On-line sample enrichment for the determination of proteins by capillary zone electrophoresis with poly(vinyl alcohol)-coated bubble cell capillaries

Field-amplified sample stacking (FASS) is used to separate basic proteins in a poly-(vinyl alcohol)-coated bubble cell capillary. To our knowledge, this is the first paper describing the on-column stacking of proteins (as cations) using FASS in bubble cell capillary. The bubble cell capillary is fabricated using a one-step method. Cetyltrimethylammonium chloride is added into the running buffer to reverse the EOF and, thus, to pump the water plug out during the sample stacking step. The effect of the water plug lengths and sample injection durations were investigated and optimized. The results obtained were compared with those for the normal capillary without bubble cell in terms of resolution and sensitivity enhancement. Under the optimal condition, this method can improve the sensitivity of the peak areas ranging from 5000- to 26 000-fold. The RSDs (n = 5) of the migration time and peak area are satisfactory (less than 0.6 and 12%, respectively). Application of the capillary electrophoresis method with bubble cell, FASS, and UV detection thereby leads to the determination of these proteins at concentrations ranging from 3 to 10 ng/mL, based on a signal-to-noise ratio of 3:1.     

Field-amplified on-line sample stacking for determination of carnosine-related peptides by capillary electrophoresis

An on-line sample stacking method, namely field-amplified sample injection, has been developed for the separation and determination of carnosine, anserine, and homocarnosine by capillary electrophoresis. Using electrokinetic injection, about 130- to 160-fold improvement of sensitivity was achieved without loss of separation efficiency when compared to conventional sample injection. For conventional injection, the samples were dissolved in running buffer and then hydrodynamically injected for 10 s (3.45 kPa). Various parameters affecting separation and sample stacking were optimized. Under optimum conditions, linear responses were obtained over two orders of magnitude and the detection limits (defined as S/N = 3) of carnosine, anserine, and homocarnosine were 1.5 x 10(-8) to 1.6 x 10(-8) mol/L.     

场放大样品堆积毛细管区带电泳检测尿液中苯丙胺类毒品

建立了毛细管区带电泳(CZE)中场放大样品堆积(FASS)技术分析尿液中苯丙胺类毒品的方法。采用体积分数30%甲醇的100 mmol/L磷酸盐(pH 3)为分离缓冲液,利用缓冲体系与样品溶液体系电导率的差异,在毛细管中浓缩样品组分,对苯丙胺、甲基苯丙胺、3,4-(亚甲二氧基)苯丙胺(MDA)、3,4-(亚甲二氧基)甲基苯丙胺(MDMA)4种毒品进行了分离和定量测定,与常规毛细管区带电泳比较,检测灵敏度提高约2000倍。采用利多卡因为内标,对添加上述4种毒品的尿液进行提取和测定,分析的相对标准偏差在15%范围之内,可检测到的上述毒品质量浓度为0.002μg/mL,相对回收率在70%~120%内。该方法可用于生物检材中苯丙胺类毒品的检测。     

Capillary electrophoresis determination of biogenic amines by field-amplified sample stacking and in-capillary derivatization

A sensitive CE method for determining biogenic amines in wines based on in-capillary derivatization with 1,2-naphthoquinone-4-sulfonate is presented. In this method, reagent and buffer solutions are introduced hydrodynamically into the capillary whereas the sample is injected electrokinetically, thus, allowing a selective preconcentration of the analytes by field-amplified sample stacking. Amines are labeled inside the capillary using a zone-passing derivatization approach in mixed tandem mode. The most relevant variables influencing on the derivatization and separation as well as significant interactions have been evaluated using experimental design. Multi-criteria decision making is utilized for the simultaneous optimization of interacting variables through overall desirability response surfaces. The validation of the method has proven an excellent separation performance and accuracy for the determination of biogenic amines such as histamine, tryptamine, phenylethylamine, tyramine, agmatine, ethanolamine, serotonin, cadaverine, and putrescine in red wines. Detection limits range from 0.02 mg/L for ethanolamine to 0.91 mg/L for serotonin. The RSDs for migration time and peak area are around 1.2 and 6.2%, respectively. Red wines from different Spanish regions have been analyzed using the proposed method.     

Combination of off-line solid-phase extraction and on-column sample stacking for sensitive determination of parabens and p-hydroxybenzoic acid in waters by non-aqueous capillary electrophoresis

For the first time, a procedure based on solid-phase extraction (SPE) for the simultaneous extraction of a group of parabens (methyl, ethyl, propyl, butyl and benzyl p-hydroxybenzoates) and p-hydroxybenzoic acid (PHBA), from environmental water samples has been developed. Analysis of the extracts was performed by non-aqueous capillary electrophoresis (NACE) coupled with diode array detection (DAD), using large-volume sample stacking (LVSS) based on the electroosmotic flow pump as on-column preconcentration technique. Several water samples, such as tap, river, and wastewater samples, were analyzed using both SPE-NACE-DAD and SPE-LVSS-NACE-DAD methods. It has been observed that in addition to SPE parameters such as sorbent material, sample pH, breakthrough volume, addition of an organic solvent and elution solvent, also sample characteristics, such as organic matter content, have influence on SPE extraction yields, especially in the case of PHBA. The presence of PHBA and some parabens was detected at trace levels in surface water samples. Concentrations up to 8.4 ng mL⁻¹ were found in raw wastewater, with the highest levels corresponding to methylparaben, propylparaben, and their main degradation product, PHBA.     

毛细管电泳-场强放大样品堆积法检测染发剂中的7种苯胺类物质

建立了毛细管电泳-场强放大样品堆积测定染发剂中4,4′-二氨基二苯甲烷、苯胺、邻甲氧基苯胺、对氨基苯甲醚、3,4-二甲基苯胺、间氨基苯酚、1-萘胺7种苯胺类物质的分析方法。在优化的缓冲溶液体系(0.15 mol/L NaH2PO4,0.015 mol/L 三乙醇胺, pH 2.3)下7种分析物在6.5 min内实现基线分离。考察了样品中添加的磷酸浓度和乙腈浓度、水柱长度、电动进样时间与电压对场强放大富集效率及重现性的影响。最佳的富集条件为: 水柱注入3.45 kPa(0.5 psi)×6 s,样品中添加40%(v/v)乙腈和0.6×10~3mol/L磷酸,进样电压与进样时间为10 kV×10 s。线性范围为3～1000 μg/L(R2＞0.996),检出限为0.26～2.75 μg/L,将已有方法的检测灵敏度提高了1～3个数量级。在2种市售黑色染发剂中均检测到间氨基苯酚,含量分别为7.32 mg/g和1.34 mg/g。平均加标回收率为74%～108%。该方法灵敏度高、快速、重现性好、成本低,可供多种样品基质中痕量苯胺类污染物及其他阳离子物质的测定借鉴使用。     

Application of capillary zone electrophoresis with large-volume sample stacking to the sensitive determination of sulfonamides in meat and ground water

A CZE method with UV-Vis detection has been established and validated for the determination of nine sulfonamides: sulfapyridine, sulfamethazine, sulfamerazine, sulfamether, sulfadiazine, sulfadimethoxine, sulfamethoxazole, sulfachlorpyridazine, and sulfamethizole. Optimum separation was obtained on a 64.5 cm x 75 microm bubble cell capillary using a buffer containing 45 mM sodium phosphate and 10% methanol at pH 7.3, with temperature and voltage of 27 degrees C and 25 kV, respectively. p-Aminobenzoic acid was used as an internal standard . Taking into account the lack of sensitivity of the UV-Vis detection, the application of an on-line preconcentration methodology, such as large-volume sample stacking with polarity switching has been proposed. This procedure combined with a solvent extraction/SPE method applied for off-line preconcentration and cleanup provides a significant improvement in the LODs, ranging from 2.59 to 22.95 mug/L for the studied compounds; the quantification of these residues being possible below the levels established by EU legislation in animal food products, such as meat. Satisfactory recoveries were also obtained in the analysis of these compounds in ground water.     

Determination of sulfonylurea herbicides in soil extracts by solid-phase extraction and capillary zone electrophoresis

Summary Sulfonylurea herbicides in soil extracts were concentrated using off-line solid-phase extraction (SPE), and determined by capillary zone electrophoresis (CZE) and UV detection. The method involves extraction of soils with 0.1 M NaHCO₃ solution and subsequent preconcentration by using C₁₈ cartridges prior to separation of the pesticide using CZE. The results show that a C₁₈ cartridge is suitable for the purification of sulfonylurea herbicides in soil extracts with the recoveries ranging from 65–103%. The separation conditions affecting the resolution and detection sensitivity was systematically investigated. The sulfonylureas were resolved well using 30 mM sodium acetate (NaAc)/acetic acid (HAc)+10% acetonitrile (ACN) buffer at pH 4.80. The calibration plots for the test solutes in the concentration of 0.2–50 mg L⁻¹ were linear with detection limits in the range of 0.05–0.10 mgL⁻¹. The proposed method has been successfully demonstrated for the determination of sulfonylurea herbicides in soil samples.     

On-line preconcentration of perfluorooctanoic acid and perfluorooctanesulfonic acid by nonaqueous capillary electrophoresis

Separation of major environmental pollutants as perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) by capillary electrophoresis is reported for the first time. It is not possible to resolve the solutes in an aqueous media. However, the use of methanol and acetonitrile as the background electrolyte (BGE) solvents allowed their rapid separation in an uncoated capillary. A major effort was put into BGE optimization in respect to both separation efficiency and detection for further on‐line preconcentration. 5 mmol.L^?1 naphthalene‐1‐sulfonic acid and 10 mmol.L^?1 triethylamine dissolved in ACN/MeOH (50:50 v/v) provided best separation and detection conditions. Next, the large‐volume sample stacking and the field‐amplified sample injection were applied and compared. Large‐volume sample stacking improved limits of detection (LODs) with regard to the standard injection by 69 times for PFOA and 143 times for PFOS with LODs of 280 and 230 nmol.L^?1, respectively. Field‐amplified sample injection improved LODs 624 times for PFOAand 806 times for PFOS with LODs 31 and 40 nmol.L^?1, respectively. Both preconcentration methods showed repeatabilities of migration times less than 1.2% RSD intraday and 6.6% RSD interday. The method was applied on PFOA and PFOS analysis in a sample of river water treated with solid‐phase extraction, which further improved LOD toward 5.6 × 10^?10 mol.L^?1 for PFOS and 6.4 × 10^?10 mol.L^?1 for PFOA and allows the method to be used for river water contamination screening or decomposition studies.     

Double‐layer poly(vinyl alcohol)‐coated capillary for highly sensitive and stable capillary electrophoresis and capillary electrophoresis with mass spectrometry glycan analysis

Glycosylation plays an important role in protein conformations and functions as well as many biological activities. Capillary electrophoresis combined with various detection methods provided remarkable developments for high‐sensitivity glycan profiling. The coating of the capillary is needed for highly polar molecules from complex biosamples. A poly(vinyl alcohol)‐coated capillary is commonly utilized in the capillary electrophoresis separation of saccharides sample due to the high‐hydrophilicity properties. A modified facile coating workflow was carried out to acquire a novel multiple‐layer poly(vinyl alcohol)‐coated capillary for highly sensitive and stable analysis of glycans. The migration time fluctuation was used as index in the optimization of layers and a double layer was finally chosen, considering both the effects and simplicity in fabrication. With migration time relative standard deviation less than 1% and theoretical plates kept stable during 100 consecutive separations, the method was presented to be suitable for the analysis of glycosylation with wide linear dynamic range and good reproducibility. The glycan profiling of enzymatically released N‐glycans from human serum was obtained by the presented capillary electrophoresis method combined with mass spectrometry detection with acceptable results.     

On‐line synergistic stacking in capillary zone electrophoresis featuring field‐amplified sample stacking and micelle to cyclodextrin stacking in the determination of two alkaloids in complicated matrix samples

A novel on‐line synergistic proconcentration strategy coupling field‐amplified sample stacking and micelle to cyclodextrin stacking for cationic analytes in capillary zone electrophoresis has been proposed and applied for the separation and determination of two alkaloids, matrine, and oxymatrine in complicated matrix samples. The approach was performed by the long injection of sample in a low‐conductivity sodium dodecyl benzene sulfonate solution followed by the injection of hydroxypropyl‐β‐cyclodextrin solution in higher conductivity. The stacking mechanism of this method has been expounded and parameters affecting stacking effect have been optimized in our study. Under the optimum experimental conditions, 169‐ and 218‐fold sensitivity improvements were achieved for matrine and oxymatrine when compared with normal injection. Analytical indicators including linearity, limits of detection, and reproducibility (intra‐ and inter‐day relative standard deviations) were evaluated. Moreover, sample matrix effect was studied using compound flavescent sophora and salicylic acid powder and spiked urine samples. The developed method is an attempt for the combination of micelle to cyclodextrin stacking with other stacking methods. It could be a good alternative choice for the determination of alkaloids in a complex sample matrix.     

Simultaneous stacking of cationic and anionic compounds in single run capillary zone electrophoresis by two-end field amplified sample injection

In order to extend the application of field amplified sample injection (FASI) in high throughput analysis, a convenient and simple procedure, namely two-end field amplified sample injection (TE-FASI), was developed for the simultaneous stacking of cationic and anionic compounds in a single run capillary zone electrophoresis (CZE). Following the capillary-filling with a buffer of high conductivity, water plug was loaded into each end of the capillary; and two high-field strength zones were generated at both heads of the column when high voltage was applied. Therefore, under suppressed EOF cations and anions can be selectively FASI stacked at anode and cathode head, respectively. After separation, the stacked anions and cations are detected by a common detector placed in the center of the capillary. Under the optimized conditions, the limits of detection for the model cationic (matrine and oxymatrine) and anionic (5-sulfosalicylic acid) compounds were determined as 0.2, 0.2 and 0.06 ng/mL, respectively. Compared with non-stacking conditions, the sensitivities of these compounds were enhanced 1003-, 1330- and 1380-fold, respectively. The results of reproducibility, linearity and real sample analysis show that the proposed procedure is promising to be applied for the simultaneous quantification detection of trace cationic and anionic analytes.     

Development and validation of a capillary electrophoresis method for the determination of phenothiazines in human urine in the low nanogram per milliliter concentration range using field-amplified sample injection

A capillary zone electrophoresis (CZE) method with ultraviolet-visible detection has been established and validated for the determination of five phenothiazines: thiazinamium methylsulfate, promazine hydrochloride, chlorpromazine hydrochloride, thioridazine hydrochloride, and promethazine hydrochloride in human urine. Optimum separation was obtained on a 64.5 cm x 75 microm bubble cell capillary using a buffer containing 150 mM tris(hydroxymethyl)aminomethane and 25% acetonitrile at pH 8.2, with temperature and voltage of 25 degrees C and 20 kV, respectively. Naphazoline hydrochloride was used as an internal standard. Field-amplified sample injection (FASI) has been applied to improve the sensitivity of the detection. Considering the influence of parameters affecting the on-line preconcentration (nature of preinjection plug, sample solvent composition, injection times, and injection voltage) and due to the significant interactions among them, in this paper we propose for the first time the application of a multivariate approach to carry out the study. The optimized conditions were as follows: preinjection plug of water for 7 s at 50 mbar, electrokinetic injection for 40 s at 6.2 kV, and 32 microm of H3PO4 in the sample solvent. Also, a solid-phase extraction (SPE) procedure is developed to obtain low detection limits and an adequate selectivity for urine samples. The combination of SPE and FASI-CZE-UV allows adequate linearities and recoveries, low detection limits (from 2 to 5 ng/mL), and satisfactory precisions (3.0-7.2% for an intermediate RSD %).     

Analysis of triazolopyrimidine herbicides in soils using field-enhanced sample injection-coelectroosmotic capillary electrophoresis combined with solid-phase extraction

In this work, a combined methodology using off-line solid-phase extraction (SPE), on-line field-enhanced sample injection (FESI) and coelectroosmotic capillary electrophoresis with UV detection (CE-UV) is developed for the trace analysis of five triazolopyrimidine sulfonanilide pesticides (i.e., flumetsulam, florasulam, cloransulam-methyl, diclosulam and metosulam). An adequate background electrolyte (BGE) was obtained for the separation of these pesticides using hexadimethrine bromide (HDB) as electroosmotic flow (EOF) modifier. This BGE consisted of 0.00042% HDB, 11 mM formic acid, 16 mM ammonium carbonate and 2.5 mM alpha-CD solution at pH 7.6. The use of this running buffer together with the FESI preconcentration method provided limits of detection (LODs) in the low microg/L range (i.e., between 13.0 and 31.5 microg/L). The optimized FESI-CE-UV method was combined with off-line SPE using C(18) cartridges and applied to the determination of the selected group of pesticides in soil samples. Recovery percentages ranged between 50 and 84% in these samples with LODs between 18 and 34 microg/kg. This work shows the great possibilities of the combined use of SPE-FESI-CE-UV to improve CE sensitivity allowing the achievement of LODs similar to other analytical techniques as GC or HPLC.     

Anionic microemulsion to solvent stacking for on‐line sample concentration of cationic analytes in capillary electrophoresis

The common SDS microemulsion (i.e. 3.3% SDS, 0.8% octane, and 6.6% butanol) and organic solvents were investigated for the stacking of cationic drugs in capillary zone electrophoresis using a low pH separation electrolyte. The sample was prepared in the acidic microemulsion and a high percentage of organic solvent was included in the electrolyte at anodic end of capillary. The stacking mechanism was similar to micelle to solvent stacking where the micelles were replaced by the microemulsion for the transport of analytes to the organic solvent rich boundary. This boundary is found between the microemulsion and anodic electrolyte. The effective electrophoretic mobility of the cations reversed from the direction of the anode in the microemulsion to the cathode in the boundary. Microemulsion to solvent stacking was successfully achieved with 40% ACN in the anodic electrolyte and hydrodynamic sample injection of 21 s at 1000 mbar (equivalent to 30% of the effective length). The sensitivity enhancement factors in terms of peak height and corrected peak area were 15 to 35 and 21 to 47, respectively. The linearity R² in terms of corrected peak area were >0.999. Interday precisions (%RSD, n = 6) were 3.3–4.0% for corrected peak area and 2.0–3.0% for migration time. Application to spiked real sample is also presented.     

Surfactant‐assisted dispersive liquid–liquid microextraction combined with field‐amplified sample stacking in capillary electrophoresis for the determination of mexiletine and lidocaine

A sensitive method for the determination of mexiletine and lidocaine using surfactant‐assisted dispersive liquid–liquid microextraction coupled with capillary electrophoresis was developed. Triton X‐100 and dichloromethane were used as the dispersive agent and extraction solvent, respectively. After the extraction, mexiletine and lidocaine were analyzed using capillary electrophoresis with ultraviolet detection. The detection sensitivity was further enhanced through the use of field‐amplified sample stacking. Under optimal extraction and stacking conditions, the calibration curves were linear over a concentration range of 0.05–1.00 μM for mexiletine and 0.03–1.00 μM for lidocaine. The limits of detection (signal‐to‐noise ratio of 3) were 0.01 and 0.01 μM for mexiletine and lidocaine, respectively. An approximately 1141‐ to 1250‐fold improvement in sensitivity was observed for the two analytes compared with the injection of a standard solution without the surfactant‐assisted dispersive liquid–liquid microextraction and field‐amplified sample stacking procedures. This developed method was successfully applied to the determination of mexiletine and lidocaine in human urine and serum samples. Both precision and accuracy for urine and serum samples were less than 8.7 and 6.7%, respectively. The recoveries of the two analytes from urine and serum samples were 54.7–64.9% and 16.1–56.5%, respectively.     

Qualitative and quantitative analysis of active flavonoids in Flos Lonicerae by capillary zone electrophoresis coupled with solid-phase extraction

Flavonoids are an important bioactive group in the commonly used herbal medicine Flos Lonicerae. A new method of capillary zone electrophoresis (CZE) coupled with solid-phase extraction (SPE) was developed for simultaneous assay of flavonoid aglycones and glycosides in Flos Lonicerae. Optimum CZE separation was achieved with a background electrolyte (BGE) solution consisting of 80 mM boric acid and 20 mM phosphate acid, adjusted to pH 8.1, with 15% acetonitrile (v/v) added, and applying a separation voltage of 28 kV. The SPE method was used for pretreating the complex matrix of botanical materials and good reproducibility was obtained when avicularin was used as internal standard. Linearity of the method was excellent with correlation coefficients (r2) in the range of 0.9995-0.9999 and detection limits were lower than 0.6 microg/mL for the four flavonoids. The obtained recoveries varied between 93 to 104% while the relative standard deviations (RSDs) were below 4.4% (n=3). The developed CZE method was successfully used for the separation of eight flavonoids and the quantification of the four flavonoids in five species of Flos Lonicerae.     

Poly(methacrylic acid-ethylene glycol dimethacrylate) monolith in-tube solid-phase microextraction applied to simultaneous analysis of some amphetamine derivatives in urine by capillary zone electrophoresis

A method based on in-tube solid-phase microextraction and capillary zone electrophoresis (CZE) was proposed for simultaneously determining four amphetamines (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, and 3,4-methylenedioxymethamphetamine) in urine. A poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary column, which can provide sufficient extraction efficiency, was introduced for the extraction of amphetamines from urine samples. The hydrophobic main chains and acidic pendant groups of the monolithic column make it a superior material for extraction of basic analytes from aqueous matrix. After extraction, the samples were analyzed by CZE. The best separation was achieved using a buffer composed of 0.1 M disodium hydrogen phosphate (adjusted to pH 4.5 with 1 M hydrochloric acid) and 20% methanol v/v, with a temperature and voltage of 25 degrees C and 20 kV, respectively. By applying electrokinetic injection with field-amplified sample stacking, detection limits of 25-34 microg/L were achieved. Excellent method of reproducibility was found over a linear range of 0.1-5 mg/L. Determination of these analytes from abusers' urine sample was also demonstrated.     

Determination of tobramycin in human serum by capillary electrophoresis with contactless conductivity detection

A study on the determination of the antibiotic tobramycin by CE with capacitively coupled contactless conductivity detection is presented. This method enabled the direct quantification of the non-UV-absorbing species without incurring the disadvantages of the indirect approaches which would be needed for optical detection. The separation of tobramycin from inorganic cations present in serum samples was achieved by optimizing the composition of the acetic acid buffer. Field-amplified sample stacking was employed to enhance the sensitivity of the method and a detection limit of 50 microg/L (S/N = 3) was reached. The RSDs obtained for migration time and peak area using kanamycin B as internal standard were typically 0.12 and 4%, respectively. The newly developed method was validated by measuring the concentration of tobramycin in serum standards containing typical therapeutic concentrations of 2 and 10 mg/L. The recoveries were 96 and 97% for the two concentrations, respectively.     

Determination of chloroacetic acids in water by capillary zone electrophoresis with field-amplified sample injection

A simple and sensitive method has been developed for the determination of chloroacetic acids and acetic acid in water using capillary zone electrophoresis under modified electroosmotic flow with indirect UV detection. Potassium hydrogen phthalate at pH 5.40 was used as background electrolyte (BGE), and hexadecyltrimethylammonium bromide was used as electroosmotic flow modifier. Field-amplified sample injection (FASI) method was used to enhance the sensitivity. Results showed that the limit of detection for these analytes was enhanced more than 15-fold and the repeatabilities were good with relative standard deviations (RSDs %) of migration time and corrected peak areas being below 0.33%, 4.45% (intra-day) and 0.87%, 9.67% (inter-day), respectively. An off-line liquid–liquid extraction (LLE) process with methyl tert-butyl ether was carried out to detect these compounds in water samples. The dissociation constants of acetic acid and monochloroacetic acid (MCA) were determined with two methods and the results obtained were consistent with the reference values.