Micellar Electrokinetic Chromatography Separation Research Articles

Development of transient trapping micellar electrokinetic chromatography coupled with mass spectrometry for steroids analysis.

An on-line sample preconcentration technique based on transient trapping (tr-trapping) in micellar electrokinetic chromatography (MEKC) was applied for steroid detection with UV (tr-trapping-UV) and electrospray ionization mass spectrometry detection (tr-trapping-ESI-MS). ESI-MS was used to improve the sensitivity in MEKC. The MEKC separation was carried out using volatile ammonium formate as a background solution to facilitate the coupling with ESI-MS. The partial introduction of a sodium dodecyl sulfate (SDS) micellar solution before the introduction of a sample solution to the capillary provided the effective preconcentration of analytes. At the same time, the SDS micelle would not enter the ESI-MS system, so its interference in ESI-MS detection was suppressed under the optimal condition, then five steroids can be separated by the developed method. In tr-trapping-ESI-MS, an acidic condition of pH 3.5 was employed to suppress the electroosmotic flow, which can avoid micellar solution migrating to the MS instrument. The developed method showed that the micellar solution requires a twofold slower time than the sample to migrate along the column, which can prohibit the cause of the problem with the MS instrument and interference signal of SDS in the steroid's detection. The tr-trapping-ESI-MS protocol showed up to 540-fold enhancements of the peak intensity and 50-fold improvement of the limit of detection compared with capillary zone electrophoresis using androsterone as a model sample.

Evaluation of HPLC and MEKC methods for the analysis of lipopeptide antibiotic iturin A produced by Bacillus amyloliquefaciens

Two methods, high-performance liquid chromatography (HPLC) and micellar elec- trokinetic chromatography (MEKC), for analysis of multicomponent lipopeptide antibiotic iturin A are described. After conducting a series of optimizations, the HPLC separation of iturin A2, A3, A4, A5, A6, A7, and A8 isomers were achieved by a NPS RP-C18 column with an isocratic elution of acetonitrile/10 mM ammonium acetate (40:60, v/v), and flow rate of 1 mL/min. On the other hand, the MEKC separation achieved at 25 °C using an applied voltage of 20 kV, and back- ground electrolyte consisting of 20 mM boric acid buffer (pH 8.72) with 50 mM sodium dodecyl sulfate (SDS) and 10% (v/v) acetonitrile. The calibration curve was linear to 20-200 µg/mL and 50-500 µg/mL for HPLC and MEKC with R 2 = 0.9954 and R 2 = 0.9967, respectively. The detec- tion limits of iturin A from the strain B128 cultivation medium were in the 2.5-7.0 µg/mL and 5.0-13.0 µg/mL ranges, and the reproducibility of within-day assay was 98.1 and 98.7%, for HPLC and MEKC, respectively (a UV detector was applied in the both cases). Both methods were selective, robust, and specific, allowing reliable quantification of iturin A, and could be useful for clinical and biomedical investigations of related antibiotics.

Extraction and preconcentration of compounds from the l-tyrosine metabolic pathway prior to their micellar electrokinetic chromatography separation

The prominent biological effects of adrenaline (A), noradrenaline (NA) and dopamine (DA) as well as the clinical importance of their metabolites (such as dihydroxyphenylacetic acid (DOPAC), methoxy‑4-hydroxyphenyl glycol (MHPG), dihydroxyphenylglycol (DHPG), metanephrine (M), normetanephrine (NM), vanillylmandelic acid (VMA), homovanillic acid (HVA)) have forced researchers to evaluate new analytical methodologies for their isolation and preconcentration from biological samples. For this reason, the three most popular extraction techniques (dispersive liquid-liquid microextraction (DLLME), solid-phase extraction (SPE), solid-phase microextraction (SPME)) were tested. Micellar electrokinetic chromatography (MEKC) – a mode of capillary electrophoresis – with a diode array detector (DAD) was applied to assess the extraction efficiency. Next, the enrichment factor (EF) of each applied method was calculated in respect to standard mixtures of the analytes at the same concentration levels. The EF results of seven selected metabolites of biogenic amines (BAs) from urine after sample preparation procedures based on twenty-five different protocols (one DLLME, thirteen SPE and eleven SPME) were calculated and compared using hierarchical cluster analysis (HCA). The SPE as well as SPME procedures were proved to be the most effective approaches for the simultaneous extraction of the chosen compounds. Moreover, an ionic liquid (IL) – 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide – added to methanol in SPME additionally could successfully improve the extraction efficiency. It was also confirmed that the HCA approach could be considered a supportive tool in the selection of a suitable sample preparation procedure for that group of endogenous substances.

Chiral micellar electrokinetic chromatographic separation for determination of L- and D-primary amines released from murine islets of Langerhans.

D-amino acids have been located in various tissues including the endocrine portion of the pancreas, the islets of Langerhans. D-Serine (D-Ser), is of particular interest since it is an agonist for the ionotropic N-methyl-D-aspartate receptors. To examine the potential release of D-Ser and other D-amino acids from islets, a chiral micellar electrokinetic chromatography method was developed by derivatizing primary amines with 2,3-naphthalenedicarboxaldehyde and to achieve resolution of the enantiomers, two surfactants were used in the separation, sodium dodecyl sulfate and sodium deoxycholate. With the optimized conditions, 7 of 13 enantiomeric pairs that were tested had greater than baseline resolution, while the resolution of numerous other L-amino acids and small molecules were maintained. For the 17 compounds that were fully resolved, limits of detection were less than 10 nM. The resulting optimized separation method produced high efficiency peaks, with an average of 300,000 theoretical plates per peak and a peak capacity of 120. The method was used to examine the release of small molecules from groups of 50 murine islets of Langerhans. A peak was detected from islets incubated with 20 mM glucose that co-migrated with a D-Ser standard, although its level was below the quantifiable limit.

Study of aldehyde oxidase by micellar electrokinetic chromatography separation of O6 -benzylguanine and 8-oxo-O6 -benzylguanine.

A separation method for O6 -benzylguanine (O6 -BG) and 8-oxo-O6 -benzylguanine (8-oxo-O6 -BG) is developed by using MEKC. This study includes the optimization of separation and incubation parameters for both off-line and on-line procedures. The BGE consisted of 25 mM sodium phosphate buffer-methanol (70:30, v/v), apparent pH 7.4, in which SDS and methyl-β-cyclodextrin were dissolved yielding final concentrations of 50 and 15 mM, respectively. Separations were performed at 15 kV using an untreated fused-silica capillary (40 cm length, effective length is 30 cm) with the detection wavelength at 195 nm. The capillary was kept at 15°C. Good performances were demonstrated for the repeatability and linearity. The LOQ was determined to be 14 μM for 8-oxo-O6 -BG (S/N = 10). The accuracy values showed a bias of +7.9% for 50 μM and -7.0% for 100 μM. Premix and transverse diffusion of laminar flow profiles (TDLFP) methods were used for on-line mixing and reaction of the substrate O6 -BG with aldehyde oxidase. Both procedures were successful in mixing as well as subsequent separation of the substrate and the metabolite, while the repeatability of TDLFP (14.7%(n= 3)) was much better than the premix technique.

Molecularly imprinted probe for solid-phase extraction of hippuric and 4-methylhippuric acids directly from human urine samples followed by MEKC analysis.

Hippuric acid (HA) and 4-methylhippuric acid (4-MHA) are metabolites as well as biological indicators for toluene and xylenes, respectively, and their determination in urine samples is very important, in order to monitor the occupational exposition to these solvents, ensuring a safe working environment. Thus, this paper describes the synthesis and characterization of a probe impregnated with molecularly imprinted polymers (MIPs) for the solid-phase extraction of HA and 4-MHA directly from untreated urine samples followed by micellar electrokinetic chromatography (MEKC) analyses. The MIP probe selectivity was compared to the non-imprinted polymer probe. The MEKC separations were carried out in 50 mmol/L sodium tetraborate pH 10.0/0.5 mmol/L cetyltrimethylammonium bromide aqueous solution, with a constant voltage of -15 kV. The system variables were optimized to provide ideal conditions for the extraction and desorption of the analytes, as well as for the MEKC analyses. The method was linear from 0.5 to 5.0 g/L for both analytes, with correlation coefficients > 0.994. Precisions and accuracies, expressed as relative standard deviation and relative error, were < 20.0 and within -15.4 to 16.6%, respectively, in accordance with the United States Food and Drug Administration recommendation. The MIP probe has proven to be simple, cheap, resistant, and synthetically reproducible, being successfully used to analyze both HA and 4-MHA from real samples.

Direct determination of lignin peroxidase released from Phanerochaete chrysosporium by in-capillary enzyme assay using micellar electrokinetic chromatography

Here we describe the application of an in-capillary enzyme assay using micellar electrokinetic chromatography (MEKC) in the determination of enzyme activity in a crude culture medium containing lignin peroxidase released from Phanerochaete chrysosporium (P. chrysosporium). The method consists of a plug–plug reaction between lignin peroxidase and its substrate, veratryl alcohol, the separation of the product, veratraldehyde, from the other components including the enzyme and the culture medium, and the determination of the enzyme activity from the peak area of veratraldehyde produced by the plug–plug reaction. This method is more sensitive than conventional spectrophotometry since the background originates from the enzyme and the culture medium can be removed via MEKC separation. Veratraldehyde was separated at −10kV in a background electrolyte containing 50mM tartrate buffer (pH 2.5) and 50mM sodium dodecyl sulfate (SDS) after a plug–plug reaction in the capillary for 5min. The calibration curve of veratraldehyde was linear up to 4pmol (500μM) with a limit to quantification of 0.026pmol (3.2μM) (SN=10). The activity of lignin peroxidase was directly measured from the peak area of veratraldehyde. The activity of lignin peroxidase released from P. chrysosporium into the medium for 7 days was successfully determined to be 3.40UL−1.

Determination of selected synthetic cannabinoids and their metabolites by micellar electrokinetic chromatography – mass spectrometry employing perfluoroheptanoic acid-based micellar phase

Perfluoroheptanoic acid was employed as a volatile micellar phase in background electrolyte for micellar electrokinetic chromatography–tandem mass spectrometry separation and determination of 15 selected naphthoyl- and phenylacetylindole- synthetic cannabinoids and main metabolites derived from JWH-018, JWH-019, JWH-073, JWH-200 and JWH-250. The influence of concentration of perfluoroheptanoic acid in background electrolytes on the separation was studied as well as the influence of perfluoroheptanoic acid on mass spectrometry detection. The background electrolyte consisted of 75mM perfluoroheptanoic acid, 150mM ammonium hydroxide pH 9.2 with 10% (v/v) propane-2-ol allowed micellar electrokinetic chromatography separation together with mass spectrometry identification of the studied parent synthetic cannabinoids and their metabolites. The limits of detection of studied synthetic cannabinoids and metabolites were in the range from 0.9ng/mL for JWH-073 to 3.0ng/mL for JWH-200 employing liquid–liquid extraction. The developed method was applied on the separation and identification of studied analytes after liquid–liquid extraction of spiked urine and serum samples to demonstrate the potential of the method applicability for forensic and toxicological purposes.

In-capillary derivatization with (−)-1-(9-fluorenyl)ethyl chloroformate as chiral labeling agent for the electrophoretic separation of amino acids

An original micellar electrokinetic chromatography (MEKC) method using in-capillary derivatization with a chiral labeling reagent was developed for the separation of amino acid (AA) derivatives. The potential of (−)-1-(9-fluorenyl)-ethyl chloroformate (FLEC) as in-capillary derivatization agent is described for the first time. Several parameters for in-capillary derivatization and subsequent MEKC separation were systematically investigated using experimental designs. Firstly experimental conditions for in-capillary derivatization were optimized using face-centered central composite design (FCCD). Mixing voltage and time as well as concentration of the labeling solution were investigated. Efficient labeling was achieved by sequential injection of AAs and FLEC labeling solution followed by the application of a voltage of 0.2kV for 570s. The background electrolyte (BGE) composition was then optimized in order to achieve selectivity. A FCCD was performed with two factors, namely the sodium dodecyl sulfate (SDS) concentration and the percentage of propan-2-ol (IPA). The separation of 12 pairs of derivatized AA (FLEC-AA) diastereomers was achieved with resolution values comprised between 3 and 20. Furthermore, an efficient derivatization and separation of 29 FLEC-AA derivatives were achieved in a single run using a buffer made up of 40mM sodium tetraborate, 21mM SDS and 8.5% IPA. The method was successfully applied to the analysis of spiked artificial cerebrospinal fluid (aCSF) sample.

Effect of low concentration sodium dodecyl sulfate on the electromigration of palonosetron hydrochloride stereoisomers in micellar electrokinetic chromatography

The effect of low concentrations of sodium dodecyl sulfate (SDS) on the separation of palonosetron hydrochloride (PALO) stereoisomers by micellar electrokinetic chromatography (MEKC) has been investigated. It was found that the addition of SDS prolongs the migration time and the migration order of four stereoisomers changes regularly with the SDS concentration. Good separations for all the four stereoisomers were achieved at appropriate SDS concentration. The effect of SDS on the electromigration (mobilities) of PALO stereoisomers has been studied, in order to explain its effect on the separation by MEKC. It was found that low concentrations of SDS added into the separation media forms negatively charged complexes with PALO stereoisomers and hence reverses their electromigration direction. Furthermore, the migration order between two enantiomeric pairs is also reversed because the enantiomeric pair with a bigger positive mobility than that of another pair turns to have a bigger negative mobility when bound with SDS. Based on these results, the effect of SDS on the MEKC separation of PALO stereoisomers was elucidated reasonably. The performance of the developed chiral MEKC method was validated by the analysis of a real sample.

Microwave-Plasma Dry-Etch for Fabrication of Conducting Polymer Microelectrodes

An inexpensive dry etch technology based on a low-pressure microwave plasma generated in a countertop microwave oven is characterized for the patterning of a conductive polymer microelectrode. The etch process is described, and the microwave-generated plasma is characterized by emission spectroscopy. The plasma is generated with an atmospheric mixture of mostly nitrogen and oxygen. A 10 μm wide band microelectrode composed of PEDOT:Tosylate, an optically transparent conductive polymer, is fabricated on a plastic substrate. Conductive polymer etch rates are approximately 280-300 nm/minute. A patterned microelectrode is characterized by atomic force microscopy. The horizontal distance of a 10-90% height of a plasma-etched 150 nm thick electrode was measured to be 360 ± 200 nm (n = 5). Electrodes are further characterized using steady-state cyclic voltammetry, and they have an electroactive area congruent with their geometric area. Finally, a complete device is assembled and used as a separation platform for biogenic amines. A microwave-etched 250 μm PEDOT:PSS electrode is employed for end-channel electrochemical detection on this microchip, where an electrophoretic separation of dopamine and catechol and a micellar electrokinetic chromatography separation of dopamine and serotonin are performed. Both mass and concentration LODs are comparable to other electrochemical detectors in an end-channel configuration. With the added advantages of easy processing, robustness, optical transparency, and low cost, we expect microwave-etched polymer films to be a viable alternative to traditional electrodes.

Establishment of Micellar Electro Kinetic Chromatography Detection Method for Puerarin in Pueraria Lobata(Willd.)Ohwi

Relative to the pharmacopoeia of high performance liquid chromatography (HPLC), establishing detection of puerarin in radix puerariae micellar electro kinetic chromatography (MEKC). Studying on buffer pH, borax concentration, separation voltage, SDS concentration of chromatographic conditions on the puerarin MEKC separation effect of influence, determined the best capillary electrophoresis conditions: test 250 nm wavelength, buffer of pH 9.0 0.1M KH2PO4solution-20 mM borax solution, separation voltage 18 kV, 40 mmol/L SDS concentration, 4.5 min retention time., grained puerarin MEKC standard equation: Y=36.522X-471.9, R2=0.9994(N=5), the puerarin concentration in 550 μg/mL to 750 μg/mL range comes out a good relationship. Get the lowest detection limit with 13.74 μg/mL. Puerarin content of the total isoflavone detected by MEKC was 45.35% that similar to 42.25% detected by HPLC. The method was stable, accurate, sensitive, possessed organic solvent consumption. The research results have applications in puerarin and its product quality evaluation system and promote isoflavonoids detection.

Micellar electrokinetic chromatography: A new simple tool for the analysis of synthetic cannabinoids in herbal blends and for the rapid estimation of their log P values

For the first time a capillary separation based on micellar electrokinetic chromatography (MEKC) with diode array detection (DAD) was developed and validated for the rapid determination of synthetic cannabinoids in herbal blends. Separations were carried out on a 30μm(ID)×40cm uncoated fused silica capillaries. The optimized buffer electrolyte was composed of 25mM sodium tetraborate pH 8.0, 30mM SDS and n-propanol 20% (v/v). Separations were performed at 30kV. Sample injection conditions were 0.5psi, 10s. Diazepam and JWH-015 were used as internal standards. The determination of the analytes was based on the UV signal recorded at 220nm, corresponding to the maximum wavelength of absorbance of the molecules, whereas peak identification and purity check were also performed on the basis of the acquisition of UV spectra between 200 and 400nm wavelengths. Under the described conditions, the separation of the compounds was achieved in 25min without any significant interference from the matrix. Linearity was assessed within a concentration range from 5 to 100μg/mL. The intra-day and inter-day imprecision values were below 2.45% for relative migration times and below 10.75% for relative peak areas. The present method was successfully applied to the direct determination of synthetic cannabinoids in 15 different herbal blend samples requiring only sample dilution. In addition, the developed MEKC separation was also applied to estimate the octanol/water partition coefficients (logP) of these new and poorly known molecules.

Rapid and accurate determination of deoxyribonucleoside monophosphates from DNA using micellar electrokinetic chromatography with a cationic surfactant additive

A micellar electrokinetic chromatography (MEKC) method for rapid and accurate determination of 2'-deoxyribonucleoside 5'-monophosphates (dNMPs), four structural elements of DNA, is described. MEKC separation at an optimized pH enabled complete separation of four dNMPs. The use of a cationic surfactant additive for MEKC led to the reversal of EOF, which enhanced the migration velocities of the negatively charged dNMPs. Under the optimized condition, full-baseline separation of the four dNMPs assuring accurate peak integration was obtained within 5 min. For the given separation condition, pH-mediated on-column sample stacking was optimized and applied to enhance sensitivity up to 6-fold. Analytical precision was improved by spiking iothalamate as an internal standard. The accuracy of dNMP quantitation was ensured with dNMP standard solutions determined by inductively coupled plasma-optical emission spectroscopy that measured phosphorous quantity. Performance of the proposed method was ultimately proven by accurate quantitation of a DNA oligonucleotide that was enzymatically hydrolyzed prior to dNMP analysis. The proposed MEKC method turned out to be a reliable analytical method for dNMPs that features high speed, high sensitivity, and high precision, and could be utilized for high-accuracy determination of the amount of DNA as well as the base composition of DNA.

Micellar electrokinetic chromatographic separation and quantitative analysis of thorium, uranium, gold, and mercury from environmental ore samples

Summary A rapid and reliable analytical procedure has been developed for the separation and analysis of dioxouranium, thorium, gold, and mercury by micellar electrokinetic chromatography (MEKC) using bis(acetylacetone)ethylenediamine (H2AA2en) as complexing reagent. Under the optimized conditions (64 mm borate buffer containing 12.25 mm SDS and 13% acetonitrile, pH 8.0, 25 kV, 311 nm as detection wavelength) the ions were separated within 5.0 min. Linear dynamic ranges were 1–5, 8–42, 10–50, and 2–40 μg mL−1, respectively, for gold, mercury, thorium, and uranium and the respective detection limits were 0.66, 3.33, 1.6, and 3.3 μg mL−1. The applicability of method has been evaluated by application to uranium and thorium from ore samples.

Using Thiol-Capped Gold Nanoparticles in the Background Solution of MEKC to Concentrate and Separate Neutral Steroids

In this study, it was aimed to use micellar electrokinetic chromatography (MEKC) to concentrate and separate neutral steroids (Δ4-androstene-3,17-dione, testosterone, progesterone, and testosterone propionate). It was aimed to study the impact of background solution (BGS) parameters on MEKC concentration and separation. These included sodium dodecylsulfate concentration, buffer concentration, pH, and the presence of organic solvent. Salt contents of the sample matrix and the operation conditions of the capillary electrophoresis system, i.e., voltage and temperature, have also been investigated. The addition of thiol-capped Au nanoparticles (NPs) to the BGS and how this affected the concentration and separation of the steroids was investigated. It was found that the migration time window of the steroids widened upon the increased concentration of these NPs present in the BGS. All of the tested steroids were separated well, showing limits of detection values ranging from 11.8 to 16.4 μg L−1.

Micellar electrokinetic chromatographic separation and analysis of thorium, uranium, gold, and mercury in environmental ore samples

Micellar electrokinetic chromatographic separation and analysis of thorium, uranium, gold, and mercury in environmental ore samples

Twenty-five years of micellar electrokinetic chromatography

Micellar electrokinetic chromatography (MEKC), which can separate neutral analytes as well as charged analytes by the capillary electrophoretic technique, was developed in 1982 and the first paper was published in 1984. The authors’ group concentrated their effort into the characterization of MEKC as a separation technique until early 1990s. Most issues in MEKC separations were successfully solved and wide applicability of MEKC was verified in 1990s. In particular, sweeping, an on-line sample preconcentration technique, was very successful for the concentration of neutral analyte as well as ionic ones. In this paper, our studies on MEKC will be summarized from the personal viewpoint of the author.

Capillary Separation: Micellar Electrokinetic Chromatography

Micellar electrokinetic chromatography (MEKC), a separation mode of capillary electrophoresis (CE), has enabled the separation of electrically neutral analytes. MEKC can be performed by adding an ionic micelle to the running solution of CE without modifying the instrument. Its separation principle is based on the differential migration of the ionic micelles and the bulk running buffer under electrophoresis conditions and on the interaction between the analyte and the micelle. Hence, MEKC's separation principle is similar to that of chromatography. MEKC is a useful technique particularly for the separation of small molecules, both neutral and charged, and yields high-efficiency separation in a short time with minimum amounts of sample and reagents. To improve the concentration sensitivity of detection, several on-line sample preconcentration techniques such as sweeping have been developed.

In-capillary derivatization and analysis of ephedrine and pseudoephedrine by micellar electrokinetic chromatography with laser-induced fluorescence detection

This paper describes an automatic rapid approach for in-capillary derivatization of ephedrine (E) and pseudoephedrine (PE) and subsequent sensitive determination of the derivatives by micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as fluorescent reagent. The unique feature of this method is the capillary being used as a small reaction chamber, in which the sample, derivatization buffer and reagent solutions were injected directly into the capillary by tandem mode, followed by an electrokinetic step (5 kV, 15 s) to enhance the mixing efficiency of analytes and reagent plugs. Standing a specified time of 1 min for reaction, the derivatives were then immediately separated and determined. Several parameters for in-capillary derivatization and subsequent MEKC separation were systematically investigated. Under these optimized conditions, a baseline separation of the two analytes was achieved within 10 min and the derivatization concentration limits of detection were found to be 4.8 ng mL −1 for E and 1.6 ng mL −1 for PE, respectively. The method was validated in terms of precision, linearity, accuracy and successfully applied for the determination of the two alkaloids in ephedra herb and its preparations.