determination of 23 nonprotein amino acids (NPAAs) and 4 protein amino acids with aromatic moiety. It involves separation by an uncoated fused-silica capillary under phosphoric acid buffer in CZE mode and by another uncoated fused-silica capillary under neutral sodium dihydrogen phosphate buffer containing sodium dodecyl sulfate in MEKC mode. Migration orders of the amino acids studied on the two separation modes under each optimum condition were very different. The repeatability of migration times measured by the CZE and MEKC was found to be better than 4.8 and 3.4%, respectively, thereby enabling to cross-check the identification of each amino acid. The method linearity and limit of detection of the CZE for each amino acid were found to be adequate for the assay of aromatic amino acids. When the present CE profiling and screening analysis in dual modes was applied to plant seeds, NPAAs such as mimosine from Mimosa pudica Linné, and 2-phenylglycine from Lindera erythrocarpa Makino were positively detected along with tryptophan, phenylalanine and tyrosine.     

Direct multicomponent analysis of beer samples constituents using micellar electrokinetic capillary chromatography

A capillary electrophoretic method was developed using micellar electrokinetic capillary chromatography (MEKC) with diode-array detection to analyze simultaneously 26 beer constituents in a single procedure, including alcohols, iso-alpha-acids, amino acids, flavonoids, isoflavonoids, a vitamin, purine and pyrimidine bases. After filtration, sample components were separated with an uncoated capillary and a 25 mM sodium borate and 110 mM SDS buffer at pH 10.5. Analyses were run at 14 kV and 8 s of hydrodynamic injection with UV detection at 210 nm and 270 nm. The proposed method was successfully applied to the direct determination of beer constituents without any sample cleanup procedures.     

Quality evaluation of Guan‐Xin‐Ning injection based on fingerprint analysis and simultaneous separation and determination of seven bioactive constituents by capillary electrophoresis

The purpose of this study was to develop a comprehensive, rapid and practical capillary electrophoresis (CE) method for quality control (QC) of Guan‐Xin‐Ning (GXN) injection based on fingerprint analysis and simultaneous separation and determination of seven constituents. In fingerprint analysis, a capillary zone electrophoresis (CZE) method with a running buffer of 30 mM borate solution (pH 9.3) was established. Meanwhile, ten batches of samples were used to establish the fingerprint electropherogram and 34 common peaks were obtained within 20 min. The RSD of relative migration times (RMT) and relative peak areas (RPA) were less than 5%. In order to further evaluate the quality of GXN injection, a micellar electrokinetic chromatography (MEKC) method was developed for simultaneous separation and determination of bioactive constituents. Seven components reached baseline separation with a running buffer containing 35 mM SDS and 45 mM borate solution (pH 9.3). A good linearity was obtained with correlation coefficients from 0.9906 to 0.9997. The LOD and LOQ ranged from 0.12 to 1.50 μg/mL and from 0.40 to 4.90 μg/mL, respectively. The recoveries ranged between 99.0 and 104.4%. Therefore, it was concluded that the proposed method can be used for full‐scale quality analysis of GXN injection.     

Analysis of artichoke (Cynara cardunculus L.) extract by means of micellar electrokinetic capillary chromatography

The main constituents of artichoke extract were separated by micellar electrokinetic chromatography (MEKC), using a buffer consisting of 100 mM sodium dodecyl sulfate (SDS) in 20 mM sodium dihydrogen phosphate, 20 mM disodium tetraborate (pH 8.6) as background electrolyte. Optimum separation voltage of 28 kV (positive polarity) and a capillary temperature of 25 degrees C gave the best analysis. The UV detection was performed at 200 nm. The method was successfully used to analyze plant and drug samples as well as for the study of artichoke antioxidant activity. The quantitative MEKC results were in good agreement to those obtained previously by reversed-phase high-performance liquid chromatography (RP-HPLC).     

Capillary electrokinetic chromatography of insulin and related synthetic analogues

With the implementation of recombinant DNA technology in the pharmaceutical industry, some synthetic insulins have been developed in order to improve the therapy of diabetes. These analogues differ only slightly in the amino acid sequence, therefore displaying a great challenge for analytical chemistry. Within the work presented in this paper, capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC) with sodium dodecylsulphate (SDS) as micelle-forming agent, and microemulsion electrokinetic chromatography (MEEKC) with microemulsions consisting of SDS, n-octane and 1-butanol were investigated for the separation of human insulin and five synthetic analogues. Best results were achieved with a solvent-modified MEKC system consisting of 100 mM sodium dodecyl sulphate and 15% acetonitrile in 10 mM borate buffer (pH 9.2). A similar system based on perfluorooctanoic acid as micelle-forming agent in ammonium acetate (pH 9.2) was successfully employed for the hyphenation with a quadrupole/time-of-flight mass spectrometer via a sheath-flow interface. In this case, detection limits at 10 mg/L could be achieved.     

Evaluation of capillary electrophoretic techniques towards systematic toxicological analysis.

Two capillary electrophoresis (CE) methods were evaluated for their suitability in systematic toxicological analysis (STA). A test set of 25 barbiturates was analysed using capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Buffers used consisted of 90 mM borate set at pH 8.4 (CZE) and 20 mM phosphate, 50 mM sodium dodecyl sulphate set at pH 7.5 (MEKC). All analyses were carried out using fused silica capillaries using an electric field strength of 52.6 kV/m. The use of a reproducible identification parameter is very important in STA as it influences the identification power (IP). To deal with the poor reproducibility of the migration time, we introduced the corrected effective mobility. Inter-day reproducibilities of the latter parameter were < 0.6% for CZE and < 0.5% for MEKC, using daily prepared buffers. The IP of the methods was expressed by calculation of the discriminating power and the mean list length. Data obtained were compared to gas chromatographic and high-performance liquid chromatographic data, and correlations between all methods were calculated. It was shown that little correlation exists between chromatographic and electrophoretic techniques. The results indicated that CE has a good identification power for the application in STA, especially when a combination of methods having a low correlation is used.     

Identification and determination of effective components in Euphrasia regelii by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) method has been developed for simultaneous determination of eukovoside, cinnamic acid and ferulic acid in Euphrasia regelii for the first time. The electrophoresis buffer was 20 mmol/L sodium borate containing 10% (v/v) methanol (pH 8.50). The effects of concentration of borate and electrolyte pH on electrophoretic behavior and separation were studied. Regression equations revealed linear relationships (correlation coefficients 0.9995-0.9998) between the peak area of each analyte and the concentration. The levels of analytes in the different parts of Euphrasia regelii were easily determined with recoveries ranging from 95.5 to 104.2%.     

Comparative study of capillary zone electrophoresis and micellar electrokinetic chromatography for the separation of twelve aromatic sulphonate compounds

Summary Two modes of capillary electrophoresis (CE), capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC), were investigated for the separation of 12 aromatic sulphonate compounds. In CZE, although the voltage applied, the buffer concentration and the pH were optimized for effective separation of the compounds studied, under the best conditions four of the five amino compounds coeluted, as did naphthalene-1-sulphonic acid and naphthalene-2-sulphonic acid. In MEKC, sodium dodecyl sulphate (SDS) and Brij 35 were chosen as the anionic and nonionic surfactants and the effect of the concentration of micelles was examined. The effect of adding methanol as the organic modifier was also investigated with each of these micellar systems. All the analytes, including the isomers, were completely separated by use of MEKC with Brij 35 but when SDS was used only 11 compounds were separated because two amino compounds coeluted.     

Sodium maleopimaric acid as pseudostationary phase for chiral separations of amino acid derivatives by capillary micellar electrokinetic chromatography

A new type of chiral surfactant, sodium maleopimaric acid (SMA), was synthesized, and employed for the enantioselective micellar electrokinetic chromatographic (MEKC) separation of amino acid enantiomers derivatized with naphthalene-2,3-dicarboxaldehyde (NDA-D/L-AAs). The effect of the surfactant concentration, type and concentration of the BGE, and buffer pH on the resolution was studied, and optimized conditions were used to evaluate the ability of this new surfactant to perform chiral separations toward NDA-D/L-AAs by MEKC. Enantiomeric separations of NDA-D/L-AAs were achieved with a running buffer consisting of 100 mM borate (pH 9.5) and 20 mM SMA in a 58.5 cm length x 50 microm id capillary. Under the conditions selected, two pairs of tested amino acid enantiomers including NDA-D/L-trptophan (Trp) and NDA-D/L-kynurenine (Kyn) were resolved.     

Separation of naturally occurring triterpenoidal saponins by capillary zone electrophoresis.

A high-performance capillary electrophoresis (HPCE) was successfully applied to the separation and quantitation of naturally occurring oleanene triterpenoidal saponins. The HPCE adapted to the separation of two pairs of disteriomeric saponins (1-2) or (3-4), obtained from Trifolium alexandrinum seeds, was based on capillary zone electrophoresis (CZE) in borate buffer with UV detection at 195 nm. An usual technique for isolation and group separation of saponins was developed as an appropriate purification step prior to determination of individual saponins by CZE. The separation parameters such as borate concentration, pH and applied voltage were varied in order to find the best compromise that complied with demands for high separation, short duration and sufficiently high detector response. The optimum running conditions were found to be 60 mM borate buffer, pH 10 and 12 kV. Under the alkaline borate electrolyte, no resolution was achieved for the saponins (1 and 3) or (2 and 4) in a single mixture, except when 20 mM beta-cyclodextrin was added to the running electrolyte. With the combined techniques of group separation, purification and CZE, a rapid and efficient method for the determination of naturally occurring diasteriomeric saponins is now available.     

Elimination of adsorption effects in the analysis of water-soluble vitamins in pharmaceutical formulations by capillary electrophoresis.

A tendency to an increase in migration times was observed when different water-soluble vitamins were analysed repeatedly in pharmaceutical preparations by capillary electrophoresis. In order to better understand the origin of this effect, the influence of the vitamins and the excipients, such as cellulose derivatives, was investigated. These studies indicated that the increase in analyte migration times was most probably due to the adsorption of different kinds of constituents to the capillary wall. Different rinsing procedures were tested in order to eliminate these unfavourable effects. A rinse of the capillary with a 25 mM sodium dodecyl sulfate (SDS) solution in the running buffer between successive runs was found to be particularly effective when the analysis was performed by free solution capillary zone electrophoresis (CZE). When the vitamins were determined by micellar electrokinetic chromatography (MEKC) using SDS as surfactant, a short capillary rinse with the running buffer was sufficient to obtain reproducible migration times. The CZE and MEKC methods developed were validated and compared. Both methods could be applied to the determination of water-soluble vitamins in different multivitamin formulations.     

Reversed-polarity capillary zone electrophoretic analysis of usnic acid.

A capillary zone electrophoretic (CZE) method for the determination of usnic acid is described for the first time. Usnic acid is an antibiotic substance from lichens. Due to its low solubility in water, a high content of methanol in CZE buffer is required. Because of the methanol in the buffer, the electroosmotic flow velocity was lower than the electrophoretic mobility of usnic acid. Accordingly, the use of reversed-polarity (with the anode on the detector side of the capillary) was necessary. The optimal buffer composition was 50 mM NaOH, 20 mM acetic acid and 5% water in methanol. The detection limit of UV detector at 290 nm for usnic acid in the injected extract was 3.5 mg/L and the relative standard deviation of the normalized peak area was 3.3% at 250 mg/L.     

Method development and validation for the analysis of didanosine using micellar electrokinetic capillary chromatography

A selective MEKC method was developed for the analysis of didanosine in bulk samples. Successful separation of didanosine from 13 of its potential impurities, derived from the various synthetic preparation procedures, was achieved. As CZE gave poor separation selectivity, MEKC was preferable. The use of EKC allowed achievement of the separation in a significantly shorter time than conventional HPLC. An anionic long-chain surfactant, lithium dodecyl sulfate (LiDS), was used as the pseudostationary phase and sodium tetraborate buffer as the aqueous phase. In order to obtain the optimal conditions and to test the method robustness, a central composite response surface modeling experiment was performed. The optimized electrophoretic conditions include the use of an uncoated fused-silica capillary with a total length of 40 cm and an ID of 50 microm, a BGE containing 40 mM sodium tetraborate and 110 mM LiDS at pH 8.0, an applied voltage of 18.0 kV, and the capillary temperature maintained at 15 degrees C. The method was found to be robust. The parameters for validation such as linearity, precision, and sensitivity are also reported. Three commercial bulk samples were analyzed with this system.     

Separation of linoleic acid oxidation products by micellar electrokinetic capillary chromatography and nonaqueous capillary electrophoresis

In this work the suitability of micellar electrokinetic capillary chromatography (MEKC) and nonaqueous capillary electrophoresis (CE) to the analysis of the primary oxidation products of linoleic acid was studied with uncoated fused-silica capillaries. The primary autoxidation products of linoleic acid are the four hydroperoxide isomers 13-hydroperoxy-cis-9, trans-11-octadecadienoic acid, 13-hydroperoxy-trans-9, trans-11-octadecadienoic acid, 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid, 9-hydroperoxy-trans-10, trans-12-octadecadienoic acid. Addition of a surfactant such as sodium dodecyl sulfate (SDS) or sodium cholate (SC) into the running buffer (20-30 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) or ammonium acetate, pH 9.5-11) was required to enhance the water solubility of the sample and selectivity of the separation. MEKC proved to be a promising new technique for the separation of the primary oxidation products of lipids giving results comparable to high performance liquid chromatography (HPLC). Partial separation of hydroperoxide isomers was also achieved using nonaqueous CE with methanol-acetonitrile-sodium cholate as running buffer.     

Determination of chlorophenols in human urine based on the integration of on-line automated clean-up and preconcentration unit with micellar electrokinetic chromatography.

A new method was developed and validated for the determination of chlorophenols in human urine by using micellar electrokinetic chromatography (MEKC) coupled via a mechanic arm to an on-line automatic clean-up and preconcentration unit for urine samples. Separation is accomplished by using a selective buffer consisting of 15 mM borate, 25 mM phosphate and 100 mM sodium dodecyl sulfate (SDS) at pH 9.1 in addition to a positive power supply of 25 kV at 18 degrees C. The proposed capillary electrophoresis (CE) method allows the separation of 11 chlorophenols within 7 min with a reproducibility as relative standard deviation (RSD) between 2.6% and 7.2%, and limits of detection (LODs) between 0.08 and 0.46 microg/mL for all chlorophenols. Urine samples were previously hydrolyzed with 37% HCl at 80 degrees C for 60 min and then cleaned on a C-18 mini-column. Recoveries ranged from 58% to 103%. The preconcentration treatment affords limits of determination between 4 and 12 ng/mL for all chlorophenols except pentachlorophenol and 4-chlorophenol, which could not be determined. The overall analysis time, including on-line clean-up, preconcentration and electrophoretic separation is 20 min per sample.     

Interlaboratory reproducibility of mobility parameters in capillary electrophoresis for substance identification in systematic toxicological analysis

An interlaboratory pilot study was performed to determine the reproducibility of mobility parameters in capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). The study was performed by an intended small number of laboratories (three) that used different brands of instruments (two). The effective mobility was corrected using standards by a method that was recently introduced to obtain a more reproducible migration parameter. A test set of 20 acidic test compounds and 5 reference compounds were analyzed during five days in each laboratory using CZE and MEKC. Buffers used consisted of 90 mM borate set at pH 8.4 (CZE) and 20 mM phosphate, 50 mM sodium dodecyl sulfate set at pH 7.5 (MEKC). Analyses were carried out using fused-silica capillaries at an electric field strength of either 52.6 kV/m or 37.5 kV/m. The interlaboratory reproducibility (mean RSD) of the effective mobility was 3.0% for CZE and 6.7% for MEKC. After applying the correction method, these values became 3.0% for CZE and 3.3% for MEKC, which is adequate for systematic toxicological analysis (STA) applications. A significant improvement of reproducibility for the calculated corrected effective mobility mu(eff)c was observed when variations are high. Therefore, it is recommended to use the correction method in interlaboratory situations, especially when instruments and capillaries from different manufacturers are used.     

A capillary zone electrophoresis for determination of thiolic peptides in biological samples

A new method to improve the analyses of thiolic peptides (cysteine, γGlu-Cys, glutathione, phytochelatins and desglycyl-phytochelatins) derivatized with monobromobimane (mBrB) in complex biological samples by CZE is described. The method involves a SPE using Sep-Pak Light C18 Cartridges after derivatization and a later CZE analysis. Elution of mBrB-thiols was achieved with 10 mM HCl + 70% methanol v/v in deionised water. Electrophoretic parameters, such as BGE pH and concentration, different organic additives (methanol and trifluoroethanol), applied voltage and capillary length were studied in order to establish suitable analytical conditions. Optimum separation of the mBrB-thiolic peptides was obtained with 100 mM sodium borate buffer at pH 7.60. The electrophoretic conditions were +15 kV, capillary length of 90 cm from inlet to detector (98 cm total length, 50 μm ID), samples were loaded into the capillary by hydrodynamic injection (50 mbar, 20 s) and detection was performed at 390 nm. The improved method showed good reproducibility, linearity and sensitivity. The LODs and LOQs estimated using a standard of GSH were 1.41 and 4.69 μM respectively.     

Optimization of solid-phase extraction and solid-phase microextraction for the determination of alpha- and beta-endosulfan in water by gas chromatography-electron-capture detection

The electrophoretic behaviour of ionizable and neutral alkylxanthines commonly used in pharmaceutical preparations was studied. The performance of various separation modes including capillary zone electrophoresis (CZE), cyclodextrin electrokinetic chromatography, and micellar electrokinetic chromatography (MEKC) with either sodium dodecyl sulfate (SDS) or bile salts as surfactants, was assessed. CZE in an alkaline medium successfully separates ionizable xanthines and dyphylline. The addition of carboxymethyl-β-cyclodextrin to the background electrolyte allows only partial resolution of neutral xanthines. Based on MEKC results, bile salts exhibit more discrimination ability than SDS to separate similar xanthines. The best results are provided by taurodeoxycholic acid, which ensures baseline separation of xanthines.