Sample Stacking Research Articles

Advances and Applications of Capillary Electrophoresis Mass Spectrometry in Food Analysis: Strategies for Online and Offline Preconcentration.

Advancements in food technology have increased the need for thorough analysis to ensure food safety, quality, and compliance with regulatory requirements. Capillary electrophoresis-mass spectrometry (CE-MS) has emerged as a powerful tool in food analysis due to its high separation efficiency, low sample consumption, and ability to handle complex matrices. However, challenges such as the use of volatile running buffers and maintaining the stability of the electrical circuit connecting the CE and MS systems have been addressed through advancements in interface designs, such as sheathless systems and optimized sheath-liquid compositions. Online and offline preconcentration techniques have significantly enhanced CE-MS sensitivity (up to 1000-fold) through stacking methods such as large volume sample stacking (LVSS) and dynamic pH junction stacking. Meanwhile, offline sample preparation techniques, such as solid-phase extraction (SPE) and liquid-based methods, are essential for removing matrix interferences and preconcentrating targeted analytes. This review explores both online and offline preconcentration methods and emphasizes the importance of CE-MS in helping researchers develop effective strategies for selecting the best preconcentration methods for food analysis.

In-Situ Charging-Discharging SEM Observation and Analysis for Si Anode in a Solid-State Battery~ Cross-Section Preparation and in-Situ SEM Observation By Stack Pressure Holder ~

Next-generation solid-state batteries are expected to offer improved capacity and safety. The increase in capacity depends on the negative electrode material, and high-capacity materials such as Si, which can absorb large amounts of Li, are the subject of research. The amount of Li transferred can be measured electrically. However, it has not been possible to visualize where Li is distributed within the anode particles or to analyze the chemical state of the anode particles in real-time.In-situ observation and analysis of sample cross sections using a scanning electron microscope (SEM) are effective in analyzing the behavior and chemical state of Li introduced into the Si anode and the interface state between the solid electrolyte and the anode during charge-discharge. During charging and discharging, the Si anode undergoes a threefold or greater volume expansion and contraction due to Li absorption and desorption, cutting off the ionic conduction path, so it is necessary to apply a sample stacking pressure to suppress the volume expansion. A stack pressure holder is designed for operando experiments of solid-state batteries in an SEM. This holder enables smooth cross-sectioning using a broad Ar ion beam and in-situ SEM observation while applying uniform pressure which minimizes the resistance across interfacial boundaries. It also reduces the risk of specimen breakage because it fixes the sample throughout the entire air-isolated workflow, from cross-section preparation to real-time observation in the SEM.Si anode composites were made by mixing Si particles, argyrodite sulfide solid electrolyte (SSE), and acetylene black (AB). The cathode composite was made by mortar mixing the ternary oxide cathode material LiNi1/3Mn1/3Co1/3O2 (NMC), SSE, and AB. Each component was put into a pelletizer, stacked, and pressurized at about 500 MPa to produce a full cell pellet. The pellet was cut into 4.8 mm squares using a precision punching tool for highly brittle materials (NOGAMIGIKEN, NC-CE-SS) and placed on the stack pressure holder, where a pressure of 25 MPa was applied. The cross-sections were prepared with an Ar ion beam (acceleration voltage: 5 kV, cooling temperature: -120 °C, processing time: 5 hr) using a cross-section preparation system (JEOL, Cooling Cross Section PolisherTM, IB-19520CCP) while applying stack pressure. In order to minimize sample deterioration due to exposure to the atmosphere, glove boxes and transfer vessels that can be closed to the atmosphere were used for the entire process from pretreatment to processing to observation. Samples transported to the SEM in the stack pressure holder were charged and discharged at 0.2C (SOC vs. NMC reference) by a charge/discharge device (MEIDEN HOKUTO, Hz-Pro).The observation results of the charge-discharge process on a cross-sectional sample under stack pressure using this holder are shown in Fig. 1. (a) shows the backscattered electron (BSE) composition image near the interface between the Si anode and SSE and the interface and Si anode particles are clearly observed. (b) shows the charge-discharge curve in this experiment, and (c) shows the BSE composition image after 30 minutes of charging (SOC 10% vs. NMC reference), in which the composition contrast of the Si negative electrode particles gradually changes from the solid electrolyte side due to Li absorption during charging. In the experiment, the sample was maintained at a fixed holding pressure from the time of CP cross-section processing to the SEM observation without changing the holder, and it can be assumed that the observed behavior is similar to the actual behavior. Energy dispersive X-ray spectroscopy (EDS) and soft X-ray spectroscopy (SXES) can also be performed during in-situ charge-discharge observations to obtain elemental distribution and chemical state analysis of lithium and Si. The changes in microstructure of the battery cross-section can also be captured, and the chemical state analysis and the evaluation of the optimum stack pressure at the pressures up to 50 MPa can be expected.AcknowledgmentsWe would like to thank Professor Nobuya Machida of Konan University for cooperation in some of the battery sample preparation. Figure 1

Catalytic gasification of a single coal char particle: An experimental and simulation study

The catalytic coal gasification technology has been widely researched and developed under the background of “Carbon peaking and carbon neutrality goals”. Currently, most of catalytic gasification experiments on coal char particles are analyzed by thermogravimetric analyzer (TGA). However, the gasification agent will be subject to diffusion resistance during the reaction because of the sample stacking, making the inherent reaction kinetics unclear. In this study, we investigated the catalytic gasification behavior of single-particle coal char using high temperature stage microscope (HTSM). With the diffusion resistance ruled out, the reaction conditions when using a HTSM are more similar to those inside a real industrial gasifier. Numerical models of the gasification reaction of single-particle coal char were further developed using the kinetic parameters obtained under HTSM. Three models were investigated, including regular spherical structured, irregular spherical structured and porous spherical structured models, representing different morphologies of coal char particles in the gasifier. The gasification characteristics of coal char particles under different K2CO3 catalyst loadings and gasification temperatures were also studied. Compared with the activation energies data of coal char particles without catalyst, the activation energies of coal char particles loaded with 2.2 %, 4.4 %, 6.6 %, and 10.0 % catalysts were reduced by 110 kJ/mol, 116 kJ/mol, 121 kJ/mol, and 126 kJ/mol, respectively. The reaction surface area affects the temperature distribution. The temperature near the irregular spherical particle is about 20 K higher than the temperature near the regular spherical particle.

Advancements in Multiple‐Step On‐Line Preconcentration Techniques for Enhanced Sensitivity in Capillary Electrophoresis

ABSTRACTMultiple‐step on‐line preconcentration, a combination of at least two stacking techniques has been developed to increase the sensitivity in capillary electrophoresis (CE) for analytes in various samples. It is usually conducted sequentially, or in some cases, synergistically, where different stacking modes occur simultaneously. Multiple‐step techniques allow simultaneous preconcentration and separation of various kinds of analytes in different complex samples in a single CE run. This review aims to provide recent advances in multiple‐step on‐line preconcentration techniques in CE. We critically review technical papers published for the last 7 years up until July 2024, subsequently organized according to the combination of the main stacking techniques, that is, field amplification, large volume sample stacking, transient isotachophoresis, micelle to solvent or micelle to cyclodextrin stacking, and others. The procedures, fundamental mechanism, analytical figures of merits achieved, and their feasibility for complicated sample matrices are reviewed.

Combination of on-line sample preconcentration by large-volume dual preconcentration by isotachophoresis and stacking (LDIS) with field-amplified sample injection (FASI) on Y-channel microchips.

In our previous study, the combination of two on-line sample preconcentration techniques, large-volume sample stacking with an electroosmotic flow (EOF) pump (LVSEP) and transient isotachophoresis (tITP), in microchip electrophoresis (MCE) was developed, which was named large-volume dual preconcentration by isotachophoresis and stacking (LDIS). LDIS was apparently effective for improving the sensitivity and the peak shape. In LDIS, however, there was a limit to the improvement of the sensitivity enhancement factor (SEF) since the amount of analytes to be concentrated was limited to the channel volume. To overcome this issue, in the present article, LDIS was coupled with field-amplified sample injection (FASI) technique on Y-shaped channel microchips. The use of a Y-channel in LDIS-FASI allowed consecutive LVSEP, FASI and tITP enrichments with a simple voltage control. In conventional LVSEP and LDIS analyses of a standard analyte, the SEFs were evaluated to be 2630 and 13,100, respectively, whereas in LDIS-FASI that was increased to 27,900 even at the FASI injection time of 0s. To achieve higher SEFs, furthermore, the FASI injection time was increased to 150s, resulting in the best SEF of 58,500. It should be emphasized that the peak width in LDIS-FASI was quite narrow, only 0.3-3.1s, while in normal LVSEP that was 13s. Furthermore, the LDIS-FASI technique was applied to the analysis of oligosaccharide mixture. Due to the focusing effect by LDIS-FASI, the resolutions were improved from 0.97-1.57 to 2.08-2.73.

Investigation of on-line electrokinetic enrichment strategies for capillary electrophoresis of extracellular vesicles

This work explores strategies for electrokinetic preconcentration of extracellular vesicles (EVs) that are potential source of biomarkers for different diseases. The first approach that led to successful preconcentration of EVs is based on large volume sample stacking (LVSS), allowing an enrichment factor of 7 for CE of EVs with long-end injection (using a capillary with an effective length of 50 cm). Attempts were also made to perform multiple cycles of LVSS, field amplified sample stacking (FASS) and field amplified sample injection (FASI), to improve EVs preconcentration performance. The focus was then put on development of capillary isotachophoresis under high ionic strengths (IS) for electrokinetic enrichment of slow migrating EVs having heterogeneous mobilities. This approach relies on the use of extremely high concentrations of the terminating electrolyte (TE) to slow down the mobility of TE co-ions, rendering them slower than those of EVs. The limit of detection for intact EVs using the developed ITP-UV method reached 8.3 × 108 EVs/mL, allowing an enrichment of 25 folds and a linear calibration up to 4 × 1010 EVs/mL. The ITP-UV and ITP-LIF approaches were applied to provide the electrokinetic signature of EVs of bovine milk and human plasma as well as to visualize more specifically intravesicular fluorescently labelled EVs. The investigation of these strategies shredded light into the challenges still encountered with electrokinetic preconcentration and separation of heterogeneous EVs sub-populations which are discussed herein based on our results and other attempts reported in the literature.

NerveTracker: a Python-based software toolkit for visualizing and tracking groups of nerve fibers in serial block-face microscopy with ultraviolet surface excitation images.

Information about the spatial organization of fibers within a nerve is crucial to our understanding of nerve anatomy and its response to neuromodulation therapies. A serial block-face microscopy method [three-dimensional microscopy with ultraviolet surface excitation (3D-MUSE)] has been developed to image nerves over extended depths ex vivo. To routinely visualize and track nerve fibers in these datasets, a dedicated and customizable software tool is required. Our objective was to develop custom software that includes image processing and visualization methods to perform microscopic tractography along the length of a peripheral nerve sample. We modified common computer vision algorithms (optic flow and structure tensor) to track groups of peripheral nerve fibers along the length of the nerve. Interactive streamline visualization and manual editing tools are provided. Optionally, deep learning segmentation of fascicles (fiber bundles) can be applied to constrain the tracts from inadvertently crossing into the epineurium. As an example, we performed tractography on vagus and tibial nerve datasets and assessed accuracy by comparing the resulting nerve tracts with segmentations of fascicles as they split and merge with each other in the nerve sample stack. We found that a normalized Dice overlap ( ) metric had a mean value above 0.75 across several millimeters along the nerve. We also found that the tractograms were robust to changes in certain image properties (e.g., downsampling in-plane and out-of-plane), which resulted in only a 2% to 9% change to the mean values. In a vagus nerve sample, tractography allowed us to readily identify that subsets of fibers from four distinct fascicles merge into a single fascicle as we move along the nerve's length. Overall, we demonstrated the feasibility of performing automated microscopic tractography on 3D-MUSE datasets of peripheral nerves. The software should be applicable to other imaging approaches. The code is available at https://github.com/ckolluru/NerveTracker.

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.

A simple and green offline-online capillary electrophoresis stacking strategy for the simultaneous determination of hydrophobic compounds in complicated samples using sodium dodecyl sulfate as the solubilizer and pseudophase

BackgroundCapillary electrophoresis is a powerful analytical method featured with high separation efficiency, minimal sample requirements, and reduced organic solvents consumption. However, its low sensitivity hinders its wide application in determination of trace analytes especially for the weakly ionized hydrophobic compounds. Offline and Online capillary electrophoresis stacking methods are more favored to enhance detection sensitivity of analytes. The determination of two sesquiterpenes and an alkaloid from the dried root of Lindera aggregata merged as an example for developing a simple, sensitive and green method for the simultaneous determination of two hydrophobic compounds in complicated matrix samples. ResultsAn offline-online capillary electrophoresis stacking strategy by integrating micro matrix solid phase dispersion with field-amplified sample stacking and micelle to cyclodextrin stacking has been developed for the simultaneous determination of dehydrocostus lactone, linderane, norisoboldine in complex matrices. The optimized parameters were set at 65 mM sodium dihydrogen phosphate, 35 % methanol, 180 s for sample injection and 210 s for cyclodextrin injection, 20 mM sodium dodecyl sulfate of sample matrix for online stacking; 1:1 sample to MCM-48, 180 s grinding time, and 1000 μL of 20 mM sodium dodecyl sulfate elution for offline procedure. Under the optimum conditions, the method showed good linearity with correlation coefficients (R2 ≥ 0.9927), low limits of detection within the range of 25–50 ng mL−1, satisfactory repeatability and reproducibility below 3.98 %, and acceptable recoveries between 94 % and 97 %. The developed method was successfully applied to two real samples, the root of L. aggregata and rat feces. SignificanceSodium dodecyl sulfate is firstly used as an eluent in micro matrix solid phase dispersion and plays a dual role throughout the analytical procedure, including extraction solvent in sample preparation and micelle pseudophase during online stacking. It brings great procedure convenience to the method. The sensitivity of this method can improve up to 1283-folds compared with the normal mode. Moreover, the overall strategy indicates satisfied green potential evaluated by greenness assessment tools.

Online preconcentration and determination of five alkaloids in Yangxinshi tablet by large-volume sample stacking and micelle to solvent stacking in cyclodextrin-modified electrokinetic chromatography.

The two-step preconcentration technique consisting of large-volume sample stacking (LVSS) and micelle to solvent stacking (MSS) in cyclodextrin-modified electrokinetic chromatography (CDEKC) was developed for the analysis of five cationic alkaloids in complex Chinese herbal prescriptions. Relevant parameters affecting separation and stacking performance were optimized separately. Under the optimal LVSS-MSS-CDEKC conditions, less analysis time and organic solvent were required, and the enhancement factors of analytes ranged from 12 to 15 compared with the normal CDEKC separation mode. Further, all validation results demonstrated good applicability and multiple alkaloids (epiberberine, dehydrocorydaline, jatrorrhizine, coptisine and berberine) in Yangxinshi tablet (YXST) have been simultaneously determined. This approach presents powerful potential for the determination of multiple components in complex preparations of Chinese medicine.

Combination of large-volume sample stacking with polarity switching and micelle electrokinetic chromatography for the analysis of anion compounds in Yangxinshi tablets.

Yangxinshi tablet (YXST) is a traditional Chinese medicine preparation characterized by its high efficacy and safety for the treatment of cardiovascular diseases. Anionic compounds have been revealed as potential active components. However, there is currently limited research regarding its quality control. We aimed to establish a strategy for the simultaneous separation and determination of five key anionic compounds in YXST. A sensitive and efficient analytical method was developed and applied for the simultaneous separation and determination of five key compounds in YXST using large-volume sample stacking with polarity switching and micelle electrokinetic chromatography (LVSS-PS-MEKC) coupled with diode array detection. Crucial parameters, including sample volume, applied voltage, composition and pH of the running buffer, concentration of organic modifier, and switching time of the polarity, were systematically evaluated and optimized using a single variable method to enhance separation performance. Furthermore, the impact of cyclodextrin and sodium dodecyl sulfate as electrolyte modifiers was also investigated. Under the optimal conditions, baseline separation of the five compounds (daidzein, puerarin, glycyrrhiztinic acid, chlorogenic acid, and salvianolic acid B) was achieved within 20 min. In comparison to the conventional MEKC mode, the constructed LVSS-PS-MEKC method exhibited a more than sixfold increase in the enrichment factor. The method was validated in terms of linearity, precision, accuracy, 24 h stability, and recovery and successfully applied to analyze YXST samples. A sensitive strategy was developed for the simultaneous separation and determination of five key anionic components in YXST, offering a robust and efficient strategy for pharmaceutical analysis.

Capillary electrophoresis tandem mass spectrometry to determine multiclass cyanotoxins in reservoir water and spinach samples

Cyanotoxins constitute a group of toxic secondary metabolites, the presence of which in any water body poses a major health risk. Moreover, advanced organisms such as edible plants exposed to these toxins, are a possible pathway for human exposure. Green analytical chemistry is demanding environmentally friendly analytical techniques. In this sense, we propose the use of capillary electrophoresis coupled to tandem mass spectrometry (CE-MS/MS) to determine a mixture of eight cyanotoxins belonging to three different classes: cyclic peptides (microcystin-LR, microcystin-RR and nodularin), alkaloids (cylindrospermopsin and anatoxin-a) and three isomeric non-protein amino acids (β-methylamino-l-alanine, 2,4-diaminobutyric acid and N-(2-aminoethyl)glycine). Separation was achieved by using an acidic background electrolyte consisting of 2 M formic acid and 20% acetonitrile in water. Parameters affecting MS/MS detection and the sheath-liquid interface were also studied. Finally, a combination of pH-junction, field-amplified sample stacking (FASS) and acid barrage as online preconcentration strategies, was employed to improve sensitivity and efficiency. The online preconcentration applied, in combination with a dual cartridge solid-phase extraction (SPE) system, allows to obtain limits of detection in the very low range of µg·L−1 for these multiclass cyanotoxins in reservoir water samples (from 0.005 to 0.10 µg·L−1). Furthermore, for the first time cyanotoxins are analysed in spinach samples through CE-MS/MS using the same SPE procedure, following lyophilisation and solid-liquid extraction with 6 mL 80 % aqueous MeOH.

Implementation of the method of encoding series lengths to provide procedures steganographic image insertion

The purpose of this article is to introduce the principle of encoding series lengths to provide inter-block data multiplexing of a hybrid steganoalgorithm. As part of the modeling, it is made the assumption that the attacker has successfully determined one of the two parameters of content processing: the size of the basic blocks (BB) and the implemented principle of series scanning. The modeling was performed on the example of one test halftone image of the «city» type. Samples of the attacked test image obtained for a short sample stack (length in 4 series) are presented. An analysis of the results of attack content when using different ways of smoothing the source images is carried out. The implementation of different smoothing ways allows to improve in the combinatorics of multiplexing series and the number of formed BB. It is emphasized that with an increased dimensionality of the BB, the combinatorics of the multiplex of series parameters is limited. The dimension of the BB and the way of organizing the series scanning are elements of the composite key of the data extractor. The use of the principle of encoding series lengths significantly reduces the computational complexity of the algorithm and creates conditions for the implementation of inter-block multiplexing procedures. It is concluded that the use of the parameter of series lengths destroys the correlation relations of the source data more than in the case of using only BB. The main elements at the stage of inter-block content processing are BB and their parameters of series lengths. The modeling results confirm the key role of the «series length» parameter in the procedure of legitimizing content extraction. The variability of the sampling order of the used parameters of the BB series significantly enhances the resistance of the content to unauthorized extraction attempts. Attention is drawn to the fact that the principle of encoding series lengths is limited in the background areas of images, which makes it possible to preserve highly informative image areas and determine the structure of visual artifacts. According to the modeling results, it is concluded that the use of the method of encoding series lengths in inter-block multiplexing procedures additionally strengthens the protection and complicates the work of the steganoanalyst and determines the further course of the attack.

Determination of α-Naphthol and β-Naphthol in water samples by capillary electrophoresis coupled with large volume sample stacking

ABSTRACT A simple, fast, and efficient on-line pre-concentration based on large volume sample stacking (LVSS-CE) was developed for the determination of α-Naphthol, and β-Naphthol (α-NAP and β-NAP) in complex matrices. Parameters that affect the analytical performance of LVSS were systematically optimised using a Box-Behnken design (injection time, pre-concentration time, and voltage of reverse polarity). Under the optimal conditions of injection time (180 s), pre-concentration time (180 s), and voltage of reverse polarity (−6 kV), the sensitivity was improved 25.9 and 38.8-fold for α-NAP and β-NAP in comparison to CE-UV. The detection limits for α-NAP and β-NAP were 6.56 and 4.63 µg L−1, and the method was successfully applied in drinking and tap water samples with recoveries from 92.98 to 97.46 with RSD less than 10.00% in all cases.

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.

On-line sample preconcentration by LVSEP-FASI with simple voltage control on Y-channel chips.

To eliminate complicated voltage controls for highly sensitive microchip electrophoresis (MCE) analyses on the basis of combining two online sample preconcentration techniques, large-volume sample stacking with an electroosmotic flow (EOF) pump (LVSEP) and field-amplified sample injection (FASI), cross-channel microchips and a multichannel high-voltage power supply were replaced to Y-channel chips and a conventional power supply designed for capillary electrophoresis, respectively. By simple switching of the electric circuit after the LVSEP-FASI sample enrichments, the focused analytes could be separated during anodic migration in a separation channel. In the LVSEP-FASI analysis of fluorescein using the Y-channel microchip, the maximum sensitivity enhancement factor (SEF) of 7400 was achieved, resulting in a 30-fold detectability increase compared to the conventional LVSEP. The developed method was applied to the oligosaccharide analysis in MCE. As a result, the SEF for maltotriose was improved from 450 to 2300 and the baseline separation of the oligosaccharides was achieved without any complicated voltage control in LVSEP-FASI on the Y-channel chips.

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.

Paper electrophoretic enrichment-assisted ultrasensitive SERS detection

To achieve sensitive detection of trace substances in fluids by surface-enhanced Raman spectroscopy (SERS), effective enrichment of molecules at subwavelength regions (hot spots) with a large enhancement is adopted. In this work, a glass fibre paper with Ag nanoparticles (AgNPs) is employed for electrodynamic enrichment of analytes in fluids by paper electrophoresis integrated with field amplification sample stacking (FASS) and capillary effects to obtain both Raman and SERS convenient and sensitive detection. With the help of electrophoretic enrichment on the glass fibre paper and surface plasmon enhancement on the AgNPs, this paper electrophoretic enrichment could improve the detection limit of Raman and SERS detection by more than an order of magnitude, even achieving a SERS detection limit of 10−17 M for Nile Blue A. Furthermore, this flexible SERS detection method can also detect trace organic contaminants at the ppt level in aquaculture and food applications.

Determination of ticagrelol in rat plasma and tablets by micellar electrokinetic chromatography coupled with large volume sample stacking: Application to a pharmacokinetic study.

A method using micellar electrokinetic chromatography coupled with large-volume sample stacking for the determination of ticagrelol was developed and validated. The analysis was performed in a fused silica capillary (41.5cm effective length, 50μm diameter) with ultraviolet detection at 195nm. The background electrolytes were 30mM phosphate buffer of pH 3.0 with 120mM sodium dodecylsulfate and 10 % (v/v) acetonitrile (120 s X 50mbar; 20°C; -18kV) and 30mM borate buffer of pH 8.5 with 75mM sodium dodecylsulfate (120 s X 50mbar; 20°C; 25kV); under acidic and alkaline conditions, respectively. The method was found to be reliable with respect to specificity, linearity of the calibration line (R2 >0.99), repeatability (relative standard deviation 2.56%-3.34%), and accuracy (recovery in the range 101.21%-102.67%). The limits of detection and quantitation were 0.032, 0.071, and 0.087, 0.188μg/mL, respectively. The method was successfully applied for the determination of ticagrelol concentrations in rat plasma and tablets with good recoveries and reproducibility. The presented method proved to be suitable for monitoring ticagrelor in rat plasma.

Method Development for Determination of Doripenem in Human Plasma via Capillary Electrophoresis Coupled with Field-Enhanced Sample Stacking and Sweeping.

In this study, we established a novel capillary electrophoresis method for monitoring the concentration of doripenem in human plasma. As a time-dependent antibiotic, doripenem maximizes its antibacterial effects and minimizes the potential for antibiotic resistance through careful therapeutic drug monitoring. Two online preconcentration techniques, field-enhanced sample stacking (FESS) and sweeping, were coupled to enhance the detection sensitivity. Briefly, an uncoated fused silica capillary (40 cm × 50 μm i.d) was rinsed with a high conductivity buffer (HCB) composed of 150 mM phosphate buffer (NaH2PO4, pH 2.5) and 20% methanol. A large sample plug prepared in a low-conductivity phosphate buffer (50 mM NaH2PO4, pH 2.5) was then hydrodynamically injected (5 psi, 80 s) into the capillary. Under an applied voltage of -30 kV, the analyte was accumulated at the FESS boundary and swept by the negatively charged micelles toward the UV detector. Plasma samples were pretreated by solid-phase extraction (SPE) to eliminate endogenous interferences. The validation results demonstrated a high coefficient of determination (r2 > 0.9995) for the regression curve with impressive precision and accuracy: relative standard deviation (RSD) <5.86% and relative error <4.63%. The limit of detection (LOD, S/N = 3) for doripenem was determined to be 0.4 μg/mL. Compared to the conventional micellar electrokinetic chromatography method, our developed method achieved a sensitivity enhancement of up to 488-fold for doripenem. Furthermore, the newly developed method successfully quantified doripenem concentrations in plasma samples obtained from patients accepting doripenem regimens, proving its application potential in the clinical realm.