Stacking Capillary Electrophoresis Research Articles

Development, synthesis, and application of magnetic layered double hydroxides (Fe3O4@SiO-LDH/DS−) as an efficient adsorbent for the removal of tetracyclines from milk samples

This work presents the development, synthesis, and application of a layered double hydroxide (LDH) coupled to magnetic particles for the removal of antibiotics as tetracyclines (TC´s): tetracycline (TC), chlortetracycline (CT), oxytetracycline (OT), and doxycycline (DT) from milk samples. The LDH synthesis conditions, reaction time (30–90 min), molar ratios Mg2+/Al3+ (7:1–1:7), interlayer anion (NO3−, Cl−, CO32−, and dodecyl sulphate (DS−)) were evaluated. Under synthesis conditions (reaction time of 30 min, Mg2+/Al3+ molar ratio of 7:1, and DS− as interlayer anion), the LDH was coupled in a magnetic solid phase microextraction (MSPμE) methodology. At the optimal extraction conditions (pH 6, 5 min of contact time, 10 mg of adsorbent), a removal percentage of 99.0 % was obtained for each tetracycline. FTIR, TGA, SEM, and adsorption isotherms were employed to characterize the optimal adsorbent. Each experiment was corroborated by large-volume sample stacking capillary electrophoresis (LVSS-CE). The adsorbent was applied directly to positive milk samples (previously tested) for TC´s removal.

Innovative analysis of diazepam, zolpidem and their main metabolites in human urine by micelle-to-solvent stacking in capillary electrophoresis

Diazepam and zolpidem are the most widely used medications for managing insomnia. However, significant concerns regarding the potential risks of misuse and abuse problems arose in many literatures. While urine analysis is a valuable diagnostic tool, a challenge arises from the fact that some parent drugs may remain undetectable in urine. This necessitates concurrent monitoring of their metabolites. Here, we described an innovative on-line sample preconcentration technique known as micelle to solvent stacking (MSS) for the analysis of diazepam, zolpidem, and their main metabolites in urine. Several key parameters warrant further discussion to optimize the MSS model, enhancing its performance in terms of sensitivity and resolution. After optimizing the conditions, we conducted a validation test, achieving high correlation coefficients (greater than 0.9977) for intra-day and inter-day regression lines. Additionally, both the relative standard deviation (RSD) and relative error (RE) remained below 6.10% and 12.55%, respectively. The limits of detection (LODs, S/N = 3) for all five analytes ranged from 2.0 to 56 ng/mL. Compared to the conventional capillary zone electrophoresis method, this new approach exhibited remarkable sensitivity enhancements, ranging from 123 to 235-fold. Upon applying this method to actual urine samples from patients, we successfully detected nordiazepam, zolpidem, and its metabolites. This simple and sensitive approach has promising applications in supporting patient medication safety and bolstering forensic investigations.

Simple and efficient removal of azo dyes from water samples by dispersive solid-phase micro-extraction employing graphene oxide with high oxygen content

ABSTRACT A technique based on dispersive solid-phase micro-extraction (DSPµE), employing graphene oxide (GO) particle with a higher oxygen content synthesised by through a modification of the Hummers method was investigated. GO particle was characterised using Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), Dubinin-Radushkevich, and Freundlich models. DSPµE parameters such pH effect, adsorbent amount, and contact time were optimised in the dye removal process. The proposed method was applied for the removal of three azo dyes in tap water samples: sunset yellow (SY), allure red (AR), and tartrazine (TAR). The water samples were previously analysed using large volume sample stacking capillary electrophoresis (LVSS-CE). Maximum removal capacity was achieved under the optimal conditions of pH solution (pH = 6.0), GO amount (10.0 mg), and contact time (10 minutes) with percentages at 99.0%. The precision was measured in terms of recovery (% desorption) and relative standard deviation from 95.5 to 98.0%, RSD (<10.0%). Two positive water samples for SY and TAR were completely free of azo dyes after application of the proposed method, whereas AR was not detected in any of the ten samples.

Ultrasound and surfactant-assisted dispersive liquid-liquid microextraction prior to poly(ethylene oxide)-mediated stacking in CE for highly sensitive determination of barbiturates in human fluids.

This study developed a facile, highly sensitive technique for extracting and quantifying barbiturates in serum samples. This method combined ultrasound and surfactant-assisted dispersive liquid-liquid microextraction with poly(ethylene oxide)-mediated stacking in capillary electrophoresis. Factors influencing the extraction and stacking performance, such as the type and volume of extraction solvents, the type and concentration of surfactant, extraction time, salt additives, sample matrix, solution pH, and composition of the background electrolyte, were carefully studied and optimized to achieve the optimal detection sensitivity. Under the optimized extraction (injecting 140μL C2 H4 Cl2 into 1mL of sample with pH 4 (5mM sodium phosphate containing 0.05mM Tween 20 and sonication for 1min) and separation conditions (150mM tris(hydroxymethyl)aminomethane-borate with pH 8.5 containing 0.5% (m/v) poly(ethylene oxide)), the limits of detection (signal-to-noise ratio=3) of five barbiturates ranged from 0.20 to 0.33ng/mL, and the calculated sensitivity improvement ranged from 868- to 1700-fold. The experimental results revealed excellent linearity (R2 >0.99), with relative standard deviations of 2.1%-3.4% for the migration time and 4.3%-5.7% for the peak area. The recoveries of the spiked serum samples were 97.1% -110.3%. Our proposed approach offers a rapid and practical method for quantifying barbiturates in biological fluids.

Enhancing Extracellular Vesicle Analysis by Integration of Large-Volume Sample Stacking in Capillary Electrophoresis with Asymmetrical Flow Field-Flow Fractionation.

Extracellular vesicles (EVs) play important roles in cell-cell communication and pathological development. Cargo profiling for the EVs present in clinical specimens can provide valuable insights into their functions and help discover effective EV-based markers for diagnostic and therapeutic purposes. However, the highly abundant and complex matrix components pose significant challenges for specific identification of low-abundance EV cargos. Herein, we combine asymmetrical flow field-flow fractionation (AF4) with large-volume sample stacking and capillary electrophoresis (LVSS/CE), to attain EVs with high purity for downstream protein profiling. This hyphenated system first separates the EVs from the contamination of smaller serum proteins by AF4, and second resolves the EVs from the coeluted, nonvesicular matrix components by CE following LVSS. The optimal LVSS condition permits the injection of 10-fold more EVs into CE compared to the nonstacking condition without compromising separation resolution. Collection and downstream analysis of the highly pure EVs after CE separation were demonstrated in the present work. The high EV purity yields a much-improved labeling efficiency when detected by fluorescent antibodies compared to those collected from the one-dimension separation of AF4, and permits the identification of more EV-specific cargos by LC-MS/MS compared to those isolated by ultracentrifugation (UC), the exoEasy Maxi Kit, and AF4. Our results strongly support that AF4-LVSS/CE can improve EV isolation and cargo analysis, opening up new opportunities for understanding EV functions and their applications in the biomedical fields.

Online preconcentration and determination of anthraquinones in Cassiae Semen tea by salting-out assisted liquid‒liquid extraction coupled with dynamic pH junction-sweeping-large volume sample stacking in capillary electrophoresis

A new strategy by coupling salting-out assisted liquid‒liquid extraction with dynamic pH junction-sweeping-large volume sample stacking in capillary electrophoresis was proposed for extraction, online preconcentration, separation and determination of trace anthraquinones in food samples. Based on the complementary advantages of salting-out assisted liquid‒liquid extraction and online capillary electrophoresis stacking, the strategy allowed for rapid extraction and sensitive analysis of anthraquinones in aqueous matrix samples. Under the optimized conditions, the proposed strategy showed good linearity (100–3000 ng/mL, r2 ≥ 0.993) with a low detection limit of 30–40 ng/mL. Sensitivity enhancement factors of up to 468-fold were reached with the optimized dynamic pH junction-sweeping-large volume sample stacking using capillary electrophoresis with a UV detector. As a proof-of-concept study, this strategy was successfully applied for analysing anthraquinones in Cassiae Semen tea, which demonstrated good potential for the efficient and sensitive determination of weakly acidic analytes in complicated sample matrices.

Ultrasensitive analysis of mirtazapine and its metabolites enantiomers in body fluids using ultrasound-enhanced and surfactant-assisted dispersive liquid–liquid microextraction followed by polymer-mediated stacking in capillary electrophoresis

A simple, rapid, and sensitive technique for measuring mirtazapine and its metabolites enantiomers in human fluids, such as urine and serum, was developed by applying ultrasound-enhanced and surfactant-assisted dispersive liquid–liquid microextraction (USA-DLLME) integrated with poly(diallyldimethylammonium chloride) (PDDAC)-mediated stacking in capillary electrophoresis (CE). The parameters that affect extraction and stacking performance, such as the extraction volume, surfactant types, surfactant concentrations, salt additives, extraction time, solution pH, and background electrolytes, were comprehensively studied and optimized to achieve optimal detection performance. Under optimal extraction conditions (injection of 120 µL of C2H2Cl4 into 1 mL of a sample solution containing 0.05 mM Brij-35 at pH 10.0) and separation conditions (0.9% PDDAC, 10 mM phosphate, pH 3.0, and 20 mM dimethyl-β-cyclodextrin), on-line CE stacking of mirtazapine-related chiral drugs was achieved by the two strategies: (i) neutral DM-β-CD sweep low concentrations of DL-NaSSA and (ii) DL-NASSA is stacked by the difference in the viscosity between the PDDAC and sample zone. An approximately 2,800–4000-fold improvement in detection sensitivity was revealed for mirtazapine, N-demethylmirtazapine, and 8-hydroxymirtazapine enantiomers. The linear ranges for the quantification of all analyte enantiomers were 1.2–150 nM, with a coefficient of determination higher than 0.99; the relative standard deviations in the migration time and peak areas for six analytes were less than 1.8% and 5.8%, respectively. The proposed system provided the limits of detection (signal-to-noise ratio of 3) of the six analytes as 0.3–0.5 nM. The recovery of the six separated analytes spiked in urine and serum samples was revealed to be 82.7%–109.5% and 91%–112.8%, respectively. This advanced technique with high sensitivity enhancement factors was successfully employed to analyze mirtazapine and its metabolites enantiomers in urine and serum samples with reliability.

A chemometric experimental design with three-step stacking capillary electrophoresis for analysis of five tobacco-specific nitrosamines in cigarette products

Tobacco-specific nitrosamines (TSNAs) as carcinogens endanger our health and life from cigarette products. However, the safe range of TSNAs levels in commercial cigarette products has not yet been established. For the purpose of safety and supervision, a three-step stacking approach including field amplified sample injection (FASI), sweeping, and analyte focusing by micelle collapse (AFMC), was developed for the simultaneous determination of five TSNAs levels in cigarette products. This approach also involved aspects of chemometric experimental design, including fractional factorial design and central composite design. After the multilevel optimization of the experimental design, the five TSNAs were well separated. The LOD (S/N = 3) values of the N´-nitrosonornicotine (NNN), N´-nitrosoanatabine (NAT), N´-nitrosoanabasine (NAB), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the FASI-sweeping-AFMC CE approach were 1.000 ng/mL, 0.500 ng/mL, 0.125 ng/mL, 1.000 ng/mL, and 0.500 ng/mL respectively. The results of relative standard deviation (RSD) and relative error (RE) were all less than 3.35%, demonstrating good precision and accuracy. Finally, this novel approach was further applied to monitor three commercial cigarette products, and a range of 250.1–336.6 ng/g for NNN, 481.6–526.7 ng/g for NAT, 82.2–247.6 ng/g for NAB, 167.7–473.7 ng/g for NNAL, and 39.4–246.7 ng/g for NNK could be observed among these. Based on these results, the novel CE stacking strategy was successfully applied for the analysis of five TSNAs levels in cigarette products and could serve as a tool for assays of quality control of nitrosamines.

A three-step stacking capillary electrophoresis of field-amplified sample injection, sweeping, and micellar collapse for determination of dabigatran and its active metabolite in human plasma.

A three-step stacking capillary electrophoresis (CE) composed of field-amplified sample injection, sweeping, and analyte focusing by micellar collapse (FASI-sweeping-AFMC) was developed to determine dabigatran (D) and its major active metabolite, dabigatran acyl-beta-d-glucuronide (DAG), in human plasma. After optimization and validation, this novel approach was further applied to monitor 5 real samples, and the 25.2-186.8 ng mL-1 D could be observed among those. Based on these results, the novel CE stacking strategy was successfully applied for the analysis of D and DAG in human plasma and could be served as a tool for clinical assays.

Ultrasensitive Analysis of Mirtazapine and its Metabolites Enantiomers in Body Fluids Using Ultrasound-Enhanced and Surfactant-Assisted Dispersive Liquid–Liquid Microextraction Followed by Polymer-Mediated Stacking in Capillary Electrophoresis

Ultrasensitive Analysis of Mirtazapine and its Metabolites Enantiomers in Body Fluids Using Ultrasound-Enhanced and Surfactant-Assisted Dispersive Liquid–Liquid Microextraction Followed by Polymer-Mediated Stacking in Capillary Electrophoresis

A Chemometric Experimental Design with Three-Step Stacking Capillary Electrophoresis for Analysis of Five Tobacco-Specific Nitrosamines in Cigarette Products

A Chemometric Experimental Design with Three-Step Stacking Capillary Electrophoresis for Analysis of Five Tobacco-Specific Nitrosamines in Cigarette Products

Electrokinetic instability due to streamwise conductivity gradients in microchip electrophoresis

We present an experimental and numerical investigation of electrokinetic instability (EKI) in microchannel flow with streamwise conductivity gradients, such as those observed during sample stacking in capillary electrophoresis. A plug of a low-conductivity electrolyte solution is initially sandwiched between two high-conductivity zones in a microchannel. This spatial conductivity gradient is subjected to an external electric field applied along the microchannel axis, and for sufficiently strong electric fields an instability sets in. We have explored the physics of this EKI through experiments and numerical simulations, and supplemented the results using scaling analysis. We performed EKI experiments at different electric field values and visualised the flow using a passive fluorescent tracer. The experimental data were analysed using the proper orthogonal decomposition technique to obtain a quantitative measure of the threshold electric field for the onset of instability, along with the corresponding coherent structures. To elucidate the physical mechanism underlying the instability, we performed high-resolution numerical simulations of ion transport coupled with fluid flow driven by the electric body force. Simulations reveal that the non-uniform electroosmotic flow due to axially varying conductivity field causes a recirculating flow within the low-conductivity region, and creates a new configuration wherein the local conductivity gradients are orthogonal to the applied electric field. This configuration leads to EKI above a threshold electric field. The spatial features of the instability predicted by the simulations and the threshold electric field are in good agreement with the experimental observations and provide useful insight into the underlying mechanism of instability.

Simultaneous determination of five metal ions by on-line complexion combined with micelle to solvent stacking in capillary electrophoresis

A direct on-line complexion combined with micelle to solvent stacking method was proposed for simultaneous determination of metal ions by capillary electrophoresis coupled diode array detector. During the experiment, a plug of complexing agent was first injected to the inlet of capillary, followed by introducing the micelle-bound metal ions. Then the metal ions produced a micelle-to-solvent stacking effect and interacted with the complexing agent under a positive voltage. Continued application of voltage, the analytes were effectively focused and separated in the capillary zone electrophoresis. Repeatability was ranged from 1.89% to 1.94% for the migration time. The detection limits were 2.66–27.9 ng mL-1 for Ni2+, Co2+, Cu2+, Hg2+ and Cd2+. Furthermore, the developed method showed a great potential for the determination of metal ions in the crayfish, beebread and Dendrobium officinale samples.

Sensitive determination of warfarin and its metabolic enantiomers in body fluids via capillary electrophoresis combined with ultrasound-assisted dispersive liquid-liquid microextraction and online sample stacking

A sensitive technique for the measurement of warfarin and its metabolic enantiomers in human fluids such as urine and serum technique was developed by applying ultrasound-assisted dispersive liquid-liquid microextraction linked with poly(ethylene oxide)-mediated stacking in capillary electrophoresis. The parameters that influence extraction and stacking performance-the type of the extraction and dispersive solvents and their volume, extraction time, salt additives, sample matrix, solution pH, and the composition of background electrolyte stacking-were carefully studied and optimized based on the considering of obtaining the best detection sensitivity. Under the optimal extraction (30 μL C2H2Cl4 and 200 μL tetrahydrofuran in 1 mL of sample solution) and separation (0.5% PEO, 200 mM Tris-borate, pH 8.5, and 5 mM dimethyl-β-cyclodextrin) conditions, the enrichment factors of D-/L-warfarins and D-/L-7-OH warfarins covered from 1758 to 1859 and their limits of detection ranged from 0.34 to 0.38 nM. Calibration-related results exhibited acceptable linearity with the coefficient of determination higher than 0.99; the relative standard deviations of the peak area were determined to 4.1%–6.3% (n = 3). The recovery of four separated analytes spiked in samples of urine and serum was found to be 93%–110% and 95%–109%, respectively. We revealed that ultrasound-assisted dispersive liquid-liquid microextraction with poly(ethylene oxide)-mediated stacking in capillary electrophoresis could be a prompt and practical method for quantifying the levels of warfarin and its metabolic enantiomers in real-world samples, especially in biological fluids.

Recent progress of sample stacking in capillary electrophoresis (2016-2018).

Electrophoretic sample stacking comprises a group of capillary electrophoretic techniques where trace analytes from the sample are concentrated into a short zone (stack). This paper is a continuation of our previous reviews on the topic and brings a survey of more than 120 papers published approximately since the second quarter of 2016 till the first quarter of 2018. It is organized according to the particular stacking principles and includes chapters on concentration adjustment (Kohlrausch) stacking, on stacking techniques based on pH changes, on stacking in electrokinetic chromatography and on other stacking techniques. Where available, explicit information is given about the procedure, electrolyte(s) used, detector employed and sensitivity reached. Not reviewed are papers on transient isotachophoresis which are covered by another review in this issue.

Molecular-sieve-based matrix solid-phase extraction combined with field-amplified sample stacking in capillary electrophoresis for the determination of three organic acids in a complex solid matrix.

A simple, convenient, and sensitive method that involves combining matrix solid-phase dispersion extraction with field-amplified sample stacking in capillary electrophoresis has been developed for the determination of organic acids in a complex solid matrix. Mesoporous molecular sieve, MCM-48, was synthesized by a hydrothermal method and selected as the adsorbent in matrix solid-phase dispersion. After fast extraction, the enriched analytes were back-extracted into a basic aqueous solution for field-amplified sample stacking in capillary electrophoresis. Parameters that affect extraction efficiency and sample stacking were optimized. Under the optimal conditions, approximately 42-, 49-, and 56-fold sensitivity enhancements were achieved for danshensu, protocatechuic acid, and cinnamic acid, respectively, when compared to normal injection. A satisfactory correlation coefficient (r>0.99) was obtained. Both intra- and interday precision were lower than 2.53%. And the limits of detection of the three organic acids ranged between 0.01 and 0.029μg/mL. Finally, the newly proposed method was successfully applied to the determination of organic acids in Fufang Danshen tablets, which indicates its great potential in analyzing organic acids in a complex matrix.

Separation and determination of corynoxine and corynoxine B using chiral ionic liquid and hydroxypropyl-β-cyclodextrin as additives by field-amplified sample stacking in capillary electrophoresis.

A sensitive, fast, and effective method, field-amplified sample stacking (FASS) in capillary electrophoresis, has been established for the separation and determination of corynoxine and corynoxine B. Hydroxypropyl-β-CD (HP-β-CD) and tetrabutylammonium-L-glutamic acid (TBA-L-Glu) were used as additives in the separation system. Electrokinetic injection was chosen to introduce sample from inlet at 10kV for 50s after a water plug (0.5psi, 4s) was injected to permit FASS. The running buffer (pH 6.1) was composed of 40mM sodium dihydrogen phosphate solution, 130mM HP-β-CD, and 10mM TBA-L-Glu and the separation voltage was 20kV. Under the optimum conditions, corynoxine and corynoxine B were successfully enriched and separated within 12min and the sensitivity was improved approximately by 700-900 folds. Calibration curves were in a good linear relationship within the range of 62.5-5.00×103 ng/mL for both corynoxine and corynoxine B. The limits of detection (S/N=3) and quantitation (S/N=10) were 14.9, 45.2ng/mL for corynoxine and 11.2, 34.5ng/mL for corynoxine B, respectively. Finally, this method was successfully applied for the determination of corynoxine and corynoxine B in the stems with hooks of Uncaria rhynchophylla and its formulations.

Simultaneous separation and analysis of camptothecin alkaloids in real samples by large-volume sample stacking in capillary electrophoresis.

Large-volume sample stacking (LVSS) is commonly used as an effective online preconcentration method in capillary zone electrophoresis (CZE). In this paper, the method LVSS combined with CZE has been proposed to analyze camptothecin alkaloids. Optimum separation can be achieved in the following conditions: pH 9.0; 25mm borate buffer containing 20 mm sulfobutylether-β-cyclodextrin and 20 mm ionic liquid 1-ethyl-3-methyllimidazole l-lactate; applied voltage 20 kV; and capillary temperature 25 °C. The LVSS was optimized as hydrodynamic injection 4 s at 5.0 psi and the polarity switching time was 0.17 min. Under the above conditions, the analytes could be separated completely in <20 min and the detector response was increased compared with conventional hydrodynamic injection. The limits of detection were between 0.20 and 0.78 μg/L. A good linearity was obtained with correlation coefficients from 0.9991 to 0.9997. The recoveries ranged from 97.72 to 103.2% and the results demonstrated excellent accuracy. In terms of the migration time and peak area, the experiment was reproducible. The experimental results indicated that baseline separation can be obtained and this method is suitable for the quantitative determination of camptothecin alkaloids in real samples.

Chiral Measurement of Aspartate and Glutamate in Single Neurons by Large-Volume Sample Stacking Capillary Electrophoresis.

d-Amino acids (d-AAs) are endogenous molecules found throughout the metazoan, the functions of which remain poorly understood. Measurements of low abundance and heterogeneously distributed d-AAs in complex biological samples, such as cells and multicellular structures of the central nervous system (CNS), require the implementation of sensitive and selective analytical approaches. In order to measure the d- and l-forms of aspartate and glutamate, we developed and applied a stacking chiral capillary electrophoresis (CE) with laser-induced fluorescence detection method. The achieved online analyte preconcentration led to a 480-fold enhancement of detection sensitivity relative to capillary zone electrophoresis, without impacting separation resolution or analysis time. Additionally, the effects of inorganic ions on sample preconcentration and CE separation were evaluated. The approach enabled the relative quantification of d-aspartate and d-glutamate in individual neurons mechanically isolated from the CNS of the sea slug Aplysia californica, a well characterized neurobiological model. Levels of these structurally similar d-AAs were significantly different in subpopulations of cells collected from the investigated neuronal clusters.

Development of a New Microextraction Fiber Combined to On-Line Sample Stacking Capillary Electrophoresis UV Detection for Acidic Drugs Determination in Real Water Samples.

A new analytical method coupling a (off-line) solid-phase microextraction with an on-line capillary electrophoresis (CE) sample enrichment technique was developed for the analysis of ketoprofen, naproxen and clofibric acid from water samples, which are known as contaminants of emerging concern in aquatic environments. New solid-phase microextraction fibers based on physical coupling of chromatographic supports onto epoxy glue coated needle were studied for the off-line preconcentration of these micropollutants. Identification and quantification of such acidic drugs were done by capillary zone electrophoresis (CZE) using ultraviolet diode array detection (DAD). Further enhancement of concentration sensitivity detection was achieved by on-line CE “acetonitrile stacking” preconcentration technique. Among the eight chromatographic supports investigated, Porapak Q sorbent showed higher extraction and preconcentration capacities. The screening of parameters that influence the microextraction process was carried out using a two-level fractional factorial. Optimization of the most relevant parameters was then done through a surface response three-factor Box-Behnken design. The limits of detection and limits of quantification for the three drugs ranged between 0.96 and 1.27 µg∙L−1 and 2.91 and 3.86 µg∙L−1, respectively. Recovery yields of approximately 95 to 104% were measured. The developed method is simple, precise, accurate, and allows quantification of residues of these micropollutants in Genil River water samples using inexpensive fibers.