Micellar electrokinetic capillary chromatography for the separation of cefalexin and its related substances

Micellar electrokinetic capillary chromatography (MEKC) was examined for analysis of cefalexin and its related substances. Good selectivity was obtained with two different buffer solutions: a sodium acetate buffer (50 mM, pH 5.25) containing sodium dodecyl sulfate (50 mM SDS) or sodium phosphate buffer (40 mM, pH 7.0) containing 100 mM SDS. Both methods permit cefalexin to be completely separated from its ten related substances within 20 min. The robustness of the method, using pH 5.25 acetate buffer, was examined by means of a full-fraction factorial design to test the influence of buffer pH, concentration of SDS and buffer concentration. The parameters for validation such as linearity, precision, limit of detection and limit of quantitation are also reported. The results show that method 1 is suitable for the analysis of cefalexin.     

Micellar electrokinetic capillary chromatography of amoxycillin and related molecules.

A micellar electrokinetic capillary chromatographic method has been developed for the qualitative assay of amoxycillin and its degradation products and clavulanic acid. Together with amoxycillin the latter acid is an important constituent in the antibiotic Augmentin. The analytical procedure is fast and analytes can be identified both from their migration times and from changes in migration time observed either at different pH values or in electropherograms run in H2O and D2O based buffers of the same acidity.     

Micellar electrokinetic capillary chromatography of algal toxins

Recent methods employed for the analysis of algal toxins have focused on high performance liquid chromatography. However these methods suffer from poor resolution, poor efficiency, and long analysis times. This study involves the investigation of a number of toxins including nodularin, microcystin LR, YR, and RR which are cyclic peptides produced by strains of blue-green algae. The electroseparation mode was micellar electrokinetic capillary chromatography (MECC) using a borate buffer containing sodium dodecyl sulfate (SDS) as the surgactant of choice. The method was optimized with standard toxin compounds and employed for the screening opf toxins in supercritical fluid extracts (SFE) of freeze-thawed algal scum samples.     

Micellar electrokinetic capillary chromatography using polymeric hollow fibers

Summary In this paper, polymeric hollow fibers prepared from pH-stable polypropylene were used as columns for micellar electrokinetic capillary chromatography (MECC). The electroosmotic flow (EOF) for polypropylene hollow fibers was evaluated in the pH range of 5.0–12.0. With untreated polypropylene hollow fibers a stabilized but enhanced EOF was achieved when SDS was used in the buffer, decreasing the separation window for uncharged substances in MECC to impractical levels. Uncharged acrylamide and charged 2-acryloylamido-2-methylpropane sulfonic acid surface modifications were used to lower the strength of the EOF, increase the separation window and prevent local overheating that could melt the column wall.     

Micellar electrokinetic chromatography and capillary electrochromatography of nitroaromatic explosives in seawater

The ability to separate nitroaromatic and nitramine explosives in seawater sample matrices is demonstrated using both MEKC and CEC. While several capillary-based separations exist for explosives, none address direct sampling from seawater, a sample matrix of particular interest in the detection of undersea mines. Direct comparisons are made between MEKC and CEC in terms of sensitivity and separation efficiency for the analysis of 14 explosives and explosive degradation products in seawater and diluted seawater. The use of high-salt stacking with MEKC results, on average, in a three-fold increase in the number of theoretical plates, and nearly double resolution for samples prepared in 25% seawater. By taking advantage of long injection times in conjunction with stacking, detection limits down to sub mg/L levels are attainable; however, resolution is sacrificed. CEC of explosive mixtures using sol-gels prepared from methyltrimethoxysilane does not perform as well as MEKC in terms of resolving power, but does permit extended injection times for concentrating analyte onto the head of the separation column with little or no subsequent loss in resolution. Electrokinetic injections of 8 min at high voltage allow for detection limits of explosives below 100 microg/L.     

Separation of beta-lactam antibiotics by micellar electrokinetic chromatography

The retention behaviour of beta-lactam antibiotics in micellar electrokinetic chromatography (EKC) was investigated. Sodium dodecyl sulphate (SDS) and sodium N-lauroyl-N-methyltaurate were used an anionic surfactants at concentrations of 0.05-0.3 M. It was found that the retention of ionic substances in micellar EKC is determined by the following three factors: the electrophoretic migration of the ionic substances, the interaction between the ionic substances and ionic surfactants and solubilization of the solute by the micellar phase. A difference in the retention behaviours of cationic substances was observed between the two anionic surfactants, which have different groups neighbouring the charge-bearing groups. The effect of an ion-pairing reagent was also investigated to make the effect of the micelle clearer. All test solutes were successfully separated by micellar EKC at SDS concentrations above 0.1 M, with theoretical plate numbers ranging from 70,000 to 260,000.     

Separation of coumarins by micellar electrokinetic capillary chromatography

Nine coumarins were successfully separated simultaneously using micellar electrokinetic capillary chromatography with 4-hydroxybenzoic acid as an internal standard. A carrier composed of buffer solution (20 mM sodium dodecyl sulfate-15 mM sodium borate-15 mM sodium dihydrogenphosphate)-acetonitrile (24:1) was found to be the most suitable electrolyte for this separation. The analysis time (22 min) was shorter than that using high-performance liquid chromatography (47 min). Contents of coumarins in the crude drug of Angelicae Tuhou Radix could be easily determined by the proposed method.     

Simultaneous separation of nitrofuran antibiotics and their metabolites by using micellar electrokinetic capillary chromatography

Mixtures comprising nitrofuran antibiotics (NFA) and nitrofuran metabolites (NFM) were resolved for the first time by using MEKC. Sodium deoxycholate (SDC) was chosen as the micelle-forming surfactant. Optimization of separation conditions was achieved by using a central composite experimental design (CCD) approach. Experimental parameters such as concentration ratio of borate to phosphate in the buffer, pH of the running electrolyte and voltage were investigated. The effect of concentration of the surfactant on resolution was significant. Under optimal conditions of 80 mM SDC, pH 9.0, (20 mM borate + 20 mM phosphate) and 16 kV, the resolution between eight consecutive peak pairs ranged from 1.9 to 11.8. Due to the absence of a UV-active chromophore in the metabolites, they were derivatized with 2-nitrobenzaldehyde (2-NBA). In order to mimic a proposed extraction procedure for the analysis of both NFA and/or derivatized NFM in a sample, aqueous samples (prederivatized with 2-NBA) were extracted by using C(18) SPE cartridges. After washing with H(2)O, the cartridges were eluted with a small portion of organic solvent with weak elution characteristics to remove excess 2-NBA (hexane was chosen). Target analytes were then recovered with ACN. Excellent reproducibility of migration time (t(mig)) was achieved for all analytes using the developed MECC approach, with absolute t(mig) <1% RSD and t(mig) ratio <0.2% RSD, and peak area ratio was 4% RSD. The LOD for each compound, calculated by extrapolating to an S/N of 3, were found to be 0.19-2.0 microg/mL.     

Micellar electrokinetic capillary chromatography of nucleic acid constituents and dinucleoside monophosphate photoproducts

Micellar electrokinetic capillary chromatography has been explored as an efficient and rapid method of separating the photoproducts of 2′-deoxyuridylyl-(3′-5′)-thymidine (i.e., cis-syn and trans-syn cyclobutane dimers, (6–4) photoproduct, and the related Dewar valence isomer) from normal nucleosides and nucleotides. Three cationic surfactants, dodecyl-, tetradecyl-, and hexadecyltrimethy-lammonium bromide were evaluated and the separations compared with those obtainable with the anionic surfactant, sodium dodecylsulfate. Optimum resolution of the dinucleoside monophosphate photoproducts was obtained using tetradecyltrimethylammonium bromide. By use of this detergent the photoproducts could also be separated from other normal constituents in less than 6 min at ?25 kV. Dodecyl- and hexadecyltrimethylammonium bromide provided some separation between the various species but sodium dodecylsulfate did not separate the UV radiation-induced products from either the parent compound or other dinucleoside monophosphates. Increased interaction between negatively charged solutes and positively charged micelles accounts for the differences.     

Micellar electrokinetic chromatography of proteins

Micellar electrokinetic capillary chromatography (MECC) of proteins is a high resolution capillary electrophoretic (CE) analysis method that utilizes the hydrophobic and electrostatic interaction of protein analytes with surfactant micelles present in the buffer medium to facilitate separation. Through the manipulation of the protein-micelle interaction by the adjustment of variables such as surfactant concentration, solution pH, ionic strength, the presence of an organic modifier and the use of coated capillaries, MECC analyses of a wide variety of proteins have been optimized. MECC has been demonstrated to provide resolution of mixtures consisting of proteins with minor structural variations and also has provided the successful quantitative analysis of protein present in complex matrices. The adoption of protein MECC as a routine analytical technique may be dependent upon the successful interface of MECC with detection methodology, such as mass spectrometry, which can provide analyte characterization information.     

Separation of anabolic steroids by micellar electrokinetic capillary chromatography

Summary The separation of seven analogous anabolic steroids was studied by micellar electrokinetic capillary chromatography (MECC). The retention order was found to be dependent on polarity. All of these steroids were well separated by the addition of organic modifiers to the separation buffer. Of the organic modifiers tested, 1-propanol gave the best separation, better than methanol or acetonitrile.     

Micellar electrokinetic capillary chromatography determination of zinc bacitracin and nystatin in animal feed

An MEKC procedure was developed for the separation of zinc bacitracin (Zn-BC) and nystatin (NYS) in mixtures and in animal feedstuff. The running buffer was 15 mM borate/19 mM phosphate, pH 8.2, containing 20 mM SDS and 10% v/v methanol. Samples were run at 25 degrees C, the applied voltage was 25 kV, and an additional pressure of 5 mbar was applied. Both analytes were detected by UV simultaneously at 215 nm, Zn-BC alone at 192 and 254 nm, and NYS alone at 305 nm. The method was shown to be specific, accurate (recoveries were 100.0 +/- 0.6% and 100.1 +/- 0.6% for Zn-BC and NYS, respectively), linear over the tested range (correlation coefficients 0.9991 and 0.9994), and precise (RSD below 1.3% for both analytes). The method was applied to determine Zn-BC and NYS as additives in animal feed.     

Separation and determination of the macrolide antibiotics (erythromycin, spiramycin and oleandomycin) by capillary electrophoresis coupled with fast reductive voltammetric detection

Separation and determination of erythromycin, spiramycin and oleandomycin by capillary zone electrophoresis coupled with fast reductive voltammetric detection using an Hg-film electrode was investigated in a simple aqueous phosphate buffer system. The influence of pH, concentration of phosphate, applied voltage, capillary length and dimension on the separation was examined and optimized. The entire separation of erythromycin, spiramycin, and oleandomycin was achieved in a 0.2 mol/L phosphate buffer system without organic modifiers. The electrochemical detection parameters, such as electrode material, applied waveform, scan rate, preconcentration potentials and preconcentration times, were investigated and discussed. This approach provides high separation efficiency and high sensitivity for all compounds, with detection limits (3 x peak-to-peak baseline noise) of 7.5 x 10(-8) mol/L for spiramycin, and 3 x 10(-7) mol/ L for erythromycin and oleandomycin. The calibration plot of peak areas for each separated peak vs. concentration of analyte was found to be linear over three orders of magnitude.     

Micellar electrokinetic capillary chromatography for fast separation and sensitive determination of melatonin and related indoleamines using end-column amperometric detection

MEKC was used in conjunction with end-column amperometric detection (AD) at a carbon disc electrode (0.3 mm diameter) for the selective and sensitive determination of melatonin and its five related indoleamines including its precursors and metabolites in the pineal gland. The introduction of a sample stacking technique in injection and the buffer additive SDS in the buffer solution system provided the rapid and sensitive analysis. Optimal buffer conditions (10 mmol/L phosphate containing 20 mmol/L SDS, pH 7.2), detection potential (+1.0 V vs. Ag/AgCl), and electrokinetic injection 10 s with the separation voltage of 24 kV were employed to achieve the baseline separation of six pineal hormones within 15 min. The peak currents and the analyte concentrations have a good linear relationship over the range of 6.0 x 10(-8) 6.0 x 10(-5 )mol/L. The detection limits for six pineal hormones by AD are 9.7 to 41.8 nmol/L (equal to 2.0 to 9.7 ng/mL) (S/N = 3), respectively. It is proved to provide about 30- to 250-fold improvement over UV, and be comparable with the sensitive fluorescence detection, which needs pre-column derivatization. The proposed method has been applied for analysis of melatonin and related indoleamines in rat pineal glands. A very simple sample pretreatment procedure, merely involving the homogenization step in perchloric acid, was enough to achieve recoveries in the range of 71 to 127% for all the analytes in the pineal gland.     

Micellar electrokinetic capillary chromatography for selected tropane alkaloid analysis in plant extract

Summary Micellar electrokinetic capillary chromatography was applied to the simultaneous analysis of six tropane alkaloids, including hyoscyamine and scopolamine. Successful results were obtained using a 30 mM boratephosphate buffer at basic pH (8.5) in the presence of 50 mM sodium dodecyl sulfate. The operating conditions, such as buffer concentration and pH, micelle concentration and organic modifier type and percentage were also discussed on the basis of the results given with a tropane alkaloid mixture. Addition of organic modifiers showed an improvement in separation efficiency and resolution. Moreover, hyoscyamine and littorine, two positional isomers, were only resolved by the addition of organic solvents such as methanol or acetonitrile. The optimized conditions were finally applied to the analysis of tropane alkaloids found in genetically transformed root cultures ofDatura candida x D. aurea. Dedicated to Professor Werner Haerdi on the occasion of his 70^th birthday.     

Micellar electrokinetic chromatography of polyamines and monoacetylpolyamines

A selective procedure for qualitative and quantitative analysis of ten polyamines by micellar electrokinetic chromatography (MEKC) was developed. Benzoylated polyamines and acetylpolyamines in micellar phase of SDS (10 mM) were separated at 25 degrees C by 20 mM borate buffer pH 8.5, containing 8% ethanol, with an applied voltage of 25 kV (5 microA) and then detected at 198 nm. The experimental factors and operational parameters were optimized by performing analysis at different surfactant concentrations, pH, voltage and temperature with and without ethanol. The repeatibility of migration times and peak heights is a peculiarity of the method here described.