Jingwu Kang

A silica monolithic column prepared by the sol-gel process for enantiomeric separation by capillary electrochromatography

A method for the preparation of a silica monolithic capillary electrochromatography (CEC) column for the separation of enantiomers has been developed. The porous silica monolith was fabricated inside a fused‐silica capillary column by using the sol‐gel process. After gelation for 24 h, hydrothermal treatment at 100°C for 24 h was performed to prevent the sol‐gel matrix from cracking. The prepared monolith was then coated with Chirasil‐β‐Dex which represents a chiral polymer prepared by grafting permethyl‐β‐cyclodextrin to polymethylsiloxane with an octamethylene spacer. Immobilization of Chirasil‐β‐Dex was performed by heat treatment at 120°C for 48 h to give a nonextractable coating. The column performance was evaluated by using racemic hexobarbital as a model compound. The efficiency of 9.2×104 theoretical plates/m for the first eluted enantiomer of hexobarbital was obtained at an optimal flow rate of the mobile phase. The effect of mobile phase composition on enantiomeric separation of hexobarbital was also investigated. The column proved to be stable for more than one hundreds of runs during a two‐months period. The enantiomers of several neutral and negatively charged chiral compounds were baseline separated on this column.

Fast chiral separation of amino acid derivatives and acidic drugs by co-electroosmotic flow capillary electrophoresis with vancomycin as chiral selector

Abstract A fast chiral separation method for acidic enantiomers, including 9-fluorenylmethyl chloroformate (FMOC) amino acid derivatives and ketoprofen, with vancomycin as chiral selector is presented. In this method, hexadimethrine bromide (HDB), a polycationic polymer, was added to the run buffer as electroosmotic flow (EOF) modifier. The reversed EOF migrated in the same direction as the anionic analytes. Consequently, the separation time was shortened. Another advantage of using HDB as buffer additive is that the adsorption of vancomycin onto the capillary wall was minimized, hence, the separation efficiency was improved. The effects of buffer pH and vancomycin concentration on separation were investigated. Base line chiral separation of 12 FMOC-amino acid derivatives, ketoprofen and drug intermediate 4,4′-dimethoxy-5,6,5′6′-bismethylenedioxybiphenyl-2,2′-dicarboxylic acid were obtained. The separation time for each enantiomers was not more than 4.5 min, and the average efficiency of 3.2·10 5 plates/m was obtained.

Analysis of bacitracin by micellar electrokinetic capillary chromatography with mixed micelle in acidic solution

A method used for quantitative analysis of bacitracin with micellar electrokinetic capillary chromatography (MEKC) is described. As capillary zone electrophoresis gave poor separation selectivity, MEKC was preferable. It was found that a zwitterionic surfactant, 3‐(N,N‐dimethylhexadecylammonium)‐propanesulfonate (PAPS) gave the best selectivity among the several surfactants studied. As the analytes tend to adsorb onto the capillary wall due to their positive charge, an acidic solution composed of Tris‐phosphate buffer at pH 2.5 was necessary to diminish such adsorption. The peak tailing caused by relatively strong ion pair interaction between the analyte and PAPS micelle could be reduced by adding nonionic surfactant Brij 35 to the PAPS solution. This phenomenon is possibly explained by a mixed micelle mechanism. In order to obtain the optimal conditions and to test the method robustness, a central composite experimental design was performed. The optimal conditions are as follows: 44 cm length of fused‐silica capillary with 50 μm inner diameter, 90 mM Tris‐phosphate buffer (pH 2.5) containing 17 mM PAPS and 0.3% w/v‐Brij 35, 18 kV applied voltage, UV detection at 192 nm and 25°C column temperature. Under the optimal conditions, more than 50 peaks could be obtained in 30 min. The method had a linearity range from 1 to 0.05 mg/mL (concentration of bacitracin A). The limit of quantitation (LOQ) and limit of detection (LOD) were 0.005 and 0.0012 mg/mL, respectively.

Analysis of polymyxin B sulfate by capillary zone electrophoresis with cyclodextrin as additive. Method development and validation.

A capillary zone electrophoresis method for analysis of polymyxin B sulfate is described. In this method, triethanolamine (TEA)-phosphate buffer at pH 2.5 was employed to reduce the adsorption of analyte onto the capillary wall. Methyl-beta-cyclodextrin (M-beta-CD) and 2-propanol (IPA) were found to be necessary for selectivity enhancement. In order to optimize the method and to control its robustness, a central composite design was performed with four parameters, i.e. concentration of M-beta-CD, TEA, IPA and buffer pH. The optimal separation conditions were as follows: capillary, 55 cm (50 microm I.D., 47 cm effective length); 130 mM TEA-phosphate buffer (pH 2.5) containing 5 mM M-beta-CD and 5% IPA; 24 kV (51 microA) applied voltage; column temperature, 20 degrees C. Further, linearity and limits of detection quantification were examined. Three commercial samples were analyzed quantitatively.

Analysis of environmental pollutants by capillary electrophoresis with emphasis on micellar electrokinetic chromatography

This review surveys analysis of environmental pollutants by capillary electrophoresis with emphasis on the commonly used pseudo-stationary-phase micelles. Other pseudo-stationary phases are also included for a systematic description. Manipulating the sensitivity, which is important for trace analysis, is also reviewed for environmental samples. For some typical environmental pollutants, systematic description of their separation and analysis is based on different pseudo-stationary phases. The potential of capillary electrophoresis in analysis of environmental pollutants is clearly demonstrated with a bright future.

Structural Analysis of Low Molecular Weight Heparin by Ultraperformance Size Exclusion Chromatography/Time of Flight Mass Spectrometry and Capillary Zone Electrophoresis

Although low molecular weight heparins (LMWHs) have been used as anticoagulant agents for over 2 decades, their structures have not been fully characterized. In this work, we propose a new strategy for the comprehensive structural analysis of LMWHs based on the combination of ultraperformance size exclusion chromatography/electrospray quadruple time-of-flight-mass spectrometry (UPSEC/Q-TOF-MS) and capillary zone electrophoresis (CZE). More than 70 components, including oligosaccharides with special structures such as 1,6-anhydro rings, saturated uronic acid at the nonreducing end and odd-numbered saccharides units were identified with UPSEC/Q-TOF-MS. Furthermore, a more detailed compositional analysis was accomplished by CZE analysis. PEG10000 and MgCl(2) were added to the background electrolyte to separate those saccharides with the nearly same charge-to-mass ratio. Baseline separation and quantification of all the building blocks of the most complex LMWH, namely, enoxaparin, which include 10 disaccharides, 1 trisaccharide, 2 tetrasaccharides, and, of particular importance, 4 1,6-anhyro derivatives, was achieved using CZE for the first time. Additionally, the peaks of oligosaccharides, in the absence of commercially available standards, were assigned on the basis of the linear correlation between the electrophoretic mobilities of oligosaccharides and their charge-to-mass ratios. These two approaches are simple and robust for structural analysis of LMWHs.

A highly sensitive method for enantioseparation of fenoprofen and amino acid derivatives by capillary electrophoresis with on-line sample preconcentration

A highly sensitive method for enantioseparation of trace fenoprofen and amino acid derivatives by capillary electrophoresis (CE) with vancomycin as the chiral selector was developed. Several CE techniques, such as the partial filling, large-volume sample stacking with EOF as pump plus anion-selective exhaustive injection (LVSEP-ASEI) were involved in the present method to improve the detection sensitivity. With on-column concentration, enantioseparation of racemic fenoprofen and six 9-fluorenylmethyl chloroformate (FMOC)-amino acid derivatives (at the concentration level of ng/mL) with the background electrolyte composed of 100 mmol/L Tris-H(3)PO(4) (pH 6.0) and 2 mmol/L vancomycin was detected readily with the UV detection at 214 nm. Successfully performing LVSEP-ASEI needs a very low EOF that could be depressed by coating the capillary with poly(dimethylacrylamide) solution. The coating also played a role to minimize the adsorption of vancomycin onto the capillary wall. Effect of the injected sample volume and the electrokinetic injection time on the peak area of the enantiomers and their resolution factor were investigated and optimized. Under the optimized conditions, more than 1000-fold enhancement in detection sensitivity compared with the normal injection was achieved.

Analysis of vancomycin and related impurities by micellar electrokinetic capillary chromatography. Method development and validation.

A fast and highly selective micellar electrokinetic capillary chromatography (MEKC) method for quantitative analysis of vancomycin and related impurities is described. Among the tested surfactants, cetyltrimethylammonium chloride (CTAC) offered the best selectivity. Another important parameter, which strongly influenced the selectivity, was buffer pH. It was found that the selectivity increased with buffer pH decreasing from 9 to 5. Using Tris‐phosphate buffer containing CTAC, satisfactory separation could be obtained in the pH range from 5.0 to 5.5. Excellent repeatability in terms of migration time and peak area could be obtained when the capillary was carefully washed between two runs. In order to obtain optimal conditions and to evaluate the method robustness, a central composite experimental design was carried out. The optimal conditions were: 44 cm length of fused‐silica capillary with 50 νm ID, 120 mM Tris‐phosphate buffer (pH 5.2) containing 50 mM CTAC, –15 kV applied voltage, UV detection at 210 nm, and a column temperature of 25°C. Under the optimal conditions, more than 20 peaks could be separated within 8 min. The method has a linearity range from 0.004 to 1.2 mg/ml (concentration of vancomycin B, active component). The limit of detection (LOD) and limit of quantitation (LOQ) were 0.4 νg/mL vancomycin, equivalent to 0.3 νg/mL vancomycin B (0.04%) and 1.1 νg/mL vancomycin, equivalent to 0.9 νg/mL vancomycin B (0.1%), respectively.

Improved capillary electrophoresis frontal analysis by dynamically coating the capillary with polyelectrolyte multilayers.

An improved CE-FA method using a polyelectrolyte multilayer (PEML) coated capillary is described. The coating was simply created by alternatively flushing the capillary with positively charged polyelectrolyte polybrene (HDB) and negatively charged polyelectrolyte dextran sulfate (DS). Human serum albumin (HSA) and 6 drugs were selected as an experimental model to evaluate such an improvement. The PEML coating was demonstrated to effectively reduce the protein adsorption on the capillary wall. Compared with the uncoated capillary, the repeatability (RSD%) of the EOF mobility and the plateau height of free drug were improved significantly from 3.7% to 0.51% and from 0.61% to 0.34%, respectively. No interference of the coating on the measurement of protein-drug binding parameters was observed.

Screening of protein kinase inhibitors in natural extracts by capillary electrophoresis combined with liquid chromatography–tandem mass spectrometry

We report a capillary electrophoresis method in conjunction with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for screening of protein kinase inhibitors (PKIs) in natural extracts. Protein kinase A (PKA), substrate 5-carboxyfluorescein-labeled kemptide (CLK) and inhibitor H-89 were employed for the method development and validation. Enzymatic inhibition assay was performed with electrophoretically mediated microanalysis technique. Once the bioactivity of a natural extract was confirmed, an assay-guided isolation and structure elucidation using LC-MS/MS were accomplished to identify the compounds which are responsible for the observed bioactivity. Totally 33 natural extracts were screened with the method, and baicalin in the extract of Radix Scutellariae was identified to be a new PKI of PKA. This result demonstrated the practical applicability of our method in screening of PKIs from natural products.