Rapid and sensitive determination of phosphoamino acids in phosvitin by N-hydroxysuccinimidyl fluorescein-O-acetate derivatization and capillary zone electrophoresis with laser-induced fluorescence detection

A method was developed for the determination of phosphoamino acids by capillary zone electrophoresis-laser-induced fluorescence detection (argon ion laser, excitation at 488 nm and emission at 520 nm) using derivatization with N-hydroxysuccinimidyl fluorescein-O-acetate (SIFA). Different variables affecting the derivatization (SIFA concentration, derivatization pH, reaction temperature and reaction time) and the separation (type, pH and concentration of buffer, applied voltage and injection mode) were investigated in detail. The optimized separation conditions were 40 mM boric acid buffer (pH 9.2) for background electrolyte, 25 kV for the separation voltage, 25 degrees C for the capillary temperature and 5 s at 0.5 psi for the sample injection. Under the optimal conditions, the SIFA-labeled phosphoamino acids were fully separated within 7 min. The detection limits ranged from 0.1 to 0.3 nM, which are the lowest values reported for capillary electrophoresis (CE) methods. The proposed methodology allowed the rapid, sensitive and selective determination of phosphoamino acids in hen egg yolk phosvitin by the standard addition method. The recovery of these compounds in real sample was 94.0-103.5%. The developed method surpasses previously published CE methods in terms of detection limit, separation time, stability and simplicity of the electrophoretic procedure.     

Species analysis of metallothionein isoforms in human brain cytosols by use of capillary electrophoresis hyphenated to inductively coupled plasma-sector field mass spectrometry

A new approach for the speciation of metallothioneins (MT) in human brain cytosols is described. The analysis is performed by application of a newly developed coupling of capillary electrophoresis (CE) with inductively coupled plasma-sector field mass spectrometry (ICP-SFMS). Isoforms of metallothioneins are separated from 30-100 microliter sample volumes by CE and the elements Cu, Zn, Cd, and S are detected by use of ICP-SFMS. The extraction of cytosols is the first step in the analytical procedure. Tissue samples from human brain are homogenized in a buffer solution and submitted to ultra-centrifugation. The supernatant is defatted and the cytosol pre-treatment is optimized for CE separation by matrix reduction. The buffer concentration and pH used for capillary electrophoretic separation of metallothionein from rabbit liver were optimized. CE with ICP-MS detection is compared to UV detection. In the electropherograms obtained from the cytosols three peaks can be assigned to MT-1, MT-2, and MT-3. As an additional method, size-exclusion chromatography (SEC) is applied. Fractions from an SEC separation of the cytosol are collected, concentrated, and then injected into the CE. The detection of sulfur by ICP-SFMS (medium resolution mode) and quantification by isotope dilution have also been investigated as a new method for the quantification of MT isoforms. The analytical procedure developed has been used for the first time in comparative studies of the distributions of MT-1, MT-2, and MT-3 in brain samples taken from patients with Alzheimer's disease and from a control group.     

Congophilicity (Congo red affinity) of different beta2-microglobulin conformations characterized by dye affinity capillary electrophoresis

The amyloidogenic protein beta-microglobulin was characterized by affinity capillary electrophoresis (CE). CE could separate conformational variants of beta2-microglobulin and with the amyloid-specific dye Congo red as a buffer additive it was possible to measure different Congo red-affinities of native and abnormally folded beta2-microglobulin. We find that native beta2-microglobulin has an intermediate affinity for Congo red at pH 7.3 and that binding involves electrostatic interactions. The conformational variant of beta2-microglobulin that appears in acetonitrile solutions binds Congo red more strongly. Affinity CE using Congo red as a buffer additive is a new, simple, fast, and quantitative micromethod for the characterization of soluble conformational intermediates of amyloidogenic proteins.     

Identification of kappa and lambda chains of the major immunoglobulin G subclasses by capillary zone electrophoresis

Immunoglobulins are present in most tissues and plasma and play crucial role in immune system. Alteration of the levels of the immunoglobulin G (IgG) subclasses (IgG1, IgG2, IgG3 and IgG4) is an indication of a disturbed immunological response. The aim of the present study was the development of a capillary electrophoresis (CE) method for the analysis of IgG subclasses in respect to their variable kappa and lambda chains. Various analytical conditions and CE modes, including capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF) and micellar electrokinetic capillary chromatography (MECC) have been thoroughly studied. CZE was found to be the most convenient way to separate IgG subclasses. Three of the human IgG subclasses were resolved using uncoated fused-silica and 50 mM phosphate, pH = 9.3, as operating buffer at 20 kV and detection at 214 nm. IgG1kappa was completely separated from IgG2kappa and IgG3kappa, whereas IgG2kappa co-migrated with IgG4kappa, which is the minor IgG subclass. Under the same conditions IgG4lambda was completely separated from IgG1lambda, IgG2lambda and IgG3lambda, enabling the identification of the various lambda chains. The developed CE method is rapid and can be applied to the identification of the major immunoglobulin G subclasses in respect to their variable kappa and lambda chains.     

Effect of sample composition on electrophoretic migration application to hemoglobin analysis by capillary electrophoresis and agarose electrophoresis

The electrophoretic migration, in routine analysis, is crucial for compound identification especially when multiple components are present in the sample. In complex or crude samples, such as those obtained from biological fluids, electrophoretic migration often does not correspond well to that of a pure standard compound. Several factors, related to the sample itself, have been identified as modulating the electrophoretic migration in zone electrophoresis both in gel and capillary electrophoresis (CE): solute mobility and concentrations, salt content, and protein interaction in the sample. Peak shape asymmetry often signals changes in migration especially when comparing samples with wide differences in concentration or those containing high ionic strength. Also, the migration of a protein can be influenced by the presence of a high concentration of another slowly migrating protein in the sample. A weak interaction during the separation between the two proteins which lead to a decreased velocity has been postulated. This was confirmed by finding a curve-linear relationship between the ratio of the two hemoglobin (Hb) variants, hemoglobin F (Hb F) and hemoglobin S (Hb S), and the distance between the two in gel electrophoresis (GE); and also by the observation of formation of a new small peak based on the analysis of hemoglobin F by capillary electrophoresis upon the addition of Hb S to the separation buffer. These factors when present together have an additive effect on the migration. As an example, Hb F, present in low but variable concentration in patients with sickle cell disease (Hb S), migrates in gel electrophoresis slightly slower than it is expected; enough to be confused with other unknown variants. However, the small peaks with different migration distances between Hb S and the adult Hb (Hb A) correlated well (r = 0.98) with Hb F performed by an alkali-denaturing assay indicating that these peaks are indeed Hb F in spite of the difference in their migration.     

Therapeutic deferoxamine and deferiprone monitoring in β‐thalassemia patients’ plasma by field‐amplified sample injection and sweeping in capillary electrophoresis

One CE method was established for detecting deferoxamine (DFO) and deferiprone (DFR) in plasma. For β‐thalassemia patients, DFO and DFR are major medicines to treat the iron overload caused by blood transfusion. Field‐amplified sample injection combined with sweeping was used for sensitivity enhancement in CE. This method was performed on an uncoated fused‐silica capillary. After liquid–liquid extraction, the plasma samples were electrokinetically injected into capillary at +10 kV for 180 s. The phosphate buffer (100 mM) containing 50 mM triethanolamine was used as the BGE (pH 6.6). Separation buffer was phosphate buffer (100 mM, pH 3.0) containing 150 mM SDS. This method showed good linearity (r ≥ 0.9960). Precision and accuracy were evaluated by the results of RSD and relative error of intrabatch and interbatch analyses, and all of the absolute values were less than 6.12%. The LODs (S/N = 3) were 200 ng/mL for DFO, and 25 ng/mL for DFR. The LOQ (S/N = 10) of DFO and DFR were 600 and 75 ng/mL, respectively. This method was applied for clinical applications of five β‐thalassemia patients.     

Globin chain separation by SDS polyacrylamide gel electrophoresis. Simple screening method for elongated hemoglobin chains

A simple method for the separation of hemoglobin chains from hemolysate or globin, by sodium dodecyl sulfate polyacrylamide gel electrophoresis, is described. The alpha, beta, and gamma chains can be clearly separated from each other. The alpha chain has the highest mobility, the beta chain has a slower mobility than the gamma chain, while the delta chain has about the same mobility as the beta chain. Hemoglobins with elongated chains can easily be detected by this method. Tak-beta, elongated by 11 residues, moves much more slowly than betaA but is much faster than alpha Constant Spring which is elongated by 31 residues. Screening of several individuals with slow-moving hemoglobins using this method led to the finding of a case with Hb Tak-beta thalassemia and other carriers of Hb Tak.     

Application of Graphene as Solid Phase Extraction Absorbent for the Determination of Parabens in Cosmetic Products by Capillary Electrophoresis

A graphene-based solid phase extraction (SPE) technique was developed for the extraction of parabens (methylparaben, ethylparaben, propylparaben, and butylparaben) in cosmetic samples, followed by determination by capillary electrophoresis (CE). The SPE extraction parameters such as eluent reagent and eluent volume, washing solution, sample pH, and flow-rate of sample loading, were investigated for satisfied recoveries. The running buffer, consisting of 25 mM borate solution (pH 10.0), was used for the separation of four parabens with the CE method within 10 min. The limits of detection were 0.14 mg/L, 0.13 mg/L, 0.15 mg/L, and 0.10 mg/L for methylparaben, ethylparaben, propylparaben, and butylparaben, respectively, and the mean recoveries obtained were between 62.6% and 100.4%. The developed method was used for the determination of parabens in real cosmetic products.     

Investigation of folic acid stability in fortified instant noodles by use of capillary electrophoresis and reversed-phase high performance liquid chromatography

A single enzyme treatment with alpha-amylase, prior to the quantification of added folic acid (FA) in fortified instant fried Asian noodles with analysis performed by capillary zone electrophoresis (CZE) and reversed-phase high performance liquid chromatography (RP-HPLC) with UV detection, is described. The method was validated and optimized for capillary electrophoresis (CE) with separation achieved using a 8 mM phosphate-12 mM borate run buffer with 5% MeOH at pH 9.5. FA was well separated from matrix components with nicotinic acid (NA) employed as an internal standard. In a comparative study, separation of FA was performed using HPLC with a mobile phase consisting of 27% MeOH (v/v) in aqueous potassium phosphate buffer (3.5 mM KH(2)PO(4) and 3.2 mM K(2)HPO(4)), pH 8.5, and containing 5 mM tetrabutylammonium dihydrogen phosphate as an ion-pairing agent. For both methods, excellent results were obtained for various analytical parameters including linearity, accuracy and precision. The limit of detection was calculated to be 2.2 mg/L for CE without sample stacking and 0.10 mg/L with high performance liquid chromatography (HPLC). Sample extraction involved homogenization and enzymatic extraction with alpha-amylase. Results indicated that FA was stable during four main stages of instant fried noodle manufacturing (dough crumbs, cut sheets, steaming and frying).     

Determination of mercaptopurine and its four metabolites by large-volume sample stacking with polarity switching in capillary electrophoresis

This study describes approaches for stacking a large volume of sample solutions containing a mixture of mercaptopurine monohydrate, 6-methylmercaptopurine, thioguanine, thioguanosine, and thioxanthine in capillary electrophoresis (CE). After filling the run buffer (60 mM borate buffer, pH 8.5), a large sample volume was loaded by hydrodynamic injection (2.5 psi, 99.9 s), followed by the removal of the large plug of sample matrix from the capillary using polarity switching (-15 kV). Monitoring the current and reversing the polarity when 95% of current recovered, the separation of anionic analytes was performed in a run buffer < 20 kV. Around 44- to 90-fold improvement of sensitivity for five analytes was achieved by large-volume stacking with polarity switching when compared with CE without stacking. This method was feasible for determination of the analytes spiked in plasma. Removing most of electrolytes from plasma is a key step for performing large-volume sample stacking. Solid-phase extraction was used for pretreatment of biological samples. To our knowledge, this study is one of few applications showing the possibilities of this stacking procedure to analyze biological samples by large-volume sample stacking with polarity switching (LVSSPS) in CE.     

Non-aqueous capillary electrophoresis with diode array and electrospray mass spectrometric detection for the analysis of selected steroidal alkaloids in plant extracts

A capillary electrophoresis (CE) assay has been developed for the quantitation and determination of the impurity profile of the potassium channel blockers 3,4-diaminopyridine and 4-aminopyridine. The compounds were separated from related substances using a capillary of 30 cm effective length, a 50 mM phosphate buffer, pH 2.5 and an applied voltage of 25 kV. The assay was validated with respect to specificity, linearity, range, limits of quantitation and detection, precision and robustness. The method allows the detection and quantitation of impurities at the 0.05% level. The feasibility of the assay was demonstrated by analyzing a commercial sample of 3,4-diaminopyridine. All known related substances could be detected in this sample with the present CE method.     

Sensitivity enhancement of chlorinated phenols by continuous flow liquid membrane extraction followed by capillary electrophoresis

This paper describes a new analytical system, based on the combination of continuous flow liquid membrane extraction (CFLME) enrichment and capillary electrophoresis (CE) separation, for analysis of chlorinated phenols in water samples. Five chlorinated phenols including 3-chlorophenol (3CP), 4-chlorophenol (4CP) 2,4-dichlorophenol (DCP), 2,4,6-trichlorophenol (TCP), and pentachlorophenol (PCP) were separated by CE with Tris/sodium dihydrogen phosphate solution containing methanol 1% (v/v) as the run buffer. CFLME related parameters were investigated and optimal enrichment was obtained by using 0.3 mol L(-1) Tris as acceptor and with a sample pH 5.0, a sample flow rate of 4.0 mL min(-1), and an enrichment sample volume of 150 mL. The detection limit (S/N= 3) was 6.9, 1.0, and 1.7 ng mL(-1) for DCP, PCP, and TCP, respectively. The reproducibility (RSD%, n = 6) was 5.7 for DCP, 2.5 for PCP, and 2.8% for TCP (n = 6). The proposed method was applied to the determination of chlorinated phenols in spiked water samples with relatively satisfactory recoveries.     

Application of a novel micro-injector in the determination of indole derivatives in the rat pineal gland by capillary electrophoresis with electrochemical detection

A novel micro-injector has been fabricated for capillary electrophoresis (CE). It was successfully used for the determination of some indole derivatives for example melatonin (MT), serotonin (5-HT), tryptophan (Trp), and 5-hydroxy-tryptophane (5-HTrp) in the rat pineal gland by capillary electrophoresis with electrochemical detection (CE-EC). CE was performed in 0.20 mol L(-1) phosphate buffer (pH 8.0). The compounds investigated can be well separated and detected within 15 min. The working electrode used was a 300-microm diameter carbon electrode positioned opposite the outlet of the capillary. The relationship between peak current and analyte concentration was highly linear in the range from 0.10 to 500 micromol L(-1); detection limits (S/N = 3) were 0.03-0.13 micromol L(-1). The proposed method has been successfully used to analyze real biological samples.     

Analysis of chitin oligosaccharides by capillary electrophoresis with laser-induced fluorescence

A method, using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection for analyzing chitin oligosaccharides is described. Chitin oligosaccharides were derivatized with 9-aminopyrene-1,4,6-trisulfonate (APTS) via reductive amination at 37 degrees C for 16 h (optimized conditions). The APTS-chitin oligosaccharides were analyzed using either an acidic citric acid-phosphate buffer or an alkaline borate buffer. The effects of buffer types, buffer pH values, and buffer concentrations on the separation were examined. The analytes were successfully separated by using a pH 4.6 citric acid-phosphate within 19 min. The APTS-derivatized chitin monosaccharide (D-glucosamine) migrated first. The analytes were also completely separated by using a pH 9.0 borate buffer within 24 min. Moreover, the specificity of enzyme digestion on chitin polysaccharides using the optimized APTS labeling procedure and the CE-LIF method was demonstrated.     

Large-volume stacking with polarity switching and sweeping for chlorophenols and chlorophenoxy acids in capillary electrophoresis

This paper describes approaches for stacking large volumes of sample solutions containing a mixture of chlorophenols and chlorophenoxyacetic acids as their anions in capillary zone electrophoresis, and compares results to standard capillary electrophoresis (CE) and normal stacking modes. In order to increase the amount of sample injected beyond the optimal conditions and maintain high resolution, the sample introduction buffer must be removed after the stacking process is completed. This is achieved by pumping the sample buffer out of the column using polarity switching. Large sample volumes are loaded by hydrodynamic injection, then stacked at the injection buffer/run electrolyte interface, followed by the removal of the large plug of low-conductivity sample matrix from the capillary column using polarity switching and finally the separation of the stacked anions in a basic buffer (pH 8.65). Around 10- and 40-fold improvement of sensitivity was achieved by normal stacking and large-volume stacking with polarity switching, respectively, when compared to the standard CE analysis. Sweeping-micellar electrokinetic capillary chromatography (MEKC) was also investigated for the purpose of comparison to the stacking technique. The method should be suitable for the analysis of these chemical compound classes in industrial chlorophenoxyacetic acid manufacture.     

Quantitation of nicotine in tobacco products by capillary electrophoresis

A simple and rapid capillary electrophoresis (CE) method was developed for the quantitation of nicotine in commercial tobacco products. The method involves a 6 min run at 30 kV, using a 50 mM phosphate buffer (pH 2.5), paraquat as internal standard, and UV detection at 260 nm. Nicotine was extracted from tobacco products in <15 min. Recoveries from spiked extracts were >95%, and the extraction efficiencies of water, 1 M HCI, 1 M acetic acid, 5 mM phosphate buffer (pH 2.5), and 1% triethanol amine were similar. Nicotine concentrations in 67 samples of cigarettes, cigars, and bidis varied between 0.37 and 2.96% (w/w). An established gas chromatography/mass spectrometry method using toluene extraction consistently yielded lower nicotine values than the CE method. Experimental evidence suggests that this is due to insufficient extraction of nicotine by toluene.     

On-line microheterogeneity analysis and rapid phenotyping of haptoglobin by capillary electrophoresis using sodium dodecyl sulfate as additive

To improve the detection sensitivity and determine phenotypes of haptoglobin (Hp), a prefilling technique was developed and tested in capillary electrophoresis (CE) with UV–vis absorbance detection. Adding 0.01% sodium dodecyl sulfate (SDS) to the protein sample and 0.1% SDS to the prefilling buffer solution, on-line stacking and microheterogeneity separation of Hp were achieved. In addition, the influences of pH, buffer concentration, sample and prefilling buffer SDS concentration upon resolution were examined. Under optimized conditions, Hp-microheterogeneity was well resolved and two phenotypes of Hp (Hp 1-1 and Hp 2-2) were differentiated. This method was applied to the analysis of sera from normal individuals and β-Thalassemia patients. After the depletion of albumin (HSA) and immunoglobulin G (IgG), this method allowed to determine two phenotypes in different individuals and to detect the decrease of Hp in β-Thalassemia patients. Featuring high efficiency, speed and simplicity, the proposed method shows great potential for use in clinical diagnosis and proteome research.     

Derivatization, extraction and concentration of amino acids and peptides by using organic/aqueous phases in capillary electrophoresis with fluorescence detection

We report a novel method that facilitates sample pretreatment and detection in amino acid analysis by coupling solvent extraction with capillary electrophoresis. Amino acids and peptides were fluorescently labeled, concentrated into an organic solvent, and then separated by capillary zone electrophoresis with fluorescence detection. To achieve this, acetophenone was first employed to dissolve the derivatizing reagent, fluorescamine. The products, which possessed both hydrophilic and hydrophobic moieties, could be extracted and concentrated into the organic phase by suppressing the deprotonation of carboxyl groups, thus enhancing the hydrophobicity of the resulting molecules through pH modification in the aqueous solution. Furthermore, by fine-tuning the pH value, individual amino acids and short peptide molecules could be separated selectively from the sample bulk. This convenient, chemically controllable concentration technique may be useful in sample concentration and purification of biologically related samples such as amino acids and short peptides.     

Rapid separation of acetophenone and its monohydroxy isomers by capillary electrophoresis

This work describes the development of a capillary electrophoresis(CE)method for the simultaneous separation of acetophenone(AP),2-hydroxyacetophenone(2-HAP),3-hydroxyacetophenone(3-HAP)and 4-hydroxyacetophenone(4-HAP)in synthetic mixtures using 10 mmol/L of sodium tetraborate buffer(pH 9.5).The aim of this work is to demonstrate the effectiveness of CE to separate AP and its monohydroxy isomers and to defne how the separations are affected by buffers,buffer pH,sample matrices and separation voltage.This method was successfully used for the trace level separation and determination of 2-HAP,3-HAP and 4-HAP in synthetic mixture and 4-HAP in spiked plasma samples.     

Velocity-difference induced focusing of xanthine and purine metabolites by capillary electrophoresis using a dynamic pH junction

Summary Velocity-difference induced focusing (V-DIF) of analytes by a dynamic pH junction represents a simple yet effective on-line preconcentration method to improve concentration sensitivity in capillary electrophoresis (CE). Differences in buffer type, pH and conductivity between sample and background electrolyte (BGE) segments of the capillary are properties used to optimize purine focusing within a multi-section electrolyte system. This method permits the injection of large volumes of sample (up to 450 nL or about 18% of capillary length), resulting in over a 50-fold improvement in sensitivity with baseline resolution. The limit of detection (S/N=3) for xanthine is determined to less than 4.0×10⁻⁸ M under optimum conditions when using UV detection. Analysis of micromolar amounts of xanthine in pooled urine is also demonstrated without sample pretreatment. A dual mechanism involving dynamic pH and isotachophoretic modes is proposed to enhance analyte focusing performance when employing buffer pH junctions based on different types of electrolyte co-ions.