Cyclodextrin-modified Micellar Electrokinetic Chromatography Research Articles

Determination of hydroxyanthraquinoids in Rhubarb by cyclodextrin-modified micellar electrokinetic chromatography using a mixed micellar system of sodium dodecyl sulfate and sodium cholate

A cyclodextrin-modified micellar electrokinetic chromatographic (CD-MEKC) method was established to determine five hydroxyanthraquinoids in Rhubarb. The five components were successfully separated by using the mixed micellar system consisting of 20 mmol/l sodium dodecyl sulfate (SDS) and 20 mmol/l sodium cholate (SC) with 10 mmol/l β-cyclodextrin in phosphate buffer (pH 10.4). The separation was optimized by adjusting buffer pH, concentrations of β-cyclodextrin and SC and applied voltage. The proposed method was validated and applied to the determination of two commercial Rhubarb samples. The results obtained were satisfactory.

Separation and Determination of Active Components in Rhubarb by Cyclodextrin-Modified Micellar Electrokinetic Chromatography Using a Mixed Micellar System

A novel method for determination of five anthraquinone derivatives and one distyrene derivative in Rhubarb by CD-MEKC with mixed micellar system of SDS and STC was developed. The separation was optimized by adjusting buffer pH, SDS, STC, and β-CD concentrations. The six components were separated successfully in buffer solution of 20 mmol/L NaOH + H3BO3 (pH 11) containing 20 mmol/L SDS and 20 mmol/L STC with 15 mmol/L β-CD. The method was examined in terms of linearity, detection limit, precision, and recovery. Two commercial Rhubarb plants and its preparation were analyzed by the proposed method.

Simultaneous determination of free and conjugated bile acids in serum by cyclodextrin-modified micellar electrokinetic chromatography

A simultaneous determination of 15 free and most conjugated forms of bile acids (BA) in serum using capillary electrophoresis is described. The optimized and validated method proposed in this work is straightforward and rapid, employing affordable equipment. A background electrolyte of 5 m M β-cyclodextrin, 5 m M 2-hydroxypropyl-β-cyclodextrin, 50 m M SDS and sodium borate–dihydrogen phosphate pH 7.0 with 10% of acetonitrile was used. The complete separation of 15 BA, not easily achievable with other methods, is performed in less than 12 min using a UV detector with good precision and accuracy. BA were extracted from pretreated serum samples using a C 18-solid-phase extraction and the recovery values ranged from 65 to 107.8%. Limits of quantitation were between 0.58 and 3.2 μ M. This method proved to be suitable to determine individual BA profiles which are more useful than total serum bile acids as indicators of metabolic disorders and hepatobiliary diseases.

Enantioselective analysis of thiobencarb sulfoxide produced by metabolism of thiobencarb by hydroxypropyl-γ-cyclodextrin modified micellar electrokinetic chromatography

Cyclodextrin-modified micellar electrokinetic chromatography was applied to the enantioseparation of thiobencarb sulfoxide, which is produced by S-oxygenation of thiobencarb, a herbicide, using γ-cyclodextrin together with sodium dodecyl sulfate. Factors affecting chiral resolution and migration time of thiobencarb sulfoxide were studied. The optimum running conditions were found to be 20 mM phosphate-5 mM borate buffer (pH 8.5) containing 60 mM hydroxypropyl-γ-cyclodextrin and 100 mM sodium dodecyl sulfate with an effective voltage of +20 kV at 20°C using direct detection at 220 nm. Under these conditions, the resolution (Rs) of racemic thiobencarb sulfoxide was approximately 1.7. Thiobencarb was treated with a rat liver microsomal fraction containing cofactors (known as S9mix). The ratio between (+)- and (-)-thiobencarb sulfoxide was found to be 15:85. It was also found that the ratio between (+)-and (-)-thiobencarb sulfoxide produced in soil spiked with thiobencarb was 3:7. These results indicated marked enantioselectivities for these metabolisms. The activities of thiobencarb, (+)- and (-)-thiobencarb sulfoxides on 5α-dihydrotestosterone-and 17β-estradiol-induced transcriptions were also investigated. Whereas thiobencarb and (+)-thiobencarb sulfoxide did not show any activities, (-)-thiobencarb sulfoxide showed significant anti-estrogenic and anti-androgenic activities, suggesting that thiobencarb sulfoxide can act as both an enantioselective anti-estrogen and an enantioselective anti-androgen.

Enantioseparation of phenothiazines in cyclodextrin-modified micellar electrokinetic chromatography

In this study, enantioseparations of five phenothiazines in cyclodextrin (CD)-modified micellar electrokinetic chromatography (MEKC) were investigated using a citrate buffer containing tetradecyltrimethylammonium bromide (TTAB) as a cationic surfactant at low pH. β-Cyclodextrin (β-CD) and hydroxylpropyl-β-CD (HP-β-CD) were selected as chiral selectors. The results indicate that the separation window is greatly enlarged by β-CD concentration and that the separability and selectivity of phenothiazines are remarkably influenced by the concentrations of both β-CD and TTAB, as well as buffer pH. The interaction of thioridazine with β-CDs is considerably reduced in the presence of TTAB micelles due to competitive complexation of thioridazine with TTAB micelles, which is pH-dependent. As a result, effective enantioseparation of thioridazine is simultaneously achievable with that of trimeprazine and promethazine or ethopropazine in MEKC with addition of either β-CD or HP-β-CD, respectively, to a micellar citrate buffer containing TTAB at pH 3.5. Better enantioresolution of thioridazine in MEKC than in capillary zone electrophoresis can be obtained.

Separation of alkamides from Echinacea purpurea extracts by cyclodextrin-modified micellar electrokinetic chromatography.

Separation of nine important alkyl methylbutyl- and isobutylamides (known as alkamides) obtained from Echinacea purpurea extracts was investigated by using cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC). Hydrophobic alkamides interact strongly with the micelles from the most common surfactants used in MEKC and this lead to predominant partition of the analytes into the micellar phase, resulting in poor resolution. The addition of neutral CDs to the alkaline (10 mM phosphate buffer pH 8.0) micellar system of sodium dodecyl sulfate (SDS), sodium cholate (SC) and sodium deoxycholate (SDC) was found to improve the separation of the studied alkamides. Among the several combinations surfactant/CD, three different systems showed to be particularly effective: SDS/hydroxypropyl-beta-CD (110 mM/100 mM) and SC/heptakis (2, 3, 6-tri-O-methyl)-beta-CD (200 mM/40 mM) which provided a complete separation of the studied compounds, and SDC/heptakis (2, 6-di-O-methyl)-beta-CD. The importance of appropriate surfactant vs. CD concentration ratio as well as that of total concentration of both surfactant and CD was considered. The optimization of the separation was performed by focussing the need for a rapid separation of nine alkamides diagnostically useful to define the fingerprint of Echinacea species.

Enantiomeric separation of primaquine, an anti-malarial drug, by cyclodextrin-modified micellar electrokinetic capillary chromatography.

Enantiomeric separation of primaquine, an anti-malarial drug, by cyclodextrin-modified micellar electrokinetic capillary chromatography.

Separation and first structure elucidation of Cremophor EL-components by hyphenated capillary electrophoresis and delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry.

The polyethoxylated heterogeneous components of the so far poorly characterized nonionic emulsifier Cremophor EL (polyoxyl 35 castor oil) (CrEL) were fractionated by cyclodextrin-modified micellar electrokinetic capillary chromatography (CD-MEKC). Due to the low UV absorbance of most of the CrEL-components an indirect UV detection was used with phenobarbital-sodium as background absorber. For a precise assignment of the resulting peaks to the corresponding components capillary electrophoresis (CE) had to be combined with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS) as detection system. For this purpose, the fractionating robot Probot was employed which enables both the on-line fractionation of the CE eluate on a MALDI target during the electrophoretic separation and the simultaneous dosage of the MALDI matrix solution. The applied CrEL amount was optimized by varying the CE injection parameters time, pressure and concentration of the sample in order to obtain homologue peak series of sufficient intensity without decreasing the separation efficiency. Evaluation of the mass spectra was performed by comparing the residue masses of the homologue peak series with the calculated residue masses of potential CrEL-components. However, the high number of polyethoxylated components leads to overlapping of homologue peak series with isobaric residue masses. These isobaric interferences were detected by a high mass accuracy of the measurements (obtained by internal calibration with polyethylene glycol (PEG) 1000 and by means of the residue mass plot, the newly developed evaluation method. The combination of these techniques allowed the first detailed structure analysis of the CrEL-components showing glycerol polyoxyethylene (POE) monoricinoleate and POE monoricinoleate to be the two main components of the emulsifier. Furthermore, the coupling of CE with DE-MALDI-TOF-MS is generally applicable to the fractionation and identification of polymers.

Determination of six components in Rhubarb by cyclodextrin-modified micellar electrokinetic chromatography using a mixed micellar system of sodium cholate and sodium taurocholate

A cyclodextrin-modified micellar electrokinetic chromatographic method was established to determine five anthraquinone derivatives and a distyrene derivative in Rhubarb. The six components were successfully separated by using the mixed micellar system consisting of 20 mmol/l SC and 20 mmol/l STC with 15 mmol/l β-CD in the BGE of 20 mmol/l borax buffer (pH 11) in less than 20 min. Effect of SC and STC concentrations, pH of BGE and concentration of β-CD on the separation was studied. The analytical performance of the method was discussed in terms of linearity response, precision, detection limits, quantitation limits and recoveries. The method developed was applied to the determination of three commercial Rhubarb samples. The results obtained agreed with those of the reported documents.

Enantioseparation of vinclozolin by gamma-cyclodextrin-modified micellar electrokinetic chromatography.

Cyclodextrin-modified micellar electrokinetic chromatography was applied to the enantioseparation of vinclozolin, which has been used as a fungicide and has an anti-androgenic activity, using gamma-cyclodextrin together with sodium dodecyl sulfate. Factors affecting the chiral resolution and migration time of vinclozolin were studied. The optimum running conditions were found to be 20 mM phosphate-5 mM borate buffer (pH 8.5) containing 50 mM gamma-cyclodextrin and 100 mM sodium dodecyl sulfate with an effective voltage of 20 kV at 20 degrees C using direct detection at 203 nm. Under these conditions, the resolution (Rs) of racemic vinclozolin was approximately 2.1. The sample was concentrated by solid-phase extraction and was fractionated by HPLC. The peak area ratio of (+)- and (-)-vinclozolins in wine was found to be 2:3, namely, not racemic, suggesting that degradation rates were different between (+)- and (-)-vinclozolins. The anti-androgenic activities of (+)- and (-)-vinclozolins on dihydrotestosterone-induced transcription were also investigated. The anti-androgenic activity of (+)-vinclozolin tended to be stronger than that of (-)-vinclozolin, suggesting the possibility that vinclozolin can act as an enantioselective anti-androgen.

Simultaneous separation of the enantiomers of cizolirtine and its degradation products by capillary electrophoresis

The simultaneous enantioselective separation of (±)-cizolirtine and its impurities: (±)- N-desmethylcizolirtine, (±)-cizolirtine- N-oxide and (±)-5-(α-hydroxybenzyl)-1-methylpyrazole was investigated by capillary electrophoresis. Electrokinetic chromatography with carboxymethyl-β-CD (CM-β-CD) and sulfobutyl-ether-β-CD was tried, showing good enantioseparation but poor chemical selectivity. The four racemic pairs were baseline separated, in a single run, by cyclodextrin-modified micellar electrokinetic chromatography. The migration buffer composition was: (60 m M hydroxypropyl-β-cyclodextrin–150 m M sodium dodecyl sulfate–50 m M disodium tetraborate, pH 9.2, in water)–butanol (95:5, v/v). Work was done to determine the effect of buffer components and their optimal concentration on selectivity. The method was validated with respect to enantioselectivity of cizolirtine as well as its degradation products and separation selectivity between the different components. Linearity, limit of detection, limit of quantitation and precision were also determined. This method is suitable for the enantiomeric purity determination and stability control of cizolirtine (racemic mixture or enantiomers) and its degradation products. Examples of electropherograms of ( R)-cizolirtine degraded under stressed conditions are shown.

Investigation of substance P transport across the blood-brain barrier

The transport of substance P (SP) was investigated using the bovine brain microvessel endothelial cell culture model of the blood-brain barrier (BBB). The samples were derivatized precolumn with naphthalene dialdehyde, then analyzed by cyclodextrin-modified micellar electrokinetic chromatography with laser-induced fluorescence detection. SP crossed the BBB in both the apical-to-basolateral and basolateral-to-apical directions through an active transport mechanism. The transport of SP from the apical side was demonstrated to be via transcytosis. The N-terminal (SP 1–4) and C-terminal (SP 3–11) fragments were also found to permeate the BBB from the apical side.

Quantitative determination of amygdalin epimers by cyclodextrin-modified micellar electrokinetic chromatography

A new capillary electrophoresis method was developed for the quantitative determination of the amygdalin epimers, amygdalin and neoamygdalin, which are biologically significant constituents in the crude drugs, namely Persicae Semen and Armeniacae Semen. The effects of surfactants, additives and other analytical parameters were studied. As a result, the resolution of two epimers was performed by cyclodextrin-modified micellar electrokinetic chromatography with a buffer containing α-cyclodextrin and sodium deoxycholate. By the application of this method, a simple, fast and simultaneous quantitative determinations of amygdalin epimers in the crude drugs (Persicae Semen and Armeniacae Semen) and the Chinese herbal prescriptions (Keishi-bukuryo-gan and Mao-to) were achieved.

Enantiomeric determination of praziquantel and its main metabolite trans-4-hydroxypraziquantel in human plasma by cyclodextrin-modified micellar electrokinetic chromatography.

A capillary electrophoresis method for the simultaneous quantitation of praziquantel and its main metabolite trans-4-hydroxypraziquantel enantiomers in human plasma was developed and validated using cyclodextrin-modified micellar electrokinetic chromatography. Sample clean-up involved a single-step liquid-liquid extraction of plasma with toluene after the addition of NaCl. The complete enantioselective analysis was obtained in less than 7 min using 2% w/v sulfated beta-cyclodextrin as chiral selector and 20 mmol/L sodium deoxycholate as surfactant, in 20 mmol/L sodium borate buffer, pH 10. A 50 microm x 42 cm uncoated fused-silica capillary was used for the analysis, performed at a voltage of 18 kV and at 20 degrees C. The calibration curves were linear over the 125-625 ng/mL concentration range. The mean recoveries for praziquantel and trans-4-hydroxypraziquantel were up to 96 and 71%, respectively, with good precision. All four enantiomers were quantified at two concentration levels (200 and 600 ng/mL) with precision and accuracy below 15%. The quantitation limit was 50 ng/mL for (-)-(R)- and (+)-(S)-praziquantel and 62.5 ng/mL for (-)-(R)- and (+)-(S)-trans-4-hydroxypraziquantel, using 1 mL of human plasma.

Hybridation of different chiral separation techniques with ICP‐MS detection for the separation and determination of selenomethionine enantiomers: chiral speciation of selenized yeast

Enantioseparation and determination of selenomethionine enantiomers in selenized yeast was investigated using chiral separation techniques based on different principles, coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS) for selenium-specific detection. High performance liquid chromatography (HPLC) on a beta-cyclodestrin (beta-CD) column, cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC), gas chromatography (GC) on a Chirasil-L-Val column, and HPLC on a Chirobiotic T column have been investigated as the chiral separation techniques. For HPLC separation on the beta-CD column, and also for CD-MEKC, selenomethionine enantiomers were derivatized with NDA/CN(-). For chiral separation by GC, selenomethionine enantiomers were converted into their N-trifluoroacetyl (TFA)-O-alkyl esters. The developed hybridation methodologies are compared with respect to enantioselectivity, sensitivity and analysis time. The usefulness of the best-suited method [HPLC (Chirobiotic T)-ICP-MS] was demonstrated by its application to the successful chiral speciation of selenium and D-and L-selenomethionine content determination in selenized yeast.

Laser-induced fluorescence detection at 266 nm in capillary electrophoresis: Polycyclic aromatic hydrocarbon metabolites in biota

The separation of five phenolic polycyclic aromatic hydrocarbon metabolites (hydroxy-PAHs) has been performed by cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) using a 30 m M borate buffer (pH 9.0) containing 60 m M sodium dodecyl sulfate and varying concentrations of γ-cyclodextrin (γ-CD). A concentration of 12.5 m M γ-CD was found to provide a baseline separation of the five hydroxy-PAHs. We applied conventional fluorescence and laser-induced fluorescence (LIF) detection, using a new, small-size, quadrupled Nd–YAG laser emitting at 266 nm. The best limits of detection, in the low ng/ml range, were achieved using LIF detection. For all analytes, linearity was observed up to ca. 100 ng/ml. As an application, conjugated pyrene metabolites in hepatopancreas samples from the terrestrial isopods Oniscus asellus and Porcellio scaber were separated and detected. Finally, flatfish bile samples from individuals exposed to polluted sediment or crude oil, which were part of an interlaboratory study, were analyzed by CD-MEKC with conventional fluorescence and LIF detection to determine the 1-hydroxypyrene concentrations.

Simultaneous determination of eleven ingredients in ophthalmic solutions by cyclodextrin-modified micellar electrokinetic chromatography with tetrabutylammonium salt

Cyclodextrin-modified micellar electrokinetic chromatography was applied to determine simultaneously 11 active ingredients in ophthalmic solutions. All the ingredients were successfully separated by using the mixed carrier system containing sodium dodecyl sulfate with β-cyclodextrin and tetrabutylammonium phosphate. The effects of the cyclodextrin type on selectivity were also examined. Excellent separation of the all ingredients was obtained by the use of dimethyl-β-cyclodextrin. The established method was validated and confirmed to be applicable to the determination of the active ingredients in a commercial ophthalmic solution. These results suggest that capillary electrophoresis can be applied to the quantitative analysis as well as qualitative analysis in pharmaceuticals.

Enantiomeric separation of selenoaminoacid derivatives by cyclodextrin-modified micellar electrokinetic chromatography using a mixed micellar system of sodium dodecyl sulphate and taurodeoxycholic acid

A new cyclodextrin-modified micellar electrokinetic chromatography (CD -MEKC) method for the enantiomeric separation and determination of the selenoaminoacids selenomethionine and selenoethionine, derivatised with 2,3-naphthalenedicarboxaldehyde (NDA) to produce cyanobenzoisoindole (CBI) derivatives, has been developed. Optical resolution was achieved in a fused-silica capillary column using 30 mmol l −1 phosphate/10 mmol l −1 boric acid (pH 7) electrophoretic buffer containing a mixed micellar system of achiral sodium dodecyl sulphate and chiral taurodeoxycholic acid surfactants among with β-cyclodextrins. Detection was followed by direct UV absorptiometric measurements at 230 nm. The concentration influence of the two chiral selectors used (β-CD and TDC micelles), the effect of applied voltage and the pH of the electrophoretic buffer on the migration time and resolution have been investigated. The analytical performance of the method is discussed in terms of detection limits, linearity of response and precision. The method developed has been applied to the determination of enantiomeric purity in a commercial sample sold as ‘pure’ l-selenomethionine.

Analysis of cis– trans isomers and enantiomers of sertraline by cyclodextrin-modified micellar electrokinetic chromatography

In this work development, optimization and validation of a cyclodextrin-modified micellar electrokinetic chromatography (CD-modified MEKC) method is proposed to resolve separation of the sertraline hydrochloride and synthesis-related substances. Sertraline hydrochloride, the cis-(1 S,4 S) enantiomer form, is used as an antidepressant therapeutic agent. A buffer concentration composed of 20 m M sodium borate, pH 9.0 with 50 m M sodium cholate, 15 m M sulfated β-cyclodextrin and 5 m M hydroxypropyl-β-cyclodextrin was found to be the most suitable background electrolyte. Quantitation of the impurities at levels of 0.1% in different samples of the bulk drug was determined. A comparison of the results with those obtained by HPLC methodology was also accomplished. The method proved appropriate for testing the purity of sertraline hydrochloride in bulk drug.

High-Speed Separation of Oleanolic acid and Ursolic acid from the Fruit of Ligustrum Lucidum ait and Crataegus by Cyclodextrin-Modified Micellar Electrokinetic Chromatography

ABSTRACT A cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) technique for determining oleanolic acid and ursolic acid in the fruit of Ligustrum lucidum Ait and Crataegus was developed. The two bio-active components were successfully separated within 4 minutes using a pH 8.5 sodium borate buffer containing 60mM SDS and 2mM γ-CD. The correlation coefficients of the linear calibration graphs for the analyses exceeded 0.9980. The effects of pH, SDS, γ-CD, sodium borate and temperature on separation were also investigated.