Analysis of combinatorial chemistry samples by micellar electrokinetic chromatography

A micellar electrokinetic chromatography (MEKC) method has been developed that can evaluate the purity of samples generated in combinatorial chemistry libraries. This method uses an open tube capillary (27 cm x 50 microm) along with a run buffer composed of sodium dodecyl sulfate (SDS), hydroxypropyl-beta-cyclodextrin, and sodium tetraborate coupled with UV detection. Neutral compounds and compounds that were insoluble in aqueous buffers could be analyzed under these conditions in approximately 3 min. The concentration of SDS and the concentration of hydroxypropyl-beta-cyclodextrin effected the separation. The affect on selectivity resulting from the addition of an organic modifier to the run buffer was examined. The low background absorbency of the run buffer made for easy detection of compounds that absorbed at low UV wavelengths. The quick analysis time made this suitable for analysis of combinatorial chemistry samples.     

Analysis of ecdysteroids by micellar electrokinetic chromatography with on-line preconcentration

In order to enhance the UV detection sensitivity, an application study of an on-line preconcentration technique for micellar electrokinetic chromatographic (MEKC) was carried out. The simultaneous determination of four test ecdysteroids, 20-hydroxyecdysone, ajugasterone C, polypodine B and ponasterone A has been investigated by using the normal stacking mode in MEKC with UV detection. The effects of anionic surfactant composition and concentration, the applied voltage, the pH buffer, the kind and the amount of organic solvent and the injection time on the analyte resolution were evaluated. The optimised conditions for the separation involved the use of a 50 mM borate as the running buffer containing 50 mM of a mixture of sodium dodecyl sulphate (SDS) and sodium cholate (SC) in the ratio of 1:1 together with a concentration of 10% (v/v) of 2-PrOH at pH 9.0. Hydrodynamic injection of 12 s at 50 mbar and separation voltage of 20 kV at temperature of 20 degrees C were employed. These conditions allowed a repeatability separation within 21 min. Concentration detection limit for the neutral analytes studied improve about an order of magnitude. The method was also applied to the determination of ecdysteroids in a real sample.     

Use of short chain alkyl imidazolium ionic liquids for on‐line stacking and sweeping of methotrexate,flinic acid and folic acid: Their application to biological fluids

Methotrexate (MTX) is widely used for the treatment of many types of cancer. Folinic acid (FNA) and folic acid (FA) were usually simultaneously supplemented with MTX to reduce the side effects of a folate deficiency. This study, for the first time, included on‐line sample preconcentration by stacking and sweeping techniques under reduced or enhanced electric conductivity in the sample region using short chain alkyl imidazolium ionic liquids (ILs) as micelle forming agents for analyte focusing. Both analyte focusing by micelle collapse (AFMC) and sweeping‐MEKC had been investigated for the comparison of their effectiveness to examine simultaneously MTX, FNA and FA in plasma and urine under physiological conditions. In sweeping‐MEKC, the sample solution without micelles was hydrodynamically injected as a long plug into a fused‐silica capillary pre‐filled with phosphate buffer containing 3.0 mol/L of 1‐butyl‐3‐methylimidazolium bromide (BMIMBr). Using AFMC, the analytes were prepared in BMIMBr micellar matrix and hydrodynamically injected into the phosphate buffer without IL micelles. The conductivity ratio between BGE and sample (γ, BGE/sample) was optimized to be 3.0 in sweeping‐MEKC and 0.33 in AFMC resulting the adequate separation of analytes within 4.0 min. To reduce the possibility of BMIMBr adsorption, an appropriate rinsing protocol was used. The limits of detection were calculated as 0.1 ng/mL MTX, 0.05 ng/mL FNA and 0.05 ng/mL FA by sweeping‐MEKC and 0.5 ng/mL MTX, 0.3 ng/mL FNA and 0.3 ng/mL FA by AFMC. The accuracy was tested by recovery in plasma and urine matrices giving values ranging between 90 and 110%. Both stacking and sweeping by BMIMBr could be successfully used for the rapid, selective and sensitive determination of pharmaceuticals in complex matrices due to its fascinating properties, including high conductivity, good thermal stability and ability to form different types of interactions by electrostatic, hydrophobic, hydrogen bonding and π–π interactions. In sweeping‐MEKC, the using of BMIMBr enhanced the γ factor, k retention factor and the injected amount of sample. Consequently, this technique offers particular potential for higher sensitivity by giving 22‐ and 5‐fold sensitivity enhancement factors (SEFs) of MTX compared to CZE and AFMC, respectively.     

Influence of high‐conductivity buffer composition on field‐enhanced sample injection coupled to sweeping in CE

The aim of this work was to clarify the mechanism taking place in field‐enhanced sample injection coupled to sweeping and micellar EKC (FESI‐Sweep‐MEKC), with the utilization of two acidic high‐conductivity buffers (HCBs), phosphoric acid or sodium phosphate buffer, in view of maximizing sensitivity enhancements. Using cationic model compounds in acidic media, a chemometric approach and simulations with SIMUL5 were implemented. Experimental design first enabled to identify the significant factors and their potential interactions. Simulation demonstrates the formation of moving boundaries during sample injection, which originate at the initial sample/HCB and HCB/buffer discontinuities and gradually change the compositions of HCB and BGE. With sodium phosphate buffer, the HCB conductivity increased during the injection, leading to a more efficient preconcentration by staking (about 1.6 times) than with phosphoric acid alone, for which conductivity decreased during injection. For the same injection time at constant voltage, however, a lower amount of analytes was injected with sodium phosphate buffer than with phosphoric acid. Consequently sensitivity enhancements were lower for the whole FESI‐Sweep‐MEKC process. This is why, in order to maximize sensitivity enhancements, it is proposed to work with sodium phosphate buffer as HCB and to use constant current during sample injection.     

On-line micellar electrokinetic chromatography-mass spectrometry: feasibility of direct introduction of non-volatile buffer and surfactant into the electrospray interface

An on-line method for the coupling of micellar electrokinetic chromatography (MEKC) and mass spectrometry (MS) is presented which allows conventional MEKC conditions to be employed without further modification. The MEKC system is coupled directly to electrospray ionization (ESI) MS using a triaxial interface. A systematic study of the influence of the surfactant concentration, the nature and concentration of buffer salts and presence of organic modifier on the interface performance indicated the feasibility of the MEKC–MS approach. Effective interfacing of MEKC was achieved with both single quadrupole and ion-trap MS instruments. Using a background electrolyte containing 20 mM sodium dodecyl sulfate (SDS) and 10 mM sodium phosphate buffer, it is demonstrated that full MEKC runs of test mixtures of mebeverine and related compounds can be monitored by ESI-MS with satisfactory sensitivity. Sub-μg/ml levels of the analytes can still be detected in full scan mode, while detection limits are in the 10–50 ng/ml range when selected ion monitoring is applied. It is shown that such sensitivity would allow full-scan MS detection of 0.1% (w/w) levels of potential impurities in mebeverine. With the ion-trap instrument successful MEKC–MS/MS experiments were carried out providing information-rich MS spectra of the related compounds. Repeated MEKC–MS analyses proved that in the course of 1 day the migration time of mebeverine remained fairly constant while the MS-signal intensity only gradually decreased to approximately 65% of its original value. Once-a-day cleaning of the first part of the ion source, which takes only 5 min, suffices to preserve an optimal interface performance for a prolonged period of time.     

Fundamental studies on electrokinetic chromatography with PEGylated phospholipid micelles.

In this study, micelles prepared from distearoylphosphatidylethanolamine with covalently attached poly(ethylene) glycol) (PEG) of molecular weight 2000 (DSPE-PEG-2000) were employed in micellar electrokinetic chromatography (MEKC) as pseudostationary phases. Since DSPE-PEG-2000 contains long hydrophobic alkyl chains, an anionic phosphate group, and hydrophilic PEG chains, the prepared micelles are expected to provide a characteristic retention behavior for both neutral and ionic compounds. As a typical example, a baseline separation of phenol and 2-naphthol was successfully achieved by using the DSPE-PEG-2000 micelles as a background electrolyte for MEKC; such success clearly shows that the micelles can retain electrically neutral compounds. The MEKC separations of anionic and cationic compounds with a DSPE-PEG-2000 micellar solution and the enantioseparation of binaphthyl compounds with mixed micelles containing bile salt are also discussed.     

表面活性剂在高效毛细管电泳中的作用

表面活性剂作为缓冲液添加剂已广泛用于高效毛细管电泳中,综述了阴离子、阳离子、两性离子、非离子及手性等多种表面活性剂在离子、中性分子、手性化合物、多肽和蛋白质分离等方面的作用,介绍了其作用机理与改善高效毛细管电泳分离的原理。     

Approaching over 10 000‐fold sensitivity increase in chiral capillary electrophoresis: Cation‐selective exhaustive injection and sweeping cyclodextrin‐modified micellar electrokinetic chromatography

A novel and simple method that combines an online concentration technique with an enantioseparation technique for capillary electrophoresis—namely, cation‐selective exhaustive injection and sweeping cyclodextrin‐modified micellar electrokinetic chromatography (CSEI‐sweeping CD‐modified MEKC)—realizes the effective enantioseparation of cationic analytes while keeping a significant increase of detection sensitivity. This technique consists of a slight modification of the basic CSEI‐sweeping MEKC. The main idea is to simply add an anionic CD as a chiral selector into the micellar buffer including sodium dodecyl sulfate, but not to change any other buffers in order to preserve the online concentration mechanism. When applied to analysis of the street drug, methamphetamine, the method achieved not only a baseline enantioseparation but also limits of detection (LODs; S/N = 3) of 70–90 pg/mL (ppt) for each isomer. This translates to a more than 10 000‐fold improvement compared to the LODs by the usual injection method. The present technique, which was made from a slight modification of CSEI‐sweeping MEKC, would give an attractive approach that is applicable to almost any analytes for which CSEI‐sweeping MEKC is applicable; all that is required is the selection of an appropriate anionic CD to be added to the micellar buffer.     

Pharmaceutical applications of micelles in chromatography and electrophoresis

This review surveys the use of micelles as separation media in chromatography and electrophoresis. Applications to pharmaceuticals whose molecular masses are relatively small are focused on in this review. In high-performance liquid chromatography (HPLC), chromatography using micelles and reversed-phase stationary phases such as octadecylsilylized silica gel (ODS) columns is known as micellar liquid chromatography (MLC). The main application of MLC to pharmaceutical analysis is the same as in ion-pair chromatography using alkylsulfonate or tetraalkylammonium. In most cases, selectivity is much improved compared with other short alkyl chain ion-pairing agents such as pentanesulfonate or octanesulfonate. Direct plasma/serum injection can be successful in MLC. Separation of small ions is also successful by using gel filtration columns and micellar solutions. In electrophoresis, especially capillary electrophoresis (CE), micelles are used as pseudo-stationary phases in capillary zone electrophoresis (CZE). This mode is called micellar electrokinetic chromatography (MEKC). Most of the drug analysis can be performed by using the MEKC mode because of its wide applicability. Enantiomer separation, separation of amino acids and closely related peptides, separation of very complex mixtures, determination of drugs in biological samples etc. as well as separation of electrically neutral drugs can be successfully achieved by MEKC. Microemulsion electrokinetic chromatography (MEEKC), in which surfactants are also used in forming the microemulsion, is successful for the separation of electrically neutral drugs as in MEKC. This review mainly describes the typical applications of MLC and MEKC for the analysis of pharmaceuticals.     

Towards a general approach for the impurity profiling of drugs by micellar electrokinetic chromatography

A general micellar electrokinetic chromatographic (MEKC) strategy for the impurity profiling of drugs was developed involving a sodium dodecyl sulfate (SDS) and a cetyltrimethylammonium bromide (CTAB) MEKC system. With this combination, in principle, each sample component passes the detector in at least one of the two MEKC systems provided that separation buffers of the same pH are used in both systems. In order to select the proper MEKC systems, the electroosmotic flow (EOF) and micelle migration time (t(mc)) were determined for separation buffers of several pH values, containing various amounts of surfactant and organic modifier. The selectivity of the MEKC systems was studied using a mixture of compounds with a wide range of physico-chemical properties. The final selection of two adequate MEKC systems for this approach was based on the requirements that the t(mc) (i.e., analysis time) of both systems was below 20 min and that the t(mc)/t(eof) ratio was above 3 or 2 for the SDS and CTAB system, respectively. Furthermore, the systems should provide high efficiency, exhibit differences in selectivity and use moderate concentrations of modifier and surfactant, so that, if needed, further optimization is possible. The selected MEKC systems contained 60 mM SDS or 10 mM CTAB, respectively, in a phosphate buffer (pH 7.5) with 10% acetonitrile. Some test compounds with extreme mobilities were used to demonstrate the suitability of the MEKC approach to detect each component of a sample. The potential of the proposed MEKC combination for impurity profiling was demonstrated by the analysis of fluvoxamine with several impurities at the 0.1% level.     

Micellar electrokinetic chromatography-mass spectrometry

The combination of micellar electrokinetic chromatography (MEKC) with mass spectrometry (MS) is very attractive for the direct identification of analyte molecules, for the possibility of selectivity enhancement, and for the structure confirmation and analysis in a MS-MS mode. The direct coupling of MEKC with MS can be hazardous due to the effect of nonvolatile MEKC surfactants on MS performance, including the loss of analyte sensitivity and ion source contamination. The possibility of off-line coupling between MEKC and matrix-assisted laser desorption/ionization (MALDI)-MS remains to be investigated. Various approaches for on-line coupling MEKC with electrospray ionization (ESI)-MS, including the use of high-molecular-mass surfactant, an electrospray-chemical ionization (ES-CI) interface, a voltage switching and buffer renewal system, partial-filling micellar plug and anodically migrating micelles, are reviewed and evaluated. The use of an ES-CI interface is most promising for routine operation of on-line MEKC-MS under the influence of nonvolatile salts and surfactants. The use of a high-molecular-mass surfactants allows the formation of a micellar phase at very low surfactant concentrations and avoids the generation of a high level of background ions in the low m/z region. Alternatively, the application of a partial-filling micellar plug and anodically migrating micelles eliminate the introduction of MEKC micelles into the ESI-MS system. It is possible to directly transfer the conventional MEKC separations to partial-filling MEKC-ESI-MS and MEKC-ESI-MS using anodically migrating micelles without any instrument modifications.     

Matrix effects and selectivity issues in LC-MS-MS

Summary Chlorobenzenes, triazine and phenylurea herbicides were separated by normal micellar electrokinetic chromatography (MEKC) and by micellar electrokinetic chromatography with reversed flow (RF-MEKC) in running buffers containing organic solvents. The relationship between the two techniques is similar to that between reversed-phase and normal-phase HPLC. Using RF-MEKC, the separation of lipophilic compounds is often improved compared to normal MEKC. The migration in MEKC and in RF-MEKC was characterised by lipophilic and polar indices. The experimental values of the lipophilic indices of the compounds tested in the two techniques were close to the indices in reversed-phase HPLC (RP-HPLC). This enables the use of the indices determined in RP-HPLC for predicting the effects of changing composition of the running buffers on migration times in MEKC and in RF-MEKC. Presented at Balaton Symposium '01 on High-Performance Separation Methods, siófok, Hungary, September 2–4, 2001     

The impact of buffers and surfactants from micellar electrokinetic chromatography on matrix-assisted laser desorption ionization (MALDI) mass spectrometry of peptides. Effect of buffer type and concentration on mass determination by MALDI-time-of-flight mass spectrometry

This paper describes the effect of various buffers, surfactants, and organic additives commonly encountered in capillary zone electrophoresis and micellar electrokinetic chromatography on the molecular weight determination of peptides by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Signal-to-noise ratio generally decreased with increasing buffer concentration without affecting mass accuracy, but the type of buffer was also important. Good spectra were obtained with an ammonium acetate buffer up to a concentration of 500 mM without impacting ionization of either peptides or other mobile phase constituents. Ionization of organic additives, such as anionic surfactants, non-ionic surfactants, and cyclodextrins was buffer dependent and presented a problem when the mass of the additive was in the range of the peptide mass. Brij-35, Tween-80, and cyclodextrins all produced prominent spectra of their own in the presence of sodium or potassium containing buffers, but not with ammonium acetate. Cationization of these neutral species with sodium or potassium ions allowed them to acquire a positive charge and produce spectra. In contrast, the ammonium ion appears to be a poor cationizating agent. Ionization of neutral surfactants was suppressed in ammonium acetate without impacting the spectra of peptides. Ammonium acetate buffers containing 30 mM sodium dodecyl phosphate also gave spectra with good signal intensity and no interference from the surfactant. Suppression of peptide ionization in MALDI was a problem when methanol, tetrabutyl amine, or poly(vinyl alcohol) were used with either ammonium acetate, sodium phosphate, and N-(2-hydroxyethyl)piperazine-N-(2-ethansulfonic acid).     

A comparison of ionic liquids to molecular organic solvents as additives for chiral separations in micellar electrokinetic chromatography

In this study, we report the effects of adding ionic liquids (ILs), as compared to adding conventional molecular organic solvents (MOSs), to aqueous buffer solutions containing molecular micelles in the separation of chiral analyte mixtures in micellar EKC (MEKC). The molecular micelle used in this study was polysodium oleyl-L-leucylvalinate (poly-L-SOLV). The ILs were 1-alkyl-3-methylimidazolium tetrafluoroborate, where the alkyl group was ethyl, butyl, hexyl, or octyl. These ILs were chosen due to their hydrophobicity, good solvating, and electrolyte properties. Thus, it was expected that these ILs would have favorable interactions with chiral analytes and not adversely affect the background current. Common CE buffers, mixed with a molecular micelle, and an IL or a MOS, were used for these chiral separations. The buffers containing an IL in the concentration range of 0.02-0.1 v/v were found to support a reasonable current when an electric field strength of 500 V/cm was applied across the capillary. However, a current break down was observed for the buffers containing more than 60% v/v MOS on application of the above-mentioned electric field. The chiral resolution and selectivity of the analytes were dependent on the concentration and type of IL or MOS used.     

A chemometric approach for the elucidation of the parameter impact in the hyphenation of field-enhanced sample injection and sweeping in capillary electrophoresis

The aim of this work was to elucidate the impacts of parameters influencing cation-selective exhaustive injection coupled to sweeping and micellar electrokinetic chromatography (MEKC). A chemometric approach using cationic compounds, acidic conditions (phosphate buffer, pH 2.3) and polyacrylamide-coated capillaries to suppress electroosmotic flow were used. It was demonstrated that the water plug was not useful because of long electrokinetic injections. If conductivity of the high conductivity buffer (HCB) and the HCB to sample conductivity ratio are sufficiently high (>1.66 S/m and >30, respectively), variations of HCB conductivity do not impact sensitivity. The length of the HCB must be long enough so that the most mobile cation remains stacked in this zone for a given injection time. SDS concentration should be as high as possible (the maximum concentration is dictated by MEKC, here 90 mM), so sensitivity is not impacted. We have shown analytes can be lost after electrokinetic injection, when the polarity of the voltage is reversed. Introducing a plug of micellar electrolyte before polarity reversal avoids these losses. Following these recommendations only injection time and sample conductivity impacted sensitivity enhancement. Sample conductivity had to be the lowest as possible and controlled in real case analyses to obtain repeatable enrichment factors.     

Separation of aromatic hydrophobic sulfonates by micellar electrokinetic chromatography

Two different buffer systems for the separation of 12 aromatic hydrophobic sulfonates by micellar electrokinetic chromatography (MEKC) were developed. The following buffer systems were used: aqueous phosphate buffers containing either cetyltrimethylammonium bromide (CTAB) or sodium dodecyl sulfate (SDS). Eleven aromatic sulfonates were simultaneously separated in less than 35 min employing 20 mM phosphate buffer, pH 7.0 containing 50 mM SDS and 10% of acetonitrile.     

Application of sweeping to micellar electrokinetic chromatography-atmospheric pressure chemical ionization-mass spectrometric analysis of environmental pollutants.

An application study of sweeping, an on-line sample concentration technique, to micellar electrokinetic chromatography (MEKC) directly combined with mass spectrometry (MS) using an atmospheric pressure chemical ionization (APCI) interface, namely MEKC-APCI-MS, was investigated to enhance the concentration sensitivity for the analysis of environmental pollutants. Under a neutral condition, around 100-fold increase in the concentration sensitivity was achieved for several aromatic amines and alkyl phthalates as test samples by sweeping-MEKC-APCI-MS compared to conventional MEKC-APCI-MS, whereas under an acidic condition, 100 to 600-fold sensitivity enhancement was gained for similar solutes. Linearity of the corrected peak area obtained in the mass chromatogram against the sample concentration was examined for 3,4-dichloroaniline and diisopropyl phthalate (DIPP). The estimated limits of detection for 3,4-dichloroaniline and DIPP were 0.6 and 0.4 ppm, respectively, in terms of the injected sample concentration.     

Investigations on the migration behaviour of purines and pyrimidines in capillary electromigration techniques with UV detection and mass spectrometric detection

Different approaches for the separation of a set of nucleosides and nucleobases using capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) are described. Several electrolyte compositions have been tested for this purpose. The addition of appropriate amounts of borate to the carrier electrolyte allowed manipulating migration orders in CZE and MEKC by selective complexation of the nucleosides. For detection either UV or two different modes of mass spectrometric (MS) detection were employed. The latter approach included a comparison of two ion sources namely electrospray ionization (ESI) and atmospheric pressure photoionization (APPI) with respect to their potential in the detection of the selected compounds. Thereby it could be demonstrated that, in particular when it comes to the analysis of real samples, APPI-MS is the better choice if MS detection of purines and pyrimidines after separation by CZE is required.     

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.     

Capillary electrophoretic determination of the constituents of Artemisiae Capillaris Herba

Two capillary electrophoretic methods, a micellar electrokinetic electrophoretic (MEKC) one and a capillary zone electrophoretic (CZE) one, were developed for the separation of 12 constituents in Artemisiae Capillaris Herba. Detection at 254 nm with 20 mM sodium dodecyl sulfate and 20 mM sodium borate buffer (pH 9.82) in MEKC or with 25 mM sodium borate and 6.75 mg/ml 2,3,6-tri-O-methyl-beta-cyclodextrin buffer in CZE was found to be the most suitable approach for this analysis. Within 42 min, the MEKC method could successfully separate 12 authentic constituents, whereof chlorogenic acid, however, appeared as a broad and split peak, and capillarisin and chlorogenic acid overlapped partially with other coexisting substances in crude extract of the herb. The CZE method could completely overcome these problems and was used to determine the amounts of capillarisin, chlorogenic acid, scopoletin and caffeic acid in the extract. The effect of buffers on the constituent separation and the validation of the two methods were discussed.