Large-volume sample sweeping with a high theoretical plate number using a coupled-capillary in capillary electrophoresis.

A large-volume sample injection (> 5 microL) with an extremely high theoretical plate number (N > 10(7)) was achieved when the sweeping-MEKC mode and a coupled-capillary (100 - 50 microm i.d.) were simultaneously used in a capillary electrophoresis (CE) separation. A low-cost and compact violet-LED ( approximately 2 mW) was used as the fluorescence excitation source. As a result, the theoretical plate numbers of the detected peaks (two model compounds: naphthalene-2,3-dicarboxaldehyde derivatized-dopamine and -norepinephrine) were 1.0 x 10(7) and 7.4 x 10(6), respectively. The limits of detection (at S/N = 3) of these were determined to be 2.8 x 10(-10) M (92 ppt) and 2.3 x 10(-10) M (83 ppt), respectively.     

Field-amplified sample injection and in-capillary derivatization for sensitivity improvement of the electrophoretic determination of histamine

The feasibility of the combination of field-amplified sample injection (FASI) and in-capillary derivatization was explored for improving sensitivity of histamine in capillary electrophoresis (CE). Naphthalene-2,3-dicarboxaldehyde (NDA) was used as derivatization reagent. The reagent and sample was introduced by tandem mode. The derivatization was accomplished by at-inlet mode with standing time of 1.5 min. The combination of FASI and in-capillary derivatization was successfully achieved with about 400-fold concentration sensitivity enhancement compared to pre-capillary derivatization at the same set-up. The detection limit of concentration for histamine reached 1.25 x 10(-11) M by CE and fluorescence detection with S/N = 3. Parameters affecting FASI and in-capillary derivatization process including sample matrix, buffer concentration and reagent injection amount, were investigated.     

High-sensitivity laser-induced fluorescence detection of native proteins in capillary electrophoresis.

A highly sensitive laser-induced (LIF) detection scheme for native, tryptophan- or tyrosine-containing proteins in capillary electrophoresis (CE) has been demonstrated. The 275.4 nm line from an argon-ion laser is used to excite native protein fluorescence. A limit of detection (LOD) (S/N = 2) of 1 x 10(-10) M for conalbumin represents a 140-fold improvement over earlier reports. With stacking at injection, the LOD is 3 x 10(-12) M. Linear dynamic ranges of at least 5 and 4 orders of magnitude for, respectively, tryptophan and bovine serum albumin are found. The practical performance and blueprint of an easily constructed, rugged, compact and user-friendly LIF detector for CE are shown.     

Determination of levofloxacin and norfloxacin by capillary electrophoresis with electrochemiluminescence detection and applications in human urine

A novel method for the determination of norfloxacin (NOR) and levofloxacin (LVX) was developed by CE separation and electrochemiluminesence detection (ECL). The methods for capillary conditioning and the effect of solvent type were studied. Parameters affecting the CE and ECL were also investigated. Under the optimum conditions, the two analytes were well separated within 9 min. The LODs (S/N = 3) in standard solution are 4.8 x 10(-7) mol/L for NOR and 6.4 x 10(-7) mol/L for LVX, respectively. The precisions of intraday and interday are less than 4.2 and 8.1%, respectively. The LOQs (S/N = 10) in real human urine samples are 1.2 x 10(-6) mol/L for NOR and 1.4 x 10(-6) mol/L for LVX, respectively. The applicability of the proposed method was illustrated in the determination of NOR and LVX in human urine samples and the monitoring of pharmacokinetics for NOR. The recoveries of NOR and LVX at different levels in human urine samples were between 84.3 and 92.3%.     

A simple and rapid flow injection chemiluminescence determination of cysteine with Ru(phen)3(2+)-Ce(IV) system

A new chemiluminescence system was developed for the determination of cysteine by flow injection system. This method is based on the reaction of L-cysteine with Ru(phen)3(2+) and Ce(IV) to produce chemiluminescence. The calibration curve was linear over the range 8.0x10(-7) to 4.0x10(-5) and 4.0x10(-5) to 1.0x10(-3) M with a detection limit of 7.0x10(-7) M (S/N=3). The relative standard deviation of 4.0x10(-6) M cysteine was found 3.5% (n=10). The influence of potential interfering substances was studied. The proposed method was successfully applied for the flow injection determination of cysteine in the real samples with minimum sampling rate of 90 sample/h.     

Discrimination of hairpin polyamides with an alpha-substituted-gamma-aminobutyric acid as a 5'-TG-3' reader in DNA minor groove

Pyrrole-imidazole (Py-Im) polyamides containing stereospecifically alpha-amino- or alpha-hydroxyl-substituted gamma-aminobutyric acid as a 5'-TG-3' recognition element were synthesized by machine-assisted Fmoc solid-phase synthesis. Their binding properties to predetermined DNA sequences containing a core binding site of 5'-TGCNCA-3'/3'-ACGN'GT-5' (N.N' = A.T, T.A, G.C, and C.G) were then systematically studied by surface plasmon resonance (SPR). SPR results revealed that the pairing of stereospecifically alpha-amino-/alpha-hydroxyl-substituted gamma-aminobutyric acids, (R or S)-alpha,gamma-diaminobutyric acid (gammaRN or gammaSN) and (R or S)-alpha-hydroxyl-gamma-aminobutyric acid (gammaRO or gammaSO), side-by-side with beta-alanine (beta) in such polyamides significantly influenced the DNA binding affinity and recognition specificity of hairpin polyamides in the DNA minor groove compared with beta/beta, beta/gamma, and gamma/beta pairings. More importantly, the polyamide Ac-Im-gammaSO-ImPy-gamma-ImPybetaPy-beta-Dp (beta/gammaSO) favorably binds to a hairpin DNA containing a core binding site of 5'-TGCNCA-3'/3'-ACGN'GT-5' (N.N' = A.T) with dissociation equilibrium constant (K(D)) of 1.9 x 10(-)(7) M over N.N' = T.A with K(D) = 3.7 x 10(-)(6) M, with a 19-fold specificity. By contrast, Ac-Im-gammaSN-ImPy-gamma-ImPybetaPy-beta-Dp (beta/gammaSN) binds to the above sequence with N.N' = A.T with K(D) = 8.7 x 10(-)(7) M over N.N' = T.A with K(D) = 8.4 x 10(-)(6) M, with a 9.6-fold specificity. The results also show that the stereochemistry of the alpha-substituent, as well as the alpha-substituent itself may greatly alter binding affinity and recognition selectivity of hairpin polyamides to different DNA sequences. Further, we carried out molecular modeling studies on the binding by an energy minimization method, suggesting that alpha-hydroxyl is very close to N3 of the 3'-terminal G to induce the formation of hydrogen bonding between hydroxyl and N3 in the recognition event of the polyamide Ac-Im-gammaSO-ImPy-gamma-ImPybetaPy-beta-Dp (beta/gammaSO) to 5'-TGCNCA-3'/3'-ACGN'GT-5' (N.N' = A.T). Therefore, SPR assays and molecular modeling studies collectively suggest that the (S)-alpha-hydroxyl-gamma-aminobutyric acid (gammaSO) may act as a 5'-TG-3' recognition unit.     

Sensitive determination of norepinephrine, synephrine, and isoproterenol by capillary electrophoresis with indirect electrochemiluminescence detection

A new and sensitive method for the determination of norepinephrine (NE), synephrine, and isoproterenol was developed by CE separation and indirect electrochemiluminescence detection (ECL) based on their quenching effects on the tris(2,2'-bipyridyl)-ruthenium(II)/tripropylamine (TPA) system. The conditions for CE separation and ECL detection were investigated in detail. Under the optimum conditions, the three analytes were well separated within 9 min. The LODs (S/N = 3) in standard solution are 2.6 x 10(-8) mol/L for NE, 6.6 x 10(-9) mol/L for synephrine and 8.4 x 10(-8) mol/L for isoproterenol, respectively. The precisions of intraday and interday are less than 4.4 and 6.1%, respectively. The LOQs (S/N = 10) in real human urine samples are 2.6 x 10(-7) mol/L for NE, 8.8 x 10(-8 ) mol/L for synephrine, and 8.8 x 10(-7) mol/L for isoproterenol, respectively. The applicability of the proposed method was illustrated in the determination of 20 human urine samples from diabetic patients and healthy persons. The results obtained indicated that the level of NE in patients (mean value 0.41 micromol/L) was higher than that in healthy persons (mean value 0.24 micromol/L).     

Determination of agmatine in biological samples by capillary electrophoresis with optical fiber light-emitting-diode-induced fluorescence detection

A capillary electrophoresis (CE)/optical fiber light-emitting diode (LED)-induced fluorescence detection method is developed for the determination of agmatine in biological samples. The agmatine was precolumn-derivatized with fluorescence tagging reagent, fluorescein isothiocyanate (FITC). Optimal separation and determination for agmatine were obtained with an electrophoretic buffer of 20 mM sodium borate (pH 9.2). Under the optimal conditions, the determination of agmatine was achieved in less than 4 min, and the detection limit was 4.1x10(-9) M (S/N = 3). The relative standard deviation (RSD) for 11 parallel determination of agmatine was less than 3.0%. The present CE-LED induced fluorescence detection method has been applied to detect agmatine in rat brain tissue, rat stomach tissue, human serum, and human urine. The level of agmatine in human urine was quantified by CE for the first time and found to be in the range 2.5-4.1x10(-7) M.     

Field-amplified sample stacking capillary electrophoresis with electrochemiluminescence applied to the determination of illicit drugs on banknotes

Capillary electrophoresis (CE) with Ru(bpy)3(2+) electrochemiluminescence (ECL) detection system was established to the determination of contamination of banknotes with controlled drugs and a high efficiency on-column field-amplified sample stacking (FASS) technique was also optimized to increase the ECL intensity. The method was illustrated using heroin and cocaine, which are two typical and popular illicit drugs. Highest sample stacking was obtained when 0.01 mM acetic acid was chosen for sample dissolution with electrokinetical injection for 6 s at 17 kV. Under the optimized conditions: ECL detection at 1.2 V, separation voltage 10.0 kV, 20 mM phosphate-acetate (pH 7.2) as running buffer, 5 mM Ru(bpy)3(2+) with 50 mM phosphate-acetate (pH 7.2) in the detection cell, the standard curves were linear in the range of 7.50x10(-8) to 1.00x10(-5) M for heroin and 2.50x10(-7) to 1.00x10(-4) M for cocaine and detection limits of 50 nM for heroin and 60 nM for cocaine were achieved (S/N = 3), respectively. Relative standard derivations of the ECL intensity and the migration time were 3.50 and 0.51% for heroin and 4.44 and 0.12% for cocaine, respectively. The developed method was successfully applied to the determination of heroin and cocaine on illicit drug contaminated banknotes without any damage of the paper currency. A baseline resolution for heroin and cocaine was achieved within 6 min.     

毛细管电泳结合压力辅助电动进样测定水样中4种酚类雌激素

在毛细管电泳的胶束电动色谱（MEKC）模式下,采用压力辅助电动进样（PAEKI）的进样方式在线富集4种酚类雌激素（PEs）。对影响PAEKI的进样电压、进样时间等进行考察,并与传统的压力进样比较。结果表明,在最优的PAEKI条件下（-9 kV,0.3 psi（约2.1 kPa）,0.4 min）,4种PEs在7 min内基线分离,线性关系良好,相关系数（r）大于0.9936,己烷雌酚和双烯雌酚的线性范围为0.05~5 mg/L、双酚A和己烯雌酚的线性范围为0.1~10 mg/L;检出限（S/N=3）为0.0071~0.017 mg/L,富集倍数为11~15。使用该MEKC-PAEKI法对自来水和湖水水样进行测定,得到定量限（S/N=10）分别为0.029~0.064 mg/L和0.033~0.079 mg/L;加标回收率为75.6%~110.1%,相对标准偏差（n=5）为4.6%~11.8%。PAEKI不需要使用其他试剂,只需对电泳仪的参数进行适当调整即可实现对分析物的在线富集,简单、快速、自动化程度高。     

Simultaneous determination of ethamsylate, tramadol and lidocaine in human urine by capillary electrophoresis with electrochemiluminescence detection

Ethamsylate, tramadol and lidocaine, partly excreted by the kidney, are generally used as hemostatic, analgesic and local anesthetic in surgery. We developed a simple and sensitive method for their simultaneous monitoring in human urine based on CE coupled with electrochemiluminescence detection by end-column mode. Under optimized conditions the proposed method yielded linear ranges from 5.0 x 10(-8) to 5.0 x 10(-5), 1.0 x 10(-7) to 1.0 x 10(-4) and 1.0 x 10(-7) to 1.0 x 10(-4) M with LODs of 8.0 x 10(-9) M (36 amol), 1.6 x 10(-8) M (72 amol) and 1.0 x 10(-8) M (45 amol) (S/N = 3) for ethamsylate, tramadol and lidocaine, respectively. The RSD for their simultaneous detection at 1.0 x 10(-6) M was 2.1, 2.8 and 3.2% (n = 7), respectively. For practical application an extraction step with ethyl acetate at pH 11 was performed to eliminate the influence of the sample ionic strength. The recoveries of ethamsylate, tramadol and lidocaine at different levels in human urine were between 87 and 95%. This method was used for simultaneous detection of ethamsylate, tramadol and lidocaine in clinic urine samples from two medicated patients. It was valuable in clinical and biochemical laboratories for monitoring these drugs for various purposes.     

Determination of ascorbic acid in individual rat hepatocyte cells based on capillary electrophoresis with electrochemiluminescence detection

A novel method to detect ascorbic acid (AA) in individual rat hepatocyte cells was developed by combining CE with electrochemiluminescence (ECL) based on tris(2,2'-bipyridine) ruthenium(II) (Ru(bpy)(3)2+). A single cell, followed by 0.1% SDS as cell lysis solution, was injected into the inlet of the separation capillary by electromigration. After optimizing the analytical conditions, the RSDs of migration time and peak height were 0.38% and 2.6% for 1.0x10(-5) M AA (n=10), respectively. The linear range of AA was from 1.0x10(-8) to 5.0x10(-5) M with a correlation coefficient of 0.9979 and the LOD (S/N=3) was estimated to be 1.0x10(-8) M. This method has been successfully applied to determine AA in single rat hepatocytes and the amount of AA in seven rat hepatocytes ranged from 16 to 62 fmol. The above results demonstrated that CE coupled with ECL is convenient, sensitive, and will become an attractive alternative method for single-cell analysis.     

A facile and sensitive chemiluminescence detection of amino acids in biological samples after capillary electrophoretic separation

It was found that native amino acids enhanced the chemiluminescence (CL) reaction between luminol and BrO(-) in an alkaline aqueous solution. This has led to the development of a facile and highly sensitive CL detection scheme for the determination of amino acids in biological samples after capillary electrophoretic (CE) separation. The CE-CL conditions were optimized. An electrophoretic buffer of 2.5 x 10(-2) M sodium borate (pH 9.4) containing 1 x 10(-4) M luminol was used. The oxidizer solution of 8 x 10(-4) M NaBrO in 0.1 M sodium carbonate buffer solution (pH 12.5) was introduced post-column. Under the optimal conditions, the detection limits were 1.0 x 10(-7) M for glutamic acid (Glu) and 1.3 x 10(-7) M (S/N = 3) for aspartic acid (Asp). The relative standard deviations (RSDs) of peak area and migration time were in the ranges of 3.8-4.3% and 1.4-1.6%, respectively. The present method was applied to the determination of excitatory amino acids (i.e., Asp and Glu) in rat brain tissue and monkey plasma. The levels of these major excitatory amino acids in monkey plasma were quantified for the first time and found to be 1.17 +/- 0.17 x 10(-5) M (mean +/- SD, n = 6) for Glu and 1.64 +/- 0.19 x 10(-6) M for Asp, which were comparable with the levels in human plasma.     

Comparison between capillary electrophoresis and liquid chromatography for the determination of diclofenac sodium in a pharmaceutical tablet

Two novel analytical methodologies using capillary electrophoresis (CE) and liquid chromatography (LC) were developed and compared for the determination of diclofenac sodium in commercial and simulated tablet formulations. The CE analysis was performed in a bare fused-silica capillary with 75 microm id and total length of 50 cm (28 cm to the detector) with a buffer solution of 20 mM sodium tetraborate, pH 9.23. The applied voltage was 20 kV, and acetaminophen was used as the internal standard (IS). The LC analysis was performed with a LiChrospher 100 RP-18 (5 microm) column and a mobile phase of methanol-diluted glacial acetic acid (0.3 parts in 2500; 75 + 25) at a flow rate of 0.9 mL/min with propylparaben as the IS. In both analyses, detection was by ultraviolet absorption at 276 nm. Under optimized conditions, the CE migration times for the diclofenac sodium standard and acetaminophen (IS) were 2.07 and 1.59 min, respectively, and the LC retention times for the diclofenac sodium standard and propylparaben (IS) were 3.98 and 2.26 min, respectively. The resolution and efficiency for CE were 14.2 and 1.6 x 10(5) plates/m, respectively, and for LC, 5.0 and 8.6 x 10(3) plates/m, respectively. Calibration curves of peak area versus concentration gave correlation coefficients of 0.9992 for CE and 0.9994 for LC. The limits of detection and quantitation were 8.40 and 25.46 microg/mL, respectively, for CE and 4.60 and 13.93 microg/mL, respectively, for LC. Coefficients of variation were 1.68 and 0.37% for CE and LC, respectively. Average recoveries obtained with CE and LC were 103.12+/-0.90 and 99.59+/-0.21%, respectively. Although both methodologies were shown to be suitable for the determination of diclofenac sodium in tablets, performing in a similar manner with regard to several aspects (linearity, recovery, and specificity), CE provided faster analysis and better column efficiency, whereas LC provided superior repeatability and sensitivity.     

Sensitive and high-throughput analyses of purine metabolites by dynamic pH junction multiplexed capillary electrophoresis: a new tool for metabolomic studies.

On-line sample preconcentration by a dynamic pH junction in conjunction with multiplexed capillary electrophoresis (CE) and UV detection represents a sensitive and high-throughput format for future metabolomic research, such as purine analysis. The optimization of purine focusing can be rapidly assessed by systematically altering the sample matrix properties, such as the buffer co-ion, pH and ionic strength using a 96-capillary array format. This method permits focusing of large sample injection volumes, resulting in over a 50-fold improvement in the concentration sensitivity. The limit of detection (S/N = 3) for purine metabolites was less than 8.0 x 10(-8) M under optimum conditions when using UV absorbance. Dynamic pH junction multiplexed CE demonstrated excellent linearity over a hundred-fold concentration range, as well as low inter-capillary precision in terms of normalized migration times and peak areas. The potential for clinically relevant high-throughput analyses of micromolar amounts of purine metabolites in urine was also demonstrated.     

Highly sensitive chemiluminescence detection of copper(II) in capillary electrophoresis with field-amplified sample injection

Field-amplified sample injection of copper(II) was investigated using capillary electrophoresis with chemiluminescence detection. The sensitivity of copper(II) has been improved markedly by the field-amplified sample injection technique and the detection limit reaches 2 x 10(-11) M. By injection of a short plug of water before sample introduction, the sensitivity can be further improved 5-fold and the detection limit reaches 4 x 10(-12) M. The relative standard deviations (n = 6) of the migration time and the peak height are 0.61% and 4.7% at 1.0 x 10(-9) M Cu(II), respectively. Parameters affecting the field-amplified sample injection, such as separation voltage and concentration of electrophoretic buffer, have been investigated.     

Rapid capillary electrophoresis time-of-flight mass spectrometry separations of peptides and proteins using a monoquaternarized piperazine compound (M7C4I) for capillary coatings

A monoquaternarized piperazine, 1-(4-iodobutyl) 4-aza-1-azoniabicyclo[2,2,2] octane iodide (M7C4I), has been evaluated as a surface derivatization reagent for CE in combination with TOF MS for the analysis of proteins, peptides, and protein digests. The M7C4I piperazine, at alkaline pH, forms a covalent bond via alkylation of the ionized silanols producing a cationic surface with a highly stable and reversed EOF. The obtained surface yields rapid separations (less than 5 min) of peptides and proteins at acidic pH with high separation efficiencies (up to 1.1 x 10(6) plates/m for peptides and up to 1.8 x 10(6) plates/m for proteins) and no observed bleeding of the coating reagent into the mass spectrometer. The simplicity of the coating procedure also enables fast (2 min) regeneration of the surface, if necessary. This is useful in the analysis of complex samples in order to prevent possible memory effects. The potential of using M7C4I-coated capillaries for MS analysis of complex samples is demonstrated by the separation of peptides, proteins, and protein digests. Even more, the spectacular thing in which large intact proteins with molecular masses over 0.5 MDa could be separated. The coating showed good ability to handle these large proteins with high efficiency and retained peak shape as demonstrated by separation of IgG(1) (150 kDa) and thyroglobulin (669 kDa).     

Differential pulse polarographic determination of ofloxacin in pharmaceuticals and biological fluids

A sensitive method is described for the determination of ofloxacin in its pure form, dosage forms and biological fluids. The proposed method depends upon the polarographic activity of ofloxacin in Britton Robinson buffers, whereby a well-defined cathodic wave is produced over the pH range 4.1-10.3. The wave was characterized as being irreversible, diffusion-controlled with limited adsorption properties. The current-concentration relationship was found to be rectilinear over the range 5x10(-5)-5x10(-4) M and 1x10(-5)-5x10(-4) M using the DC(t) and DPP modes respectively, with a minimum detectability (S/N=3) of 3x10(-7) M. The proposed method was successfully applied to the determination of ofloxacin in tablets and biological fluids. The results obtained were found to be in agreement with those obtained by a reference method.     

Simultaneous determination of phosphonate, phosphate, and diphosphate by capillary electrophoresis using in-capillary complexation with Mo(VI)

This study describes the simultaneous determination of phosphonate, phosphate, and diphosphate by CE with direct UV detection, based on in-capillary complexation with Mo(VI). When a mixture of phosphonate, phosphate, and diphosphate was injected into a capillary containing 3.0 mM Mo(VI), 0.05 M malonate buffer (pH 3.0) and 45% v/v CH3CN, three well-defined peaks, due to the migration of the corresponding polyoxomolybdate anions, were separated. The respective calibration graphs were linear in the concentration range of 2 x 10(-6)-2 x 10(-4) M for phosphonate, 1 x 10(-6)-5 x 10(-5) M for phosphate, and 1 x 10(-6)-2 x 10(-4) M for diphosphate; the correlation coefficients were better than 0.9990. The present CE method is successfully applied to the simultaneous determination of phosphonate, phosphate, and diphosphate in tap water.