Estimation of acidity constants,ionic mobilities and charges of antimicrobial peptides by capillary electrophoresis

Capillary electrophoresis (CE) was employed for the determination of thermodynamic acidity constants (pK_a) and actual ionic mobilities of polycationic antimicrobial peptides (AMPs). The effective electrophoretic mobilities of AMPs were measured by CE in a series of the background electrolytes within a wide pH range (2.00–12.25), at constant ionic strength (25 mM) and ambient temperature, using polybrene coated fused silica capillaries to suppress sorption of cationic AMPs to the capillary wall. Eventually, Haarhoff–Van der Linde peak fitting function was used for the determination of correct migration times of some AMPs peaks that were distorted by electromigration dispersion. The measured effective mobilities were corrected to 25°C. Mixed acidity constants, , and actual ionic mobilities, m_i, of AMPs were determined by the nonlinear regression analysis of pH dependence of their effective mobilities. The values were recalculated to thermodynamic pK_as using the Debye–Hückel theory. Thermodynamic pK_a of imidazolium group of histidine residues was found to be in the range 3.72–4.98, pK_a of α‐NH₃⁺ group was in the range 6.14–6.93, and pK_a of ε‐NH₃⁺ group of lysine spanned the interval 7.26–9.84, depending on the particular amino acid sequence of the AMPs. Actual ionic mobilities of AMPs with positive charges from one to six elementary units achieved values (9.8 – 36.5) × 10^?9 m²V^?1s^?1.     

Determination of egg white lysozyme by on-line coupled capillary isotachophoresis with capillary zone electrophoresis

An on-line coupled capillary isotachophoresis - capillary zone electrophoresis method for the determination of lysozyme in selected food products is described. The optimized electrolyte system consisted of 10 mM NH(4)OH + 20 mM acetic acid (leading electrolyte), 5 mM epsilon -aminocaproic acid +5 mM acetic acid (terminating electrolyte), and 20 mM epsilon -aminocaproic acid +5 mM acetic acid +0.1% m/v hydroxypropylmethylcellulose (background electrolyte). A clear separation of lysozyme from other components of acidic sample extract was achieved within 15 min. Method characteristics, i.e., linearity (0-50 micrograms/mL), accuracy (recovery 96+/-5%), intra-assay (3.8%), quantification limit (1 microgram/ml), and detection limit (0.25 microgram/mL) were determined. Low laboriousness, sufficient sensitivity and low running costs are important attributes of this method. The developed method is suitable for the quantification of the egg content in egg pasta.     

Determination of brominated phenols in water samples by on-line coupled isotachophoresis with capillary zone electrophoresis

A method for determination of nine brominated phenols as environmental risk compounds was developed by on-line coupled capillary isotachophoresis and capillary zone electrophoresis (ITP–CZE). For ITP step, 1 × 10⁻² mol L⁻¹ hydrochloric acid with 3 × 10⁻² mol L⁻¹ ammediol pH 9.1 was used as the leading electrolyte, and 3 × 10⁻² mol L⁻¹ β-alanine with 2 × 10⁻² mol L⁻¹ sodium hydroxide pH 10.05 was used as the terminating electrolyte. As the background electrolyte for CZE separation, 2.5 × 10⁻² mol L⁻¹ β-alanine with 2.5 × 10⁻² mol L⁻¹ lysine pH 9.6 was used. All electrolytes contained 0.05% or 0.1% (m/v) hydroxyethylcellulose to suppress the electroosmotic flow. UV detection at wavelength 220 nm was used. Detection limits in order of tens of nmol L⁻¹ were achieved. Good repeatability of migration times (less than 0.33% RSD) and good repeatability of peak areas (less than 7.19% RSD) at concentration level 5 × 10⁻⁸ mol L⁻¹ were observed. Developed ITP–CZE method was applied to determination of brominated phenols in spiked tap and river water samples.     

Determination of quinine in beverages by online coupling capillary isotachophoresis to capillary zone electrophoresis with UV spectrophotometric detection

The present study illustrates the possibilities of capillary isotachophoresis (CITP) online coupled with capillary zone electrophoresis (CZE) and hyphenated with fiber-based spectrophotometric diode array detection (DAD) for the direct, highly reliable, and ultrasensitive determination of quinine (QUI) in real multicomponent ionic matrices (beverages). Here, the CITP provided an effective online sample pretreatment (preseparation and preconcentration) prior to the CZE separation. Due to the CITP sample preconcentration, a simple UV-visible absorbance spectrophotometric detection was sufficient for obtaining very low concentration limits of detection (~2.3 ng/mL). Enhanced separation selectivity due to the combination of different separation mechanisms (CITP vs. CZE) enabled to obtain a pure analyte zone, suitable for its detection and quantitation in the directly injected real samples. The spectrophotometric DAD, unlike single wavelength UV detection, enabled to characterize the purity (i.e. spectral homogeneity) of the analyte zone and preliminary data indicate structurally related compounds via characteristic spectra recorded in the interval of 200-600 nm. The proposed CITP-CZE-DAD method was characterized by favorable performance parameters (sensitivity, linearity, precision, recovery, accuracy, robustness, and selectivity) and successfully applied to the control of QUI and potential QUI impurities in commercial beverages. This method is proposed as a routine automatized method for the highly reliable quality food control.     

Determination of some heavy metal cations in molten snow by transient isotachophoresis/ capillary zone electrophoresis

Online combination of transient ITP and CZE is employed for the determination of Cd(II), Pb(II), Cu(II), Ni(II), and Zn(II). Acetic acid is used for creating the transient isotachophoretic state. alpha-Hydroxyisobutyric acid and 4-aminopyridine are used as BGEs for the separation and indirect UV detection. At optimum conditions, the method allows to determine the metals at levels of 40-120 microg/L, about 50 times more sensitive than conventional CZE. In combination with a 20-fold evaporative concentration, the method is suitable for environmental monitoring of the heavy metals in snow samples.     

Analysis of therapeutic peptides in human urine by combination of capillary zone electrophoresis-electrospray mass spectrometry with preparative capillary isotachophoresis sample pretreatment

The presented study deals with the off-line coupling of preparative isotachophoresis (pITP) with on-line combination of capillary zone electrophoresis with electrospray mass spectrometric detection (CZE-ESI-MS) used for the analysis of therapeutic peptides (anserine, carnosine, and buserelin) in complex matrix (urine). Preparative capillary isotachophoresis, operating in a discontinuous fractionation mode in column-coupling configuration, served as a sample pretreatment technique to separation, and fractionation of mixture of therapeutic peptides present in urine at low concentration level. The fractions isolated by pITP procedure were subsequently analyzed by capillary zone electrophoresis with electrospray mass spectrometric detection. Acetic acid at 200 mmol L(-1) concentration served as background electrolyte in CZE stage and it is compatible with MS detection in positive ionization mode. In pITP fractionation procedure, sodium cation (10 mmol L(-1) concentration) as leading ion and beta-alanine as terminating ion (20 mmol L(-1) concentration) were used. While using CZE-ESI-MS, the limits of detection were 0.18 μg mL(-1) for carnosine, 0.17 μg mL(-1) for anserine and 0.64 μg mL(-1) for buserelin in water and 0.19 μg mL(-1) for carnosine, 0.50 μg mL(-1) for anserine and 0.74 μg mL(-1) for buserelin in 10 times diluted urine, respectively. The cleaning power of pITP sample pretreatment was proved as the peptides provided the higher MS signals at lower concentration levels resulting from the minimized matrix effects. The quality of obtained MS/MS spectra was very good so that they can provide information about the structure of analytes, and they were used for verification of the analytes identities. The pITP pretreatment improved the detection limits of the analyzed therapeutic peptides at least 25 times compared to the CZE-ESI-MS itself.     

Determination of inorganic anions in saliva by capillary isotachophoresis

Nitrite, nitrate, iodide and thiocyanate have been quantified in non-smoker and smoker saliva by capillary isotachophoresis (CITP). Hydrochloric acid (10 mmol l⁻¹) adjusted with histidine to pH 6.0 plus 6% poly(vinylpyrrolidone) was used as the leading electrolyte (LE) and 5 mmol l⁻¹ acetic acid as the terminating electrolyte (TE). Linearity was observed from 0.005 to 0.500 mmol l⁻¹ with a coefficient of determination (r²) of 0.999. The separation of anions was achieved in less than 19 min. The minimal sample pretreatment and relatively low running cost make isotachophoresis good alternative to existing methods.     

Determination of imazalil by on-line coupled capillary isotachophoresis with capillary zone electrophoresis

A simple, rapid and reproducible capillary isotachophoretic on-line coupled with capillary zone electrophoresis (CITP-CZE) method for the determination of IMz in food packaging extracts and its residues in apples is described. A good separation of the IMZ from other sample constituents was achieved within 15 minutes without any sample clean up. Method characteristics (linearity, accuracy, intra-assay and detection limit) were determined. Less amount of time involved, sufficient sensitivity and low running cost are the important attributes of CITP-CZE method.     

Analysis of enantiomers in biological matrices by charged cyclodextrin-mediated capillary zone electrophoresis in column-coupling arrangement with capillary isotachophoresis

The possibility to apply charged chiral selector as buffer additive in capillary zone electrophoresis (CZE) on-line coupled with capillary isotachophoresis (CITP) was studied. Enantioseparations and determinations of trace (ng/ml) antihistaminic drugs [pheniramine (PHM), dimethindene (DIM), dioxopromethazine (DIO)] present in samples of complex ionic matrices (urine) served as model examples. A negatively charged carboxyethyl-β-cyclodextrin (CE-β-CD) was used as a chiral selector in analytical CZE stage following upon a sample pretreatment by CITP (preconcentration of the analytes from 5 to 20-times diluted urine samples, partial sample clean up removing macroconstituents from the sample matrices). A high recognition capability of the oppositely charged CE-β-CD was demonstrated by enantioselective retardation of the drugs in presence of micro-and semi-macroconstituents migrating in CZE stage and detectable by UV detector. In this way, enantiomers of the drugs could be easily separated and determined. Due to lack of interferences between the drugs and sample-matrix constituents in presence of charged CE-β-CD, demands on both spacers in CITP step and multiple column-switching were minimized. CITP-CZE method with charged selector appeared to be a useful analytical approach for the trace enantiomers in complex ionic matrices as it combined enhanced separation selectivity and sample loadabitlity with high separation efficiency and provided favorable performance parameters including sensitivity, linearity, precision, accuracy/recovery and robustness with minimal demands on sample preparation. Analysis of urine sample taken from a patient treated by PHM, showing concentration profile of PHM enantiomers and their metabolites, illustrated potentialities of the method in clinical research.     

Investigation of the effect of ionic strength of Tris-acetate background electrolyte on electrophoretic mobilities of mono-, di-, and trivalent organic anions by capillary electrophoresis

The effect of ionic strength of the background electrolyte (BGE) composed of tris(hydroxymethyl)aminomethane (Tris) and acetic acid on the electrophoretic mobility of mono-, di- and trivalent anions of aliphatic and aromatic carboxylic and sulfonic acids was investigated by capillary zone electrophoresis (CZE). Actual ionic mobilities of the above anions were determined from their CZE separations in Tris-acetate BGEs of pH 8.1 to 8.2 in the 3 to 100 mM ionic strength interval at constant temperature (25 degrees C). It was found that the ionic strength dependence of experimentally determined actual ionic mobilities does not follow the course supposed by the classical Onsager theory. A steeper decrease of actual ionic mobilities with the increasing ionic strength of BGE and a higher estimated limiting mobility of the anions than that found in the literature could be attributed to the specific behavior of the Tris-acetate BGEs. Presumably, not only a single type of interaction of anionic analytes with BGE constituents but rather the combination of effects, such as ion association or complexation equilibria, seems to be responsible for the observed deviation of the concentration dependence of the actual ionic mobilities from the Onsager theory. Additionally, several methods for the determination of limiting ionic mobilities from CZE measured actual ionic mobilities were evaluated. It turned out that the determined limiting ionic mobilities significantly depend on the calculation procedure used.     

Determination of thermodynamic acidity constants and limiting ionic mobilities of weak electrolytes by capillary electrophoresis using a new free software AnglerFish

Thermodynamic acidity constants (acid or acid-base dissociation constants, sometimes called also as ionization constants) and limiting ionic mobilities (both of them at defined temperature, usually 25°C) are the fundamental physicochemical characteristics of a weak electrolyte, that is, weak acid or weak base or ampholyte. We introduce a novel method for determining the data of a weak electrolyte by the nonlinear regression of effective electrophoretic mobility versus buffer composition dependence when measured in a set of BGEs with various pH. To correct the experimental data for zero ionic strength we use the extended Debye-Hückel model and Onsager-Fuoss law with no simplifications. Contrary to contemporary approaches, the nonlinear regression is performed on limiting mobility data calculated by PeakMaster's correction engine, not on the raw experimental mobility data. Therefore, there is no requirement to perform all measurements at a constant ionic strength of the set of BGEs. We devised the computer program AnglerFish that performs the necessary calculations in a user-friendly fashion. All thermodynamic pKa values and limiting electrophoretic mobilities for arbitrarily charged substances having any number of ionic forms are calculated by one fit. The user input consists of the buffer composition of the set of BGEs and experimentally measured effective mobilities of the inspected weak electrolyte.     

Assay of acebutolol in pharmaceuticals by analytical capillary isotachophoresis

Acebutolol [N-{3-acetyl-4-[(2-hydroxy-3-(isopropylamino)propoxy]phenyl} butanamide] is a cardioselective beta-blocker with a potent anti-hypertensive and antiarrhythmic effect. The optimised operational system of electrolytes for the newly developed ITP separation of acebutolol consisted of 10mM potassium acetate +10mM acetic acid (pH 4.65) as the leading electrolyte and 10mM beta-alanine with pH approximately 4 (adjusted with acetic acid) as the terminating electrolyte. The driving and detection currents were 75 and 20 microA, respectively and the analysis took approximately 13 min. Under these conditions the effective mobility of acebutolol was determined as 20.7 x 10(-9) m2 V(-1) s(-1). The calibration dependence was rectilinear in the range 0.14-1.4 mg ml(-1) of acebutolol base (r = 0.9995); relative standard deviation (RSD) values were 1.1% and 1.2% (n = 6) when determining 0.42 and 0.98 mg ml(-1) of acebutolol in a pure standard solution. The method, with the limit of detection (LOD) of 0.04 mg ml(-1) and limit of quantification (LOQ) of 0.12 mg ml(-1), was applied to the assay of acebutolol in Sectral tablets, Acecor tablets, Apo-acebutol tablets (nominal content 400 mg of acebutolol per tablet) and Acebirex tablets (nominal content 200 mg of acebutolol per tablet) with RSD = 0.7-1.7% (n = 6). No interference from any excipients present in the tablets was observed. The recoveries ranged from 98.8% to 102.4% as found by the standard addition technique.     

Determination of total phosphorus in soya food samples by capillary isotachophoresis (cITP)

Capillary isotachophoresis (cITP) was applied for the determination of total phosphorus in soya food. The leading electrolyte was 8 mM HCl adjusted with β-alanine to pH=3.55 plus 3 mM bis-tris-propane and 0.2% hydroxyethylcellulose, whereas the terminating electrolyte 5 mM citric acid. Obtained results were compared with the spectrophotometric method. The correlation coefficients were 0.9996 for cITP and 0.9986 for standard method indicating the satisfactory precision of the calibration curves. The separation of anions was achieved within 25 min. Accuracy was determined using standard reference material (non-fat milk powder) and recovery assay based on standard additions method. Obtained results were discussed in respect to the accuracy and statistical parameters. Satisfactory values of recovery ranged between 99.22 and 99.85%, whereas R.S.D.<1% what demonstrate the advantage of cITP method in the routine analyses of phosphorus content in food samples.     

毛细管区带电泳法测定粉针剂中头孢拉定的含量

用毛细管区带电泳法测定头孢拉定的含量 ,未涂层毛细管柱 (75 μm×48.5cm ,有效长度 40cm) ,电压 2 8kV ,检测波长 2 3 0nm ,温度 2 0℃ ,进样 5×1 0 3Pa× 3s。运行缓冲液为 2 5mmol/L硼砂缓冲液。方法的线性范围 3 1 .2 2μg/mL～ 749.2 8μg/mL ,检测限为 1 .1 7μg/mL。     

High-sensitive analysis of DNA fragments by capillary gel electrophoresis using transient isotachophoresis preconcentration and fluorescence detection

In this report aimed on further development of a high-sensitivity capillary gel electrophoresis (CGE) method for analysis of DNA fragments, we firstly explored online transient isotachophoresis (tITP) preconcentration combined with fluorescence detection (FD). The fluorescence signal (excitation: 488 nm; emission: 590 nm) was generated using the intercalating dye of ethidium bromide (EB). It was found when the leading electrolyte (LE) was injected behind the sample zone, such a special tITP mode has significant advantages to solve the bubble formation issue and to improve the analytical performance stability. Two standard DNA samples, a 50 bp DNA step ladder and the φX174/HaeIII digest, were used to evaluate the qualitative and quantitative abilities of the tITP-FD approach. A highly diluted sample (10,000-fold in the water, e.g. the φX174/HaeIII digest diluted from 500 μg/ml to the 50 ng/ml level) was enriched and detected; the LOD was down to 0.09 ng/ml for the 72 bp fragment, apparently improved more than 1000-fold in comparison with UV detection. Although the RSD of peak areas (n = 3) was around 15.5% for the sample was electrokinetically injected, good linearity of peak area response showed that the proposed method is suitable for quantitative analysis.     

Application of 1-alkyl-3-methylimidazolium-based ionic liquids in separation of bioactive flavonoids by capillary zone electrophoresis

Room-temperature ionic liquids (ILs) are liquids that are constituted entirely of ions and can provide a solvent environment quite unlike any other available at room temperature. They continue to attract considerable interest in the chemistry research community as they are good solvents for a wide range of both inorganic and organic materials. In this study, a CZE method has been established for resolving natural flavonoids, quercetin, kaempferol and isorhamnetin in the Chinese herbal extract from Hippophae rhamnoides and its medicinal preparation (Sindacon Tablet). In this method, 1-alkyl-3-methyl-imidazolium-based ILs are used as the additive, and the effects of the alkyl group, imidazolium counterion (anionic part), along with the concentration of IL are investigated and discussed. Baseline separation, high efficiencies and symmetrical peaks of the three flavonoids were obtained. The separation mechanism seems to be the hydrogen-bonding interaction between the imidazolium cations of IL and the flavonoids.     

Separation and determination of enkephalin-related peptides using capillary electrophoresis

CZE with UV-absorption detection has been used for the separation and determination of enkephalin-related peptides. The experimental conditions, such as pH and concentration of running buffer, applied voltage, injection method, and time, were investigated in detail. Excellent separation efficiency could be obtained for ten enkephalin-related peptides with a 50 microm (ID) x 58 cm capillary using sodium dihydrogen phosphate as the running buffer (pH 3.11) when 20 kV of applied voltage was used. The concentration detection limits were found to be in the range of 0.31-1.94 microg/mL (defined as S/N = 3). The proposed method has been applied to analyze the spiked cerebrospinal fluid (CSF) sample, and the results showed that CZE is a powerful technique for separation and detection of the above biological peptides.     

毛细管区带电泳法测定消毒剂中三氯新

建立了消毒剂中三氯新的毛细管电泳分析方法。探讨了缓冲介质和电泳参数对三氯新测定的影响。以15mmol/LNa2HPO4(pH6.0)-乙腈(V(Na2HPO4)∶V(乙腈)=50∶50)为电泳缓冲液,三氯新在12kV电压下电泳,于254nm检测波长处测定,6min可以完成分析。本方法的检出限为0.04mg/L,线性范围0.04～2.00mg/mL(r=0.997),加标回收率在90.9%～108.2%范围内,测定值的相对标准偏差分别为峰高7.7%,迁移时间5.5%。将本法与高效液相色谱法进行比较,样品测定结果的相对误差小于10%。将所建立的方法已用于消毒剂样品中三氯新的测定。     

Fractionation of glycoforms of recombinant human erythropoietin by preparative capillary isotachophoresis

This feasibility study deals with the use of preparative capillary isotachophoresis (CITP), operating in a discontinuous fractionation mode, to the separations and isolations of glycoforms of recombinant human erythropoietin (rhEPO). The preparative CITP separations were monitored by capillary zone electrophoresis (CZE) with a hydrodynamically closed separation unit. Such a CZE system, suppressing fluctuations of the migration data linked with fluctuations of EOF and hydrodynamic flow, made possible to evaluate and compare the preparative CITP separations performed within a longer time frame. Preparative CITP, carried out in the separation unit with coupled columns of enhanced sample loadability, separating 100 microg of rhEPO in a run lasting ca. 30 min, gave the production rate higher than 55 ng/s for the rhEPO glycoforms. The preparative separations included valve isolations of the glycoforms from the ITP stack into four or six fractions. Such numbers of the fractions corresponded to typical numbers of the major glycoform peaks as resolved in CZE of rhEPO. With respect to close effective mobilities of the glycoforms and a multicomponent nature of rhEPO, the fractions contained mixtures of glycoforms with the dominant glycoforms enriched 10-100-fold, relative to the original rhEPO sample.