Determination of inorganic oxyhalides in drinking water by on-line coupled capillary isotachophoresis— capillary zone electrophoresis

Some oxyhalides can be found in drinking waters as inorganic disinfection byproducts. An on-line coupled capillary isotachophoresis—capillary zone electrophoresis (CITP-CZE) method was developed for the analysis of chlorate, chlorite and bromate in water. The optimized CITP-CZE electrolyte system consisted of the following: 10 mM—HCl+20 mM—β-Alanine (leading electrolyte), 5 mM—succinic acid (terminating electrolyte), and 10 mM—succinic acid +5 mM—β-Alanine +0.1% HPMC (carrier electrolyte). A clear separation of oxyhalides from other components of drinking water was achieved within 25 min. Method characteristics, i.e., linearity (0–200 ng/mL), accuracy (88–110%), intra-assay (3–5%), quantification limit (5–15 ng/mL), and detection limit (2–5 ng/mL), were determined. Minimum labor requirements, sufficient sensitivity and low running cost are important attributes of this method. It was found that the developed method is useful for the routine analysis of oxyhalides in water.     

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 aristolochic acid by capillary zone electrophoresis

A simple and rapid capillary zone electrophoresis (CZE) method for the determination of aristolochic acid (AA) in dietary supplements and selected herbs is described. A clear separation of AA from other sample constituents was achieved within 5 minutes without any sample clean up. A mixture of 20 mM-morpholinethanesulphonic acid+10 mM-BisTrisPropane+0.2% hydroxyethylcelullose in 10% methanol serves as a background electrolyte. The linearity, accuracy, intra-assay and detection limit of the developed method are 200–6000 ng/mL, 95–103%, 3.5%, and 50 ng/ml, respectively. Ease of use, sufficient sensitivity and low running cost are the most important attributes of the CZE method. The proposed CZE method was compared with HPLC.     

Determination of chlorite in drinking water by on-line coupling of capillary isotachophoresis and capillary zone electrophoresis

An isotachophoresis (ITP)–capillary zone electrophoresis (CZE) combination was used for the determination of chlorite in drinking waters. No sample preparation is needed and no interfering by other anions in tap water was observed. The reached limits of detection with conductivity detector were 0.012–0.017 mg l⁻¹. By four-fold sample loading with a 30 μl valve, 0.005 mg l⁻¹ of chlorite was determined with R.S.D.=3.3%. The concentrations of 0.05 and 0.20 mg l⁻¹ were measured with R.S.D. of 2.2 and 2.7%, respectively. The recoveries of chlorite from drinking water were 96–106% in the range of 0.02–0.20 mg l⁻¹. The R.S.D. values of migration times (inter-day) were up to 1.3%. The time for analysis is about 15 min.     

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

An on-line coupled capillary isotachophoresis--capillary zone electrophoresis (cITP-CZE) method for the determination of domoic acid in shellfish and algae is described. The optimised cITP-CZE electrolyte system was 10 mM HCl + 20 mM beta-alanine (BALA) + 0.05% hydroxyethylcellulose (leading electrolyte), 5 mM caproic acid (terminating electrolyte) and 20 mM caproic acid + 20 mM BALA + 0.1% HPMC (background electrolyte). A clear separation of the domoic acid from the other components of methanolic sample extract was achieved within 25 min. Method characteristics, i.e., linearity (0-200 microg/l), accuracy (recovery 101+/-3%), intra-assay repeatability (2.4%) and detection limit (1.5 microg/l) were determined. Speed of analysis, low laboriousness, high sensitivity and low running cost are the typical attributes of the cITP-CZE method. Developed method was successfully applied to analysis of shellfish samples and food supplements containing algae extract.     

Analysis of 5-methyltetrahydrofolate in human blood, serum and urine by on-line coupling of capillary isotachophoresis and zone electrophoresis

The predominant circulating folate coenzyme in plasma/serum, 5‐methyltetrahydrofolate (5‐MTHF) was determined in human blood, serum and urine using a method based on the hyphenation of capillary ITP and zone electrophoresis. Measurements were done with a commercially available instrument for capillary isotachophoresis equipped with a column‐switching system. The choice of electrolytes was limited by the instability of 5‐MTHF and volatility of electrolytes for the potential coupling of the instrumentation with MS detector. To get an insight into the separability of individual sample components in an isotachophoretic analysis, we constructed zone existence diagrams for isotachophoretic electrolyte systems having a leading electrolyte composed of acetate and ammonium of pH 4.5 and 7.0, hydrocarbonate and ammonium, pH 7.8, chloride and ammonium, pH 5.6, and chloride and creatinine, pH 5.0, with hydroxide ion as the terminator. For isotachophoretic preseparation, the non‐volatile leading electrolyte with good buffering capacity composed of 1×10⁻² M HCl and 2.5×10⁻² M creatinine, pH 5.0, and terminating electrolyte composed of 1×10⁻² M MES was selected as the most suitable. The optimum BGE for CZE analysis from the standpoint of analyte stability, separability and volatility for MS coupling was 1×10⁻² M acetate with 3.5×10⁻² M ammonium, pH 4.5. Using this combination of electrolytes, LODs reached with optical detection at 220 nm were 1.6×10⁻⁷ M in human blood, 1.1×10⁻⁷ M in human serum and 4.7×10⁻⁶ M in human urine. Estimated content of 5‐MTHF in blood and serum samples of women following oral daily administration of 0.8 mg of folic acid was 1.2×10⁻⁵ and 5.8×10⁻⁶ M, respectively.     

Determination of glycyrrhizin in liqueurs by on-line coupled capillary isotachophoresis with capillary zone electrophoresis

An on-line coupled capillary isotachophoresis-capillary zone electrophoresis method for the determination of glycyrrhizin in liqueurs is described. The optimised electrolyte system was 5 mM HCl+11 mM varepsilon-aminocaproic acid+0.05% hydroxyethylcellulose+30% methanol (leading electrolyte), 5 mM caproic acid+30% methanol (terminating electrolyte) and 20 mM caproic acid+10 mM histidine+0.1% hydroxyethylcellulose+30% methanol (background electrolyte). Method characteristics, i.e., linearity (20-500 ng/ml), accuracy (recovery 99+/-4%), intra-assay repeatability (2%), intermediate repeatability (3.8%) and detection limit (8 ng/ml) were determined. Speed of analysis, low laboriousness, high sensitivity and low-running cost are the typical attributes of the capillary isotachophoresis-capillary zone electrophoresis method. Developed method was successfully applied to analysis of liqueurs with liquorice extract and some foods (sweets and food supplements) containing liquorice. Found levels of glycyrrhizin in liqueurs, sweets and food supplements varied between 1-16 mg/l, 850-1050 mg/kg and 1.6-1.8 g/kg, respectively.     

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.     

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.     

Comparison of hydrodynamically closed two‐dimensional capillary electrophoresis coupled with ultraviolet detection and hydrodynamically open capillary electrophoresis hyphenated with mass spectrometry in the bioanalysis of varenicline

Two capillary electrophoresis methods for monitoring renally excreted varenicline, a highly effective drug prescribed for smoking cessation, in human urine were developed and compared. A method combining capillary electrophoresis with mass spectrometry was proposed for the fast analysis of varenicline (analysis time up to 7 min). Here, mass spectrometry was a prerequisite for achieving high sensitivity and selectivity of the analysis suitable for the quantification of a 15 ng/mL level of varenicline in un‐pretreated urine matrices. An alternative approach, two‐dimensional (column‐coupled) capillary electrophoresis with enhanced sample load capacity and ultraviolet detection, was proposed as a low‐cost alternative to capillary electrophoresis with mass spectrometry. The isotachophoresis on‐line sample treatment included simple elimination of the major matrix constituents and stacking of the sample in a large volume so that threefold lower quantitation limits could be easily achieved in comparison to the capillary electrophoresis with mass spectrometry. On the other hand, longer analysis time (ca. 4.5‐fold) and more complex electrolyte system in the coupled zone electrophoresis step (including two additives enhancing separation selectivity, i.e. isopropanol and cyclodextrin) were prerequisites for the complete separation of varenicline from the sample matrix. Anyway, both the developed methods were validated according to the Food and Drug Administration guidelines showing favorable performance parameters, suitable for their routine biomedical use.     

Determination of effective charges and ionic mobilities of polycationic antimicrobial peptides by capillary isotachophoresis and capillary zone electrophoresis

Capillary ITP (CITP) and CZE were applied to the determination of effective charges and ionic mobilities of polycationic antimicrobial peptides (AMPs). Twelve AMPs (deca‐ to hexadecapeptides) containing three to seven basic amino acid residues (His, Lys, Arg) at variable positions of peptide chain were investigated. Effective charges of the AMPs were determined from the lengths of their ITP zones, ionic mobilities, and molar concentrations, and from the same parameters of the reference compounds. Lengths of the ITP zones of AMPs and reference compounds were obtained from their CITP analyses in cationic mode using leading electrolyte (LE) composed of 10 mM NH₄OH, 40 mM AcOH (acetic acid), pH 4.1, and terminating electrolyte (TE) containing 40 mM AcOH, pH 3.2. Ionic mobilities of AMPs and singly charged reference compounds (ammediol or arginine) were determined by their CZE analyses in the BGE of the same composition as the LE. The effective charges numbers of AMPs were found to be in the range 1.65–5.04, i.e. significantly reduced as compared to the theoretical charge numbers (2.86–6.99) calculated from the acidity constants of the analyzed AMPs. This reduction of effective charge due to tightly bound acetate counterions (counterion condensation) was in the range 17–47% depending on the number and type of the basic amino acid residues in the AMPs molecules. Ionic mobilities of AMPs achieved values (26.5‐38.6) × 10⁻⁹ m²V⁻¹s⁻¹ and in most cases were in a good agreement with the ratio of their effective charges and relative molecular masses.     

乙醛酸和草酸的毛细管区带电泳分析

建立了同时测定乙醛酸和草酸的毛细管区带电泳法。考察了缓冲溶液的种类、浓度和pH以及分离电压等因素对分离结果的影响。在缓冲溶液为20 mmol/L硼砂-5.5 mmol/L邻苯二甲酸氢钾(pH 9.0)、分离电压20 kV、检测波长212 nm的优化条件下,11 min内即可实现对目标物的分离。乙醛酸和草酸分别在0.8～20 g/L和1.2～20 g/L范围内线性关系良好,相关系数分别为0.9993和0.9975;方法的检出限分别为0.2和0.4 g/L(信噪比为3);样品的加标回收率为98.3%～102.5%,相对标准偏差为0.35%～0.61%。该方法操作简便、快速、成本低廉,已应用于实际样品的分析,并获得了令人满意的结果。     

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.     

Separation and determination of magnolol and honokiol inMagnolia officinalis bark by capillary zone electrophoresis

The effects of pH on separation parameters such as migration mobility, resolution, sensitivity, column efficiency and peak shape were emphatically studied. Better separation of magnolol and honokiol using capillary zone electrophoresis was achieved by optimizing pH in the range 5.0–11.7. The influences of applied voltage and temperature were also investigated. We adopted a better sample extraction procedure by which higher contents of honokiol and magnolol with sample compositions unchanged were obtained. The analysis was performed with direct UV detection using a 10 mM borate-10 mM phosphate buffer at pH of 11.6. The method was successfully applied to the simultaneous determination of magnolol and honokiol inMagnolia officinalis bark within 9 min.     

Determination of chelating agents and metal chelates by capillary zone electrophoresis

Aminopolycarboxylic acids such as diethylenetriaminepentaacetic acid (DTPA) are commonly used as chelating agents in many pulp and paper industries, particularly as scavengers of metal ions which catalyze the decomposition of hydrogen peroxide used as a bleaching agent. Concern for the effect of waste DTPA in the aquatic environment has led to a need for the development of methods to determine its levels in waste water. This paper describes the determination of free DTPA and several metal-DTPA complexes in water and waste water by capillary zone electrophoresis. The optimization of separation conditions included the selection of an appropriate carrier electrolyte composition (pH, organic solvents, ion-pairing reagents) and the systematic investigations of selective complexation of free DTPA as well as metal exchange reactions for metal-DTPA complexes in order to achieve selective and sensitive direct UV detection. The determination of DTPA in waste water from a paper mill was possible in the low ppm range.     

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.     

毛细管区带电泳法同时测定饮料中16种食品添加剂

建立了毛细管区带电泳法测定饮料中酸性红92、专利蓝V、荧光素二钠、酸性红1、靛蓝胭脂红、亮黑、丽春红6R、日落黄、苋菜红、柠檬黄等10种人工合成色素和苯甲酸、山梨酸、对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、对羟基苯甲酸丙酯、对羟基苯甲酸丁酯等6种防腐剂的分析方法。考察了缓冲溶液种类、浓度、pH值及运行电压、温度等对分离的影响,确定最佳电泳条件为: 未涂层弹性石英毛细管柱(46 cm×50 μm),缓冲溶液为70 mmol/L硼酸(pH=9.5)(含体积分数为4%的乙腈),检测波长220 nm,电泳电压30 kV,进样时间5 s,电泳温度25 ℃。该法用于测定市售饮料样品得到满意结果: 在1～250 mg/L范围内线性关系良好,相关系数不小于0.9938,回收率在95.8%与108.7%之间。该法简便、准确,能够满足食品添加剂的常规检测要求。